Specification
NOVEL CONJUGATES. PREPARATION THEREOF, AND THERAPEUTIC USE
THEREOF
The present invention relates to cryptophycin conjugates, to compositions containing them and to
their therapeutic use, especially as anticancer agents. The invention also relates to the process
for preparing these compounds and to the cryptophycin derivatives themselves.
[Technical Field]
Cryptophycins are secondary metabolites belonging to the class of depsipeptide macrocycles
produced by cyanobacteria of the genus Nostoc. Their name refers to the fact that they are highly
cytotoxic towards yeasts of the genus Cryptococcus. The first representative of this class of
molecules, cryptophycin-1 (C-1), was isolated in 1990 from cyanobacterium Nostoc sp (ATCC
53789) (see EiBler S., et al., Synthesis 2006, 22, 3747-3789). The structure, the general formula
and the numbering of the carbon atoms of these compounds, as described in WO 98/08505, are
recalled below:
Cryptophycins C-1 and C-52, which are characterized by an epoxide function represented below,
have anticancer properties. Phase II clinical trials in lung cancer were conducted with C-52
(LY 355073): see Edelman M.J., et al., Lung Cancer 2003, 39, 197-199; Sessa C, et al.,
Eur. J.Cancer 2002, 38, 2388-96. Cryptophycin C-55, a prodrug of C-52, is itself characterized by
a chlorohydrin function instead of the epoxide function (Bionpally R.R., et al., Cancer Chemother
Pharmacol 2003, 52, 25-33). C-55 proved to be very active, but is not stable in solution.
Derivatives of chlorohydrin glycinate type such as the compound C-55 Gly have also been
described as gaining in stability (Liang J., et al., Investigational New Drugs 2005, 23, 213-224).
[Technical Problem]
Conjugate chemistry has been known for many years and has been applied to several families of
cytotoxic agents, for instance the maytansinoids (WO 04/103 272), taxanes (WO 06/061 258),
tomaymycins (WO 09/016 516), leptomycins (WO 07/144 709), CC-1065 and analogues thereof
(WO 2007/102 069); see also, with regard to conjugates, Monneret C, et al., Bulletin du Cancer.
2000, 87(11), 829-38; Ricart A.D., et al., Nature Clinical Practice Oncology 2007, 4, 245-255.
However, it has not been applied to cryptophycin derivatives conjugated to antibodies or to other
targeting agents.
The technical problem that the present invention intends to solve is that of proposing novel
conjugates based on cryptophycin derivatives, and also novel cryptophycin derivatives that are
suitable for being conjugated.
[Prior Art]
EP 0 830 136 and WO 98/08505 describe cryptophycin derivatives but do not describe
cryptophycin conjugates. WO 98/08505 describes cryptophycin derivatives of formula (A):
(A) in which Ar may represent a group Ar' of
formula: in which R54 represents H, a group (C1-C6)alkyl, (C1-C6)elkyl(R57.R57'R,57"),
aryl, phenyl, heterocycloalkyl, a halogen atom, COOR57, P03H, SO3H, SO2R58. NR59R60,
NHOR6i, NHOR61', CN, NO2, OR62, ChbORg;', CH2NRagRog', (C1-C6)alkylOR100.
SR63; R55 and R56 represent H, a group (C1-C6)alkyl, C(R57.R57'.R57"), aryl, phenyl,
heterocycloalkyl, a halogen atom, COOR57, PO3H, SO3H, SO2R58, NR59R60, NHOR61, NHCHR61',
CN, NO2, OR62, CH2OR62', CH2OCOR95, CH2NR9gR96', (C1-C6)alkylOR100, (C1-C6)alkyiNR59R60.
WO 98/08505 especially describes the following compounds:
which are characterized by the end group -NHC(=O)Ot-Bu. WO 98/08505 does not specify that
these compounds are suitable for or intended to be conjugated.
US 2007/0 213 511 describes calicheamicin immunoconjugates. Among these, mention is made
of MYLOTARG® (or CMA-676), which is a calicheamicin immunoconjugate used in the treatment
of AML (anti-CD33-calicheamicin). See also in respect of conjugates: WO 2006/042 240.
Al-awar R.S., et al., J.Med.Chem. 2003, 46(14), 2985-3007 and Al-awar R.S., et al., Mol.Cancer
Ther. 2004, 3(9), 1061-1067 describe cryptophycin derivatives, and also their in vivo evaluations.
WO 08/010 101 describes an anti-EphA2 monoclonal antibody and also the corresponding
conjugates comprising one or more molecules of a cytotoxic compound attached to the
monoclonal antibody. WO 08/047 242 describes an anti-CD38 monoclonal antibody and also the
corresponding conjugates comprising one or more molecules of a cytotoxic compound attached
to the monoclonal antibody. The cytotoxic compound may be chosen from maytansinoids,
taxanes, tomaymycins, leptomycins and CC-1065 and analogues thereof.
WO 2009/126 934 describes anti-CD70 antibodies and their conjugates with cytotoxic
compounds; cryptophycin is mentioned among the cytotoxic agents. WO 2009/134 976 describes
conjugates with an optimized degree of substitution for delivering the required amount of
cytotoxic agent into the cell; cryptophycin is mentioned among the cytotoxic agents.
WO 2005/116 255 describes conjugates of aptamers and of a cytotoxic agent that may be a
cryptophycin (see [0037] and Table 2), the linker possibly comprising a PEG chain ([0038]). More
particularly, the cryptophycin Cryp-NH2 is described:
and also its conjugates of aptamers with the following linkers: NHS-PEG-erythritol, pNP-PEG-
erythritoi, NHS-PEG-octaPEG, pNP-PEG-octaPEG and PEG-comb (Tables 3 and 4). The nature
of the sequence on the phenyl ring (-CH2O-C(=O)-CMe2-CH2NH-...) is different from that which is
envisaged in the present invention. WO 2006/096 754 also describes conjugates of aptamers and
of cytotoxic agent that may also be a cryptophycin.
WO 2009/002 993 describes cytotoxic conjugates of formula B-L-A comprising hydrophilic linkers,
for example the linker of formula:
The cytotoxic agent may be a cryptophycin (page 46), but the point of attachment of the linker is
not specified. An example of a conjugate is ECO262:
whose linker and point of attachment are different from those envisaged in the present invention.
[Definitions]
The following definitions apply:
• conjugate: a cell binding agent to which is covalently attached at least one molecule of a
cytotoxic compound;
• cell binding agent: a molecule with affinity for a biological target: it may be, for example, a
ligand, a protein, an antibody, more particularly a monoclonal antibody, a protein or antibody
fragment, a peptide, an oligonucleotide or an oligosaccharide. The function of the binding agent
is to direct the biologically active compound as a cytotoxic agent towards the biological target.
Preferably, the binding agent is not an aptamer;
• biological target: an antigen (or group of antigens) preferably located at the surface of cancer
cells or stromal cells associated with this tumour; these antigens may be, for example, a growth
factor receptor, an oncogene product or mutated "tumour suppressant" gene product, an
angiogenesis-related molecule, an adhesion molecule;
• linker: a group of atoms that can covalently attach a cytotoxic compound to the binding agent;
• alkyl group: a saturated aliphatic hydrocarbon-based group obtained by removing a hydrogen
atom from an alkane. The alkyl group may be linear or branched. Examples that may be
mentioned include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, 2,2-
dimethylpropyl and hexyl groups;
• cycloalkyl group: a cyclic alkyl group comprising between 3 and 8 carbon atoms engaged in
the cyclic structure. Examples that may be mentiond include cyclopropyl, cyclobutyl, cyclopentyl
and cyclohexyl groups;
• heterocycloalkyl group: a cycloalkyl group comprising at least one neteroatom (O, S or N)
engaged in the ring and connected to the carbon atoms forming the ring;
• alkoxy group: a group -O-alkyl, in which the aikyl group is as defined above;
• alkanoyloxy group: a group -O-CO-alkyl, in which the alkyl group is as defined above;
• alkylene group: a saturated divalent group of empirical formula -CnH2n-, obtained by removing
two hydrogen atoms from an alkane. Examples that may be mentioned include methylene
(-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-) and
hexylene (-CH2CH2CH2CH2CH2CH2-) groups or the following branched groups
• preferably, the alkylene group is of the formula -(CH2)n-, n representing an integer;
• in the ranges of values, the limits are included (e.g. a range of the type "n ranging from 1 to 6"
or "between 1 and 6" includes limits 1 and 6).
Abbreviations used
EtOAc: ethyl acetate; ALK: (CrCi2)alkylene group, more particularly (C1-C6)alkylene, more
particularly of the form -(CH2)n-, n being an integer from 1 to 12 and preferably from 1 to 6; aq.:
aqueous; TLC: thin-layer chromatography; MSC: methanesulfonyl chloride; crypto denotes the
unit of formula crypto especially denoting one of the D1-Ds
cryptophycin derivatives described later or a cryptophycin derivative of an example; Rf: retention
factor; DAR: degree of substitution (drug-antibody ratio); DBU: 1,8-diazabicyclo[5.4.0]undec-7-
ene; DCC: N,N'-dicyclohexylcarbodiimide; DCM: dichloromethane; DEAD: diethyl
azodicarboxylate; DIC: N,N'-diisopropylcarbodiimide; DIPEA: N,N-diisopropylethylamine; DMA:
dimethylacetamide; DMAP: 4-dimethylaminopyridine; DME: dimethoxyethane; DMF:
dimethylformamide; DMSO: dimethyl sulfoxide; EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-
dihydroquinoline; EDCI: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; EDTA: ethylene-
diaminetetraacetic acid; eq.: equivalent; Fmoc: fluorenylmethoxycarbonyl; HOBt:
1-hydroxybenzotriazole; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; mCPBA:
m-chloroperbenzoic acid; NHS: N-hydroxysuccinimide; NMP: N-methylpyrrolidinone; AP:
atmospheric pressure; PABAC: "para-aminobenzyl alcohol carbonate"; RP: reduced pressure;
SEC: steric exclusion chromatography; SPE: solid-phase extraction; RT: room temperature;
TBDMS: terf-butyldimethylsilyl; TCEP: tris(2-carboxyethyl)phosphine hydrochloride; TEA:
triethylamine; TFA: trifluoroacetic acid; TIPS: triisopropylsilyl; THF: tetrahydrofuran; tR: retention
time.
[Detailed Description of the invention]
The invention relates to a binding agent to which is attached at least one cryptophycin derivative
of formula (I):
in which:
■ R1 represents a halogen atom and R2 represents a group -OH, an acyl group derived from an
amino acid AA or a group (C1-C4)alkanoyloxy;
or alternatively R1 and R2 form an epoxide unit;
• AA denotes a natural or unnatural amino acid;
■ R3 represents a group (C1-C6)alkyl;
■ R4 and R5 both represent H or together form a double bond CH=CH between C13 and C14;
■ R6 and R7 represent, independently of each other, H or a group (C1-C6)alkyl;
■ Rs and R9 represent, independently of each other, H or a group (C1-C6)alkyl;
• R10 represents at least one substituent of the phenyl nucleus chosen from: H, a group -OH,
(C1-C4)alkoxy, a halogen atom or a group -NH2) -NH(C1-C6)alky! or -N(C1-C6)alkyl2;
■ R11 represents at least one substituent of the phenyl nucleus chosen from H and a group (Cr
C4)alkyl;
the binding agent and the cryptophycin derivative being covalently attached, the attachment
being in the ortho (o), meta (m) or para (p) position of the phenyl nucleus bearing the unit CR1.
ortho (o), meta (m) or para (p) positions:
Rt represents a halogen atom, more particularly CI, R3 represents a group (C1-C6)alkyl, more
particularly Me, R6 and R7 represent, independently of each other, H or a group (C1-C6)alkyl;
more particularly, they represent, independently of each other, H or a group Me, R8 and R9
represent, independently of each other, H or a group (C1-C6)alkyl; more particularly, R8
represents H and R9 represents isobutyl.
R10 represents at least one substituent of the phenyl nucleus chosen from H, a group OH, (C1-
C4)alkoxy or a halogen atom. It may also be a group -NH2, -NH(C1-C6)alkyl or -N(C1-C6)alkyl2
such as, for example, -NH2 or -NMe2, preferably in position 3 or 4 on the phenyl nucleus. More
particularly, the phenyl nucleus comprises two substituents in positions 3 and 4 on the phenyl
nucleus. Preferably, it is 3-CI and 4-methoxy. R11 represents at least one substituent of the
plenyl nucleus chosen from H and a group (C1-C4)alkyl; more particularly H.
More particularly, one of the substituents R1 to R11 may be chosen from those described in the
examples. Each substituent R1 to R11 may also adopt one of the spatial configurations (e.g. R or
S or alternatively Z or E) as described in the examples.
AA represents a natural or unnatural amino acid. It may be an α, β or γ amino acid. Mention may
be made especially of the following amino acids: alanine (Ala), β-alanine, 2-amino-2-
cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine (Arg), aspartic acid (Asp), cysteine
(Cys), glutamine (Gin), glutamic acid (Giu), glycine (Gly), histidine (His), isoleucine (lie), leucine
(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine
(Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val), y-aminobutyric acid, α,α-dimethyl-y-
aminobutyric acid, β,β-dimethyl-y-aminobutyric acid, ornithine (Orn), citrulline (Cit) and also the
protected forms of the said amino acids (e.g. acetyl, formyl, tosyl, nitro). Preferably, it is a natural
amino acid. More particularly, it is glycine.
R.) and R2 may also together form an epoxide unit.
The attachment between the cryptophycin derivative and the binding agent is produced by means
of a linker L positioned in an ortho (o), meta (m) or para (p) position of the phenyl nucleus bearing
the unit CR1; thus, the cryptophycin derivative capable of conjugation has the formula (II):
The attachment to the binding agent takes place at the other end of the linker L on a reactive
group present on the binding agent. Thus, L comprises at ieast one reactive chemical group
(RCG1) that is reactive towards a reactive chemical group (RCG2) present on the binding agent.
The reaction between RCG1 and RCG2 ensures the attachment of the compound of formula (II)
to the binding agent by formation of a covalent bond. Thus, the cryptophycin derivative of formula
(II) is able to be conjugated to a binding agent.
The cryptophycin derivatives of the present invention, including those that are illustrated, may
exist in the form of bases or of acid-addition salts, especially of pharmaceutically acceptable
acids.
Examples of RCG1 that may be mentioned include:
(i)the reactive group -SZa for which Za represents H or the group -SRa and Ra represents a
group (C1-C6)alkyl, (C3-C7)cycloalkyl, aryl, heteroaryl or (C3-C7)heterocycloalkyl;
(ii) the reactive group -C(=O)-ZbRb for which Zb represents a single bond, -O- or -NH-, more
particularly -O-, and Rb represents H or a group (C1-C6)alkyl, (C3-C7)cycloalkyl, aryl, heteroaryl or
(C3-C7)heterocycloalkyl;
(iii) one of the following reactive groups: -CI, -N3, -OH, -NH2, the maleimido
haloacetamido reactive group with R12 representing H or a group (C1-C6)alkyl, more
particularly Me in the case of the compounds of formula (III):
comprising the group G= -(CH2)nY with Y= -CI, -N3, -OH, -NH2, with R12
representing H or a group (CrCeJalkyl, more particularly Me;
(iv) the reactive group maleimido or haloacetamido with Ri2 representing H or
a group (C1-C6)alkyl, more particularly Me.
More particularly, -SZa may represent -SH or -SS(C1-C6)alkyl, especially -SSMe or -SS-
heteroaryl, especially (Xi and X2 being defined hereinbelow). More
particularly, -ZbRb may represent -OH, -OCH3, -OCH2CH=CH2)
or the group in which Gl represents at least one electroinductive group such
as -NO2 or -Hal, especially -F. They may be, for example, the following groups:
or . Another type of group -C(=O)ZbRb is the following: The reactive groups
-SH and show good reactivity. More particularly, RCG1 may be chosen from one of
those described in the examples.
Examples of RCG2 that may be mentioned include e-amino groups of lysines borne by the side
chains of the lysine residues that are present at the surface of an antibody, the saccharide groups
of the hinge region or the thiols of cysteines by reducing intra-chain disulfide bonds (Garnett
M.C., et al., Advanced Drug Delivery Reviews 2001, 53, 171-216). More recently, other
approaches have been considered, for instance the introduction of cysteines by mutation
(Junutula J.R., et al., Nature Biotechnology 2008, 26, 925-932; WO 09/026 274) or the
introduction of unnatural amino acids allowing other types of chemistry (de Graaf A.J., et al.,
Bioconjugate Chem. 2009, Publication Date (Web): February 3, 2009 (Review); DOI:
10.1021/bc800294a ; WO 2006/069 246 and according to Chin J.W., et al., JACS 2002, 124,
9026-9027 (technology ReCode®)). These modes of attachment used with antibodies are
applicable to all of the known binding agents as a function of their structure.
It is also possible to chemically modify the binding agent so as to introduce novel reactive
chemical groups RCG2. Thus, it is well known to those skilled in the art how to modify an
antibody with the aid of a modifying agent (see especially WO 2005/077 090 page 14). The
modification makes it possible to improve the conjugation reaction and to use a wider variety of
groups RCG1.
Modifying agents for introducing disulfide groups
The modifying agent may be an activated ester NHS of formula in which R
represents a group (C1-C6)alkyl, aryl, heteroaryl, (C3-C7)cycloalkyl, (C3-C7)heterocycloalkyl and
ALK represents a group (C1-C6)alkylene; for example, it is possible to use N-succinimidyl
pyridyldithiopropionate (SPDP) or N-succinimidyl pyridyldithiobutyrate (SPDB or the N-hydroxy-
succinimidyl ester of 4-(2-pyridyldithio)butanoic acid) so as to introduce dithiopyridyl reactive
groups RCG2 (see Bourdon M.A., et al., Biochem. J. 1978, 173, 723-737 ; US 5 208 020) which
may then react with a reactive chemical group RCG1 of the type -SH present on the linker of the
cryptophycin derivative so as to form a novel -S-S- bond (see Ex. 9 for a conjugate with a
disulfide bond). The N-hydroxysuccinimide group preferentially reacts on the amino groups
present on the antibody so as to form amide bonds. Another example of a modifying agent is
described in WO 2004/016 801 of formula or a pegylated
analogue of formula described in WO 2009/134 976 or a
sulfonic analogue of formula described in WO 2009/134 977 in which:
- X3, X4, X5 and X6 represent H or a (d-C6)alkyl group,
- X1 and X2 represent -H, -CONXsXg, -NO2, -X8 and -X9 representing H or a group (C1-C6)alkyl,
- X7 represents -SO3"M+ or H, or alternatively a quaternary ammonium group,
- a denotes an integer from 0 to 4 and b denotes an integer ranging from 0 to 2000 and preferably
between 1 and 200; a and b may take all the values between, respectively, 0 and 4 or between 0
and 2000.
Modifying agents for introducing maleimido groups
Another example of a modifying agent is succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-
carboxylate (SMCC), a similar compound described in EP 0 306 943 or a sulfo-SMCC
(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Other examples that may
be mentioned include: such as N-succinimidyl 3-maleimidopropanoate;
such as N-succinimidyl 6-(3-maleimidopropionamido)hexanoate;
b being an integer between 0 and 2000 and preferably between 1
and 200 (b may take ail the values between 0 and 2000), such as N-succinimidyi 3-(2-{2-[3-
maleimidopropionylamino]ethoxy}ethoxy)propanoate or SM(PEG)2; such
as maleimidoethyl N-succinimidyl succinate; such as N-succinimidyl 4-(4-
maleimidophenyl)butanoate, or - such as N-succinimidyl 3-maleimidobenzoate.
Nfl&difyinq agents for introducing thiol groups
Another example of a modifying agent described in WO 90/06774 is of the formula
in which:
- Hal represents a halogen atom;
- X10 represents a halogen atom or a group COOX14, nitro, unsubstituted or halogenated (Cr
C8)alkyl, unsubstituted or halogenated (CrC8)alkoxy, unsubstituted or halogenated (C2-
C8)alkenyl, unsubstituted or halogenated (C2-C8)alkynyl, unsubstituted (C3-C8)cycloalkyl, aryl that
is unsubstituted or substituted with one to three substituents selected from amino, halogen atom,
unsubstituted or halogenated (CrC8)alkyl group, unsubstituted or halogenated (CrC8)alkoxy;
- each of the X11t X12 and X13 independently represents a hydrogen atom or may represent X3;
orXio and Xn together form a ring (C2-C5)alkylene, which is unsubstituted or substituted with one
to five groups (C1-C4)alkyl;
or X-io and Xn form, together with Xi2, a ring (CrC5)alkylene, which is unsubstituted or
substituted with one to five groups (C1-C4) alkyl;
and X14 is -H or a group (CrC8)alkyl.
Preferably, Hal represents a chlorine or bromine atom. The table below presents possibilities for
X1O-X13:
An example of a preferred iminothiolane is the following:
Modifying agents for introducing haloacetamido groups
Another example of a modifying agent is succinirnidyl-4-(N-iodoacetyl)aminobenzoate
(SIAB) , or similar compounds, among which are succinimidyl-N-iodoacetate
(SIA) succinimidyl-N-bromoacetate (SBA), or succinimidyl-3-(N-
bromoacetamido)propionate (SBAP) or a similar pegylated compound described in
WO 2009/134 976 b being as described previously. Figures 1 and 2
illustrate the modification of an amino group of a binding agent with SPDP or with the preferred
iminothiolane above.
Thus, it is possible to introduce onto the binding agent disulfide groups RCG2 (-SSR), especially
of pyridyldisulfide type in the case where RCG1 represents -SH.
Similarly, it is possible to introduce onto the binding agent thiol groups RCG2 (-SH), for example
with an iminothiolane, in the case where RCG1 represents disulfide (i.e. RCG1= -SZa with Za?H,
for example In both cases, the covalent bond that forms by reaction between
RCG1 and RCG2 is a cleavable disulfide bond.
It is also possible, in the case where RCG1 represents -SH, to introduce onto the surface of the
binding agent RCG2 groups of maleimido type or haloacetamido type (e.g. bromo- or
iodoacetamido Reciprocally, it is possible to introduce onto the binding agent thiol
groups RCG2 (-SH), for example with an iminothiolane, in the case where RCG1 represents
In this case, the covalent bond that forms by reaction between RCG1 and
RCG2 is an uncleavable sulfide bond.
tmre particularly, in the case where RCG1 is of the type (iii) above, it is possible to chemically
modify the binding agent using an adequate modifying agent or to introduce one or more
unnatural amino acids so as to introduce the adequate functions RCG2. For example:
- with a function -N3: RCG2 may be a group -C=CH;
- with a function -OH or -NH2: RCG2 may be a carboxylic acid function;
- with a function -CI: RCG2 may be a group -SH.
In the case where RCG1 represents a maleimido or haloacetamido reactive
group with R12 representing H or (C1-C6)alkyl, more particularly Me, the cryptophycin derivative
may be represented by the formula (Ila) or (lib) below:
c
(L* represents a fragment of a linker L such that L= -L*-maleimido or L= -L*-haloacetamido)
The cryptophycin derivative may be, in the series C-52 and C-1, one of the following DrD8:
or an equivalent unit described in one of the examples.
More particularly, L is in the para position of the unit RC^.
Process for preparing the cryptophycin derivatives
The compounds of formula (II) are prepared according to Scheme 1 starting with a cryptophycin
derivative of formula (III) and a linker precursor (LP):
G represents the group -CH=CH2, -(CH2)nY or -(d-CeJalkylene-Y with n being an integer ranging
from 1 to 6 and Y representing -OH, -SH, -CI, -OLG in which LG denotes a leaving group, for
instance a mesylate (OMs) or tosylate group, or alternatively Y
represents -N3, -NH2, -COOH, -NR12-CH2-CECH in which R12 represents H or a group (Cr
C6)alkyl, more particularly a methyl group. The linker precursor LP has the function of introducing
the linker L into the cryptophycin derivative after reaction between the group G and a chemical
function present on LP.
G may also represent the group (i.e. Y represents the group chosen
from one of the following 9 groups (Ri2 and R'i2 represent H or a group (C1-C6)alkyl):
In Scheme 1, several steps and/or reactions may be necessary to arrive at the cryptophycin
derivative (II) starting from the cryptophycin derivative (III). Thus, for example, in the case where
Za=H, it is preferred to introduce a linker L for which Za=-S(C1-C6)alkyl using the corresponding
linker precursor, and then to reduce the disulfide function -SS(C1-C6)alkyl to a thiol function -SH.
It^s possible to use in order to do this, for example, TCEP: see in this respect Burns J.A., et al.,
J.Org.Chem. 1991, 56(8), 2648-2650. This conversion -SS(C1-C6)alkyl * -SH may apply, for
example, to the linkers LM and L21.23 of Table II.
Similarly, in the case where ZbRb= , a linker L for which ZbRb=-O-allyl may be introduced
using the corresponding linker precursor, followed by deprotecting the function -COOH and
introducing . Deprotection may be performed by treatment with a palladium catalyst, for
example Pd(PPh3)4 in the presence of a "scavenger" amine, for example morpholine; the
activation may be performed with N,N'-disuccinimidyl carbonate in the presence of a base, for
example DIPEA or with NHS in the presence of a coupling agent, for example DCC. This
conversion of a group ZbRb into another group ZbRb (e.g. -O-al!yl - may be applied to
obtain other groups ZbRb, especially those described above.
In the case where Ri represents a halogen atom and R2 represents an acyl group, it is preferred
first to prepare a compound of formula (II) for which R2 represents a group -OH (once the linker
has been introduced), and then to introduce the acyl group using the corresponding acylating
compound.
Schemes 1' and 1" similarly illustrate the preparation of a cryptophycin derivative comprising a
linker comprising, respectively, a maleimido or haloacetamido group (L* represents a fragment of
a linker such that L= -L*-maleimido or L= -L*-haloacetamido).
T%ese derivatives are obtained by reaction between a cryptophycin derivative comprising a linker
L' comprising an amino or thiol group and a modifying agent for introducing, respectively, a
maleimido or haloacetamido group.
Examples of reactions between the group G and a chemical function present on LP
• nucleophilic substitution between a linker precursor LP bearing an amine function -NH- (an
amine salt may also be suitable for use) and G=-(CH2)nCI or -(CH2)nOMs (see, for example,
Table II, LP^, LP7a, LPs-io, LP21-23): this reaction may be performed in a polar aprotic solvent in
the presence of a base, for instance TEA or DIPEA. See Ex. 1, compound 7 or Ex. 15,
compound 48;
• acylation between a linker precursor LP bearing a carbamoyl halide function and
G=-(CH2)nOH (see, for example, Table II, LP5): this reaction may be performed in a polar
aprotic solvent in the presence of an amine base, for instance TEA.
According to one variant, it is also possible to react a linker precursor LP bearing an amine
function -NH- and G=-(CH2)nO-C(=O)-O-(4-nitrophenyl) obtained from G=-(CH2)nOH and
p-nitrophenyi ohioroformate (activation of the alcohol in the form of carbonate) according to the
scheme below (R12= H or (C1-C6)alkyl):
-NHR12 + crypto-(CH2)nO-C(=O)-O-(4-nitrophenyl) -* crypto-(CH2)nO-C(=O)-NR12-
• the activation of an alcohol in the form of carbonate may also be used to react a linker
precursor bearing a function -OH and G=-(CH2)nNH2 or -(CH2)nOH to obtain, respectively, a
carbamate function (-O-C(=O)-NH-) or carbonate (-O-C(=O)-O-) according to the following
respective schemes:
-O-C(=O)-O-(4-nitrophenyl) + crypto-(CH2)nNH2 -> crypto-(CH2)nNH-C(=O)-O-
-OH + crypto-(CH2)nO-C(=O)-O-(4-nitrophenyl) -> crypto-(CH2)nO-C(=O)-O-.
(see, for example, Table II, LP6a-6b. LP24-2s)
«esterification between a linker precursor LP bearing an acid function -COOH and
G=-(CH2)nOH (see, for example, Table II, LP-|4b): this reaction may be performed in a polar
aprotic solvent in the presence of an amine base, for instance DMAP and a coupling agent, for
example DCC;
• amidation between a linker precursor LP bearing an acid function -COOH and G=-(CH2)nNH2
(see, for example, Table II, LP14a): this reaction may be performed in a polar aprotic solvent in
the presence of a coupling agent, for example EDCI or HOBt;
• amidation between a linker precursor LP bearing a function -NH2 and G=-(CH2)nCOOH (see,
for example, Table II, LP7c): this reaction may be performed in a polar aprotic solvent in the
presence of a coupling agent, for example EDCI or HOBt;
11,3-dipolar cycloaddition (also known as "click" chemistry) between a linker precursor LP
bearing an alkyne terminal function and G=-(CH2)nN3 or alternatively a linker precursor LP
bearing an azide function and G=-(CH2)nNR12-CH2C=CH (see, for example, Table II, LP15.t8):
this reaction may be performed in a polar solvent in the presence of Cu(l) as catalyst (see in
this respect the Huisgen cycloaddition: Rostovtsev V.V., et al., Angew. Chem. Int. Ed. 2002, 41,
2596-2599; Tornoe C.W., et al., J. Org. Chem. 2002, 67, 3057-3064);
• metathesis between a linker precursor LP bearing an ethylenic end function and G=-CH=CH2
(see Table II, LP19, LP20): this reaction may be performed in a polar aprotic solvent in the
presence of a second-generation Grubbs catalyst (CAS No. 246047-72-3, see in this respect
Poeylaut-Palena A.A., et al., J. Org. Chem. 2008, 73, 2024-2027).
As regards the linker L
The linker L may be chosen from one of the following:
-G' X (CR13Ri4MOCH2CH2)y(CR15Ri6)u Q RCG1;
-G' X (CR13Ri4)t(OCH2CH2)y-Y'-(CRi5Ri6)u Q RCd;
-G' X (CRi3Ri4)t(CRi7=CRi8)(CRi5Ri6)u(OCH2CH2)y Q RCG-i;
-G' X (CR13Ri4)t(OCH2CH2)y(CR17=CR18)(CR15Ri6)u Q RCG1;
-G' X (CRi3Ri4)rPhenyl-(CR15Ri6)u V Q RCGi; -G' X (CR13R14)rfuryl-(CR15Ri6)u V Q RCG1;
-G' X (CR13R14)t-oxazolyl-(CR15Ri6)u Y' Q RCG1; -G' X (CR13R14)rthiazolyl-(CR15Ri6)u V Q RCd;
-G' X (CRi3Ri4)rthienyl-(CRi5Ri6)u Y" Q RCG1; -G' X (CR13Ru)rimiclazolyl-(CRi5Ri6)u Y' Q RCd;
-G' X (CRi3R14)rpiperazinyl-CO(CRi5Ri6)u Y' Q RCGi; -G' X (CR13R14)rpiperidyl-methyl-NR12-
CO(CR15Ri6)u Y' Q RCG1; -G' X (CR13R14)rpiperidyl-(CR15Ri6)u Y* Q RCG1;-G" X (CR13R14)r
piperidyl-NR12-(CR15Ri6)u Y' Q RCGi;-G" X (CR13R14)rtriazolyl-(CR15R16)u Y' Q RCG1;-G' X
(CRi3R14)t-triazolyl-(CR15Ri6)u Y' Q RCGi;
-G' X (CRi3R14)rphenyl-(CR15R16)u Q RCd; -G' X (CRi3R14)rfuryl-(CR15R16)u Q RCd, -G' X
(CR13Ri4)t-oxazolyl-(CR15R16)u Q RCG^ -G' X (CR13R14)rthiazolyl-(CR15Ri6)u Q RCGT; -G' X
(CR13Ri4)rthienyl-(CR15R16)u Q RCG^ -G' X (CR13R14)rimidazolyl-(CR15Ri6)u Q RCGT; -G' X
(CRi3Ri4)rpiperaziny!-(CR15Ri6)u Q RCG^ -G' X (CR13Ri4)rpiperidyl-(CR15Ri6)u Q RCG1; -G' X
(CRi3R14)rpiperidyl-methyl-NR12-(CR15Ri6)u Q RCG1; -G' X (CR^R-i^-piperidyl-NR^^CR^RieJu
Q RCG1; -G' X (CR^RuX-triazolyl^CR^RieJu Q RCG1;
or
-G" Y (CR13R14)t(OCH2CH2)y(CR15Ri6)u Q RCG1;
-G" Y (CR13R14MOCH2CH2)y-Y'-(CR15R16)u Q RCd;
-G" Y (CR^R^MCRi^CR^XCRisRieWOCHzCH^y Q RCG1;
-G" Y (CR13R14)t(OCH2CH2)y(CR17=CR18)(CRi5Ri6)u Q RCG1;
-G" Y (CR13R14)t-phenyl-(CR15Ri6)u Y' Q RCd; -G" Y (CR^R^rfuryKCR^Ri^u Y Q RCG,; -G"
Y (CR13R14),-oxazolyl-(CR15R16)u Y Q RCG^ -G" Y (CR13Ri4)t-thiazolyl-(CR15R16)u Y Q RCG,; -
G" Y (CR13Ri4)t-thienyl-(CR15Ri6)u Y Q RCG^ -G" Y (CR13R14)rimidazolyl-(CR15Ri6)u Y' Q RCd;
-C^Y (CRi3Ri4)rPiperazinyl-CO(CR15Ri6)u Y* Q RCGi: -G" Y (CRi3Ri4)rPiperidyl-methyl-NR12-
CO(CR15R16)u r Q RCd; -G" Y (CR^RuX-piperidyKCR^RieJu Y' Q RCG1; -G" Y (CR13Ri4)r
piperidyl-NR12-(CRi5Ri6)u V Q RCG1; -G" Y (CR13Ri4)rtriazoiyl-(CRi5Ri6)u V Q RCG,;
-G" Y (CR13Ri4)t-phenyl-(CR15R16)u Q RCd; -G" Y (CR13Ri4)rfuryl-(CR15Ri6)u Q RCGi, -G" Y
(CR13Ri4)rOxazolyl-(CR15Ri6)u Q RCG,; -G" Y (CRi3R14)rthiazolyl-(CR15R16)u Q RCd; -G" Y
(CR13R14X-thienyl-(CR15Ri6)u Q RCd; -G" Y (CRi3Ri4)t-imidazolyl-(CR15Ri6)u Q RCd; -G" Y
(CR13R14)rpiperazinyl-(CRi5Ri6)u Q RCG1; -G" Y (CRi3R14)rpiperazinyl-(CR15Ri6)u Q RCG^ G" Y
(CR13R14)rpiperidyl-(CRi5Ri6)u Q RCG1; -G" Y (CR13R14)t-piperidyl-methyl-NR12-(CRi5Ri6)u Q
RCG1; -G" Y (CR^R^t-piperidyl-NR^CR^RieX, Q RCG1; -G" Y (CR13Ri4)t-triazolyl-(CR15Ri6)u Q
RCG1;;
G' represents a group -CH=CH- or -(CH2)n-;
G" represents a group -(CH2)n-;
n represents an integer ranging from 1 to 6;
X represents a single bond or a group -CO-, -COO- or -CONR12-, the group CO being attached to
G';
Y represents a group -O-, -OCO-, -OCOO-, -OCONR12-, -NR12-, -NR12CO-, -NR12CONR'12-,
-NR-|2COO- or -S(O)q-, the atom 0 or the group NR12 being attached to G";
Y' represents a group -O-, -OCO-, -OCOO-, -OCONR12-, -NR12-, -NR12CO-, -NR12CONR'12-,
-NR12COO-, -S(O)q-, -CO-, -COO-, or -CONR12-;
R12, R'i2> R13, R14, R15 and R16, R17 and Ri8 represent, independently of each other, H or a group
(d-QOalkyl;
t, u and y represent integers that may range from 0 (in the case of the absent group) to 20 and
such that t+u+y is greater than or equal to 1;
q represents an integer that may be 0,1 or 2;
Q represents a single bond, a group (CrC10)alkylene or a group (OCH2CH2)i, i being an integer
ranging from 1 to 20, more particularly from 1 to 10, even more particularly from 1 to 8 or from 1
to 6, and even more particularly from 2 to 5. i may take each of the values of these ranges, and
may especially be 2, 3,4 or 5.
In the case of the linker of formula -G" Y (CR13R14)t(OCH2CH2)y-Y'-(CR15R16)Ll Q RCGL if y is 0
(no PEG group) and if Q represents a single bond, then u cannot be 0. More particularly, linkers
comprising the terminal group -NR12-C(=O)-O- (Y'=NR12; u=O; Q=single bond and
RCG!= -C(=O)ZbRb) are excluded.
y represents an integer ranging from 0 to 20, more particularly from 1 to 20, even more
particularly from 1 to 10, from 1 to 8 or from 1 to 6, and even more particularly from 2 to 5. y may
take each of the values of these ranges, and may especially be 2, 3,4 or 5.
Some of these linkers have been described in patent applications WO 07/085 930 and
WO 09/016 516.
The linker L may be chosen from one of those of formula (IV):
in which:
• (AA)W represents a sequence of w amino acids AA connected together via peptide bonds;
• w represents an integer ranging from 1 to 12 and preferably from 1 to 6;
• n represents an integer ranging from 1 to 6;
• D represents one of the following units:
for which:
R12 represents H or a group (C1-C6)alkyl;
Ri9> R20. R21 and R22 represent, independently of each other, H, a halogen atom, -OH, -CN or
a group (C1-C4)alkyl;
T attached to (CH2)n represents NR12 or O;
V1 represents O, S or NR12;
V2 represents CR^ or N;
V3, V4 and V5 are chosen, independently of each other, from CR22 and N.
An example of D2 is as follows:
AA denotes a natural or unnatural amino acid, more particularly chosen from: alanine (Ala), (3-
alanine, 2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine (Arg), aspartic
acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His),
isoleucine (lie), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro),
se%ie (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val), y-aminobutyric acid,
a,a-dimethyl-y-aminobutyric acid, p,p-dimethyl-y-aminobutyric acid, ornithine (Orn), citrulline (Cit).
The sequence (AA)W has the formula:
in which R23 represents a residue of one of the amino acids described
above. Examples of sequences are as follows: Gly-Gly, Phe-Lys, Val-Lys, Val-Cit, Phe-Phe-Lys,
D-Phe-Phe-Lys, Gly-Phe-Lsy, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, lle-Cit, Trp-Cit, Phe-Ala, Ala-
Phe, Gly-Gly-Gly, Gly-Ala-Phe, Gly-Val-Cit, Gly-Phe-Leu-Cit, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu.
The linker precursors are those comprising the corresponding -OH units:
WO 2005/082 023 (see especially pages 61-64) describes how to obtain certain linker precursors.
The preparations of the linker precursors LP25 and LP26 described below may also be used for
obtaining other similar linker precursors comprising another sequence (AA)w.
The linker L may also be chosen from one of those described in Table II or from the illustrated
compounds. In all the formulae of linkers, NR12 or NR'12 more particularly represents NH or NMe.
Preparation of the compounds of formula (III)
in the case where G=-(CHz)nOH or -CH=CH>
Pi is prepared according to the teaching of patent applications WO 98/08505, WO 00/23429 or
WO 00/34252 and also the following publications: Rej R., et al., J. Org. Chem. 1996, 61, 6289-
6295; Salamonczyk G.M., et al., J. Org. Chem. 1996, 61, 6893-6900 or J. Med. Chem.1999, 42
(14), 2588-2603 (incorporated herein by reference). In pages 158-159 of "The isolation,
characterization and development of a novel class of potent antimitotic macrocyclic
depsipeptides: the cryptophycins", Chap.9, in "Anticancer agents from natural products",
Taylor&Francis, CRC press book, ISBN=O-8493-1863-7 are given in the synthetic schemes for
preparing the various cryptophycin fragments (A, B, C and D) and for producing Pi. Rej R., et al.,
J. Org. Chem. 1996, 61, 6289-6295 describes in Schemes 1-6 the route of access to one of the
cryptophycin derivatives of Figure 1, but these schemes may apply to the preparation of Pi using
suitable starting reagents.
Pi allows the preparation of other cryptophycin derivatives with the aid of the steps detailed
hereinbelow:
Step (i): opening of the epoxide ring of Pi in acidic medium so as to obtain the diol function.
Concentrated perchloric acid may be used, for example;
Step fli): oxidative cleavage of the diol using, for example, sodium periodate;
Step flip: Wittig reaction using a suitable phosphonium halide, for example a bromide, and a
strong base, for instance BuLi;
Step (iv): Corey-Chaykovsky epoxidation reaction involving a chiral sulfonium salt, for example a
triflate, in the presence of a base, for instance KOH;
Step (v): deprotection of the silyl ether using, for example, a tetrabutylammonium fluoride
solution.
4-(Triisopropylsiloxymethyl)benzyltriphenylphosphonium bromide is obtained from
1-(bromomethyl)-4-(triisopropylsiloxymethyl)benzene (CAS No. 934667-38-6), the preparation of
which starting from 1,4-benzenedimethanol (CAS No. 589-29-7, commercial product) is described
by Potter R.G., et al., Organic Letters 2007, 9(7), 1187-1190. The compounds for which Rn
represents a group (C1-C4)alkyl are obtained in a similar manner starting with the corresponding
diol, which is either a commercial product or is obtained by Friedel-Crafts C-alkylation starting
with 1,4-benzenedimethanol.
Starting with 1-(bromomethyl)-4-(triisopropylsiloxymethyl)benzene (CAS No. 135408-73-0), the
preparation of which is described in Scheme 4a of EP 0 496 548 or on page 83 of the article by
Nevill C.R. Jr., et al., Bioorganic & Med. Chem. Lett. 1991, 7(1), 83-86, the corresponding
phosphonium bromide may be obtained. The compounds for which R-n represents a group (Cr
C4)alkyl are obtained in a similar manner from a compound equivalent to compound 1 described
on page 83 of the article by Nevill C.R. Jr., et al., Bioorganic & Med. Chem. Lett. 1991, 7(1), 83-
86, which is either a commercial product or is obtained by Friedel-Crafts C-alkylation starting with
p-tolylacetic acid.
(1R,4R,5R,6R)-4,7,7-Trimethyl-6-(4-vinylbenzyl)-6-thionia-bicyclo[3.2.1]octane
trifluoromethanesulfonate used in step (iv) is obtained from (1R,4R,5R)-isothiocineole (see
Aggarwal V. et al., JACS 2010, 732, 1828-1830), the preparation of which from (R)-limonene
(CAS No. 95327-98-3, commercial product) is described in this same reference.
Triphenyl(p-vinylbenzyl)phosphonium bromide (CAS No. 118766-51-1) is obtained from the
corresponding bromo derivative (see Drefahl G., et al., Chem.Ber. 1961, 94(8), 2002-2010), the
preparation of which starting with 4-vinylbenzyl alcohol (CAS No. 1074-61-9, commercial product)
is described in the article by Shimomura O., et al.,Tetrahedron 2005, 61,1216O-12167.
Starting with P7 or P8, which are compounds of formula (III) for which G=-(CH2)nOH, other
compounds of formula (III) containing other groups G may be obtained.
Sorting with the group G=-CH2OH, the groups G=-CH2CI or -CH2N3 may be obtained:
- the introduction of-CI may be performed in the presence of CMS: see Ex. 1, compound 2;
- the azidation may be performed in the presence of diphenylphosphorazide (PhO)2P(=O)N3 and
a base, for example DBU. Scheme 3 describes these reactions for the case n=1, but it may also
apply for n>1.
Starting with the group G=-(CH2)2OH, the group G=-CH2COOH may be obtained via an oxidation.
Scheme 4 describes a double oxidation: 1st oxidation using the Dess-Martin reagent (see
"Encyclopedia of Reagents for Organic Synthesis"; Paquette L. A., Ed.; Wiley: Chichester, UK,
1995, Vol. 7, 4982^987 or Boeckman R.K. Jr., et al., J.J. "The Dess-Martin Periodinane" Org.
Synth. 2004, 10, 696-702) followed by a 2nd oxidation of Pinnick type in the presence of 2-methyl-
2-butene (Pinnick H.W., Tetrahedron 1981, 37, 2091-2096). Scheme 4 describes these reactions
for the case of a starting compound for which n=2, but it may also apply for n>2.
Starting with the group G=-CH2N3, the preparation of which is described in Scheme 3, the group
G =-CH2NH2 may be obtained with the aid of a reduction reaction using a phosphine such as
TCEP. In this respect, see: Faucher A.-M. et al., Synthetic Comm. 2003, 33, 3503-3511:
Recording to one variant, starting with the group G=-CH2OH, the group G=-CH2NH2 may be
obtained with the aid of a Mitsunobu reaction using triphenylphosphine and DEAD. In this
respect, see: Mitsunobu O., Synthesis 1981, 1-28; Hughes D.L., Org. Reactions 1992, 42, 335-
656; Hughes D.L., Org. Prep. 1996, 28, 127-164. Schemes 5 and 5' describe the case n=1, but
they may also apply for n>1.
in the case where G=-(CHz)n-NRi£-CHzC=CH
Starting with the group G=-(CH2)nCI, the group G=-(CH2)n-NR12-CH2-CHCH may be obtained with
the aid of a nucleophilic substitution using the compound of formula NHR12-CH2-C=CH (Ri2=H:
propargylamine; Ri2=(C1-C6)alkyl: prepared according to Mock W.L., et al., J.Org. Chem. 1989,
54 (22), 5302-8).
Starting with the group G=-(CH2)nCI, the group G=-(CH2)nSH may be obtained via a direct
functionalization with the aid of tetrabutylammonium trimethylsilylthiolate prepared in situ from
tetrabutylammonium fluoride and hexamethyldisilathiane according to Hu J. et al., J. Org. Chem.
1999, 64, 4959-4961 (see Ex. 8). Scheme 6 describes this reaction for the case n=1, but it may
also apply for n>1. In the course of this reaction, the intermediate dimer having the formula below
may be formed:
P3 allows the preparation of other cryptophycin derivatives according to Scheme 7:
Step (i): Wittig reaction using a suitable phosphonium halide, for example a bromide, and a strong
base, for instance BuLi;
Step (ii): Kumada coupling using a suitable Grignard reagent, for example an alkoxymagnesium
bromide protected in silyl ether form, in the presence of a palladium or nickel catalyst (see, for
example, Organic Letters 2009, 11, 5686-5689 or Synthesis 2009, 141, 2408-2412).
Steps (iii) to (v) are described in Scheme 2 and step (vi) is described in Scheme 6.
(4-Bromobenzyl)triphenylphosphonium bromide is a commercial product (CAS No. 51044-13-4).
The alkoxymagnesium bromides protected in silyl ether form may be prepared from the
corresponding bromo alcohols by protection of the alcohol function with the appropriate
chlorosilane, followed by formation of the organomagnesium reagent in the presence of
magnesium in an anhydrous polar aprotic solvent, for instance THF (see, for example, Organic
Letters 2005, 7,183-186). The linear or branched bromo alcohols containing 1 to 6 carbon atoms
are commercially available, for instance 3-bromo-1-propanol (CAS No. 627-18-9) or 1-bromo-2-
propanol (CAS No. 19686-73-8) or may be prepared from the corresponding bromo esters or
bromo ketones according to methods described in the literature. The chlorosilane may be, for
example, terf-butyldimethylchlorosilane (CAS No. 18162-48-6) or triisopropylchlorosilane (CAS
No. 13154-24-0).
In addition to Scheme V which describes a method for preparing cryptophycin derivatives
o
-(CH;)r,-h/j
comprising a maleimido unit, starting with the group G=-CH2OH, the group G= $ may be
obtained via a Mitsunobu reaction in the presence of triphenylphosphine and DEAD according to
Matuszak N. etal., J. Med. Chem. 2009, 52,741O-7420. Scheme 8 describes this reaction for the
case n=1, but it may also apply for n>1.
Schemes 1-8 above are given for a linker in the para position, but may identically apply for the
ortho or meta positions. Similarly, they are given for a cryptophycin derivative, but may also apply
to the preparation of other derivatives of formula (II), especially Di-D8.
More particularly, in the case of C-52, the following compounds may be used, the preparations of
which are described in Al-awar R.S., et al., J.Med.Chem. 2003, 46, 2985-3007 or in
WO 98/08505:
Moreover, starting with compound 31b for which G=-CH2OH, it is possible to obtain the
compounds for which G=-CH2CI or -CH2N3:
• G=-CH2CI: see Example 1, compound 2;
• G=-CH2N3: the conversion of -CH2OH into -CH2N3 may be performed in a polar aprotic
solvent in the presence of diphenylphosphorazide and a base such as DBU, see Example 19,
compound 60;
• G=-CH2-maleimido: the conversion of -CH2OH into -CH2-maieimido may be performed in a
polar aprotic solvent in the presence of maleimide, triphenylphosphine and DEAD.
The teaching of J.Med.Chem. 2003, 46, 2985-3007 may apply to other cryptophycin derivatives
comprising other substituents R6-R9.
Preparation of the linker precursors LP
LP may be one of the following:
Step (Q: activation of the acid using NHS; the activation is performed at RT in the presence of a
coupling agent, for instance 1-(3-dimethyiaminopropyl)-3-ethylcarbodiimide hydrochloride
dissolved in an anhydrous aprotic solvent such as DCM. The procedure may be based on the
conditions of Example 1, compound 4.
Step (ii): amidation with piperidine N-Boc; the peptide coupling is performed in a polar aprotic
solvent at RT in the presence of a base, which may be a tertiary amine, for instance TEA or
DIPEA. The solvent may be DMF. The procedure may be based on the conditions of Example 1,
compound 5.
Step (iii): deprotection of the amine using an acid solution, for example hydrochloric acid (for
example as a solution in dioxane). The procedure may be based on the conditions of Example 1,
compound 6.
The starting acid, for example 4-methyl-4-(methyldithio)pentanoic acid, may be commercially
available or prepared from a halocarboxylic acid by successive treatments with potassium
thioacetate and a derivative of methanethiosulfonate type. See also US 2 719 170.
Step (0: reductive amination with an aldehyde; the reaction is performed at RT in an anhydrous
polar aprotic solvent such as THF in two steps: formation of an intermediate complex in the
presence of titanium isopropoxide followed by in situ reduction with a reducing agent, for instance
sodium cyanoborohydride. The procedure may be based on the conditions of Example 5,
compound 17.
Step (ii): deprotection of the amine with an acid solution, for example hydrochloric acid (for
example as a solution in dioxane). The procedure may be based on the conditions of Example 5,
compound 18.
The starting aldehyde, for example 2-methyl-2-(methyldithio)propanal, may be commercially
available or prepared by oxidation of an alcohol bearing a disulfide unit obtained from a suitably
protected halogenated alcohol (for example in silyl ether form) by successive treatments with
potassium thioacetate and a derivative of methanethiosulfonate type.
Step (i): formation of an oxime; the aldehyde described previously is dissolved in a polar protic
solvent such as ethanol and then treated with O-methylhydroxylamine hydrochloride at reflux in
the presence of a base such as sodium hydroxide. The procedure may be based on the
conditions of Example 3, compound 11.
Step (ii): reduction of the oxime; the oxime is reduced via a treatment at reflux with a solution of
borane-dimethyl sulfide in an anhydrous polar aprotic solvent such as THF. The procedure may
be based on the conditions of Example 3, compound 12.
in the case where R-^H
Step (0: reductive amination with an aldehyde; the reaction is performed at RT in an anhydrous
polar aprotic solvent such as THF in the presence of a reducing agent, for instance sodium
triacetoxyborohydride.
This linker is prepared in a similar manner to that presented for LP2.
Step fl): reductive amination with an aldehyde; the reaction is performed at RT in an anhydrous
polar aprotic solvent such as THF, in two steps: formation of an intermediate complex in the
presence of titanium isopropoxide, followed by in situ reduction with a reducing agent such as
sodium cyanoborohydride. The procedure may be based on the conditions of Example 5,
compound 17.
Step (ii): deprotection of the amine with an acid solution, for example hydrochloric acid (for
example in solution in dioxane). The procedure may be based on the conditions of Example 5,
compound 18.
Step (\): protection of the amine; the reaction is performed at RT in a polar aprotic solvent such as
DCM by treating the amine with di-tert-butyl dicarbonate in the presence of a base, for instance
TEA.
Step (ii): conversion of the alcohol to a bromide; the reaction is performed at RT in a polar aprotic
solvent such as THF, by treating the alcohol function with CBr4 in the presence of a phosphine,
for example triphenylphosphine; see in this respect Appel R. Angew. Chem. Int. Ed. Engl. 1975,
14, 801-811 or Desmaris N., et al., Tetrahedron Letters 2003, 44(41), 7589-7591.
Step (NO: substitution of the bromide with thioacetate; the reaction is performed at RT in an
anhydrous polar aprotic solvent such as DMF, using potassium thioacetate as nucleophile.
Step (iv): formation of the disulfide bond; the reaction is performed in an anhydrous polar protic
solvent such as methanol in the presence of a base, for instance sodium methoxide and a
reagent comprising a pyridyl disulfide unit.
According to one variant:
Step (v): activation of the alcohol in mesylate form; the reaction is performed in an anhydrous
polar aprotic solvent such as DCM by treatment with mesyl chloride in the presence of a base, for
instance TEA.
Step (vi): formation of the free thiol; the reaction is performed in a refluxing polar protic solvent
such as an ethanol/water mixture, in two successive steps: displacement of the mesylate with
thiourea, followed by in situ hydrolysis of the isothiouronium salt by adding a base such as NaOH.
Step (viiV. activation of the thiol in the form of pyridyl disulfide; the reaction is performed at RT in a
polar protic solvent such as ethanol, by treatment with a reagent comprising a pyridyl disulfide
unit in the presence of an acid such as acetic acid.
Step (viiQ: deprotection of the amine with an acid solution, for example hydrochloric acid (for
example in solution in dioxane).
Step (ix): activation of the amine in the form of carbamoyl chloride; the reaction is performed in an
anhydrous polar aprotic solvent such as DCM, by treatment with diphosgene in the presence of a
base such as TEA.
Lp6 Rbzb-oc *u- (OCH2CH2)-OH prepared according to the scheme below:
in the case where ALK=CH?CH?
Route A:
Step (0: deprotection with a solution of hydrochloric acid (for example solution in dioxane) or of
trifluoroacetic acid.
Step (iO: protection of the carboxylic acid in allylic ester form; the reaction is performed at RT in a
polar aprotic solvent such as DMF in the presence of allyl bromide and of a base such as
potassium carbonate.
Step flip: elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF, by treatment of an unsaturated acid protected in ester form with the
alkoxide generated by the action of sodium in catalytic amount.
in the case where ALK^CHgCH?
anh a base such as DMAP. The procedure may be based on the conditions of Example 14,
compound 42.
Step (ii): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of
trifluoroacetic acid. The procedure may be based on the conditions of Example 14, compound 43.
Step (iii): alkylation of the nitrogen atom; the reaction is performed in an anhydrous polar aprotic
solvent such as THF by treatment with a base such as NaH in the presence of a reagent bearing
a nucleofugal group such as an alkyl halide. The procedure may be based on the conditions of
Example 15, compound 46.
Step (iv): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF or DMF by treatment of a halogenated ester with the alkoxide of a
benzophenone-imine-PEG-alcohol generated via the action of NaH or potassium naphthalenide
as described in WO 2007/127 440;
Step (v): saponification of the ester; the reaction is performed by reacting the ester with lithium
hydroxide in the presence of water.
Step (vi): protection of the amine; the reaction is performed at RT in a polar aprotic solvent such
as DCM by treating the amine with di-te/f-butyl dicarbonate in the presence of a base, for
instance TEA.
The amino-PEG-acids are commercially available for i=3,5,6,10 or may be prepared from tert-
butyl acrylate and the corresponding amino-PEG-alcohol.
The amino-PEG-alcohols are commercially available, for example for i=3, 4, 7, 8 or may be
prepared from the PEG diols, which are commercially available for i=3 to 12, according to the
procedure described in US 7 230 101. The protection of the amine function with benzophenone
may be performed by azeotropic dehydration in the presence of a Lewis acid such as BF3
etherate.
Step 0'): deprotection of the starting compound using a solution of hydrochloric acid (for example
solution in dioxane) or of trifluoroacetic acid. The procedure may be based on the conditions of
Example 16, compound 50.
Step (ii): protection of the carboxylic acid function in allylic ester form; the reaction is performed at
RT in a polar aprotic solvent such as DMF in the presence of allyl bromide and a base such as
potassium carbonate. The procedure may be based on the conditions of Example 16, compound
51.
gfep (in): activation of the alcohol in mesylate form; the reaction is performed in an anhydrous
polar aprotic solvent such as DCM by treatment with mesyl chloride in the presence of a base
such as TEA.
Step (iv): reaction between the mesylate function and the amine function of the compound
(alkylation); the reaction is performed at RT in an anhydrous polar aprotic solvent such as
DCM in the presence of a base such as TEA. The procedure may be based on the conditions of
Example 16, compound 52.
Step (v): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF or DMF by treatment of a halogenated ester with the alkoxide of a PEG diol
monoprotected in tetrahydropyran (THP) ether form. The preparation of this type of
monoprotected PEG diol is well described in the literature: see, for example, Richard A. et al.
Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou E.G., etal. Tetrahedron 2003, 59, 9083-9090.
The PEG alcohols comprising an acid function protected in ferf-butyl ester form are commercially
available (for instance fe/t-butyl 12-hydroxy-4,7,1O-trioxadodecanoate) or prepared from ferf-butyl
acrylate and a PEG diol. The starting PEG diols are commercially available for i= 3 to 12.
Step (i): alkylation of the amine; the reaction is performed in an anhydrous polar aprotic solvent
such as acetonitrile with an ally! haloalkylcarboxylate, for instance allyl bromoacetate, in the
presence of a base, for instance TEA. The procedure may be based on the conditions of
Example 13, compound 38.
Step (ii): deprotection of the amine using an acid solution, for example hydrochloric acid (for
example in solution in dioxane). The procedure may be based on the conditions of Example 13,
compound 39.
The allyl haloalkylcarboxylate may be obtained from allyl alcohol and the corresponding haloacyl
halide and is commercially available for ALK=-(CH2)i-6- (for instance bromoacetyl bromide or 4-
butanoyl chloride).
LPio prepared according to the schemes below:
in the case where ALK=CH?CHg
in the case where ALK* ChbCH?
Step (i): opening of the cyclic anhydride; the reaction is performed at RT in an anhydrous polar
aprotic solvent such as DCM in the presence of a base such as TEA.
Step (ii): protection of the carboxylic acid in allylic ester form; the reaction is performed at RT in a
polar aprotic solvent such as DCM in the presence of allyl alcohol, a coupling agent such as EDCI
and a base such as DMAP.
Step (iii): deprotection using a solution of hydrochloric acid (for example solution in dioxane).
Step (iv): peptide coupling; the reaction between the carboxylic acid and the amine is performed
at RT in a polar aprotic solvent such as DCM in the presence of a coupling agent such as the
DIC/HOBt system.
Step (v): saponification of the methyl ester; the reaction is performed at RT in a mixture of polar
solvents such as a THF/water mixture in the presence of lithium hydroxide.
The diacids monoprotected in methyl ester form are commercially available for ALK=-(CH2)i-6-
(such as the monomethyl ester of 1,6-hexanedioic acid).
Step (i): formation of the amide and activation of the acid; the two steps are performed
successively in a polar aprotic solvent such as DCM: reaction between the amine function and
the N-hydroxysuccInimidyl haloacetate, followed by in situ addition of a coupling agent such as
D/C. The procedure may be based on the conditions of Example 17, compound 56.
in the case where Ri?^H
Step (ii): protections of the carboxylic acid in methyl ester form and of the amine in
trifluoroacetamide form; the reaction is performed in two successive steps in a polar aprotic
solvent such as DCM: protection of the acid by treatment with trimethylsilyldiazomethane in the
presence of methanol, followed by protection of the amine by addition of trifluoroacetic anhydride
and of a base such as TEA.
Step (iii): alkylation of the amine and saponification of the ester; the reaction is performed in two
successive steps in an anhydrous polar aprotic solvent such as THF: alkylation of the amine by
treatment with a base such as NaH in the presence of a reagent bearing a nucleofugal group, for
instance an alkyl halide R12Hal, followed by addition of lithium hydroxide and water.
Step (0: following step (iii), the reactions of step (i) for the case where R12=H are repeated.
in the case where ALK^CHgCH?
in the case where Ri?=H
Step (iv): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF or DMF by treatment of a halogenated ester with the alkoxide of a
benzophenone-imine-PEG-alcohol generated via the action of NaH or potassium naphthalenide
(cf. WO 2007/127 440);
Step (v): selective cleavage of the imine by hydrogenation in the presence of palladium-on-
charcoal (cf. Wessjohann, L. et al., Synthesis 1989, 5, 359-63);
Sfep (vi): protection of the amine by addition of trifluoroacetic anhydride and of a base such as
TEA.
The amino-PEG-acids are commercially available for i=3, 5, 6, 10 or may be prepared from tert-
butyl acrylate and the corresponding amino-PEG-alcohol.
The amino-PEG-alcohols are commercially available, for example for i=3, 4, 7, 8 or may be
prepared from the PEG diols, which are commercially available for i=3 to 12, according to the
procedure described in US 7 230 101. The protection of the amine function with benzophenone
may be performed by azeotropic dehydration in the presence of a Lewis acid such as BF3
etherate.
Step (i): activation of the alcohol in mesylate form; the reaction is performed in an anhydrous
polar aprotic solvent such as DCM by treatment with mesyl chloride in the presence of a base
such as TEA.
Step (ii): mesylate/halogen exchange; the reaction is performed in a refluxing polar aprotic
solvent such as acetone, with a sodium halide such as sodium iodide.
Step (NO: deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of
trifluoroacetic acid.
Step (iv): activation of the acid; the reaction is performed at RT in a polar aprotic solvent such as
DEM by treatment with NHS in the presence of a coupling agent such as DCC.
Step (v): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF by treatment of an unsaturated acid protected in ester form with the alkoxide
generated by the action of sodium in catalytic amount.
Step (vi): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF or DMF by treatment of a halogenated ester with the alkoxide of PEG diol
monoprotected as the tetrahydropyran (THP) ether. The preparation of this type of
monoprotected PEG diol is well described in the literature: see, for example, Richard A. et al.
Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou E.G., et al. Tetrahedron 2003, 59, 9083-9090.
The intermediate formed is selectively hydrolysed at pH 5 to the hydroxy ester.
The PEG alcohols comprising an acid function protected in ferf-butyl ester form are commercially
available (for instance terf-butyl 12-hydroxy-4,7,1O-trioxadodecanoate) or prepared from ferf-butyl
acrylate and a PEG diol. The starting PEG diols are commercially available for i=3 to 12.
Step (i): deprotection using a solution of hydrochloric acid (e.g. solution in dioxane) or of
trifluoroacetic acid. In the latter case, trifluoroacetate of the hydroxy function may be formed. It is
cleaved during the following step (ii).
Step (ii): protection of the carboxylic acid in methyl ester form; the reaction is performed at RT in
a polar aprotic solvent such as methanol, by treatment with trimethylsilyldiazomethane.
Step (iii): activation of the alcohol in mesylate form; the reaction is performed in an anhydrous
polar aprotic solvent such as DCM by treatment with mesyl chloride in the presence of a base
such as TEA.
Step (iv): formation of the free thiol and saponification of the methyl ester; the reaction is
reformed in a refluxing polar protic solvent, such as an ethanol/water mixture, in two successive
steps- displacement of the mesylate with thiourea, followed by in situ hydrolysis of the
isothiouronium salt by addition of a base such as sodium hydroxide.
Step (v): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF by treatment of an unsaturated acid protected in ester form with the alkoxide
generated by the action of sodium in catalytic amount.
Step (vi): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF or DMF by treatment of a halogenated ester with the alkoxide of the PEG
diol monoprotected as the tetrahydropyran (THP) ether. The preparation of this type of
monoprotected PEG diol is well known in the literature: see, for example, Richard A. et al. Chem.
Eur. J. 2005, 11, 7315-7321 or Sakellariou E.G., et al. Tetrahedron 2003, 59, 9083-9090.
The linker with n = 8 (3-[2-mercaptoethoxyhepta(ethyleneoxy)]propionic acid) is commercially
available. The PEG alcohols comprising an acid function protected in terf-butyl ester form are
commercially available (for instance ferf-butyl 12-hydroxy-4,7,1O-trioxadodecanoate) or prepared
from ferf-butyl acrylate and a PEG diol. The starting PEG diols are commercially available for i=3
to 12.
o
X
LP|4 ra>zboc-ALK-(ocH2cH2)j+1 OH prepared according to the scheme below:
in the case where ALK=CH?CI-l£
Step (0: elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent, such as THF, by treatment of an unsaturated acid protected in ester form with the
alkoxide generated by the action of sodium in catalytic amount.
Step (ii): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of
ti%)uoroacetic acid. In the latter case, trifluoroacetate of the alcohol function optionally present on
the structure may be formed. This trifluoroacetate is cleaved during the following step (iii).
Step flip: protection of the carboxylic acid in methyl ester form; the reaction is performed at RT in
a polar aprotic solvent such as methanol, by treatment with trimethylsilyldiazomethane.
Step (iv): saponification of the methyl ester; the reaction is performed at RT in a mixture of polar
solvents such as a THF/water mixture in the presence of lithium hydroxide.
Step (v): protection of the carboxylic acid in allyl ester form; the reaction is performed at RT in a
polar aprotic solvent such as DCM in the presence of allyl alcohol, a coupling agent such as EDCI
and a base such as DMAP.
Step (vp: elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF or DMF by treatment of a halogenated ester with the alkoxide of the PEG
diol monoprotected as the tetrahydropyran (THP) ether. The preparation of this type of
monoprotected PEG diol is well described in the literature: see, for example, Richard A. et al.
Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou E.G., et al. Tetrahedron 2003, 59, 9083-9090.
The starting PEG diols are commercially available for i=3 to 12.
Step (p: nucleophilic substitution; the reaction is performed in an anhydrous polar aprotic solvent
such as THF, in the presence of a base such as NaH and of an alkynyl halide such as propargyl
bromide or 4-bromo-1-butyne. The procedure may be based on the conditions of Example 20,
compound 63.
Step HP: deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of
tnftuoroacetic acid. The procedure may be based on the conditions of Example 20, compound 65.
Step (iii): activation of the carboxylic acid in NHS ester form; the reaction is performed at RT in a
polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent
such as supported DCC.
Step (iv): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF by treatment of an unsaturated acid protected in ester form with the alkoxide
generated by the action of sodium in catalytic amount. The procedure may be based on the
conditions of Example 20, compound 64.
Step (v): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF or DMF by treatment of a halogenated ester with the alkoxide of the PEG
diol monoprotected as the tetrahydropyran (THP) ether. The preparation of this type of
monoprotected PEG diol is well described in the literature: see, for example, Richard A. et al.
Chem. Eur. J. 2005, 11, 7315-7321 orSakellariou E.G., eta!. Tetrahedron 2003, 59, 9083-9090.
The PEG alcohols comprising an acid function protected in tert-butyl ester form are commercially
available (for instance ferf-butyl 12-hydroxy-4,7,1O-trioxadodecanoate) or prepared from terf-butyl
acrylate and a PEG diol. The starting PEG diols are commercially available for i=3 to 12.
Step (0: elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF by treatment of an unsaturated acid protected in ester form with the alkoxide
generated by the action of sodium in catalytic amount.
Step (ii): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of
trifluoroacetic acid.
Step (iii): activation of the carboxylic acid in NHS ester form; the reaction is performed at RT in a
polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent
such as supported DCC.
Step (iv): elongation of the hydroxyazido PEG chain; the reaction is performed in an anhydrous
polar aprotic solvent such as THF or DMF by treatment of a halogenated ester with the alkoxide
of hydroxyazido PEG.
The azido PEG alcohols are commercially available or may be prepared from the corresponding
PEG alcohols that are commercially available for i=3 to 12.
Step (0: activation of the carboxylic acid in NHS ester form; the reaction is performed at RT in a
polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent
such as supported DCC.
The acids bearing an acetylenic group are commercially available for ALK=-(CH2)m- with m=1 to
10 (for instance 3-butynoic acid).
Step (0: nucleophilic substitution of the halide by the azide; the reaction is performed in a polar
aprotic solvent such as acetone, in the presence of sodium azide.
Step (ii): saponification of the methyl ester; the reaction is performed at RT in a mixture of polar
solvents such as a THF/water mixture in the presence of lithium hydroxide.
Step (iii): activation of the carboxylic acid in NHS ester form; the reaction is performed at RT in a
polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent
such as supported DCC.
The methyl esters bearing a haloalkyl unit are commercially available for ALK = -(CH2)m- with
m=1 to 6 (for instance methyl bromoacetate).
Step (i): nucleophilic substitution; the reaction is performed in an anhydrous polar aprotic solvent
such as THF in the presence of a base such as NaH and of an alkenyl halide such as allyl
bromide or 4-bromo-1 -butene.
Step (ii): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of
trifluoroacetic acid.
Step (NO: activation of the carboxylic acid in NHS ester form; the reaction is performed at RT in a
polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent
such as supported DCC.
Step (iv): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF by treatment of an unsaturated acid protected in ester form with the alkoxide
generated by the action of sodium in catalytic amount.
Step (v): elongation of the PEG chain; the reaction is performed in an anhydrous polar aprotic
solvent such as THF or DMF by treatment of a halogenated ester with the alkoxide of the PEG
diol monoprotected as the tetrahydropyran (THP) ether. The preparation of this type of
monoprotected PEG diol is well described in the literature: see, for example, Richard A. et al.
Chem. Eur. J. 2005, 11, 7315-7321 or Sakellariou E.G., et al. Tetrahedron 2003, 59, 9083-9090.
The PEG alcohols comprising an acid function protected in te/f-butyl ester form are commercially
available (for instance fert-butyl 12-hydroxy-4,7,1O-trioxadodecanoate) or prepared from terf-butyl
acrylate and a PEG diol. The starting PEG diols are commercially available for i=3 to 12.
Step (i): activation of the carboxylic acid in NHS ester form; the reaction is performed at RT in a
polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent
such as supported DCC.
The acids bearing an ethylenic group are commercially available for ALK=-(CH2)m- with m= 1 to
10 (for instance 3-butenoic acid).
LP2i zas-ALK-CH2-NRl2-(CH2CH2o)rcH2CH2-NHR'12 prepai-ed according to the schemes below:
in the case where Ry? and R'^H
Step fl): activation of the alcohol in tosylate form; the reaction is performed in a polar aprotic
solvent such as DCM by treatment with tosyl chloride in the presence of silver oxide and
potassium iodide. The procedure may be based on the conditions of Example 6, compound 23.
Step (ii): nucleophilic substitution of the tosylate; the reaction is performed in a polar aprotic
solvent such as acetonitrile by treatment with sodium azide. The procedure may be based on the
conditions of Example 6, compound 24.
Step (iiO: reduction of the azide; the reaction is performed in a polar solvent such as a THF/water
mixture in the presence of triphenylphosphine. The procedure may be based on the conditions of
Example 6, compound 26.
Step (iv): reductive amination with an aldehyde; the reaction is performed at RT in an anhydrous
polar aprotic solvent such as DCM in the presence of a reducing agent such as sodium
triacetoxyborohydride and, if necessary, acetic acid as catalyst. The procedure may be based on
the conditions of Example 6, compound 27.
Step (v): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of
trifluoroacetic acid. The procedure may be based on the conditions of Example 6, compound 29.
Step (vi): protection of the NHBoc function; the reaction is performed in a polar aprotic solvent
such as THF or DMF by treatment with 1 equivalent of base such as sodium hydride, followed by
a benzyl halide such as benzyl chloride.
Step (vii): cleavage of the benzyl group and reduction of the azido function; the reaction is
performed in a protic solvent such as methanol with hydrogen in the presence of a catalyst such
as palladium hydroxide.
Step (viii): alkylation of the amine; the reaction is performed in an anhydrous polar aprotic solvent
such as THF by treatment with a base such as sodium hydride in the presence of a reagent
bearing a nucleofugal group such as an alkyl halide. The procedure may be based on the
conditions of Example 6, compound 25.
The amino-PEG-alcohols optionally protected with a Boc group on the amine function are
commercially available (for instance N-Boc-aminoethoxyethoxyethanol or 1-amino-3,6,9-
trioxaundecanyl-11-ol) or may be prepared from PEG diols, which are commercially available for
i=3 to 12, according to the procedure described in US 7 230 101. The aldehyde ZaSS-ALK-CHO,
for example 2-methyl-2-(methyldithio)propanal, is commercially available or may be prepared by
oxidation of an alcohol bearing a disulfide unit obtained from a suitably protected halogenated
alcohol (for example in silyl ether form) by successive treatments with potassium thioacetate and
a derivative of methanethiosulfonate type.
Step (i): activation of the alcohol in tosylate form; the reaction is performed in a polar aprotic
solvent such as DCM by treatment with tosyl chloride in the presence of silver oxide and
potassium iodide. The process may be based on the conditions of Example 6, compound 23.
Step Hi): nucleophilic substitution of the tosylate; the reaction is performed in a polar aprotic
solvent such as acetonitrile by treatment with sodium azide. The process may be based on the
conditions of Example 6, compound 24.
Step (iii): reduction of the azide; the reaction is performed in a polar solvent such as a THF/water
mixture in the presence of triphenylphosphine. The procedure may be based on the conditions of
Example 6, compound 26.
Step (iv): peptide coupling; the reaction is performed in a polar aprotic solvent such as
dimethylformamide in the presence of coupling agents such as the N,N'-
diisopropylcarbodiimide/1-hydroxybenzotriazole system and of a base such as TEA.
Step (v): deprotection using a solution of hydrochloric acid (for example solution in dioxane) or of
trifluoroacetic acid. The procedure may be based on the conditions of Example 7, compound 32.
Step (vi): protection of the function NHBoc; the reaction is performed in a polar aprotic solvent
such as THF or DMF by treatment with 1 equivalent of base such as sodium hydride, followed by
a benzyl halide such as benzyl chloride.
Step (vii): cleavage of the benzyl group and reduction of the azido function; the reaction is
performed in a protic solvent such as methanol, with hydrogen in the presence of a catalyst such
as palladium hydroxide.
Step (viii): alkylation of the amine by reductive amination with an aldehyde; the reaction is
performed at RT in an anhydrous polar aprotic solvent such as DCM in the presence of a
reducing agent such as sodium triacetoxyborohydride and, if necessary, acetic acid as catalyst.
Step (ix): alkylation of the NHBoc group; the reaction is performed in an anhydrous polar aprotic
solvent such as THF by treatment with a base such as sodium hydride in the presence of a
reagent bearing a nucleofugal group such as an alkyl halide. The procedure may be based on the
conditions of Example 6, compound 25.
The amino-PEG-alcohols protected or otherwise with a Boc group on the amine function are
commercially available (for instance N-Boc-aminoethoxyethoxyethanol or 1-amino-3,6,9-
trioxaundecanyl-11-ol) or may be prepared from the PEG diols, which are commercially available
for i=3 to 12, according to the procedure described in US 7 230 101. The carboxylic acid ZaS-
ALK-CO2H, for example 4-methyl-4-(methyldithio)pentanoic acid, may be commercially available
or prepared from a halogenated carboxylic acid via successive treatments with potassium
thioacetate and a derivative of methanethiosulfonate type.
LP23 zas-(CH2CH2°)rCH2CH2-NHR12 prepared according to the schemes below:
Step (0: activation of the alcohol in mesylate form; the reaction is performed in an anhydrous
polar aprotic solvent such as DCM by treatment with mesyl chloride in the presence of a base
such as TEA.
Step (ii): formation of the free thiol; the reaction is performed in a refluxing polar protic solvent
such as an ethanol/water mixture in two successive steps: displacement of the mesylate with
thiourea, followed by in situ hydrolysis of the isothiouronium salt by addition of a base such as
sodium hydroxide.
Step (in): protection of the thiol; the reaction is performed in a mixture of polar solvents such as
an ethanol/water mixture with a reagent comprising a methanethiosulfonate function, for instance
methyl methanethiosulfonate, in the presence of a base such as sodium carbonate.
Step (iv): deprotection using a solution of hydrochloric acid (for example a solution in dioxane) or
of trifluoroacetic acid.
Step M: alkylation of the amine; the reaction is performed in an anhydrous polar aprotic solvent
such as THF by treatment with a base such as sodium hydride in the presence of a reagent
bearing a nucleofugal group, such as an alkyl halide.
The amino-PEG-alcohols protected or not protected with a Boc group on the amine function are
commercially available (for instance N-Boc-aminoethoxyethoxyethanol or 1-amino-3,6,9-
tnoxaundecanyl-11-ol) or may be prepared from PEG diols, which are commercially available for
i=3 to 12, according to the procedure described in US 7 230 101.
Step (0: activation of the Fmoc-L-valine in the form of the NHS ester; the reaction is performed in
an anhydrous polar aprotic solvent such as THF Dy treatment with NHS in the presence of a
coupling agent such as DCC.
Step (iiV. peptide coupling between Fmoc-L-valine-NHS and L-citrulline; the reaction is performed
in a polar solvent such as a dimethoxyethane/THF/water mixture in the presence of a base such
as sodium bicarbonate.
Step (iip: peptide coupling with 4-aminobenzyl alcohol; the reaction is performed in a polar
solvent such as a DCM/methanol mixture in the presence of a coupling agent such as EEDQ.
Step (iv): deprotection of the amine Fmoc; the reaction is performed in a polar solvent such as a
DCM/methanol mixture in the presence of a base such as diethylamine.
Step (v): activation of the carboxylic acid in NHS ester form; the reaction is performed in a polar
aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent such as
EDCI hydrochloride.
Step (vi): peptide coupling between the dipeptide and the NHS ester; the reaction is performed at
RT in a polar aprotic solvent such as a DCM/acetonitrile mixture.
The diacids monoprotected in allylic ester form are commercially available for n=2 (monoallyl
succinate) or may be prepared by transesterification of the methyl or t-butyl monoesters, which
are commercially available for n = 2 to 6.
Step (0: activation of Fmoc-L-valine in NHS ester form; the reaction is performed in an anhydrous
polar aprotic solvent such as THF by treatment with NHS in the presence of a coupling agent
such as DCC.
Step (ii): peptide coupling between Fmoc-L-valine-NHS and L-citrulline; the reaction is performed
in a polar solvent such as a DME/THF/water mixture in the presence of a base such as sodium
bicarbonate.
Step (iii): peptide coupling with 4-aminobenzyl alcohol; the reaction is performed in a polar
solvent such as a DCM/methanol mixture in the presence of a coupling agent such as EEDQ.
Step (iv): deprotection of the amine Fmoc; the reaction is performed in a polar solvent such as a
DCM/methanol mixture in the presence of a base such as diethylamine.
Step (v): activation of the carboxylic acid in NHS ester form; the reaction is performed at RT in a
polar aprotic solvent such as DCM by treatment with NHS in the presence of a coupling agent
such as supported DCC.
Step (vQ: peptide coupling between the dipeptide and the NHS ester; the reaction is performed at
RT in a polar aprotic solvent such as a DCM/acetonitrile mixture.
The PEG diacids monoprotected in allylic form are prepared according to the description of the
preparation of the linker L14.
2,5-Dioxopyrrolidin-1-yl bromoacetate and iodoacetate are commercial products, whose CAS
numbers are, respectively, 42014-51-7 and 39028-27-8.
Process for preparing the conjugate
The conjugate is obtained via the process that consists in:
(i) placing in contact and leaving to react an optionally buffered aqueous solution of the binding
agent and a solution of the cryptophycin derivative of formula (II);
(ii) and then in optionally separating the conjugate formed in step (i) from the cryptophycin
derivative and/or from the unreacted binding agent and/or from any aggregates formed.
More particularly, in step (ii) the conjugate from step (i) is separated only from the unreacted
cryptophycin derivative and from any aggregates formed, and any unreacted binding agent is left
in the solution.
The function of the placing in contact is to react the chemical groups RCG1 and RCG2 in order to
ensure attachment of the cryptophycin derivative to the binding agent by formation of a covalent
bond; preferably
■ when RCG1 represents -SZa: the binding agent is modified with a modifying agent so as
to introduce onto the binding agent suitable groups RCG2, especially those described in
the 2nd column of the Table I:
o disulfide chemical groups in the case where RCG1 represents -SH;
o thiol chemical groups in the case where RCG1 represents -SZa with Za?H;
o maleimido or iodoacetamido chemical groups in the case where RCG1 represents
-SH;
In the case of an antibody (MAb), the formulae of the conjugates are found in the 4th
column of Table I;
■ when RCG1 represents -C(=O)-ZbRb: the reaction preferably takes place on the amino
functions of the binding agent, especially the e-amino groups borne by the side chains of
the lysine (Lys) residues of an antibody. In the case of an antibody (MAb), a conjugate of
the following formula is obtained in this case: MAb-[NH-C(=O)-L*-Crypto]d with L* =
fragment of a linker L comprising RCG1=-C(=O)-ZbRb and such that L= -L*C(=O)-ZbRb;
■ in the presence of a cryptophycin derivative of formula (III) with G= -(CH2)nY, the binding
agent comprises groups -SH when Y= -CI, groups -CsCH when Y= -N3 or carboxylic acid
groups when Y= -OH or -NH2;
■ in the presence of a cryptophycin derivative comprising a reactive chemical group RCG1
of maleimido or haloacetamido type, the binding agent comprises thiol chemical groups.
The term "aggregates" means associations that may form between two or more binding agents,
the binding agents possibly having been modified by conjugation. Aggregates are liable to form
under the influence of a wide variety of parameters such as a high concentration of binding agent
in the solution, the pH of the solution, high shear forces, the number of grafted dimers and their
hydrophobic nature, the temperature (see the references cited in the introduction of J. Membrane
Sci. 2008, 318, 311-316), the influence of some of them not, however, having been clearly
elucidated. In the case of proteins or antibodies, reference may be made to AAPS Journal,
"Protein Aggregation and Bioprocessing" 2006, 8(3), E572-E579. The aggregate content may be
determined via known techniques such as SEC (see in this respect Analytical Biochemistry 1993,
212 (2), 469^80).
The aqueous solution of the binding agent may be buffered, for example with buffers such as
potassium phosphate or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer) or
a mixture of buffers such as buffer A described later. The buffer depends on the nature of the
binding agent. The cryptophycin derivative is dissolved in a polar organic solvent, for example
DMSO or DMA.
The reaction takes place at a temperature generally of between 20 and 40°C. The reaction time
may vary between 1 and 24 hours. The reaction between the antibody and the cryptophycin
derivative may be monitored by SEC with a refractometric and/or ultraviolet detector in order to
determine its degree of progress. If the degree of substitution is insufficient, the reaction can be
left for longer and/or cryptophycin derivative can be added. Reference may be made to the
general method given in the examples section for further details regarding particular conditions.
Particular embodiments are described in Examples 9,10,11,25,26 and 27.
A person skilled in the art has at his disposal various chromatographic techniques for the
separation of step (ii): the conjugate may be purified, for example, by steric exclusion
chromatography (SEC), by adsorption chromatography (for instance ion exchange, IEC), by
hydrophobic interaction chromatography (HIC), by affinity chromatography, by chromatography
on mixed supports such as ceramic hydroxyapatite, or by HPLC. Purification by dialysis or
diafiltration may also be used.
After step (i) or (ii), the solution of the conjugate may undergo an ultrafiltration and/or diafiltration
step (iii). After these steps, the conjugate in aqueous solution is thus obtained.
Antibody
The antibody (see in this respect, Janeway et al. "Immunobiology", 5th edition, 2001, Garland
Publishing, New York) may be chosen from those described especially in WO 04/043 344, WO
08/010 101, WO 08/047 242, WO 05/009 369 (anti CA6) or WO 2010/014 812. The antibody may
optionally be modified with a modifying agent so as to promote the attachment of the
cryptophycin derivative (see above). The antibody may especially be monoclonal, polyclonal or
multispecific. It may also be an antibody fragment. It may also be a murine, human, humanized or
chimeric antibody.
Conjugate
A conjugate generally comprises from about 1 to 10 cryptophycin derivatives covalently attached
to the binding agent (this is the degree of grafting or "drug-to-antibody ratio" or "DAR"). This
number varies as a function of the nature of the binding agent and of the cryptophycin derivative,
and also of the operating conditions used in the conjugation process (for example the number of
equivalents of cryptophycin derivative relative to the binding agent, the reaction time, the nature
of the solvent and of any cosolvent). Placing of the binding agent and the cryptophycin derivative
in contact leads to a mixture comprising several conjugates that are individually distinguished
from each other by different DARs; optionally the unreacted binding agent; optionally aggregates.
The DAR that is determined on the final solution thus corresponds to an average DAR.
In the case where the binding agent is an antibody, UV spectroscopy may be a method used for
determining the DAR. This method is based on that presented in Antony S. Dimitrov (ed), LLC,
2009, "Therapeutic Antibodies and Protocols", vol. 525, 445, Springer Science. It consists in
measuring the absorbance of a solution of conjugate after the separation step (ii) at two
wavelengths noted X1 and 72. The following molar extinction coefficients of the naked antibody
and of the cryptophycin derivative measured prior to conjugation are used.
The absorbances of the conjugate solution at X1 and 12 (A^) and (A^) are measured either on
the corresponding peak of the SEC spectrum (this allows calculation of a "DAR(SEC)") or by
using a standard UV spectrophotometer (this allows calculation of a "DAR(UV)"). The
absorbances may be expressed in the form:
AM =(cDxeDX1) + (cAX8A?li)
A^ = (CDXEDX2) + (CAX8AX2)
for which equations:
• cD and cA denote, respectively, the concentrations in the solution of the part of the
conjugate relative to the cryptophycin derivative and the part of the conjugate relative to
the antibody;
• eD xi and eD u denote, respectively, the molar absorption coefficients for the cryptophycin
derivative before conjugation at the two wavelengths A,1 and 12, these coefficients
measured on the compounds of formula (II) of the type SZa with Za=-SMe or of the
type -C(=O)-ZbRb with ZbRb= OMe or OCH2-CH=CH2;
• eA M and eA X2 denote, respectively, the molar absorption coefficients of the naked
antibody at the wavelengths M and X2.
The term "naked antibody" means the antibody to which no cryptophycin derivative is attached,
i.e. the antibody before the conjugation step.
Resolution of these two equations leads to:
The average DAR thus corresponds to cD/cA. In the case of cryptophycin derivatives, the
wavelength M= 280 nm may be considered, and, depending on the nature of the cryptophycin
derivative, X2 is chosen in the specific wavelength range 246 nm - 252 nm. The DAR(UV) is
preferably greater than 0.5, more particularly between 1 and 10 and even more particularly
between 2 and 7.
The conjugate may be used as an anticancer agent. Owing to the presence of the binding agent,
the conjugate is made highly selective towards tumour cells rather than healthy cells. This makes
it possible to direct the cryptophycin derivative in an environment similar thereto or directly
therein; (in this respect, see the following publications that describe the use of monoclonal
antibody conjugates in cancer treatment: "Antibody-drug conjugates for cancer therapy" Carter
P.J., et al., Cancer J. 2008, 14, 154-169; "Targeted cancer therapy: conferring specificity to
cytotoxic drugs" Chari R., Ace. Chem. Res. 2008, 41, 98-107). It is possible to treat solid or liquid
cancers. The conjugate may be used alone or in combination with at least one other anticancer
agent.
The conjugate is formulated in the form of a buffered aqueous solution at a concentration
generally of between 1 and 10 mg/ml. This solution may be injected in perfusion form per se or
may be rediluted to form a perfusion solution.
[Examples]
Analytical methods used
High-pressure liquid chromatography - mass spectrometry (LCMS)
Method A1
The analysis is performed on a Waters ZQ machine and an XBridge C18 2.5 urn (3*50 mm)
column at 70°C with a flow rate of 0.9 ml/min, an elution gradient (7 min) of (A) water/0.1 % formic
acid and of (B) acetonitrile/0.1% formic acid (gradient: from 5% to 100% B over 5.3 minutes; 5.5
minutes: 100% B; 6.3 minutes: 5% B) and an electrospray ionization in positive and/or negative
mode.
Method A2
The analysis is performed on a Waters UPLC-SQD machine and an Acquity BEH C18 1.7 urn
(2.1x50 mm) column at 50°C with a flow rate of 1 ml/min, an elution gradient (2 minutes) of (A)
vteter/0.1% formic acid and of (B) acetonitrile/0.1% formic acid (gradient: from 5% to 50% B over
0.8 minute; 1.2 minutes: 100% B; 1.85 minutes: 100% B; 1.95 minutes: 5% B) and electrospray
ionization in positive and/or negative mode.
Method A3
The analysis is performed on a Waters UPLC-SQD machine and an Acquity BEH Ci8 1 .7 urn
(2.1x50 mm) column at 70°C with a flow rate of 1 ml/min, an elution gradient (2 minutes) of (A)
water/0.1% formic acid and of (B) acetonitrile/0.1% formic acid (gradient: from 5% to 50% B over
1 minute; 1.3 minutes: 100% B; 1.45 minutes: 100% B; 1.75 minutes: 5% B) and electrospray
ionization in positive and/or negative mode.
Method A4
The analysis is performed on a Waters ZQ machine and a Phenomenex Kinetex C18100A 2.6 urn
(3x50mm) column at 45°C with a flow rate of 1 ml/minute, an elution gradient (6 minutes) of (A)
water/0.1% formic acid and of (B) acetonitrile/0.1% formic acid (gradient: 6% B: 0.8 minute; from
6% to 100% B over 4.1 minutes; 4.8 minutes: 100% B; 5.O-6.0 minutes: 6% B) and electrospray
ionization in positive and/or negative mode.
Method A5
The analysis is performed on a Waters ZQ machine and a Phenomenex Kinetex C18 2.6 urn
(3x1000mm) column at 50°C with a flow rate of 0.8 mi/minute, an elution gradient (8 minutes) of
(A) water/0.1% formic acid and of (B) acetonitrile/0.1% formic acid (gradient: 4% B: 0.15 minute;
from 4% to 100% B over 6.85 minutes; 7.1 minutes: 100% B; 7.4-8.2 minutes: 4% B) and
electrospray ionization in positive and/or negative mode.
Mass spectrometry (MS)
The spectra were acquired by direct introduction onto a Waters GCTof machine (direct
introduction without LC).
Steric exclusion chromatography - high resolution mass spectrometry (SEC-HRMS)
The analysis may necessitate a prior step of deglycosylation of the conjugate. This is performed
by adding to the conjugate solution 2% by volume of a solution of enzyme PNGase F (prepared
by making up to 100 ml a flask of 100 units of lyophilizate of N-glycanase enzyme with MilliQ
water). The solution is homogenized by vortex and incubated at 37°C for 19 hours. The
deglycosylated sample is ready to be analyzed by SEC-HRMS. The chromatographic analysis is
performed on an Agilent HP1100 machine and a Waters Biosuite 250 HR SEC 4 pm
(4.6>«300 mm) column at 30°C with a flow rate of 0.4 ml/minute and an isocratic elution of (A)
25 mM ammonium formate pH=7/(B) acetonitrile 70/30 for 15 minutes. The mass spectrometry is
performed on a Waters QTOF II machine with electrospray ionization in positive mode. The mass
spectra are deconvoluted with the Waters MaxEntl software.
Steric exclusion chromatography (SEC HPLC)
The analysis is performed on a Merck Lachrom Elite HPLC machine with an L2455 DAD
spectrophotometric detector and a Tosoh Bioscience TSKgel G3000 SWXL 5 urn (7.8x300 mm)
column with a flow rate of 0.5 ml/minute and an isocratic elution of 30 minutes with a pH 7 buffer
containing 0.2 M of KCI, 0.052 M of KH2P04, 0.107 M of K2HP04 and 20% by volume of
isopropanol.
1H nuclear magnetic resonance (NMR)
The 1H NMR spectra were acquired on a Bruker Avance spectrometer, either of model DRX-300,
DRX-400, DRX-500 or DMX-600. The chemical shifts are given in ppm.
General method used for preparing the conjugates in the case of the cryptophycin
derivatives comprising a linker L ending with -SZg
Method in two successive steps
1st step
The antibody is first modified with an activated NHS ester, so as to introduce onto its surface
pyridyldisulfide groups. A solution of antibody hu2H11 in an aqueous pH 6.5 buffer containing
0.05 M of potassium phosphate and 0.05 M of NaCI (referred to as buffer A) is treated with 5 to
10 eq. of the NHS activated ester dissolved in DMA such that the final antibody concentration is
between 5 and 10 mg/ml and the percentage of DMA in the aqueous buffer is 5%. The reaction is
continued for 2 hours at RT. The mixture is deposited onto a filtration column on gel (Sephadex™
G25 matrix, GE Healthcare) pre-equilibrated in an aqueous pH 8 buffer containing 0.05 M of
HEPES, 0.05 M of NaCI and 2 mM of EDTA. The modified antibody is eluted with the pH 8
HEPES buffer, collected and then assayed by UV spectrometry so as to determine the antibody
concentration of the sample and the number of pyridyldisulfide groups. A specimen of the
modified antibody is treated with dithiothreitol so as to reduce the disulfide bond, and the
pyridine-2-thione released is assayed by spectrometry (extinction coefficients: 6343 nm: 8080 M"
1cm"1, £280 nm: 5100 M"1cm"1 for pyridine-2-thione, and e28onm: 208380 M"W for the antibody). On
average, from 3 to 6 pyridyldisulfide groups are grafted per antibody molecule.
2nd step
The modified antibody solution from the 1st step is diluted in the aqueous pH 8 buffer described
above and then treated with a solution of the cryptophycin derivative (5 eq.) such that the final
antibody concentration is 3 mg/ml and the percentage of DMA in the aqeous buffer is 20%; the
number of equivalents of cryptophycin derivative is expressed relative to the number of
pyridyldisulfide molecules introduced during the first step. The reaction is continued overnight at
aire or with stirring at about 2000 rpm. The mixture is analyzed by SEC HPLC so as to
determine the degree of grafting of the cryptophycin derivative onto the antibody. If the degree of
substitution is insufficient, the mixture is treated with an additional 1 to 5 eq. of cryptophycin
derivative in DMA for 3 hours at 30°C or with stirring at about 2000 rpm. The mixture is filtered
through a Millex®-SV 5 urn filter (PVDF membrane, Durapore, Millipore) and then purified by gel
filtration using a Superdex 200 pg matrix (HiLoad 16/60 desalting column, GE Healthcare) pre-
equilibrated with an aqueous pH 6.5 buffer containing 0.01 M of phosphate, 0.14 M of NaCI and
10% to 20% of NMP. The fractions containing the conjugated antibody in monomer form are
collected, pooled and concentrated on Amicon Ultra-15 (Ultracel 10k or 50k membrane, Millipore)
to a concentration of between 2 and 5 mg/ml. A change of buffer is finally performed so as to
remove the organic solvent from the conjugate storage buffer. The conjugate is deposited on a
gel filtration column composed of a Sephadex™ G25 matrix (Nap-5, -10, PD-10, Hiprep 26/10
desalting columns, GE Healthcare) pre-equilibrated with an aqueous buffer whose composition
and pH are suited to each conjugate. The final conjugate is assayed by UV spectrometry using
the extinction coefficients determined for the antibody and the corresponding cryptophycin
derivative so as to measure the antibody concentration and the average cytotoxic number per
antibody. The degree of substitution may also be calculated from the deconvolution of the SEC-
HRMS spectrum of the conjugate.
"One-pot" two-step method
The antibody is first modified by an activated NHS ester, so as to introduce onto its surface
pyridyldisulfide groups. A solution of antibody hu2H11 in an aqueous pH 6.5 buffer containing
0.05 M of potassium phosphate and 0.05 M of NaCI is diluted with the pH 6.5 phosphate buffer
and an aqueous 1N solution of HEPES such that the final proportion of initial pH 6.5 phosphate
buffer and of HEPES is 96/4, in order to obtain a pH=7.5-8. This antibody solution is treated with
5 to 10 eq. of the NHS activated ester dissolved in DMA such that the final antibody concentration
is between 5 and 10 mg/ml and the percentage of DMA in the aqueous buffer is 5%. The reaction
is continued for 2 hours at RT. The antibody solution thus modified is directly diluted with a 96/4
mixture of pH 6.5 phosphate buffer and of HEPES and then treated with a solution of the
cryptophycin derivative (4 eq.) in DMA such that the final antibody concentration is 3 mg/ml and
the percentage of DMA in the aqueous buffer is 20%; the number of equivalents of cryptophycin
derivative is expressed relative to the number of equivalents of NHS activated ester introduced
during the first step. The reaction is continued overnight at 30°C or with stirring at about
2000 rpm. The mixture is analyzed by SEC HPLC so as to determine the degree of grafting of the
cryptophycin derivative onto the antibody. If the degree of substitution is insufficient, the mixture
is treated with a further 1 to 5 eq. of cryptophycin derivative in DMA for 3 hours at 30°C or with
stirring at about 2000 rpm. The mixture is filtered through a Millex®-SV 5 urn filter (PVDF
membrane, Durapore, Millipore) and then purified by gel filtration using a Superdex 200 pg matrix
(HiLoad 16/60 desalting column, GE Healthcare) pre-equilibrated in an aqueous pH 6.5 buffer
containing 0.01 M of phosphate, 0.14 M of NaCI and 10% to 20% of NMP. The fractions
containing the conjugated antibody in monomer form are collected, pooled and concentrated on
Amicon Ultra-15 (Ultracel 10k or 50k membrane, Millipore) to a concentration of between 2 and
5 mg/ml. A change of buffer is finally performed so as to remove the organic solvent from the
conjugate storage buffer. The conjugate is deposited on a gel filtration column composed of a
Sephadex™ G25 matrix (Nap-5, -10, PD-10, Hiprep 26/10 desalting columns, GE Healthcare)
pre-equilibrated with an aqueous buffer whose composition and pH are suited to each conjugate.
The final conjugate is assayed by UV spectrometry using the extinction coefficients determined
for the antibody and the corresponding cryptophycin derivative so as to measure the antibody
concentration and the average cytotoxic number per antibody. The degree of substitution may
also be calculated from the deconvolution of the SEC-HRMS spectrum of the conjugate.
General method used for preparing the conjugates in the case of cryptophycin derivatives
comprising a linker ending with -C(=Q)ZhRh
A solution of antibody hu2H11 in an aqueous pH 8 buffer containing 0.05 M of HEPES, 0.05 M of
NaCI and 2 mM of EDTA or composed of a 96/4 mixture of an aqueous pH 6.5 buffer containing
0.05 M of potassium phosphate and 0.05 M of NaCI/1N HEPES is treated with an excess of a
solution in DMA on the cryptophycin derivative such that the final antibody concentration Is
3 mg/ml and the percentage of DMA in the aqueous buffer is 20%. The reaction is continued for 3
hours at 30°C or with stirring at about 2000 rpm. The mixture is analyzed by SEC HPLC so as to
determine the degree of grafting of cytotoxic agent on the population of monomer antibodies. If
the degree of substitution is insufficient, the mixture is treated with a further 1 to 5 eq. of
cryptophycin derivative in DMA for 3 hours at 30°C or with stirring at about 2000 rpm. The mixture
is filtered through a Millex®-SV 5 urn filter (PVDF membrane, Durapore, Millipore) and then
purified by gel filtration using a Superdex 200 pg matrix (HiLoad 16/60 desalting column, GE
Healthcare) pre-equilibrated in an aqueous pH 6.5 buffer containing 0.01 M of phosphate, 0.14 M
of NaCI and 10% to 20% of NMP. The fractions containing the conjugated antibody in monomer
form are collected, pooled and concentrated on Amicon Ultra-15 (Ultracel 10k or 50k membrane,
Millipore) to a concentration of between 2 and 5 mg/ml. A change of buffer is finally performed so
as to remove the organic solvent from the conjugate storage buffer. The conjugate is deposited
on a gel filtration column composed of a SephadexTM G25 matrix (Nap-5, -10, PD-10 columns or
Hiprep 26/10 desalting column, GE Healthcare) pre-equilibrated with an aqueous buffer whose
composition and pH are suited to each conjugate. The final conjugate is assayed by UV
spectrometry using the extinction coefficients determined for the antibody and the corresponding
cryptophycin derivative, so as to measure the antibody concentration and the degree of grafting.
The degree of substitution may also be calculated from the deconvolution of the SEC-HRMS
spectrum of the conjugate.
The methods described for the case of the antibody hu2H 11 may also be similarly applied to
other antibodies, and also to other binding agents.
Example 1: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-
{4-[4-(4-mercapto-4-methylpentanoyl)piperazin-1-ylmethyl]phenyl}oxiranyl)ethyl]-6,6-
dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 2: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-16-{(S)-1-[(R)-3-(4-chloro-
methylphenyl)oxiranyl]ethyl}-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-
2,5,9,12-tetraone
Compound 1 (30 mg; 42.9 umol, prepared according to Al-awar R.S., et al., J.Med.Chem. 2003,
46, 2985-3007) is dissolved in anhydrous DMF (2 ml) and the mixture is cooled to 0°C, followed
by addition of TEA (107 umol) and then CMS (64.6 umol). After 15 minutes, the bath is removed
and stirring is continued for 12 hours at RT. The mixture is diluted by adding EtOAc (2 ml) and the
organic phase is washed with water (2x1 ml), with saturated aqueous NaHCO3 solution (1 ml)
and with saturated aqueous NaCI solution (1 ml). The organic phase is dried over MgSO4 and,
after filtering and evaporating off the solvents under reduced pressure, product 2 is obtained in
the form of a colourless oil that crystallizes (25 mg; 81%). 1H NMR (500 MHz, DMSO-c/6): 0.77 -
0.83 (m, 6 H); 1.02 (s, 3 H); 1.04 - 1.07 (m, 3 H); 1.14 (s, 3 H); 1.29 - 1.36 (m, 1 H); 1.54 - 1.63
(m, 2 H); 1.80 - 1.87 (m, 1 H); 2.24 - 2.33 (m, 1 H); 2.63 - 2.73 (m, 2 H); 2.96 - 3.06 (m, 3 H);
3.28 - 3.32 (m, 1 H); 3.83 (s, 3 H); 3.93 (d, J = 1.6 Hz, 1 H); 4.27 (ddd, J = 11.3, 8.0, 3.6 Hz, 1 H);
4.78 (s, 2 H); 4.93 (dd, J = 9.6, 3.6 Hz, 1 H); 5.13 (dd, J = 10.8, 5.1 Hz, 1 H); 5.81 (d, J = 14.8 Hz,
1 H); 6.49 (ddd, J = 15.0, 11.2, 3.7 Hz, 1 H); 7.07 (d, J = 8.5 Hz, 1 H); 7.18 (dd, J = 8.5, 1.9 Hz, 1
H); 7.23 (d, J = 9.6 Hz, 1 H); 7.29 (d, J = 1.9 Hz, 1 H); 7.34 (d, J = 8.2 Hz, 2 H); 7.47 (d, J = 8.2
Hz, 2 H); 8.35 (d, J = 8.2 Hz, 1 H). LCMS (A1): ES m/z = 713 [M + H]+; m/z = 715 [M - H]~: fo
= 5.17 min.
Compound 4: 2,5-Dioxopyrrolidin-1-yl 4-methyl-4-methyldisulfanylpentanoate
To a solution, purged with argon, of compound 3 (3.05 g, 15.7 mmol, prepared according to WO
2007/085 930) in DCM (30 ml) are successively added NHS (17.3 mmol) and EDCI chloride
(17.3 mmol). The mixture is stirred for 3 hours at RT and then washed with pH 6 phosphate buffer
(2 x 30 ml) and then with saturated NaCI solution (30 ml), dried over MgSO4 and concentrated to
dryness. The amber-coloured oil obtained, which crystallizes, is washed with a 75/25
heptane/EtOAc mixture and filtered through a sinter funnel to give compound 4 in the form of a
white solid (2.08 g, 45%). The filtrate is concentrated to dryness and the crude product obtained
is purified by chromatography on silica gel, eluting with a heptane/EtOAc mixture from 50/50 to
0/100. The fractions containing the expected product are concentrated to dryness and taken up in
isopropyl ether (5 ml); the precipitate is filtered through a sinter funnel to give the expected
compound 4 (1 g, 22%). 1H NMR (400 MHz, DMSO-d6): 1.29 (s, 6 H); 1.92 to 1.98 (m, 2 H); 2.41
(s, 3 H); 2.72 to 2.78 (m, 2 H); 2.81 (s, 4 H). LCMS (A4): El, m/z = 291 [M + H]+.
Compound 5: fert-Butyl 4-(4-methyl-4-methyldisulfanylpentanoyl)piperazine-1 -carboxylate
Compound 4 (200 mg, 686 umol), 1-Boc-piperazine (686 umol), TEA (755 umol) and DMF
(1.6 ml) are placed in a Wheatton tube. The mixture is stirred at RT overnight and then diluted
with EtOAc (5 ml), washed with water (2 x5 ml) dried over MgSO4 and concentrated to dryness.
The crude product is taken up in isopropyl ether (3 ml); the precipitate is filtered through a sinter
funnel to give compound 5 (213 g, 86%). 1H NMR (400 MHz, DMSO-c/6): 1.27 (s, 6 H); 1.41 (s, 9
H); 1.73 to 1.87 (m, 2 H); 2.34 to 2.41 (m, 2 H); 2.40 (s, 3 H); 3.23 to 3.36 (partially masked m, 4
H); 3.39 to 3.45 (m, 4 H). LCMS (A2): ES m/z = 363 [M + H]*; m/z = 307 [M + H - C4H8r; tR =
1.08 min.
Compound 6: 4-Methyl-4-methyldisulfanyl-1-piperazin-1-ylpentan-1-one hydrochloride
To a solution of compound 5 (213 mg, 588 umol) in dioxane (4.4 ml) is added a 4M solution of
HCI in dioxane (4.4 ml). Stirring is continued for 4 hours at RT. The mixture is filtered through a
sinter funnel and the solid obtained is rinsed with dioxane (2 ml) and then with isopropyl ether
(2 ml) to give compound 6 (132 mg, 75%) in the form of a cream-coloured solid. 1H NMR (500
\Jlhz, DMSO-cfe): 1.27 (s, 6 H); 1.73 to 1.85 (m, 2 H); 2.37 to 2.45 (m, 2 H); 2.40 (s, 3H); 2.98 to
3.15 (m, 4 H); 3.62 to 3.73 (m, 4H); 9.39 (broad m, 2 H). LCMS (A1): ES m/z = 263 [M + H]+; tR =
2.40 min.
Compound 7: (E)-(3S, 10R, 16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-16-[(S)-1 -
((2R,3R)-3^4-[4-(4-methyM-methyldisulfanylpentanoyl)piperazin-1-ylmethyl]phenyl}oxiranyl)-
ethyl]-1,4-dioxa-8,11 -diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 1 (20.3 mg; 29.0 umol) is dissolved in anhydrous DMF (0.77 ml) and TEA (72.6 umol),
and then CMS (43.6 umol) is added. After 12 hours at RT, the product 2 formed is not isolated,
and TEA (58.0 umol) and then 4-methyl-4-methyldisulfanyl-1-piperazin-1-ylpentan-1-one
hydrochloride 6 (34.8 umol) are added. The mixture is stirred for a further 72 hours at RT and
then diluted with EtOAc (10 ml). The organic phase is washed with water (2x2 ml), with saturated
aqueous NaHCO3 solution (2 ml) and with saturated aqueous NaCI solution (2 ml). After drying
over MgSO4 and filtering, the solvents are evaporated off under reduced pressure. The crude
reaction product is purified by chromatography on silica gel, eluting with a 99/1 to 98/2
DCM/methanol mixture. A white solid, 7, is obtained (5.7 mg; 21%). TLC (DCM 90/MeOH 10):
Rf=O.6; 1H NMR (400 MHz, DMSO-d6): 0.75 - 0.81 (m, 6 H); 1.01 (s, 3 H); 1.05 (d, J=6.8 Hz, 3
H); 1.12 (s, 3 H); 1.26 (s, 6 H); 1.28 - 1.33 (m, 1 H); 1.52 - 1.61 (m, 2 H); 1.76 - 1.83 (m, 2 H);
2.27 - 2.38 (m, 4 H); 2.39 (s, 3 H); 2.64 - 2.74 (m, 2 H); 2.95 - 3.05 (m, 2 H); 3.24 - 3.34 (m, 6
H); 3.44 (br. s., 4 H); 3.49 (s, 2 H); 3.81 (s, 3 H); 3.88 (d, J=1.7 Hz, 1 H); 4.22 - 4.29 (m, 1 H);
4.92 (dd, J=9.9, 3.5 Hz, 1 H); 5.08 - 5.15 (m, 1 H); 5.81 (d, J=14.2 Hz, 1 H); 6.48 (ddd, J=15.2,
11.3, 3.5 Hz, 1 H); 7.05 (d, J=8.6 Hz, 1 H); 7.17 (dd, J=8.4, 2.3 Hz, 1 H); 7.22 (d, J=9.3 Hz, 1 H);
7.25 - 7.34 (m, 5 H); 8.34 (d, J=8.1 Hz, 1 H); LCMS (A1): ES m/z = 943 [M + H]+; m/z = 941 [M -
H]";tR = 4.03 min.
Example 1: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-16-[(S)-1-
((2R,3R)-3-{4-[4-(4-mercapto-4-methylpentanoyl)piperazin-1-ylmethyl]phenyl}oxiranyl)ethyl]-1,4-
dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
Product 7 (9.6 mg; 10.2 umol) is dissolved in a mixture of ethanol (1.2 ml)/water (1 ml) and the
mixture becomes cloudy. TCEP (25.4 umol) is then added and the mixture is stirred for 5 hours at
RT. The mixture is diluted by adding EtOAc and the organic phase is washed with a 1/1 mixture
in water and saturated aqueous NH4CI solution (1 ml). After drying the organic phase over
MgSO4, filtering and evaporating off the solvents under reduced pressure, the final product, Ex. 1,
is obtained in the form of a white solid (6.5 mg; 71%). TLC (DCM 90/MeOH 10): Rf = 0.56; 1H
NMR (500 MHz, DMSO-cf6): 0.76 - 0.81 (m, 6 H); 1.01 (s, 3 H); 1.06 (d, J = 6.8 Hz, 3 H); 1.13 (s,
3 H); 1.24 (s, 6 H); 1.27 - 1.31 (m, 1 H); 1.56 - 1.64 (m, 2 H); 1.73 - 1.85 (m, 3 H); 2.26 - 2.33
(m, 3 H); 2.36 - 2.45 (m, 4 H); 2.63 - 2.75 (m, 2 H); 2.95 - 3.06 (m, 3 H); 3.34 - 3.36 (m, 1 H);
3.42 - 3.51 (m, 6 H); 3.82 (s, 3 H); 3.89 (s, 1 H); 4.22 - 4.29 (m, 1 H); 4.92 (dd, J = 9.8, 3.4 Hz, 1
H); 5.12 (dd, J = 10.8, 4.9 Hz, 1 H); 5.81 (d, J = 15.2 Hz, 1 H); 6.48 (ddd, J = 15.0, 11.4, 3.4 Hz, 1
H); 7.06 (d, J = 8.3 Hz, 1 H); 7.18 (dd, J = 8.3, 1,5 Hz, 1 H); 7.24 (d, J = 9.8 Hz, 1 H); 7.26 - 7.36
(m, 5 H); 8.37 (d, J = 7.8 Hz, 1 H); LCMS (A2): ES m/z = 897 [M + H]+; m/z = 895 [M - H]_; tR =
0.97 min.
Example 2: (E)-(3S,6R,1 OR, 16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1 -
((2R,3R)-3-{4-[4-(4-mercapto-4-methylpentanoyl)piperazin-1-ylmethyl]phenyi}oxiranyi)-
ethyl]-6-methyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 9: (E)-(3S,6R,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-16-{(S)-1-[(R)-3-(4-chloro-
methylphenyl)oxiranyl]ethyl}-3-isobutyl-6-methyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-
2,5,9,12-tetraone
Compound 8 (49 mg; 71.4 umol, which may be prepared according to Al-awar R.S., et a!.,
J.Med.Chem. 2003, 46, 2985-3007) is dissolved in anhydrous DCM (5 ml) and the mixture is
cooled to 0°C, followed by addition of DIPEA (428 umol) and then CMS (214 umol). The mixture
is allowed to return to RT and stirring is continued for 40 hours at RT. The mixture is hydroiyzed
with 5 ml of water and the aqueous phase is extracted with DCM (3x5 ml). The organic phases
are combined, washed with saturated aqueous NaHCO3 solution (10 ml) and with saturated
aqueous NaCI solution (10 ml) and dried over MgSO4. After filtering and evaporating off the
solvents under reduced pressure, the crude product is purified by chromatography on silica gel,
eluting with a 100/0 to 90/10 DCM/MeOH mixture. Compound 9 is obtained in the form of a white
solid (37 mg; 79%). 1H NMR (500 MHz, DMSO-cfe): 0.78 (d, J = 6.6 Hz, 3 H); 0.80 (d, J = 6.6 Hz,
3 H); 1.01 (d, J = 6.9 Hz, 3 H); 1.05 (d, J = 6.9 Hz, 3 H); 1.30 (m, 1 H); 1.54 (m, 1 H); 1.60 (m, 1
H); 1.82 (m, 1 H); 2.27 (m, 1 H); 2.60 to 2.78 (m, 3 H); 2.97 to 3.05 (m, 2 H); 3.15 (m, 1 H); 3.41
(m, 1 H); 3.81 (s, 3 H); 3.92 (d, J = 1.6 Hz, 1 H); 4.26 (ddd, J = 3.8 and 8.2 and 11.5 Hz, 1 H),
4.77 (s, 2 H); 4.88 (dd, J = 3.8 and 9.6 Hz, 1 H); 5.12 (ddd, J = 1.5 and 5.3 and 11.2 Hz, 1 H);
5.80 (dd, J = 1.5 and 15.0 Hz, 1 H); 6.47 (ddd, J = 3.7 and 11.2 and 15.0 Hz, 1 H); 7.05 (d, J =
8.5 Hz, 1 H); 7.17 (dd, J = 2.2 and 8.5 Hz, 1 H); 7.23 (dd, J = 2.7 and 9.1 Hz, 1 H); 7.29 (d, J =
2.2 Hz, 1 H); 7.32 (d, J = 8.2 Hz, 2 H); 7.45 (d, J = 8.2 Hz, 2 H); 8.34 (d, J = 8.2 Hz, 1 H). LCMS
(A2): ES m/z = 703 [M + H]+; m/z = 701 [M - Hp m/z = 747 [M - H + HCO2Hr base peak; tR =
1.17 min.
Compound 10: (E)-(3S,6R, 10R, 16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-6-methyl-16-[(S)-
1-((2R,3R)-3-{4-[4-(4-methyl-4-methyldisulfanylpentanoyl)piperazin-1-ylmethyl]phenyl}oxiranyl)-
ethyl]-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 9 (36.6 mg; 52 umol) is dissolved in anhydrous acetonitrile (3 ml), followed by
successive addition of DiPEA (260 umol) and Compound 6 (156 pmol). The reaction medium is
stirred for 20 hours at RT and then hydrolyzed by addition of 4 ml of water. The aqueous phase is
extracted with EtOAc (3x4 ml) and the organic phases are combined, washed with saturated
aqueous NaHCO3 solution (5 ml) and with saturated aqueous NaCI solution (5 ml). After drying
over MgSO4 and filtering, the solvents are evaporated off under reduced pressure. The crude
reaction product is purified by chromatography on silica gel, eluting with a 100/0 to 95/5
DCM/methanol mixture. Compound 10 is obtained in the form of a white solid (39 mg; 80%). 1H
NMR (500 MHz, DMSO-d6): 0.78 (d, J = 6.6 Hz, 3 H); 0.80 (d, J = 6.6 Hz, 3 H); 1.01 (d, J = 6.9
Hz, 3 H); 1.05 (d, J = 6.9 Hz, 3 H); 1.26 (s, 6 H); 1.30 (m, 1 H); 1.53 (m, 1 H); 1.60 (m, 1 H); 1.75
to 1.84 (m, 3 H); 2.26 to 2.37 (m, 7 H); 2.39 (s, 3 H); 2.61 to 2.77 (m, 3 H); 2.96 to 3.05 (m, 2 H);
3.15 (m, 1 H); 3.38 (partially masked m, 1 H); 3.44 (m, 4 H); 3.48 (s, 2 H); 3.81 (s, 3 H); 3.88 (d, J
= 1.6 Hz, 1 H); 4.27 (ddd, J = 4.0 and 8.1 and 11.5 Hz, 1 H); 4.88 (dd, J = 3.8 and 9.6 Hz, 1 H);
5.12 (ddd, J = 1.6 and 5.3 and 11.5 Hz, 1 H); 5.80 (dd, J = 1.6 and 15.1 Hz, 1 H); 6.47 (ddd, J =
3.6 and 11.5 and 15.1 Hz, 1 H); 7.05 (d, J = 8.5 Hz, 1 H); 7.17 (dd, J = 2.2 and 8.5 Hz, 1 H); 7.23
(dd, J = 2.6 and 9.2 Hz, 1 H); 7.27 (d, J = 8.4 Hz, 2 H); 7.29 (d, J = 2.2 Hz, 1 H); 7.32 (d, J = 8.4
Hz, 2 H); 8.35 (d, J = 8.1 Hz, 1 H). LCMS (A2): ES m/z = 929 [M + H]+; m/z = 927 [M - H]-; m/z =
973 [M - H + HCO2H]" base peak; tR= 0.99 min.
Example 2: (E)-(3S,6R,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-6-methyl-16-[(S)-1-
((2R,3R)-3-{4-[4-(4-mercapto-4-methylpentanoyl)piperazin-1-ylmethyl]phenyl}oxiranyl)ethyl]-1,4-
dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
Product 10 (34 mg; 36.6 umol) is dissolved in a mixture of ethanol (4.3 ml)/water (3.6 ml) and the
mixture turns cloudy. TCEP (146 umol) is then added, the mixture becomes colourless, and is
stirred for 2 hours at RT. The mixture is diluted by adding EtOAc (20 ml) and the organic phase is
washed with a 1/1 mixture of water and saturated aqueous NH4CI solution (20 ml). The aqueous
phase is extracted with 2x20 ml of EtOAc and the organic phases are combined and washed with
saturated NaCI solution (20 ml). After drying over MgSO4, filtering and evaporating off the
solvents under reduced pressure, the crude product is purified by chromatography on silica gel,
eluting with a 98/2 to 90/10 DCM/MeOH mixture. The compound Ex. 2 is obtained in the form of a
white solid (28.2 mg; 87%). 1H NMR (500 MHz, DMSO-d6): 0.77 (d, J = 6.6 Hz, 3 H); 0.79 (d, J =
6.6 Hz, 3 H); 1.01 (d, J = 6.9 Hz, 3 H); 1.04 (d, J = 6.9 Hz, 3 H); 1.27 (m, 1 H); 1.31 (s, 6 H); 1.53
(m, 1 H); 1.59 (m, 1 H); 1.77 (m, 2 H); 1.82 (m, 1 H); 2.24 to 2.32 (m, 3 H); 2.34 to 2.44 (m, 4 H);
2.62 to 2.76 (m, 4 H); 2.98 to 3.04 (m, 2 H); 3.15 (m, 1 H); 3.41 (m, 1 H); 3.46 (m, 4 H); 3.48 (s, 2
H); 3.81 (s, 3 H); 3.88 (d, J = 2.0 Hz, 1 H); 4.26 (ddd, J = 3.4 and 8.3 and 11.5 Hz, 1 H); 4.88 (dd,
J = 3.7 and 10.0 Hz, 1 H); 5.12 (ddd, J = 2.0 and 5.3 and 11.1 Hz, 1 H); 5.80 (dd, J = 2.0 and
15.0 Hz, 1 H); 6.47 (ddd, J = 3.7 and 11.1 and 15.0 Hz, 1 H); 7.05 (d, J = 8.3 Hz, 1 H); 7.17 (dd, J
= 2.0 and 8.3 Hz, 1 H); 7.23 (dd, J = 2.9 and 9.3 Hz, 1 H); 7.27 (d, J = 8.5 Hz, 2 H); 7.29 (d, J =
2.0 Hz, 1 H); 7.32 (d, J = 8.5 Hz, 2 H); 8.35 (d, J = 8.3 Hz, 1 H). LCMS (A2): ES m/z = 883 [M +
H]+; m/z = 881 [M - H]_; tR = 0.92 min.
Example 3: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-
{4-[(2-mercapto-2-methylpropylamino)methyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-
dioxa-8,11 -diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 11: 2-Methyl-2-methyldisulfanylpropionaldehyde O-methyloxime
5 g (33.3 mmol) of 2-(methyldithio)isobutyraldehyde are dissolved in 50 ml of ethanol, under an
inert atmosphere. A suspension of 5.56 g (66.54 mmol) of O-methylhydroxylamine chloride in
50 ml of ethanol and then 6.65 ml (66.54 mmol) of NaOH are successively added. The cloudy
white mixture is refluxed overnight. After cooling to RT, the mixture is poured into 500 ml of water.
The aqueous phase is extracted with EtOAc (3x175 ml), and the combined organic phases are
washed with saturated NaCI solution (200 ml) and dried over MgSO4. After filtering and
concentrating under reduced pressure, compound 11 is obtained in the form of a colourless oil
(5.89 g, 32.9 mmol).
Compound 12:2-Methyl-2-methyldisulfanylpropylamine
1.78 g (9.9 mmol) of oxime ether 11, 19 ml of anhydrous THF and 99.5 ml of a 2M solution of
borane-methyl sulfide in THF are successively introduced, at RT, into a round-bottomed flask
purged with argon. The mixture is refluxed for 16 hours. The reaction is then stopped and the
mixture is allowed to cool to RT, followed by cautious dropwise addition of 100 ml of MeOH at
0°C while the foam forms, and then at RT. The mixture is evaporated under reduced pressure to
give about 2.8 g of a yellow oil, which is taken up in 30 ml of a 5 to 6N solution of HCI in
isopropanol. The mixture is refluxed for 1 hour and then left at RT overnight. After evaporating
under reduced pressure, the residue obtained is taken up in 80 ml of 1N HCI and then extracted
with diethyl ether (3x20 ml). The aqueous phase is treated with aqueous 30% ammonia until a pH
of 12.5-13 is obtained, i.e. 12 ml (pale purple aqueous phase) and then extracted again with
3x20 ml of ether. The organic phases are combined, dried over MgSO4, filtered and evaporated
to dryness under reduced pressure to give 760 mg of crude product. This residue is finally
purified by chromatography on silica gel, eluting with a 100/0 to 90/10 DCM/methanol mixture.
Compound 12 is obtained in the form of a pale yellow oil (301 mg, 20%). LCMS (A2): ES m/z =
152 [M + H]+;tR = 0.27 min.
Compound 13: (E)-(3S, 10R, 16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1 -((2R, 3R)-
3-{4-t(2-methyl-2-methyldisulfanylpropylamino)methyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-
dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
To a solution, purged with argon, of 25 mg (35 umol) of compound 2 in 3.1 ml of anhydrous DMF
are successively added, at RT, 9.8 ul (70 umol) of TEA and 6.3 mg (42 umol) of compound 12.
Stirring is continued at 40°C. After reaction for 72 hours, some starting compound 2 remains, so a
further 9.8 pi (70 pmol) of TEA and 6.3 mg (42 umol) of the amine 12 are added. After a further
72 hours at 40°C, some compound 2 still remains: 6.3 mg (42 pmol) of the amine 12 are added
and stirring is continued at40°C. One day later, the reaction is complete. The mixture is diluted in
10 ml of EtOAc, washed with 2x10 ml of water, with 10 ml of saturated NaHCO3 solution and
10 ml of saturated NaCI solution. After drying the organic phase over MgSO4, it is filtered and
then evaporated under reduced pressure to give 30 mg of crude product. This residue is purified
by chromatography on silica gel, eluting with a 98/2 to 93/7 DCM/methanol mixture. Compound
13 is obtained in the form of a white powder (23.3 mg, 81%). TLC (DCM 90/ MeOH 10): Rf=O.55;
1H NMR(500 MHz, DMSO-c/6): 0.76 to 0.81 (m, 6 H); 1.02 (s, 3 H); 1.07 (d, J = 6.9 Hz, 3 H); 1.13
(s, 3 H); 1.27 to 1.33 (m, 7 H); 1.52 to 1.63 (m, 2 H); 1.82 (sxt, J = 6.8 Hz, 1 H); 2.06 (broad s, 1
H); 2.25 to 2.34 (m, 1 H); 2.37 (s, 3 H); 2.57 (s, 2 H); 2.66 to 2.76 (m, 2 H); 2.97 to 3.06 (m, 3 H);
3.33 to 3.38 (m, 1 H); 3.75 (s, 2 H); 3.83 (s, 3 H); 3.88 (d, J = 1.6 Hz, 1 H); 4.27 (ddd, J = 3.7 and
8.0 and 11.4 Hz, 1 H); 4.93 (dd, J = 3.7 and 9.7 Hz, 1 H); 5.12 (dd, J = 5.8 and 11.3 Hz, 1 H);
5.82 (dd, J = 1.5 and 15.2 Hz, 1 H); 6.49 (ddd, J = 3.8 and 11.3 and 15.2 Hz, 1 H); 7.07 (d, J =
8.5 Hz, 1 H); 7.18 (dd, J = 1.9 and 8.5 Hz, 1 H); 7.23 (dd, J = 2.6 and 9.5 Hz, 1 H); 7.26 (d, J =
8.2 Hz, 2 H); 7.30 (d, J = 1.9 Hz, 1 H); 7.35 (d, J = 8.2 Hz, 2 H); 8.35 (d, J = 8.0 Hz, 1 H). LCMS
(A2): ES m/z = 832 [M + H]+; m/z = 830 [M - Hf; tR = 0.96 min.
Example 3: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-{4-
[(2-mercapto-2-methylpropylamino)methyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-8,11-
diazacyclohexadec-13-ene-2,5,9,12-tetraone
Product 13 (10.6 mg; 12.8 umol) is dissolved in a mixture of ethanol (1.5 ml)/water (1.26 ml).
TCEP (9.1 mg, 31.9 umol) is then added and the mixture is stirred for 2 hours 30 minutes at RT.
The mixture is diluted in 15 ml of EtOAc and the organic phase is washed with a 1/1 mixture of
water and saturated aqueous NH4CI solution (5 ml). After drying the organic phase over MgSO4,
filtering and evaporating off the solvents under reduced pressure, the crude product obtained is
finally purified by chromatography on silica gel, eluting with a 98/2 to 92/8 DCM/methanol mixture.
The compound Ex. 3 is obtained in the form of a white solid (6.4 mg; 63%). TLC (DCM 90/MeOH
10): Rf=O.47; 1H NMR (500 MHz, DMSO-c/6): 0.80 to 0.86 (m, 6 H); 1.06 (s, 3 H); 1.11 (d, J = 6.9
Hz, 3 H); 1.18 (s, 3 H); 1.31 to 1.38 (m, 7 H); 1.57 to 1.67 (m, 2 H); 1.87 (sxt, J = 6.8 Hz, 1 H);
2.29 to 2.38 (m, 1 H); 2.56 (s, 2 H); 2.70 to 2.80 (m, 2 H); 3.00 to 3.11 (m, 3 H); 3.39 to 3.43 (m, 1
H); 3.82 (s, 2 H); 3.87 (s, 3 H); 3.92 (d, J = 1.9 Hz, 1 H); 4.31 (ddd, J = 3.6 and 8.0 and 11.5 Hz, 1
H); 4.97 (dd, J = 3.7 and 9.7 Hz, 1 H); 5.17 (dd, J = 5.8 and 11.3 Hz, 1 H); 5.86 (dd, J = 1.4 and
15.1 Hz, 1 H); 6.54 (ddd, J = 3.8 and 11.3 and 15.1 Hz, 1 H); 7.11 (d, J = 8.7 Hz, 1 H); 7.23 (dd, J
= 1.9 and 8.7 Hz, 1 H); 7.27 (dd, J = 2.7 and 9.6 Hz, 1 H); 7.31 (d, J = 8.0 Hz, 2 H); 7.34 (d, J =
1.9 Hz, 1 H); 7.42 (d, J = 8.0 Hz, 2 H); 8.40 (d, J = 8.0 Hz, 1 H). LCMS (A2): ES m/z = 786 [M +
H]+; m/z = 784 [M - H]~; tR = 0.92 min.
Example 4: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-
{4-[({2-mercapto-2-methylpropyl}methylamino)methyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-
1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 14: Methyl[2-methyl-2-methyldisulfanylprop-(E)-ylidene]amine
1 g (6.66 mmol) of 2-(methyldithio)isobutyraldehyde are dissolved in 10 ml of anhydrous THF,
under an inert atmosphere. A 2M solution of methylamine in THF (33.3 ml, 66,6 mmol) is added
and the mixture is then stirred for 5 hours at RT. It is diluted in 50 ml of EtOAc, washed with water
(30 ml) and with saturated NaCI solution (30 ml) and dried over MgSO4. After filtering and
concentrating under reduced pressure, compound 14 is obtained in the form of a pale yellow oil
(1.01 g, 93%). 1H NMR (400 MHz, chloroform-d): 1.46 (s, 6 H); 2.36 (s, 3 H); 3.32 (s, 3 H); 7.52
(s, 1 H).
Compound 15: 2-Methyl-2-methyldisulfanylpropylamine
A solution of compound 14 (1.01 g, 6.185 mmol) in 30 ml of THF is purged with argon and cooled
to 0°C, followed by addition, at 0°C, of 1.44 g (6.80 mmol) of sodium triacetoxyborohydride.
Stirring is continued for 15 hours at RT: some starting imine remains; 1.44 g (6.80 mmol) of
sodium triacetoxyborohydride and 354 pi (6.185 mmol) of acetic acid are added at 0°C and
stirring is then continued for 3 hours at RT. The mixture is diluted in 50 ml of EtOAc and washed
with water (50 mi). The pH of the aqueous phase is adjusted to about 12 by adding 14 mi of
aqueous 1N sodium hydroxide solution, and it is then extracted with diethyl ether (3x50 ml). The
organic phases are combined, washed with saturated NaCI solution (50 ml) and dried over
MgSO4. After filtering and concentrating under reduced pressure, compound 15 is obtained in the
form of a colourless oil (695 mg, 68%). 1H NMR (400 MHz, DMSO-cfe): 1.25 (s, 6 H); 1.47 (broad
m, 1 H); 2.31 (s, 3 H); 2.38 (s, 3 H); 2.53 (m, 2 H). LCMS (A2): ES m/z = 166 [M + H]+; tR = 0.28
min.
Compound 16: (E)-(3S, 10R, 16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-{(S)-1 -[(2R,3R)-3-
(4-{[(2-methyl-2-methyldisulfanylpropyl)methylamino]methyl}phenyl)oxiranyl)ethyl]-6,6-dimethyl-
1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 16 may be obtained by nucleophilic substitution of the chloro group of the derivative 2
with the amine 15 by applying the method described for the preparation of compound 30.
Example 4: (EH3S,10R,16S)-1O-(3-Chioro-4-methoxybenzyl)-3-isobutyl-16-{(S)-1-[(2R,3R)-3-(4-
{[(2-mercapto-2-methylpropyl)methylamino]methyl}phenyl)oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-
8,11 -diazacyclohexadec-13-ene-2,5,9,12-tetraone
Example 4 may be obtained by applying the method described for the preparation of Example 6
to compound 16.
Example 5: (E)-(3S,10R,16S;-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-
{4-[4-(2-;nercapto-2-methylpropyl)piperazin-1-ylmethyl]phenyl}oxiranyl)ethyl]-6,6-Jimethyl-
1,4»rlioxa-8,11 -diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 17: ferf-Butyl 4-(2-methyl-2-methyldisulfanylpropyl)piperazine-1 -carboxylate
To a solution, purged with argon, of 1-Boc-piperazine (1.0 g, 5.37 mmol) in anhydrous THF
(20 ml) are added 2-(methyldithio)isobutyraldehyde (5.37 mmol) and titanium(IV) isopropoxide
(6.71 mmol). The mixture is stirred for 20 minutes at RT, and a further addition of 2-(methyldithio)-
isobutyraldehyde (5.37 mmol) and of titanium(IV) isopropoxide (6.71 mmol) is performed. Stirring
is continued for 2 hours, and then 12 ml of ethanol are added and stirring is continued for 5
minutes. Sodium cyanoborohydride (5.37 mmol) is added and the mixture is stirred for 1 hour,
followed by addition of a further 5.37 mmol of sodium cyanoborohydride. Stirring is continued for
1 hour. The mixture is concentrated to dryness and then taken up in EtOAc. Water is added,
causing the formation of a precipitate, which is filtered off on a sinter funnel and rinsed with
EtOAc and water. The soiid obtained is dissolved in aqueous 1N HCI solution (50 ml) and the
medium is neutralized with aqueous 5N NaOH solution and then extracted with DCM. The
organic phases are combined, washed with saturated NaCI solution, dried over MgSO4, filtered
and concentrated under reduced pressure. The crude product is purified by chromatography on
silica gel, eluting with a 100/0 to 97/3 DCM/methanol mixture. Compound 17 is obtained in the
form of a pale yellow oil (650 mg; 40%). 1H NMR (400 MHz, DMSO-afe): 1.26 (s, 6 H); 1.39 (s, 9
H); 2.39 (s, 3 H); 2.42 (s, 2H); 2.44 to 2.48 (m, 4 H); 3.27 (partially masked m, 4 H). LCMS (A1):
ES m/z = 321 [M + H]+; m/z = 265 base peak; tR = 3.02 min.
Compound 18:1-(2-Methyl-2-methyldisulfanylpropyl)piperazine hydrochloride
To a solution of compound 17 (322 mg, 1.01 mmol) in dioxane (13 ml) is added a 4M solution of
HCI in dioxane (5 ml). Stirring is continued for 16 hours at RT. The mixture is filtered through a
sinter funnel and the solid obtained is rinsed with dioxane to give compound 18 (231 mg, 90%) in
the form of a white powder. 1H NMR (400 MHz, DMSO-cfe): 1.31 (broad s, 6 H); 2.42 (s, 3 H);
2.59 to 3.38 (very broad m, 10 H); 8.78 (broad m, 2 H). LCMS (A2): ES m/z = 221 [M + Hf; tR =
0.46 min.
Compound 19: (E)-(3S, 10R, 16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobuty!-6,6-dimethyl-16-[(S)-
i-((2R,3R)-3-{4-[4-(2-methyl-2-methyldisulfanylpropyl)piperazin-1-ylmethy|!phenyl}oxiranyl)ethyl]-
1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
To a solution, purged with argon, of compound 2 (15.02 mg, 20.93 umol) in anhydrous DMF
(0.9 ml) is added a solution of compound 18 (25.12 umol) and TEA (52.3 umol) in DMF (1 ml).
The mixture is stirred at 40°C under argon. After 24 hours, 25.12 umol of compound 18 and
31.4 umol of TEA are added. After a further 24 hours at 40°C, the reaction is complete. The
mixture is diluted with EtOAc (6 ml) and washed with water (2x6 ml), with saturated NaHCO3
solution (6 ml) and with saturated NaCI solution (6 ml). After drying the organic phase over
MgSO4, it is filtered and then evaporated under reduced pressure to give 46 mg of crude product.
The residue is purified by chromatography on silica gel, eluting with a 100/0 to 95/5
DCM/methanol mixture. Compound 19 is obtained in the form of a white powder (5.2 mg, 28%).
1H NMR (500 MHz, DMSO-cfe): 0.76 (d, J = 6.4 Hz, 3 H); 0.78 (d, J = 6.4 Hz, 3 H); 1.01 (s, 3 H);
1.06(d, J = 6.8 Hz, 3H); 1.13 (s, 3 H); 1.25 (s, 6 H); 1.27 to 1.33 (m, 1 H); 1.51 to 1.63 (m, 2 H);
1.76 to 1.85 (m, 1 H); 2.23 to 2.32 (m, 1 H); 2.37 (m, 4 H); 2.39 (s, 3 H); 2.41 (s, 2 H); 2.53 to
2.56 (m, 4 H); 2.63 to 2.76 (m, 2 H); 2.94 to 3.06 (m, 3 H); 3.34 (partially masked m, 1 H); 3.40 to
3.48 (m, 2 H); 3.82 (s, 3 H); 3.87 (d, J = 2.0 Hz, 1 H); 4.26 (ddd, J = 3.8 and 7.8 and 11.3 Hz, 1
I4T; 4.92 (dd, J = 3.8 and 9.8 Hz, 1 H); 5.07 to 5.14 (m, 1 H); 5.81 (d, J = 15.2 Hz, 1 H); 6.48 (ddd,
J = 3.8 and 11.3 and 15.2 Hz, 1 H); 7.06>(d, J = 8.5 Hz, 1 H); 7.18 (dd, J = 2.2 and 8.5 Hz, 1 H);
7.21 to 7.32 (m, 6 H); 8.35 (d, J = 8.3 Hz, 1 H). LCMS (A1): ES m/z = 901 [M + H]+; m/z = 451
base peak; tR = 4.33 min.
Example 5: (E)-(3S, 10R.16S)-1O-(3-Chloro-4-methoxybenzyl)-3~isobutyl-16-[(S)-1 -((2R,3R)-3-{4-
[4-(2-mercapto-2-methylpropyl)piperazin-1-ylmethyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-
8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
Product 19 (5.7 mg; 6.3 umol) is dissolved in a mixture of ethanol (0.8 ml)/water (0.5 ml). TCEP
(4.54 mg, 15.83 umol) is then added and the mixture is stirred for 1 hour at RT. The mixture is
diluted in 7 ml of EtOAc and the organic phase is washed with a 1/1 mixture of water and
saturated aqueous NH4CI solution (7 ml). After drying the organic phase over MgSO4, filtering
and evaporating off the solvents under reduced pressure, the crude product obtained is purified
by chromatography on silica gel, eluting with a 99/1 to 95/5 DCM/methanol mixture. The
compound Ex. 5 is obtained in the form of a white solid (2.95 mg; 55%). 1H NMR (500 MHz,
DMSO-cfe): 0.77 (d, J = 6.3 Hz, 3 H); 0.78 (a, J = 6.3 Hz, 3 H); 1.02 (s, 3 H); 1.06 (d, J = 6.9 Hz, 3
H); 1.13 (s, 3 H); 1.27 (s, 6 H); 1.29 to 1.34 (m, 1 H); 1.52 to 1.63 (m, 2 H); 1.82 (m, 1 H); 2.29
(m, 1 H); 2.37 (s, 2 H); 2.39 to 2.41 (m, 4 H); 2.57 to 2.62 (m, 4 H); 2.65 to 2.76 (m, 2 H); 2.96 to
3.06 (m, 3 H); 3.34 (partially masked m, 1 H); 3.46 (s, 2 H); 3.83 (s, 3 H); 3.88 (d, J = 2.2 Hz, 1
H); 4.27 (ddd, J = 3.7 and 8.0 and 11.5 Hz, 1 H); 4.93 (dd, J = 3.7 and 9.7 Hz, 1 H); 5.12 (m, 1 H);
5.82 (d, J = 15.2 Hz, 1 H); 6.49 (ddd, J = 3.7 and 11.5 and 15.2 Hz, 1 H); 7.07 (d, J = 8.5 Hz, 1
H); 7.18 (dd, J = 2.2 and 8.5 Hz, 1 H); 7.21 to 7.33 (m, 6 H); 8.36 (d, J = 8.0 Hz, 1 H). LCMS (A1):
ES m/z = 855 [M + H]+; m/z = 428 [M + 2H]2+ base peak; tR = 4.13 min.
Example 6: (E)-(3S)10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-
{4-[4-({2-[2-(2-{2-methyl[2-mercapto-2-methylpropyl]aminoethoxy}ethoxy)ethoxy]ethyl}-
methylamino)methyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-8,11 -
diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 20: 2-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}ethyl 4-toluenesulfonate
To a solution, purged under argon and cooled to 0°C, of 5 g (25.74 mmol) of tetraethylene glycol
in 68.7 ml of DCM are successively added portionwise, so as to maintain suitable stirring, 8.95 g
(38.61 mmol) of silver oxide and 5.40 g (28.31 mmol) of tosyl chloride. 855 mg (5.15 mmol) of Kl
are added portionwise so as to maintain the temperature of the mixture below 5°C. Stirring is
continued for 1 hour while keeping the temperature below 5°C. After warming to RT, the mixture
is filtered through Clarcel, the residue is rinsed with DCM and the filtrate is then concentrated to
dryness under reduced pressure. The crude product is purified by chromatography on silica gel,
using a 99/1 to 95/5 DCM/methanol mixture as eluent. Compound 20 is obtained in the form of a
colourless oil (5.4 g, 60%). 1H NMR (400 MHz, DMSO-cfe): 2.43 (s, 3 H); 3.40 (m, 2 H); 3.44 to
3.52 (m, 10 H); 3.58 (m, 2 H); 4.11 (m, 2 H); 4.53 (t, J = 5.4 Hz, 1 H); 7.48 (d, J = 8.3 Hz, 2 H);
7.78 (d, J = 8.3 Hz, 2 H). LCMS (A2): ES m/z = 349 [M + H]+; m/z = 371 [M + Na]+; tR = 0.69 min.
Compound 21:1-Azido-3,6-9-trioxaundecan-11-ol
To a solution of 5.4 g (15.51 mmol) of compound 20 in 40.6 ml of anhydrous acetonitrile are
added 1.34 g (20.63 mmol) of NaN3 and the mixture is then refluxed for 6 hours 30 minutes. After
cooling to RT, the mixture is filtered through Clarcel and concentrated under reduced pressure.
Some starting compound 20 remains: the crude product is taken up in 25 ml of anhydrous
acetonitrile and 230 mg (3.5 mmol) of NaN3 are added. The mixture is refluxed for 5 hours. After
cooling to RT, it is filtered through Clarcel and concentrated to dryness under reduced pressure to
give compound 21 in the form of a pale yellow oil (3.37 g, 99%). 1H NMR (400 MHz, DMSO-c/6):
3.37 to 3.43 (m, 4 H); 3.45 to 3.58 (m, 10 H); 3.60 (m, 2 H); 4.54 (t, J = 5.1 Hz, 1 H). LCMS (A2):
ES m/z = 220 [M + H]+; m/z = 242 [M + Na]+ base peak; tR = 0.39 min.
Compound 22: N-Boc-aminoethoxyethoxyethoxyethanol
To a solution, rendered inert under argon, of 320 mg of 10% palladium-on-charcoal in 5 ml of
EtOAc is added a solution of 3.25 g (14.8 mmol) of compound 21, 6.46 g (29.6 mmol) of terf-butyl
dicarbonate and 4.13 ml (29.6 mmol) of TEA in 45 mi of EtOAc. The reaction is performed for 17
hours at 30°C under a hydrogen pressure of 2 bar. After cooling to RT and atmospheric pressure,
the mixture is filtered through Clarcel and concentrated under reduced pressure. The crude
product is purified by chromatography on silica gel, using a 98/2 to 90/10 DCM/methanol mixture
aT eluent. Compound 22 is obtained in the form of a colourless oil (2.92 g, 67%). 1H NMR (400
MHz, DMSO-cfe): 1.37 (s, 9 H); 3.06 (q, J = 6.1 Hz, 2 H); 3.37 (t, J = 6.1 Hz, 2 H); 3.42 (m, 2 H);
3.46 to 3.53 (m, 10 H); 4.54 (broad t, J = 5.1 Hz, 1 H); 6.71 (broad t, J = 6.1 Hz, 1 H). LCMS (A1):
ES m/z = 316 [M + Na]+; m/z = 194 base peak; tR = 2.81 min.
Compound 23: 2-{2-[2-(2-tert-Butoxycarbonylaminoethoxy)ethoxy]ethoxy}ethyl 4-toluenesulfonate
To a solution, purged with argon, of 3.06 g (10.42 mmol) of compound 22 in 20 ml of DCM are
added 2.53 ml (31.26 mmol) of pyridine. The mixture is cooled to 0°C, followed by dropwise
addition of a solution of 2.98 g (15.63 mmol) of tosyl chloride in 10 ml of DCM. Stirring is
continued for 15 hours at RT. The mixture is diluted in 20 ml of DCM, washed with saturated
NaHCO3 solution (30 ml), with water (2x30 ml) and with saturated NaCI solution (30 ml) and dried
over MgSO4. After filtering and concentrating under reduced pressure, the crude product is
purified by chromatography on silica gel, using a 100/0 to 90/10 DCM/methanol mixture as eluent.
Compound 23 is obtained in the form of a pale yellow oil (4.38 g, 94%). 1H NMR (400 MHz,
DMSO-d6): 1.37 (s, 9 H); 2.42 (s, 3 H); 3.05 (q, J = 6.1 Hz, 2 H); 3.36 (t, J = 6.1 Hz, 2 H); 3.46 (m,
8 H); 3.58 (m, 2 H); 4.10 (m, 2 H); 6,71 (broad t, J = 6.1 Hz, 1 H); 7.48 (d, J = 7.8 Hz, 2 H); 7.78
(d, J = 7.8 Hz, 2 H). LCMS (A2): ES m/z = 448 [M + H]+; m/z = 470 [M + Na]+; m/z = 348 base
peak; m/z = 492 [M - H + HCO2H]"; tR = 1.00 min.
Compound 24: terf-Butyl (2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl)carbamate
To a solution of 4.38 g (9.79 mmol) of compound 23 in 25 ml of acetonitrile are added 846 mg
(13.02 mmol) of sodium azide and the mixture is then refluxed for 6 hours. A large proportion of
starting compound remains: after cooling to RT, 1.7 g (26.13 mmol) are added to the mixture.
Stirring is continued at reflux for 24 hours. After cooling to RT, the mixture is filtered through
Clarcel and concentrated under reduced pressure. The crude product is purified by
chromatography on silica gel, using a 98/2 to 90/10 DCM/methanol mixture as eluent. Compound
24 is obtained in the form of a pale yellow oil (2.16 g, 69%). 1H NMR (400 MHz, DMSO-d6): 1.37
(s, 9 H); 3.06 (q, J = 6.1 Hz, 2 H); 3.34 to 3.43 (m, 4 H); 3.46 to 3.58 (m, 8 H); 3.60 (m, 2 H); 6.70
(broad t, J = 6.1 Hz, 1 H). LCMS (A2): ES m/z = 341 [M + Na]+; tR = 0.81 min.
Compound 25: ferf-Butyl (2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}ethyl)methylcarbamate
To a solution, purged with argon and cooled to 0°C, of 2.06 g (6.47 mmol) of compound 24 in
25 ml of anhydrous THF are successively added, at 0°C, 854 mg (21.35 mmol) of 60% NaH as a
dispersion in mineral oil (portionwise) and 886 pi (14.23 mmol) of methyl iodide. Stirring is
continued for 1 hour at 0°C and then for 16 hours at RT. The mixture is filtered through Clarcel,
washed with THF and concentrated under reduced pressure. The crude product is purified by
chromatography on silica gel, using a 99/1 to 90/10 DCM/methanol mixture as eluent. Compound
25 is obtained in the form of a colourless oil (1.67 g, 78%). 1H NMR (400 MHz, DMSO-d6): 1.39
(s, 9 H); 2,80 (broad s, 3 H); 3.29 (partially masked t,J = 5.6 Hz, 2 H); 3.38 (m, 2 H); 3.46 to 3.56
(m, 10 H); 3.60 (t, J = 5.4 Hz, 2 H).
Compound 26: te/t-Butyl (2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)methylcarbamate
To a solution, purged with argon, of 1.66 g (4.99 mmol) of compound 25 in 20 ml of THF are
successively added 1.31 g (4.994 mmol) of triphenylphosphine and 108 pi (5.99 mmol) of water.
Stirring is continued for 25 hours 30 minutes and the mixture is then concentrated to dryness and
purified by SFE filtration on an SCX cartridge (Varian) conditioned and washed with methanol
and then eluted with a 0.5 N solution of aqueous ammonia in methanol. Compound 26 is
obtained in the form of a colourless oil (1.23 g, 80%). 1H NMR (400 MHz, DMSO-afe): 1.39 (s, 9
H); 2.65 (t, J = 5.9 Hz, 2 H); 2.80 (broad s, 3 H); 3.29 (t, J = 5.9 Hz, 2 H); 3.36 (t, J = 5.7 Hz, 2 H);
3.45 to 3.56 (m, 10 H). LCMS (A2): ES m/z = 307 [M + H]+; tR = 0.49 min.
Compound 27: ferf-Butyl methyl[2-(2-{2-[2-(2-methyl-2-methyldisuifanylpropyiamino)ethoxy]-
ethoxy}ethoxy)ethyl]carbamate
To a solution, purged with argon, of 131 mg (426 pmol) of compound 26 in 3 ml of DCM are
successively added 64 mg (426 pmol) of 2-methyldithioisobutyraldehyde, 126 mg (596 pmol) of
sodium triacetoxyborohydride and 24.4 pi (426 pmol) of acetic acid. Stirring is continued for 6
hours at RT under argon, the reaction is quenched by addition of 1 ml of aqueous 1N sodium
hydroxide solution and then the mixture is extracted with 10 ml of diethyl ether. After
concentrating under reduced pressure, the crude product is purified by chromatography on silica
gel, using a 90/10 DCM/isopropanol mixture as eluent, which does not make it possible to
separate out the expected product from the residual starting amine. The product obtained is
again purified by RP18 reverse-phase chromatography using a 95/5 to 5/95 water/acetonitrile
mixture as elient. Compound 27 is obtained in the form of a colourless oil (67 mg, 36%). 1H NMR
(400 MHz, DMSO-cf6): 1.25 (s, 6 H); 1.39 (s, 9 H); 1.58 (broad m, 1 H); 2.38 (s, 3 H); 2.59 (s, 2
H); 2.68 (t, J = 5.6 Hz, 2 H); 2.80 (broad s, 3 H); 3.46 (t, J = 5.6 Hz, 2 H); 3.48 to 3.54 (m, 12 H).
LCMS (A1): ES m/z = 441 [M + H]+; tR = 3.29 min.
Compound 28: terf-Butyl methyl{2-[2-(2-{2-[rnethyl(2-methyl-2-rnethyldisulfanylpropyl)amino]-
ethoxy}ethoxy)ethoxy]ethyl}carbamate
To a solution, purged with argon and cooled to 0°C, of 65 mg (148 umol) of compound 27 in 1 ml
of DCM are successively added 19.5 mg (487 umol) of NaH as a 60% dispersion in a mineral oil
and 20 ul (325 umol) of methyl iodide. Stirring is continued for 45 minutes at 0°C and then for 72
hours at RT. The mixture is filtered through Clarcel and then concentrated under reduced
pressure/The crude product is purified by chromatography on silica gel, using a 99/1 to 90/10
DCM/methanol mixture as eluent. Compound 28 is obtained in the form of a colourless oil (43 mg,
64%). 1H NMR (400 MHz, DMSO-d6): 1.25 (s, 6 H); 1.39 (s, 9 H); 2.32 (s, 3 H); 2.39 (s, 3 H); 2.54
(m, 2 H); 2.61 (t, J = 6.1 Hz, 2 H); 2.80 (broad s, 3 H); 3.42 to 3.54 (m, 14 H). LCMS (A2): ES m/z
•- 455 [M + H]+; tR = 0.77 min.
Compound 29: Methyl(2-{2-[2-(2-methylaminoethoxy)ethoxy]ethoxy}ethyl)(2-methyl-2-methyl-
disulfanylpropyl)amine
To a solution of 43 mg (95 umol) of compound 28 in 1.5 ml of DCM are added 351 ul of TFA.
Stirring is continued for 5 hours at RT and the mixture is then concentrated to dryness. The crude
product is purified by SPE filtration on an SCX cartridge (Varian) conditioned and washed with
methanol, and then eluted with a 0.5 N solution of aqueous ammonia in methanol. Compound 29
is obtained in the form of a colourless oil (25 mg, 75%). 1H NMR (400 MHz, DMSO-d6): 1.25 (s, 6
H); 1.75 (broad m, 1 H); 2.27 (s, 3 H); 2.32 (s, 3 H); 2.39 (s, 3 H); 2.53 (m, 2 H); 2.57 to 2.64 (m,
4 H); 3.44 (t, J = 5.7 Hz, 2 H); 3.46 to 3.55 (m, 10 H). LCMS (A2): ES m/z = 355 [M + H]+; tR =
0.30 min.
Compound 30: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-
{4-[4-({2-[2-(2-{2-methyl[2-methyl-2-methyldisulfanylpropyl]aminoethoxy}ethoxy)ethoxy]-
ethyl}methylamino)methyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-
13-ene-2,5,9,12-tetraone
To a solution, purged with argon, of 15.3 mg (21.3 pmol) of compound 2 in 1 ml of anhydrous
acetonitrile are successively added 18.6 pi (106.6 pmol) of DIPEA and a solution of 22.7 mg
(64 pmol) of compound 29 in 1 ml of anhydrous acetonitrile. Stirring is continued for 24 hours
under argon at 40°C. After cooling to RT, the mixture is diluted with 7 ml of EtOAc, washed with
water (2x3 ml), with saturated NaHCO3 solution (3 ml) and with saturated NaCI solution (3 ml)
and dried over MgSO4. After filtering and concentrating under reduced pressure, the crude
product is purified by chromatography on silica gel, using a 90/10 DCM/methanol mixture as
eluent. Compound 30 is obtained in the form of a colourless solid (14.6 mg, 66%). 1H NMR (500
MHz, DMSO-d6): 0.76 (d, J = 6.1 Hz, 3 H); 0.76 (d, J = 6.1 Hz, 3 H); 1.00 (s, 3 H); 1.04 (d, J = 6.8
Hz, 3 H); 1.12 (s, 3 H); 1.24 (s, 6 H); 1.29 (m, 1 H); 1.50 to 1.62 (m, 2 H); 1.81 (m, 1 H); 2.15 (s, 3
H); 2.27 (m, 1 H); 2.31 (s, 3 H); 2.38 (s, 3 H); 2.45 to 2.54 (partially masked m, 4 H); 2.60 (t, J =
6.1 Hz, 2 H); 2.63 to 2.75 (m, 2 H); 2.92 to 3.06 (m, 3 H); 3.25 to 3.35 (partially masked m, 1 H);
3.44 to 3.54 (m, 14 H); 3.81 (s, 3 H); 3.87 (d, J = 2.0 Hz, 1 H); 4.25 (ddd, J = 3.9 and 8.3 and 11.7
Hz, 1 H); 4.91 (dd, J = 3.4 and 9.8 Hz, 1 H); 5.11 (ddd, J = 1.5 and 5.7 and 11.5 Hz, 1 H); 5.80
(dd, J = 1.5 and 15.5 Hz, 1 H); 6.47 (ddd, J = 3.5 and 11.5 and 15.5 Hz, 1 H); 7.05 (d, J = 8.8 Hz,
1 H); 7.17 (dd, J = 2.0 and 8.8 Hz, 1 H); 7.20 to 7.26 (m, 3 H); 7.27 to 7.33 (m, 3 H); 8.35 (d, J =
8.3 Hz, 1 H). LCMS (A2): ES m/z = 1035 [M + H]+; m/z = 518 [M + 2H]2+ base peak; tR = 0.86 min.
Example 6: (E)(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-{4-
[4-({2-[2-(2-{2-methyl[2-mercapto-2-methylpropyl]aminoethoxy}ethoxy)ethoxy]ethyl}methylamino)-
methyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
tetraone
To a solution of 10.9 mg (10.5 pmol) of compound 30 in 1.24 ml of ethanol is added a solution of
12.06 mg (42.1 pmol) of TCEP in 1.04 ml of water. Stirring is continued for 4 hours at RT. The
mixture is diluted with 6 ml of EtOAc, washed with a 1/1 mixture of water/NH4Cisat (6 ml) and with
saturated NaCI solution (6 ml), and dried over MgSO4. After filtering and concentrating under
reduced pressure, the crude product is purified by chromatography on silica gel, using a 99/1 to
90/10 DCM/methanol mixture. Compound Ex. 6 is obtained in the form of a white solid (7.36 mg,
71%). 1H NMR (500 MHz, DMSO-cf6): 0.76 (d, J = 5.9 Hz, 3 H); 0.78 (d, J = 5.9 Hz, 3 H); 1.00 (s,
3 H); 1.04 (d, J = 6.8 Hz, 3 H); 1.12 (s, 3 H); 1.25 (s, 6 H); 1.28 (m, 1 H); 1.51 to 1.62 (m, 2 H);
1.80 (m, 1 H); 2.15 (s, 3 H); 2.27 (m, 1 H); 2.34 (s, 3 H); 2.44 (s, 2 H); 2.51 (t, J = 6.4 Hz, 2 H);
2.60 (s, 1 H); 2.63 (t, J = 6.4 Hz, 2 H); 2.66 to 2.74 (m, 2 H); 2.93 to 3.04 (m, 3 H); 3.25 to 3.37
(partially masked m, 1 H); 3.45 to 3.55 (m, 14 H); 3.81 (s, 3 H); 3.87 (d, J = 1.5 Hz, 1 H); 4.25
(ddd, J = 3.7 and 8.0 and 11.5 Hz, 1 H); 4.91 (dd, J = 3.4 and 9.8 Hz, 1 H); 5.11 (ddd, J = 1.5 and
5.6 and 11.5 Hz, 1 H): 5.80 (dd, J = 1.5 and 15.2 Hz, 1 H); 6.47 (ddd, J = 3.9 and 11.5 and 15.2
Hz, 1 H); 7.05 (d, J = 8.8 Hz, 1 H); 7.17 (dd, J = 2.2 and 8.8 Hz, 1 H); 7.20 to 7.26 (m, 3 H); 7.27
to 7.32 (m, 3 H); 8.35 (d, J = 8.0 Hz, 1 H), LCMS (A1): ES m/z = 989 [M + Hj+; m/z = 495 [M +
2H]2+ base peak; tR = 3.24 min.
Example 7 : (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-
3-{4-[4-({2-[2-(2-{4-methyl-4-methyldisulfanylpentanoylaminoethoxy}ethoxy)-
ethoxy]ethyl}methylamino)methyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-8,11-
diazacyclohexadec-13-ene-2,5,9,12-tetraone
Compound 31: ferf-Butyl methyl[2-(2-{2-[2-(4-methyl-4-methyldisulfanylpentanoylamino)ethoxy]-
ethoxy}ethoxy)ethyl]carbamate
270 mg (649 umol) of compound 4, 199 mg (649 umol) of compound 26 are dissolved in 1.5 m! of
DMF, and 100 ul (714 umol) of TEA are then added to the mixture. Stirring is continued for 16
hours at RT. The mixture is diluted with 10 ml of EtOAc, washed with water (2x5 ml) and with
saturated NaCI solution (5 ml) and dried over MgSO4. After filtering and concentrating under
reduced pressure, the crude product is purified by chromatography on silica gel, using a 98/2 to
90/10 DCM/methanol mixture as eluent. Compound 31 is obtained in the form of a colourless oil
(251 mg, 80%). 1H NMR (400 MHz, DMSO-cfe): 1.24 (s, 6 H); 1.39 (s, 9 H); 1.79 (m, 2 H); 2.16
(m, 2 H); 2.40 (s, 3 H); 2,80 (broad s, 3 H); 3.18 (q, J = 5.9 Hz, 2 H); 3.39 (t, J = 5.9 Hz, 2 H); 3.45
to 3.54 (m, 12 H); 7.88 (broad t, J = 5.9 Hz, 1 H). LCMS (A2): ES m/z = 483 [M + H]+; m/z = 505
[M + Na]+; m/z = 527 [M - H + HCO2Hr; tR = 1.04 min.
Compound 32: N-(2-{2-[2-(2-Methylaminoethoxy)ethoxy]ethoxy}ethyl)-4-methyl-4-methyl-
disulfanylpentamide
To a solution of 251 mg (520 umol) of compound 31 in 8 ml of DCM are added 1.93 ml (26 mmol)
of TFA. Stirring is continued for 3 hours at RT and the mixture is then concentrated to dryness.
The crude product is dissolved in a minimum amount of DCM and then entrained several times
with toluene. The crude product is purified by SPE filtration on an SCX cartridge (Varian)
conditioned and washed with methanol and then eluted with a 0.5 N solution of aqueous
ammonia in methanol. Compound 32 is obtained in the form of a colourless oil (159 mg, 80%). 1H
NMR (400 MHz, DMSO-o6): 1.24 (s, 6 H); 1.79 (m, 2 H); 2.15 (m, 2 H); 2.27 (s, 3 H); 2.40 (s, 3
Hj; 2.58 (t, J = 5.7 Hz, 2 H); 3.18 (q, J = 5.7 Hz, 2 H); 3.39 (t, J = 5.7 Hz, 2 H); 3.44 (t, J = 5.7 Hz,
2 H); 3.47 to 3.54 (m, 8 H); 7.89 (broad t, J = 5.7 Hz, 1 H), LCMS (A1): ES m/z = 383 [M + Hf; tR
= 2.68 min.
Example 7: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-{4-
[4-({2-[2-(2^4-methyW-methyldisulfanylpentanoylaminoethoxy}ethoxy)ethoxy]ethyl}methyl-
amino)methyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-
2,5,9,12-tetraone
Example 7 may be obtained by nucleophilic substitution of the chloro group of derivative 2 with
the amine 32 by applying the method described for the preparation of compound 30, followed by
reduction of the disulfide by applying the method described for the preparation of Example 6.
Example 8: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-16-{(S)-1-[(2R,3R)-3-
(4-mercaptomethylphenyl)oxiranyl]ethyl}-6,6-dimethyl-1,4-dioxa-8,11 -diazacyclohexadec-
13-ene-2,5,9,12-tetraone
Compound 33: dimer of Example 8
Compound 2 (24.5 mg; 34.1 umol) is dissolved in anhydrous THF (2.5 ml) and the mixture is
cooled to -10°C, followed by addition of hexamethyldisilathiane (44.3 umol) and then a 1M
solution of tetrabutylammonium fluoride in THF (40.9 umol). The mixture is cooled to room
temperature and stirring is continued for 1 hour 30 minutes. The mixture is diluted by adding
EtOAc (5 ml) and the organic phase is washed with saturated aqueous NH4CI solution (5 ml). The
aqueous phase is extracted with EtOAc (2x5 ml). The organic phases are combined and washed
with saturated aqueous NaCI solution (5 ml). After drying over MgSO4 and filtering, the solvents
are evaporated off under reduced pressure. The crude reaction product is purified by
chromatography on silica gel, eluting with a 100/0 to 97/3 DCM/methanol mixture as eluent. A
white solid, compound 33, is obtained (19 mg; 78%). 1H NMR (400 MHz, DMSO-d6): 0.78 (m, 12
H); 1.00 (s, 6 H); 1.04 (d, J = 6.8 Hz, 6 H); 1.11 (s, 6 H); 1.30 (m, 2 H); 1.49 - 1.63 (m, 4 H); 1.82
(m, 2 H); 2.26 (m, 2 H); 2.63 - 2.72 (m, 4 H); 2.93 - 3.05 (m, 6 H); 3.25 - 3.37 (partially masked
m, 2 H); 3.81 (s, 6 H); 3.82 (s, 4 H); 3.89 (d, J = 2.0 Hz, 2 H); 4.25 (ddd, J = 3.7r 8.0 and 11.5 Hz,
2 H); 4.91 (dd, J = 3.7, 9.6 Hz, 2H); 5.10 (ddd, J = 1.3, 5.3 and 10.8 Hz, 2 H); 5.79 (dd, J = 1.3,
15.3 Hz, 2 H); 6.47 (ddd, J = 3.7, 10.8 and 15.3 Hz, 2 H); 7.05 (d, J = 8.6 Hz, 2 H); 7.17 (dd, J =
2.0, 8.6 Hz, 2 H); 7.22 (dd, J = 2.5, 9.3 Hz, 2 H); 7.26 - 7.32 (m, 10 H); 8.34 (d, J = 8.0 Hz, 2 H),
LCMS (A2): ES m/z = 1427 [M + H]+; tR= 1.31 min.
Example 8: (E)-(3S, 10R, 16S)-1O-(3-Chloro^-methoxybenzyl)-3-isobutyl-16-{(S)-1 -[(2R,3R)-3-(4-
mercaptomethylphenyl)oxiranyl]ethyl}-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-
2,5,9,12-tetraone
Compound 33 (11 mg; 7.7 umol) is dissolved in methanol (8.8 ml). TCEP (76.7 umol) dissolved in
2.2 ml of water is then added and the mixture is stirred for 2 hours at RT. The mixture is diluted in
20 ml of EtOAc and the organic phase is washed with a 1/1 mixture of water and of saturated
aqueous NH4CI solution (20 ml). The aqueous phase is extracted with EtOAc (2x15 ml). The
organic phases are combined, washed with saturated aqueous NaCI solution (15 ml). After drying
over MgSO4 and filtering, the solvents are evaporated off under reduced pressure. The crude
reaction product is purified by chromatography on silica gel, eluting with a 100/0 to 95/5
DCM/methanol mixture. A white solid, Ex. 8, is obtained (4.7 mg; 31%). 1H NMR (400 MHz,
DMSO-d6): 0.79 (d, J = 6.4 Hz, 6 H); 1.00 (s, 3 H); 1.05 (d, J = 6.9 Hz, 3 H); 1.12 (s, 3 H); 1.30
(m, 1 H); 1.50 - 1.63 (m, 2 H); 1.80 (m, 1 H); 2.27 (m, 1 H); 2.62 - 2.75 (m, 2 H); 2.84 (broad t, J
= 6.5 Hz, 1 H); 2.93 - 3.06 (m, 3 H); 3.34 (partially masked m, 1 H); 3.73 (broad d, J = 6.5 Hz, 2
H); 3.81 (s., 3 H); 3.87 (d, J = 1.6 Hz, 1 H); 4.25 (ddd, J = 3.7, 8.0 and 11.4 Hz, 1 H); 4.91 (dd, J =
3.6, 9.6 Hz, 1 H); 5.11 (ddd, J = 1.3, 5.3 and 11.4 Hz, 1 H); 5.79 (dd, J = 1.3, 15.2 Hz, 1 H); 6.47
(ddd, J = 3.4, 11.3 and 15.2 Hz, 1 H); 7.05 (d, J = 8.5 Hz, 1 H); 7.17 (dd, J = 1.9, 8.5 Hz, 1 H);
7.22 (dd, J = 2.3, 9.5 Hz, 1 H); 7.25 (d, J = 8.3 Hz, 2 H); 7.28 (d, J = 1.9 Hz, 2 H); 7.35 (d, J = 8.3
Hz, 2 H); 8.34 (d, J = 8.0 Hz, 2 H). LCMS (A2): ES m/z = 715 [M +D H]+; m/z = 713 [M - H]"; tR =
1.18 min.
According to the general two-step method, 10.4 mg (0.071 umol, 1.174 ml) of naked antibody
hu2H11 with an initial concentration of 8.86 mg/ml are treated with 7 eq. of the N-hydroxy-
succinimidyl ester of 4-(2-pyridyldithio)butanoic acid (0.16 mg, 0.496 umol) dissolved in 34.2 pi of
DMA such that the final antibody concentration is 8 mg/ml in the mixture. After purification, 2.2 ml
of modified antibody hu2H11 at a concentration of 4.28 mg/ml (9.42 mg, 91%) are obtained with,
on average, 4.68 pyridyldisulfide molecules per antibody. 1.68 ml (7.2 mg, 0.049 pmol) of
modified antibody hu2H11 are treated with 1.03 mg of (E)-(3S,10R,16S)-1O-(3-chloro-4-methoxy-
benzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-{4-[4-(4-mercapto^-methylpentanoyl)piperazin-1-
ylmethyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-
tetraone (compound Ex. 1, 1.148 umol) dissolved in 101.2 pi of DMA. After purification on
Superdex in the presence of 10% NMP and concentration on Amicon Ultra-15, the final change of
buffer is performed in an aqueous pH 6.5 buffer containing 0.01 M of phosphate and 0.14 M of
NaCI. 1.5 ml of conjugate Ex. 9 are thus obtained at a concentration of 1.1 mg/ml with, on
average, 3 cryptophycin derivatives per antibody (HRMS) and a monomer purity of 99.9%.
Example 10: hu2H11-SPDB-Ex2
According to the one-step general method, 13.5 mg (0.092 umol, 1.318 ml) of naked antibody
hu2H11 at an initial concentration of 10.24 mg/ml are treated with 6 eq. of the N-hydroxy-
succinimidyl ester of 4-(2-pyridyldithio)butanoic acid (0.18 mg, 0.551 pmol) dissolved in 38.4 pi of
DMA such that the final antibody concentration is 9 mg/ml in the mixture. After stirring for 2 hours
at about 2000 rpm at RT, 1.333 ml (12.0 mg, 0.081 pmol) of the mixture of modified antibody
hu2H11, 1.760 ml of pH 7.5-8 buffer, 543 pi of DMA and then 1.73 mg of (E)-(3S,6R,10R,16S)-
1O-(3-chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1 -((2R,3R)-3-{4-[4-(4-mercapto-4-methyl-
pentanoyl)piperazin-1-ylmethyl]phenyl}oxiranyl)ethyl]-6-methyl-1,4-dioxa-8,11-diazacyclohexa-
dec-13-ene-2,5,9,12-tetraone (compound Ex. 2, 1.958 pmol) dissolved in 182 pi of DMA are
successively added. After purification on Superdex in the presence of 20% of NMP and
concentration on Amicon Ultra-15, the final buffer change is performed in an aqueous pH 6.5
buffer containing 0.01 M of histidine, 10% of sucrose (w/v) and 5% of NMP (v/v). 1.5 ml of
conjugate Ex. 10 are thus obtained at a concentration of 2.83 mg/ml with, on average, 3.7
cryptophycin derivatives per antibody and a monomeric purity of 98.8%.
Example 11: hu2H11-SPDB-Ex5
According to the one-step general method, 9.45 mg (0.064 pmol, 0.923 ml) of naked antibody
hu2H11 at an initial concentration of 10.24 mg/ml are treated with 6eq. of the N-hydroxy-
succinimidyl ester of 4-(2-pyridyldithio)butanoic acid (0.13 mg, 0.398 pmol) dissolved in 26.88 |_JI
of DMA, such that the final antibody concentration is 9 mg/ml in the mixture. After stirring for 2
hours at about 2000 rpm at RT, 1.0 ml (9.0 mg, 0.061 pmol) of the reaction medium of modified
antibody hu2H11, 1.45 ml of pH - 7.5-8 buffer, 265 M' of DMA and then 1.26 mg of
(E)(3S, 10R, 16S)-1O-(3-chloro-4-methoxybenzyl)-3-isobutyl-16-[(S)-1 -((2R,3R)-3-{4-[4-(2-
mercapto-2-methylpropyl)piperazin-1-ylmethyl]phenyl}oxiranyl)ethyl]-6,6-dimethyl-1,4-dioxa-8,11-
diazacyclohexadec-13-ene-2,5,9,12-tetraone (compound Ex. 5, 1.472 umol) dissolved in 285 pi
of DMA are successively added. After purification on Superdex in the presence of 20% of NMP
and concentration on Amicon Ultra-15, the final buffer change is formed in an aqueous pH 6.5
buffer containing 0.01 M of histidine, 10% of sucrose (w/v) and 5% of NMP (v/v). 2.5 ml of
conjugate Ex. 12 are thus obtained at a concentration of 1.70 mg/ml with, on average, 3.7/3.1
cryptophycin derivatives (UV/HRMS) per antibody and a monomer purity of 98.0%.
Example 12: hu2H11-SPDB-Ex6
According to the one-step general method, 10.31 mg (0.070 umol, 1.006 ml) of naked antibody
hu2H11 at an initial concentration of 10.24 mg/ml are treated with 6 eq. of the N-hydroxy-
succinimidyl ester of 4-(2-pyridyldithio)butanoic acid (0.14 mg, 0.429 umol) dissolved in 29.32 pi
of DMA, such that the final antibody concentration is 9 mg/ml in the mixture. After stirring for 2
hours at about 2000 rpm at RT, 1.595 ml of pH = 7.5-8 buffer, 291 pi of DMA and then 1.60 mg of
(E)-(3S,10R,16S)-1O-(3-chloro4-methoxybenzyl)-3-isobutyl-16-[(S)-1-((2R,3R)-3-{4-[4-({2-[2-(2-
{2-methyl[2-mercapto-2-methylpropyl]aminoethoxy}ethoxy)ethoxy]ethyl}methylamino)methyl]-
pheny^oxiranyOethylJ-e.e-dimethyl-l^-dioxa-S.H-diazacyclohexadec-IS-ene^.S.g.^-tetraone
(compound Ex. 6, 1.617 pmol) dissolved in 314 pi of DMA are successively added to 1.1 ml
(9.9 mg, 0.067 pmol) of the mixture of modified antibody hu2H11. After purification on Superdex
in the presence of 20% of NMP and concentrating on Amicon Ultra-15, the final buffer change is
performed in an aqueous pH 6.5 buffer containing 0.01 M of histidine, 10% of sucrose (w/v) and
5% of NMP (v/v). 3 ml of conjugate Ex. 12 at a concentration of 1.97 mg/ml are thus obtained
with, on average, 3.4 cryptophycin derivatives per antibody (HRMS) and a monomer purity of
99.8%.
Example 13: 2,5-Dioxopyrrolidin-1-yl (4-{4-[(2R,3R)-3-((S)-1-{(E)-(3S)10R,16S)-1O-[3-chloro-
4-methoxybenzyl]-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexa-
dec-13-en-16-yl}ethyl)oxiranyi]benzyl}piperazin-1-yl)acetate
Compound 34: ferf-Buty! 4-methoxycarbonylmethylpiperazine-1-carboxylate
200 mg (1.07 mmol) of terf-butyl piperazine-1-carboxylate are dissolved in anhydrous acetonitriie
(10 ml). TEA (1.07 mmol) and then methyl bromoacetate (1.61 mmol) are then added. The white
suspension is stirred for 48 hours at RT, followed by addition of saturated aqueous NaHCC>3
solution (10 ml). The aqueous phase is extracted with DCM (3x10 ml) and the organic phases are
combined, washed with saturated aqueous NaCI solution and dried over MgSCv After filtering
and evaporating off the solvents under reduced pressure, the crude product is obtained. This
crude product is purified by chromatography on silica gel, eluting with a 98/2 DCM/methanol
mixture. The expected product 34, a colourless oil, is thus obtained (174.8 mg; 63%). TLC (DCM
90 /MeOH 10): Rf = 0.66; 1H NMR (300 MHz, DMSO-d6): 1.39 (s, 9 H); 2.40 to 2.48 (m, 4 H); 3.25
(s, 2 H); 3.28 to 3.33 (partially masked m, 4 H); 3.61 (s, 3 H).
Compound 35: Mechyl piporazin-1-ylacetate hydrochloride
Compound 34 (174 mg; 0.67 mmol) is dissolved in anhydrous dioxane (5 ml) and a 4 M solution
of HCI in dioxane (0.02 mmol) is added. The mixture is stirred for 5 hours at RT and the
suspension is then filtered on a sinter funnel. The solid thus obtained is washed with dioxane
(2 ml) and then with isopropyl ether (2 ml), and then dried under vacuum. A beige-coloured solid,
35, is obtained (131 mg; 100%). 1H NMR (300 MHz, DMSO-d6): 2.88 to 2.97 (m, 4 H); 3.09 to
3.19 (m, 4 H); 3.53 to 3.59 (m, 2 H); 3.65 (s, 3 H); 8.65 to 9.22 (broad m, 2 H).
Compound 36: Methyl (4-{4-[(2R,3R)-3-((S)-1-{(E)-(3S,10R,16S)-1O-[3-Chloro-4-methoxy-
benzyl]-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11 -diazacyclohexadec-13-en-16-
yljethyl )oxi ra nyl] benzyl}pi perazin-1-yl )acetate
The derivative 1 (20 mg; 28.6 umol) is dissolved in anhydrous DCM (1 ml), and TEA (71.5 umol)
and then CMS (45.8 umol) are added. After 12 hours at RT, the product 2 formed is not isolated.
TEA (85.7 umol) and then methyl piperazin-1-ylacetate hydrochloride 35 (42.8 umol) are added.
Trie mixture is stirred for a further 72 hours at RT, followed by addition of anhydrous DMF (1 ml)
and Nal (30 umol). The mixture is stirred for 48 hours at 45°C, followed by dilution with EtOAc (5
ml). The organic phase is washed with water (2x2 ml), with saturated aqueous NaHCO3 solution
(2 ml) and with saturated aqueous NaCI solution (2 ml). After drying over MgSO4 and filtering, the
solvents are evaporated off under reduced pressure. The crude reaction product is purified by
chromatography on silica gel, eluting with a 100/0 to 98/2 DCM/methanol mixture. Compound 36
is obtained in the form of a white solid (8.2 mg; 34%). TLC (DCM 90/MeOH 10): Rf = 0.45; 1H
NMR (400 MHz, DMSO-d6): 0.77 (d, J = 6.1 Hz, 3 H); 0.78 (d, J = 6.1 Hz, 3 H); 1.00 (s, 3 H); 1.05
(d, J = 7.1 Hz, 3 H); 1.12 (s, 3 H); 1.27 to 1.32 (m, 1 H); 1.50 to 1.60 (m, 2 H); 1.74 to 1.84 (m, 1
H); 2.21 to 2.30 (m, 1 H); 2.37 (broad s, 4 H); 2.64 to 2.74 (m, 2 H); 2.94 to 3.06 (m, 3 H); 3.18 to
3.52 (partially masked m, 9 H); 3.60 (s, 3 H); 3.81 (s, 3 H); 3.87 (d, J = 2.0 Hz, 1 H); 4.20 to 4.31
(m, 1 H); 4.91 (dd, J = 3.8 and 9.9 Hz, 1 H); 5.11 (m, 1 H); 5.80 (d, J = 15.2 Hz, 1 H); 6.48 (ddd, J
= 3.8 and 11.2 and 15.2 Hz, 1 H); 7.05 (d, J = 8.3 Hz, 1 H); 7.17 (dd, J = 2.2 and 8.3 Hz, 1 H);
7.20 to 7.33 (m, 6 H); 8.34 (d, J = 8.1 Hz, 1 H). LCMS (A1): ES m/z = 839 [M + H]+; m/z = 420 [M
+ 2H]2+ (base peak); m/z = 837 [M - H]~; m/z = 883 [M + HCO2H - H]~ (base peak); tR = 3.57
min.
Compound 37: Allyl bromoacetate
288 mg (4.95 mmol) of allyl alcohol are dissolved in 25 ml of DCM. The solution is cooled to 0°C
and then 760 ul (5.45 mmol) of TEA and 3.03 mg (24.8 umol) of DMAP are added. The mixture is
stirred at 0°C and 1.0 g (4.95 mmol) of bromoacetyl bromide is then added. The reaction is
continued overnight at RT. Water is added to the mixture; the aqueous phase is washed with
DCM. The organic fractions are combined, washed with water and with saturated NaCI solution
and dried over MgSO4. After filtering and evaporating off the solvents under reduced pressure,
product 37 is obtained (747 mg, 84%). 1H NMR (400 MHz, DMSO-cfe): 4.18 (s, 2 H); 4.64 (td, J =
1.5 and 5.4 Hz, 2 H); 5.25 (qd, J = 1.5 and 10.5 Hz, 1 H); 5.35 (qd, J = 1.5 and 17.2 Hz, 1 H);
5.92 (tdd, J = 5.4 and 10.5 and 17.2 Hz, 1 H).
Compound 38: ferf-Butyl 4-allyloxycarbonylmethylpiperazine-1-carboxylate
To a solution of 200 mg (1.07 mmol) of 1-Boc-piperazine in 8 ml of acetonitrile are added 150 ul
(1.07 mmol) of TEA and 250 mg (1.40 mmol) of allyl bromoacetate 37. The mixture is stirred at
RT overnight. The reaction is stopped by adding saturated NaHCO3 solution. The mixture is
extracted with EtOAc (3 times); the organic phases are combined, washed with saturated NaCI
solution and dried over MgSO4. After filtering and evaporating off the solvent under reduced
pressure, the crude product is purified by chromatography on silica gel, eluting with a 100/0 to
95/5 DCM/methanol mixture. The expected product 38 is obtained in the form of a yellow oil
(314 mg; 100%). 1H NMR (400 MHz, DMSO-cfe): 1.39 (s, 9 H); 2.45 to 2.48 (m, 4 H); 3.25 to 3.34
(partially masked m, 6 H); 4.57 (td, J = 1.5 and 5.6 Hz, 2 H); 5.22 (qd, J = 1.5 and 10.3 Hz, 1 H);
5.30 (qd, J = 1.5 and 17.1 Hz, 1 H); 5.83 to 5.98 (m, 1 H). LCMS (A2):ES m/z = 285 [M + H]+; m/z
= 229 base peak; tR = 0.51 min.
Compound 39: Allyl piperazin-1-ylacetate hydrochloride
314 mg (1.10 mmol) of compound 38 are dissolved in 12.6 ml of dioxane, followed by addition of
5.5 ml (22.0 mmol) of a 4M solution of HCI in dioxane. Stirring is continued overnight at RT. The
mixture is concentrated to dryness to give the expected compound 39 in the form of a yellow oil
(260 mg; 100%). 1H NMR (400 MHz, DMSO-cfe): 2.79 to 2.88 (m, 4 H); 3.07 to 3.15 (m, 4 H); 3.48
to 3.51 (m, 2 H); 4.60 (td, J = 1.5 and 5.6 Hz, 2 H); 5.23 (qd, J = 1.5 and 10.3 Hz, 1 H); 5.32 (qd,
J = 1.5 and 7.4 Hz, 1 H); 5.86 to 5.98 (m, I H); 8,74 (broad s, 2 H). LCMS (A2): ES m/z = 185 [M
+ H]+;tR= 0.19 min.
Compound 40: Allyl (4-{4-[(2R,3R)-3-((S)-1 -{(E)-(3S, 10R, 16S)-1O-[3-chloro-4-methoxybenzyl]-3-
isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl}ethyl)-
oxiranyl]benzyl}piperazin-1-yl)acetate
To a solution, purged with argon, of compound 2 (28.3 mg, 39.5 umol) in anhydrous acetonitrile
(2.5 ml) is added a solution of compound 39 (118.5 umol) and TEA (198 umol) in anhydrous
acetonitrile (1 ml). Stirring is continued for 24 hours at 40°C. The mixture is diluted in EtOAc
(10 ml). The organic phase is washed with water (10 ml), with saturated aqueous NaHCC>3
solution (10 ml) and saturated aqueous NaCI solution (10 ml). After drying over MgSO4 and
filtering, the solvents are evaporated off under reduced pressure. The crude reaction product is
purified by chromatography on silica gel, eluting with a 98/2 DCM/methanol mixture. Compound
40 is obtained in the form of a white solid (19.2 mg; 56%). 1H NMR (500 MHz, DMSO-cfe): 0.75 (d,
J = 6.3 Hz, 3 H); 0.77 (d, J = 6.3 Hz, 3 H); 1.00 (s, 3 H); 1.05 (d, J = 6.9 Hz, 3 H); 1.12 (s, 3 H);
1.25 to 1.33 (m, 1 H); 1.50 to 1.61 (m, 2 H); 1.74 to 1.84 (m, 1 H); 2.27 (dt, J = 11.3 and 14.2 Hz,
1 H); 2.32 to 2.42 (m, 4 H); 2.52 (partially masked m, 4 H); 2.64 to 2.74 (m, 2 H); 2.94 to 3.05 (m,
3 H); 3.25 (s, 2 H); 3.32 to 3.35 (partially masked m, 1 H); 3.40 to 3.48 (m, 2 H); 3.81 (s, 3 H);
3^7 (d, J = 1.6 Hz, 1 H); 4.25 (ddd, J = 3.7 and 8.0 and 11.6 Hz, 1 H); 4.56 (td, J = 1.5 and 5.5
Hz, 2 H); 4.91 (dd, J = 3.7 and 9.7 Hz, 1 H); 5.11 (m, 1 H); 5.21 (qd, J = 1.5 and 10.5 Hz, 1 H);
5.30 (qd, J = 1.5 and 17.3 Hz, 1 H); 5.80 (d, J = 16.2 Hz, 1 H); 5.86 to 5.95 (m, 1 H); 6.47 (ddd, J
= 3.8 and 11.2 and 15.2 Hz, 1 H); 7.05 (d, J = 8.5 Hz, 1 H); 7.17 (dd, J = 2.2 and 8.5 Hz, 1 H);
7.19 to 7.32 (m, 6 H); 8.34 (d, J = 8.0 Hz, 1 H). LCMS (A2): ES m/z = 865 [M + H]+; m/z = 433.5
[M + 2H]2+ (base peak); m/z = 863 [M - H]~; m/z = 909 [M + HCO2H - H]-; tR = 0.92 min.
Compound 41: (4-{4-[(2R,3R)-3-((S)-1-{(E)-(3S,10R,16S)-1O-[3-Chloro-4-methoxybenzyl]-3-
isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl}ethyl)-
oxiranyl]benzyl}piperazin-1 -yl)acetic acid
To a solution under argon of compound 40 (12.8 mg, 14.8 umol) in anhydrous THF are added
tetrakis(triphenylphosphine)pailadium (1.48 umol) and morpholine (148 umol). After reaction for 3
hours, the mixture is concentrated to dryness and taken up in 5 ml of DCM. The organic phase is
washed with 1 ml of 0.1 N HCI solution in 3 ml of water (pH=5), dried over MgSCU, filtered and
evaporated to give compound 41 (6.7 mg, 55%). 1H NMR (500 MHz, DMSO-cfe): 0.76 (d, J = 6.0
Hz, 3 H); 0.78 (d, J = 6.0 Hz, 3 H); 1.00 (s, 3 H); 1.05 (d, J = 6.9 Hz, 3 H); 1.12 (s, 3 H); 1.28 (m,
1 H); 1.52 to 1.59 (m, 2 H); 1.76 to 1.84 (m, 1 H); 2.21 to 2.32 (m, 1 H); 2.42 (d, J = 6.6 Hz, 4 H);
2.58 to 2.76 (m, 6 H); 2.97 to 3.09 (m, 3 H); 3.14 (broad s, 2 H); 3.33 (masked m, 1 H); 3.46
(broad s, 2 H); 3.81 (s, 3 H); 3.87 (d, J = 1.8 Hz, 1 H); 4.20 to 4.29 (m, 1 H); 4.89 to 4.93 (m, 1 H);
5.06 to 5.14 (m, 1 H); 5.80 (d, J = 15.2 Hz, 1 H); 6.43 to 6.50 (m, 1 H); 7.05 (d, J = 8.5 Hz, 1 H);
7.17 (dd, J = 2.2 and 8.5 Hz, 1 H); 7.20 to 7.32 (m, 6 H); 8.34 (d, J = 8.0 Hz, 1 H). LCMS (A2): ES
m/z = 825 [M + H]+; m/z = 413 [M + 2H]2+ (base peak); m/z = 823 [M - Hf; tR = 0.86 min.
Example 13: 2,5-Dioxopyrro!idin-1 -yl (4-{4-[(2R,3R)-3-((S)-1 -{(E)-(3S, 10R, 16S)-1O-[3-chioro-4-
methoxybenzyl]-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-
en-16-yl}ethyl)oxiranyl]benzyl}piperazin-1 -yl)acetate
Example 13 may be obtained by activating the acid 41 according to the method described for
Example 18.
Example 14: 2,5-Dioxopyrrolidin-1 -yl (2-{2-[2-(2-{4-[(2R,3R)-3-((S)-1 -{(E)-{3S,1 OR, 16S)-1O-[3-
chloro-4-methoxybenzyl]-3-isobutyl-6,6-dimethyl-2,5,9,12-tPtraoxo-1,4-dioxa-8,11-diaza-
cyclohexadec-13-en-16-yl}ethyl)oxiranyl]benzylamino}ethoxy)ethoxy]ethoxy}ethoxy)-
propanoate
Compound 42: Allyl 3-(2-{2-[2-(2-tert-butoxycarbonylaminoethoxy)ethoxy]ethoxy}ethoxy)-
propanoate
To a solution of Boc-15-amino-4,7,10,13-tetraoxapentadecanoic acid (50 mg, 137 umol) in
1.25 ml of DCM are successively added EDCI hydrochloride (164.2 umol), DMAP (13.7 umol)
and allyl alcohol (164.2 umol). The mixture is stirred at RT for 16 hours and then evaporated to
dryness. The crude product is purified by chromatography on silica gel, eluting with a 100/0 to
95/5 DCM/methanol mixture. Compound 42 is obtained in the form of a colourless oil (37.5 mg;
67%). 1H NMR (400 MHz, DMSO-cfe): 1.37 (s, 9 H); 2.57 (t, J = 6.1 Hz, 2 H); 3.06 (q, J = 6.1 Hz, 2
H); 3.37 (t, J = 6.1 Hz, 2 H); 3.44 to 3.53 (m, 12 H); 3.64 (t, J = 6.1 Hz, 2 H); 4.55 (td, J = 1.6 and
5.4 Hz, 2 H); 5.20 (qd, J = 1.6 and 10.5 Hz, 1 H); 5.30 (qd, J = 1.6 and 17.3 Hz, 1 H); 5.90 (tdd, J
= 5.4 and 10.5 and 17.3 Hz, 1 H); 6.70 (broad t, J = 6.1 Hz, 1 H). LCMS (A2) ES m/z = 406 [M +
H]+; m/z = 428 [M + Na]+; m/z = 306 base peak; tR = 0.91 min.
Compound 43: Allyl 3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propanoate hydrochloride
To a solution of compound 42 (37.5 mg, 92.5 umol) in 2 ml of dioxane are added 460 pi
(1.85 mmol) of a 4M solution of hydrogen chloride in dioxane. Stirring is continued at RT
overnight and the reaction medium is then evaporated to dryness to give compound 43 in the
form of a colourless oil (31 mg, quantitative). 1H NMR (400 MHz, DMSO-c/6): 2.58 (t, J = 6.1 Hz, 2
H); 2.96 (t, J = 5.4 Hz, 2 H); 3.48 to 3.53 (m, 8 H); 3.55 to 3.58 (m, 4 H); 3.60 (t, J = 5.4 Hz, 2 H);
3.65 (t, J = 6.1 Hz, 2 H); 4.56 (td, J = 1.6 and 5.4 Hz, 2 H); 5.21 (qd, J = 1.6 and 10.5 Hz, 1 H);
5.30 (qd, J = 1.6 and 17.3 Hz, 1 H); 5.91 (tdd, J = 5.4 and 10.5 and 17.3 Hz, 1 H); 7.91 (broad m,
3 H). LCMS (A2): ES m/z = 306 [M + H]+; tR = 0.42 min.
Compound 44: Allyl (2-{2-[2-(2-{4-[(2R,3R)-3-((S)-1-{(E)-(3S,10R,16S)-1O-[3-chloro-4-methoxy-
benzyl]-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-
yl}ethyl)oxiranyl]benzylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
To a solution, purged with argon, of compound 2 (12.7 mg, 17.7 umol) in 1.48 ml of anhydrous
acetonitrile are successively added 22.2 pi of TEA (159 pmol) and 30.2 mg of compound 43
(88.4 pmol). Stirring is continued at 40°C for 24 hours. Some starting compound 2 remains;
22.2 pi of TEA (159 pmol) and 30.2 mg of compound 43 (88.4 pmol) are added to the mixture and
stirring is continued at 40°C for a further 48 hours. 2 ml of water are added to the mixture, which
is then extracted with 2x2 ml of EtOAc. The organic phases are combined, washed with saturated
NaHCO3 solution (2 ml) and with saturated sodium chloride solution (2 ml) and dried over MgSO4.
After filtering and concentrating under reduced pressure, the crude reaction product is purified by
chromatography on silica gel, eluting with a 100/0 to 95/5 DCM/methanol mixture. Compound 44
is obtained in the form of a white solid (4.8 mg; 27%). 1H NMR (500 MHz, DMSO-ofe): 0.77 (m, 6
H); 1.00 (s, 3 H); 1.05 (d, J = 6.9 Hz, 3 H); 1.12 (s, 3 H); 1.29 (m, 1 H); 1.49 to 1.60 (m, 2 H); 1.81
(m, 1 H); 2.26 (m, 1 H); 2.56 (t, J = 6.1 Hz, 2 H); 2.61 to 2.73 (m, 4 H); 2.93 to 3.04 (m, 3 H); 3.28
to 3.38 (partially masked m, 1 H); 3.45 to 3.53 (m, 14 H); 3.63 (t, J = 6.1 Hz, 2 H);3.72 (s, 2 H);
3.81 (s, 3 H); 3.86 (broad s, 1 H); 4.25 (ddd, J = 3.4 and 8.2 and 11.4 Hz, 1 H); 4.55 (dm, J = 5.4
Hz, 2 H); 4.90 (dd, J = 3.9 and 9.8 Hz, 1 H); 5.11 (ddd, J = 1.4 and 5.4 and 11.2 Hz, 1 H); 5.19
(dm, J = 10.8 Hz, 1 H); 5.29 (dm, J = 17.2 Hz, 1 H); 5.79 (dd, J = 1.4 and 15.2 Hz, 1 H); 5.90 (m,
1 H); 6.47 (ddd, J = 3.9 and 11.2 and 15.2 Hz, 1 H); 7.05 (d, J = 8.8 Hz, 1 H); 7.17 (dd, J = 2.0
and 8.8 Hz, 1 H); 7.21 (m, 1 H); 7.24 (d, J = 8.3 Hz, 2 H); 7.28 (d, J = 2.0 Hz, 1 H); 7.33 (d, J =
8.3 Hz, 2 H); 8.35 (d, J = 8.2 Hz, 1 H). LCMS (A2): ES m/z = 986 [M + H]+; m/z = 493.5 [M +
2H]2+ base peak; m/z = 984 [M - H]'; m/z = 1030 [M - H + HCO2H]" base peak; tR = 0.95 min.
Example 14: 2,5-Dioxopyrrolidin-1-yl (2-{2-[2-(2-{4-[(2R,3R)-3-((S)-1-{(E)-(3S,10R,16S)-1O-[3-
chloro-4-methoxybenzyi]-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-
hexadec-13-en-16-yl}ethyl)oxiranyl]benzylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
Example 14 may be obtained by deprotecting compound 44 according to the method described
for compound 41 and by activating the acid obtained according to the method described for
Example 18.
Example 15: 2,5-Dioxopyrrolidin-1 -yl (2-{2-[2-(2-{4-[(2R,3R)-3-((S)-1 -{(E)-(3S, 10R,16S)-1O-[3-
chloro^-methoxybenzyll-S-isobutyl-e.e-dimethyl^.S.g.^-tetraoxo-l^-dioxa-S.H-diaza-
cyclohexadec-13-en-16-yl}ethyl)oxiranyl]benzylmethylamino}ethoxy)ethoxy]ethoxy}-
ethoxy)propanoate
Compound 45: 3-[2-(2-{2-[2-(fe/f-Butoxycarbonylmethylamino)ethoxy]ethoxy}ethoxy)ethoxy]-
propanoic acid
520 mg (1.423 mmol) of Boc-15-amino-4,7,10,13-tetraoxapentadecanoic acid are dissolved in
14 ml of anhydrous THF and the mixture is then cooled to 0°C, followed by addition, by spatula,
of 85.4 mg (2.135 mmol) of sodium hydride. Stirring is continued for 10 minutes at 0°C, and then
150.6 pi (2.419 mmol) of methyl iodide are added at 0°C. The temperature is allowed to return to
RT and stirring is continued for 2 hours. 8 ml of water are added to the mixture, the pH of which is
then acidified by addition of acetic acid to obtain pH=4. It is extracted with 3x10 ml of EtOAc. The
organic phases are combined, washed with 10 ml of saturated NaCI solution and dried over
MgSO4. After filtering and concentrating to dryness under reduced pressure, compound 45 is
obtained in the form of a colourless oii (414 mg, 77%). 1H NMR (400 MHz, DMSO-cfe): 1.38 (s, 9
H), 2.43 (t, J = 6.4 Hz, 2 H); 2.80 (broad s, 3 H); 3.29 (t, J = 5.9 Hz, 2 H); 3.45 to 3.52 (m, 14 H);
3.60 (t, J = C.4 Hz, 2 H); 12.01 (broad m, 1 H). LCMS (A2): ES m/z = 402 [M ^ Na]+; m/z = 378
[M - H]~; m/z = 280 base peak; tR = 0.95 min.
Compound 46: Ally! 3-[2-(2-{2-[2-(tert-butoxycarbonylmethylamino)ethoxy]ethoxy}ethoxy)ethoxy]-
propanoate
To a solution of 540 mg (1.423 mmol) of compound 45 in 15 ml of anhydrous DCM are
successively added 327 mg (1.71 mmol) of EDCI, 17.4 mg (142 pmol) of DMAP and 116 pi
(1.71 mmol) of allyl alcohol. Stirring is continued for 15 hours at RT and the mixture is then
concentrated to dryness. The crude product obtained is purified by chromatography on silica gel,
eluting with a 100/0 to 90/10 DCM/methanol mixture. Compound 46 is obtained in the form of a
colourless oil (337 mg; 56%). 1H NMR (400 MHz, DMSO-ofe): 1.38 (s, 9 H); 2.57 (t, J = 6.1 Hz, 2
H); 2.80 (broad s, 3 H); 3.22 to 3.33 (partially masked m, 2 H); 3.44 to 3.54 (m, 14 H); 3.64 (t, J =
6.1 Hz, 2 H); 4.55 (broad d, J = 4.9 Hz, 2 H); 5.20 (broad d, J = 10.3 Hz, 1 H); 5.30 (broad d, J =
17.1 Hz, 1 H); 5.90 (m, 1 H). LCMS (A2) : ES m/z = 442 [M + Na]+; m/z = 320 base peak; tR =
0.98 min.
Compound 47: AIM 3-(2-f2-r2-(2-methvlaminoethoxv)ethoxv1ethoxv}ethoxy)p'-opanoate
To a solution of 337 mg (0.802 mmol) of compound 46 in 20 ml of DCM are added 1.19 ml
(16.04 mmol) of TFA. Stirring is continued for 3 hours at RT and the mixture is then concentrated
to dryness under reduced pressure. The crude product is purified by SPE filtration on an SCX
cartridge (Varian) conditioned and washed with methanol, and then eluted with a 0.5 N solution of
aqueous ammonia in methanol. Compound 47 is obtained in the form of a colourless oil (208 mg,
81%). 1H NMR (400 MHz, DMSO-cfe): 2.27 (s, 3 H); 2.54 to 2,61 (m, 4 H); 3.44 (t, J = 5.6 Hz, 2
H); 3.47 to 3.52 (m, 12 H); 3.65 (t, J = 6.2 Hz, 2 H); 4.56 (broad d, J = 5.4 Hz, 2 H); 5.20 (broad d,
J - 10.5 Hz, 1 H); 5.30 (broad d, J = 17.2 Hz, 1 H); 5.90 (m, 1 H). LCMS (A2): ES m/z = 320 [M +
H]+; tR = 0.42 min.
Compound 48: Allyl (2-{2-[2-(2-{4-[(2R,3R)-3-((S)-1 -{(E)-(3S, 10R, 16S)-1O-[3-chloro-4-methoxy-
benzyl]-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-
yl}ethyl)oxiranyl]benzylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
To a solution, purged with argon, of compound 2 (40 mg, 55.7 umol) in 5 ml of anhydrous
acetonitrile are successively added 48.5 ul of DIPEA (279 umol) and 53 mg of compound 47
(167 umol). Stirring is continued at 40°C for 15 hours; 5 ml of water are added to the mixture,
which is then extracted with 4x5 ml of EtOAc. The organic phases are combined, washed with
saturated NaHCO3 solution (5 ml) and with saturated NaCI solution (5 ml) and dried over MgSO4.
After filtering and concentrating under reduced pressure, the crude reaction product is purified by
chromatography on silica gel, eluting with a 100/0 to 90/10 DCM/methanol mixture. Compound 48
is obtained in the form of a colourless solid (45.3 mg; 80%). 1H NMR (500 MHz, DMSO-d6): 0.76
(d, J = 5.9 Hz, 3 H); 0.78 (d, J = 5.9 Hz, 3 H); 1.00 (s, 3 H); 1.04 (d, J = 6.8 Hz, 3 H); 1.12 (s, 3
H);1.29(m, 1 H); 1.50 to 1.61 (m, 2 H); 1.80 (m, 1 H); 2.15 (s, 3 H); 2.28 (m, 1 H); 2.52 (t, J = 6.4
Hz, 2 H); 2.56 (t, J = 6.4 Hz, 2 H); 2.63 to 2.73 (m, 2 H); 2.94 to 3.06 (m, 3 H); 3.25 to 3.35
(partially masked m, 1 H); 3.48 (s, 2 H); 3.49 to 3.51 (m, 12 H); 3.52 (t, J = 6.4 Hz, 2 H); 3.63 (t, J
= 6.4 Hz, 2 H); 3.81 (s, 3 H); 3.87 (d, J = 1.5 Hz, 1 H); 4.25 (ddd, J = 3.4 and 8.3 and 11.5 Hz, 1
H); 4.55 (broad d, J = 5.0 Hz, 2 H); 4.91 (dd, J = 3.7 and 9.5 Hz, 1 H); 5.11 (ddd, J = 1.5 and 5.7
and 11.5 Hz, 1 H); 5.19 (dm, J = 10.3 Hz, 1 H); 5.29 (dm, J = 17.6 Hz, 1 H); 5.80 (dd, J = 1.5 and
15.2 Hz, 1 H); 5.90 (m, 1 H); 6.47 (ddd, J = 3.7 and 11.5 and 15.2 Hz, 1 H); 7.05 (d, J = 8.5 Hz, 1
H); 7.17 (dd, J = 2.0 and 8.5 Hz, 1 H); 7.22 (dd, J = 2.9 and 9.8 Hz, 1 H); 7.25 (d, J = 8.3 Hz, 2
H); 7.28 (d, J = 2.0 Hz, 1 H); 7.30 (d, J = 8.3 Hz, 2 H); 8.35 (d, J = 8.3 Hz, 1 H). LCMS (A2): ES
m/z = 1000 [M + H]+; m/z = 500.5 [M + 2H]2+; m/z = 1044 [M - H + HCO2H]"; tR = 1.01 min.
Compound 49: (2-{2-[2-(2-{4-[(2R,3R)-3-((S)-1 -{(E)-(3S, 10R, 16S)-1O-[3-Chloro-4-methoxy-
benzyl]-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11 -diazacyclohexadec-13-en-16-
yl}ethyl)oxiranyl]benzylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoicacid
19 mg (18.9 umol) of compound 48 are dissolved in 3.8 ml of anhydrous THF and purged with
argon. 6 ul (18.9 umol) of diethylamine are added to the mixture, which is stirred for 15 minutes at
RT, followed by addition of 4.45 mg (19.8 umol) of palladium(ll) acetate and 18.89 mg of
supported triphenylphosphine. Stirring is continued for 6 days at RT: some starting material
remains, but the reaction no longer proceeds. The mixture is filtered, concentrated to dryness,
taken up in 2 ml of anhydrous THF and treated with 10 pi (31.5 umol) of diethylamine and 10 mg
of tetrakis(triphenylphosphine)palladium for 1 hour. The mixture is hydrolyzed with 2 ml of
aqueous 2M sodium hydrogensulfate solution and extracted with 3x2 ml of DCM. The organic
phases are combined, washed with saturated NaCI solution (2 ml) and dried over MgSO4. After
filtering and concentrating under reduced pressure, the crude product is purified Dy
r.hromatography on diol-grafted silica gel, eluting with a 98/2 to 90/10 DCM/methanol mixture.
Compound 49 is obtained in the form of a colourless solid (5.4 mg; 30%). "H NMR (500 MHz,
DMSO-cf6): 0.76 (t, J = 5.9 Hz, 3 H); 0.78 (t, J = 5.9 Hz, 3 H); 1.00 (s, 3 H); 1.04 (d, J = 6.8 Hz, 3
H); 1.12 (s, 3 H); 1.31 (m, 1 H); 1.51 to 1.62 (m, 2 H); 1.80 (m, 1 H); 2.15 (s, 3 H); 2.28 (m, 1 H);
2.42 (t, J = 6.4 Hz, 2 H); 2.52 (t, J = 6.4 Hz, 2 H); 2.64 to 2.73 (m, 2 H); 2.94 to 3.04 (m, 3 H);
3.25 to 3.44 (partially masked m, 3 H); 3.45 to 3.51 (m, 12 H); 3.53 (t, J = 6.4 Hz, 2 H); 3.59 (t, J =
6.4 Hz, 2 H); 3.81 (s, 3 H); 3.87 (d, J = 2.0 Hz, 1 H); 4.25 (ddd, J = 4.2 and 7.8 and 11.5 Hz, 1 H);
4.91 (dd, J = 3.4 and 9.8 Hz, 1 H); 5.11 (dd, J = 5.4 and 11.2 Hz, 1 H); 5.80 (d, J = 15.2 Hz, 1 H);
6.47 (ddd, J = 3.7 and 11.2 and 15.2 Hz, 1 H); 7.05 (d, J = 8.3 Hz, 1 H); 7.17 (dd, J = 2.0 and 8.3
Hz, 1 H); 7.21 to 7.27 (m, 3 H); 7.28 to 7.32 (m, 3 H); 8.38 (broad d, J = 7.8 Hz, 1 H); 11.22 (very
broad m, 1 H). LCMS (A2): ES m/z = 960 [M + H]+; m/z = 480.5 [M + 2H]2+ base peak; m/z = 958
[M - H]"; tR = 0.90 min.
Example 15: 2,5-Dioxopyrrolidin-1-yl (2-{2-[2-(2-{4-[(2R,3R)-3-((S)-1-{(€H3S,10R,16S)-1O-[3-
chloro-4-methoxybenzyl]-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-
hexadec-13-en-16-yl}ethyl)oxiranyl]benzylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
Example 15 may be prepared by activating the acid 49 according to the method described for
Example 18.
Example 16: 2,5-Dioxopyrrolidin-1-yl (a^-p^^R.SRJ-S-KSJ-l-aEJ^SS.IOR.ieSJ-IO^-
chloro^-methoxybenzy^-S-isobutyl-e.S-dirnethyl^.S.Q^-tetraoxo-l^-dioxa-S.I-J-diaza-
cyclohexadec-13-en-16-yl)ethyl]oxiranyl}benzylpiperazin-1-yl)ethoxy]ethoxy}ethoxy)-
propanoate
Compound 50: 3-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}propanoic acid
I 0 ° 50
To a solution of 300 mg (1.08 mmol) of ferf-butyl 12-hydroxy-4,7,1O-trioxadodecanoate in 6 ml of
DCM are added 1.6 ml (21.56 mmol) of TFA. Stirring is continued at RT for 3 hours. The mixture
is concentrated to dryness, taken up in a minimum amount of DCM and entrained several times
with toluene to give compound 50 in the form of a pale yellow oil (240 mg, quantitative).
Compound 51: Ally! 3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propanoate
To a solution of 240 mg (1.08 mmol) of compound 50 in 3 ml of DCM are successively added
248 mg (1.29 mmol) of EDCI, 13.2 mg (1.29 mmol) of DMAP and 88 ul (1.29 mmol) of allyl
alcohol. Stirring is continued at RT for 15 hours and the mixture is then concentrated to dryness
under reduced pressure, and the crude product purified by chromatography on silica gel using as
eluent a 98/2 to 90/10 DCM/methanol mixture. Compound 51 is obtained in the form of a
colourless oil (144 mg, 51%). 1H NMR (400 MHz, DMSO-d6): 2.57 (t, J = 6.2 Hz, 2 H); 3.41 (m, 2
H); 3.46 to 3.52 (m, 10 H); 3,65 (t, J = 6.2 Hz, 2 H); 4.52 (t, J = 5.5 Hz, 1 H); 4.56 (m, 2 H); 5.20
(dm, J = 10.5 Hz, 1 H); 5,50 (dm, J = 17.4 Hz, 1 H); 5.90 (m, 1 H).
Compound 52: terf-Butyl 4-(2-{2-[2-(2-allyloxycarbonylethoxy)ethoxy]ethoxy}ethyl)piperazine-1 -
carboxylate
To a solution, cooled to 0°C, of 94 mg (357 umol) of compound 51 in 3.7 ml of DCM are
successively added 125 pi (893 umol) of TEA and 30.4 pi (393 pmol) of mesyl chloride, and
stirring is continued at RT for 1 hour. The mixture is concentrated to dryness under reduced
pressure and then taken up in 5 ml of acetonitrile. 249 pi (1.785 mmol) of TEA and 200 mg
(1.071 mmol) of Boc-piperazine are added to the solution and the mixture is stirred and heated
for 15 hours at 40°C. After cooling to RT, the mixture is concentrated to dryness and purified by
chromatography on silica gel, using a 98/2 to 90/10 DCM/methanol mixture as eluent. Compound
52 is obtained in the form of a colourless oil (69 mg, 45%). 1H NMR (400 MHz, DMSO-d6): 1.39
(s, y H); 2.33 (m, 4 H); 2.46 (t, J = 5.9 Hz, 2 H); 2.57 (t, J = 6.2 Hz, 2 H); 3.28 (partially masked
m, 4 H); 3.45 to 3.53 (m, 10 H); 3.64 (t, J = 6.2 Hz, 2 H); 4.55 (m, 2 H); 5.20 (dm, J = 10.5 Hz, 1
H); 5.30 (dm, J = 17.2 Hz, 1 H); 5.90 (m, 1 H).
Compound 53: Allyl 3-{2-[2-(2-piperazin-1-ylethoxy)ethoxy]ethoxy}propanoate
To a solution of 110 mg (256 umol) of compound 52 in 10 ml of DCM are added 380 ul
(5.11 mmol) of TFA. Stirring is continued for 24 hours at RT. The mixture is concentrated to
dryness, taken up in a minimum amount of DCM and entrained several times with toluene. The
crude product is purified by SPE filtration on an SCX cartridge (Varian) conditioned and washed
with methanol and then eluted with a 0.5 N solution of aqueous ammonia in methanol. Compound
53 is obtained in the form of a colourless oil (47 mg, 55%). 1H NMR (400 MHz, DMSO-d6): 2.58 (t,
J = 6.2 Hz, 2 H); 3.10 to 3.36 (broad m, 6 H); 3.45 to 3.78 (partially masked m, 16 H); 4.56 (m, 2
H); 5.20 (dm, J = 10.5 Hz, 1 H); 5.30 (dm, J = 17.2 Hz, 1 H); 5.90 (m, 1 H); 9.00 (broad m, 1 H).
Compound 54: Allyl (2-{2-[2-(44(2R,3R)-3-[(S)-1-((E)-(3S,10R,16S)-1O-{3-chloro-4-methoxy-
benzyl}-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-
yl)ethyl]oxiranyl}benzylpiperazin-1-yl)e,+ioxy]ethoxy}ethoxy)propanoate
Compound 54 may be obtained by nucleophilic substitution of the chloro group of derivative 2
with the amine 53 by applying the method described for the preparation of compound 30.
Compound 55: (2-{2-[2-(4-{(2R,3R)-3-[(S)-1-((E)-(3S,10R,16S)-1O-{3-Chloro-4-methoxybenzyl}-3-
isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11 -diazacyclohexadec-13-en-16-yl)ethyl]-
oxiranyl}benzylpiperazin-1-yl)ethoxy]ethoxy}ethoxy)propanoicacid
Compound 55 may be obtained according to the method described for Compound 41.
Example 16: 2,5-Dioxopyrrolidin-1 -yl (2-{2-[2-(4-{(2R,3R)-3-[(S)-1 -((E)-(3S, 10R, 16S)-1O-{3-
chloro-4-methoxybenzyl}-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-
hexadec-13-en-16-yl)ethyl]oxiranyl}benzylpiperazin-1-yl)ethoxy]ethoxy}ethoxy)propanoate
Example 16 may be obtained according to the method described for Example 18.
Example 17: 2,5-Dioxopyrrolidin-l-yl 3-(2-{2-[2-(2-{2-[4.(4-{4-[(2R)3R)-3-((S)-1 -{(E)-
(10R,16S)-1O-{3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-
dioxa-8,11-diazacyclohexadec-13-en-16-yl}ethyl)oxiranyl]benzyl}piperazin-1-yl)-1,1-
dimethyl-4-oxobutylsulfanyl]acetylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
Compound 56: 2,5-Dioxopyrrolidin-1 -yl 3-(2-{2-[2-(2-{2-bromoacetylamino}ethoxy)ethoxy]ethoxy}-
ethoxy)propanoate
To a solution of 3-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethoxy)propionic acid (671 mg,
2.53 mmo!) in DCM (10 ml) is added a solution of 2,5-dioxopyrrolidin-1-yl bromoacetate
(2.53 mmol) in 4.7 ml of DCM. Stirring is continued for 15 minutes at RT and DCC is then added
to the mixture. After reaction for 4 hours, the mixture is filtered through a sinter funnel and the
filtrate is then evaporated and purified by chromatography on silica gel, eluting with a 99/1 to 94/6
DCM/methanol mixture. The oil obtained (800 mg) is again purified by chromatography on a
cyano-grafted silica gel, eluting with a 99/1 DCM/methanol mixture. Compound 56 is obtained in
the form of a colourless oil (611 mg, 50%). 1H NMR (500 MHz, DMSO-afe): 2.81 (s, 4 H); 2.92 (t, J
= 5.9 Hz, 2 H); 3.23 (q, J = 5.9 Hz, 2 H); 3.43 (t, J = 5.9 Hz, 2 H); 3.48 to 3.55 (m, 12 H); 3.72 (t, J
= 5.9 Hz, 2 H); 3.85 (s, 2 H); 8.30 (broad t, J = 5.9 Hz, 1 H). LCMS (A2): ES m/z = 483 [M + H]+;
m/z = 481 [M - HJ"; tR = 0.51 min.
Compound 7: (E)-(3S,10R.16S)-1O-(3-Chloro-4-methoxvbenzyl)-3-isobutyl-6,6-dimethyl-16-r(S)-1-
((2R,3R)-3-{4-[4-(4-methyl-4-methyldisulfanylpentanoyl)piperazin-1-ylmethyl]phenyl}oxiranyl)-
ethyl]-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetraone
To a solution, purged with argon, of compound 2 (19.8 mg, 27.6 umol) in anhydrous acetonitrile
(2.5 ml) are successively added TEA (138 umol) and 4-methyl-4-methyldisulfanyl-1-piperazin-1-
y/pentan-1-one hydrochloride 6 (83 (jmol). Stirring is continued at 40°C for 24 hours and the
mixture is then diluted in EtOAc (10 ml). The organic phase is washed with water (2x10 ml), with
saturated aqueous NaHCO3 solution (10 ml) and with saturated aqueous NaCI solution (10 ml).
After drying over MgSO4 and filtering, the solvents are evaporated off under reduced pressure.
The crude reaction product is purified by chromatography on silica gel, eluting with a 99/1 to
90/10 DCM/methanol mixture. A white powder, 7, is obtained (19.4 mg; 75%). TLC (DCM 90 /
MeOH 10): Rf = 0.6; 1H NMR (400 MHz, DMSO-cfe): 0.75 -0.81 (m, 6 H); 1.01 (s, 3 H); 1.05 (d, J
= 6.8 Hz, 3 H); 1.12 (s, 3 H); 1.26 (s, 6 H); 1.28 - 1.33 (m, 1 H); 1.52-1.61 (m, 2 H); 1.76 - 1.83
(m, 2 H); 2.27 - 2.38 (m, 4 H); 2.39 (s, 3 H); 2.64 - 2.74 (m, 2 H); 2.95 - 3.05 (m, 2 H); 3.24 -
3.34 (m, 6 H); 3.44 (br. s., 4 H); 3.49 (s, 2 H); 3.81 (s, 3 H); 3.88 (d, J = 1.7 Hz, 1 H); 4.22 - 4.29
(m, 1 H); 4.92 (dd, J = 9.9, 3.5 Hz, 1 H); 5.08 - 5.15 (m, 1 H); 5.81 (d, J = 14.2 Hz, 1 H); 6.48
(ddd, J = 15.2, 11.3, 3.5 Hz, 1 H); 7.05 (d, J = 8.6 Hz, 1 H); 7.17 (dd, J = 8.4, 2.3 Hz, 1 H); 7.22
(d, J = 9.3 Hz, 1 H); 7.25 - 7.34 (m, 5 H); 8.34 (d, J = 8.1 Hz, 1 H). LCMS (A1): ES m/z = 943 [M
+ H]+; m/z = 941 [M - Hf; tR = 4.03 min.
Note: Compound 7 may also be prepared from G=OMs (see Example 1)
Compound Ex. 1: (E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-16-
[(S)-1-((2R,3R)-3-{4-[4-(4-mercapto-4-methylpentanoyl)piperazin-1-ylmethyl]phenyl}oxiranyl)-
ethyl]-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2l5,9,124etraone
Compound 7 (17.9 mg; 18.97 umol) is dissolved in a mixture of ethanol (2.2 ml)/water (1.8 ml),
and the mixture turns cloudy. TCEP (47.4 umol) is then added and the mixture is stirred for 3
hours at RT and then diluted by adding EtOAc (20 ml) and the organic phase is washed with a
1/1 mixture of water and saturated aqueous NH4CI solution (20 ml) and then with 20 ml of
saturated NaCI solution. After drying the organic phase over MgSO4, filtering and evaporating off
the solvents under reduced pressure, the product Ex. 1 is obtained in the form of a white solid
(15.6 mg; 92%). TLC (DCM 90 / MeOH 10): Rf = 0.56; 1H NMR (500 MHz, DMSO-cfe): 0.76 - 0.81
(m, 6 H); 1.01 (s, 3 H); 1.06 (d, J = 6.8 Hz, 3 H); 1.13 (s, 3 H); 1.24 (s, 6 H); 1.27 - 1.31 (m, 1 H);
1.56 - 1.64 (m, 2 H); 1.73 - 1.85 (m, 3 H); 2.26 - 2.33 (m, 3 H); 2.36 - 2.45 (m, 4 H); 2.63 - 2.75
(m, 2 H); 2.95 - 3.06 (m, 3 H); 3.34 - 3.36 (m, 1 H); 3.42 - 3.51 (m, 6 H); 3.82 (s, 3 H); 3.89 (s, 1
H); 4.22-4.29 (m, 1 H); 4.92 (dd, J = 9.8, 3.4 Hz, 1 H); 5.12 (dd, J = 10.8, 4.9 Hz, 1 H); 5.81 (d, J
= 15.2 Hz, 1 H); 6.48 (ddd, J = 15.0, 11.4, 3.4 Hz, 1 H); 7.06 (d, J = 8.3 Hz, 1 H); 7.18 (dd, J =
8.3, 1.5 Hz, 1 H); 7.24 (d, J = 9.8 Hz, 1 H); 7.26 - 7.36 (m, 5 H); 8.37 (d, J = 7.8 Hz, 1 H). LCMS
(A2): ES m/z = 897 [M + H]+; m/z = 895 [M - H]~; tR = 0.97 min.
ExampJeJZ: 2,5-Dioxopyrrolidin-1 -yl 3-(2-{2-[2-(2-{2-[4-(4-{4-[(2R,3R)-3-((S)-1-{(EH10R,16S)-1O-
(3^chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-
hexadec-13-en-16-yl}ethyl)oxiranyl]benzyl}piperazin-1 -yl)-1,1 -dimethyl-4-oxo-butylsulfanyl]-
acetylamino}ethoxy)ethoxy]ethoxy}ethoxy)propanoate
To a solution, purged with argon, of compound Ex. 1 (15.6 mg, 17.8 umol) in anhydrous
acetonitrile (1.0 ml) are successively added DIPEA (19.12 umol) and compound 56 (19.12 umol).
Stirring is continued at RT for 4 hours, and a further 29.5 umol of DIPEA are then added, and
stirring is continued for 16 hours. The next day, a further 29.5 umol of compound 56 and of
DIPEA are added and the mixture is heated to 50°C. After reaction for a further 24 hours,
22.6 umol of compound 56 and 29.5 umol of DIPEA are added and the mixture is heated at 60°C
for a further 24 hours. The heating is then stopped and stirring is continued at RT for 64 hours.
The mixture is diluted in 10 ml of EtOAc and the organic phase is washed with 2x10 ml of water
and then with 10 ml of saturated NaCI solution. After drying the organic phase over MgSO4,
filtering and evaporating off the solvents under reduced pressure, the crude product is purified by
chromatography on silica gel, eluting with a 99/1 to 90/10 DCM/methanol mixture. The product
Ex. 17 is obtained in the form of a colourless solid (9.2 mg; 41%). 1H NMR (500 MHz, DMSO-c/6):
0.77 to 0.82 (m, 6 H): 1.02 (s, 3 H): 1.06 (d, J = 6.9 Hz, 3 H): 1.13 (s, 3 H): 1.23 (s, 6 H): 1.26 to
1.35 (m, 1 H): 1.52 to 1.64 (m, 2 H): 1.68 to 1.77 (m, 2 H): 1.78 to 1.86 (m, 1 H): 2.26 to 2.33 (m,
3 H): 2.35 to 2.41 (m, 4 H): 2.68 to 2.76 (m, 2 H): 2.82 (s, 4 H): 2.93 (t, J = 6.0 Hz, 2 H): 2.96 to
3.07 (m, 4 H): 3.13 (s, 2 H): 3.20 (q, J = 5.8 Hz, 2 H): 3.33 (m, 1 H): 3.38 to 3.57 (m, 18 H): 3.73
(t, J = 5.8 Hz, 2 H): 3.83 (s, 3 H): 3.89 (s, 2 H): 4.27 (m, 1 H): 4.93 (dd, J = 3.8 and 10.0 Hz, 1 H):
5.13 (m, 1 H):5.82(d, J= 15.4 Hz, 1 H): 6.49 (ddd, J = 3.8 and 11.1 and 15.4 Hz, 1 H): 7.07 (d, J
= 8.8 Hz, 1 H): 7.15 to 7.37 (m, 7 H): 8.01 (t, J = 5.8 Hz, 1 H): 8.35 (d, J = 8.0 Hz, 1 H). LCMS
(A2): ES m/z = 1299 [M + H]+: m/z = 650 [M + 2H]2+: m/z = 1297 [M - H]~: tR = 0.92 min.
Example 18: 2,5-Dioxopyrrolidin-1-yl 3-(2-{2-[2-(2-{4-[(2S,3S)-3-((S)-1-{(E)-(3S)10R,16S)-1O-
[3-chloro^-methoxybenzyl]-3w'sobutyl-6,63R)-3-[(S)-1 -((E)-(10R,16S)-
lO^S-chloro^-methoxybenzylJ-S-isobutyl-e.e-dimethyl^.S.S.^-tetraoxo-l^-dioxa-S.H-
diazacyclohexadec-13-en-16-yl)ethyl]oxiranyl}benzyl)-1,2,3-triazol-1-yl]methoxy}ethoxy)-
ethoxy]ethoxy}ethoxy)propanoate
Compound 63: 2-{2-[2-(2-Prop-2-ynyloxyethoxy)ethoxy]ethoxy}ethanol
To a solution, purged with argon and cooled to 0°C, of 1 g (5.15 mmol) of tetraethylene glycol in
25 ml of anhydrous THF are added 144 mg (3.60 mmol) of NaH as a 60% dispersion in a mineral
oil. Stirring is continued for 30 minutes at 0°C, and 194 ul (2.58 mmol) of propargyl bromide are
then added. Stirring is continued at RT for 15 hours. The mixture is concentrated under reduced
pressure and the crude product is then purified by chromatography on silica gel, using a 98/2 to
90/10 DCM/methanol mixture as eluent. Compound 63 is obtained in the form of a colourless oil
(789 mg, 65%). 1H NMR (400 MHz, DMSO-d6): 3.40 (t, J = 2.4 Hz, 1 H); 3.42 (t, J = 5.5 Hz, 2 H);
3.48 (q, J = 5.5 Hz, 2 H); 3.51 to 3.58 (m, 12 H); 4.14 (d, J = 2.4 Hz, 2 H); 4.53 (t, J = 5.5 Hz, 1
H). LCMS (A2): ES m/z = 233 [M + H]+; tR= 0.38 min.
Compound 64: ferf-Butyl 3-(2-{2-[2-(2-prop-2-ynyloxyethoxy)ethoxy]ethoxy}ethoxy)propanoate
To a solution, purged with argon, of 790 mg (3.40 mmol) of compound 63 in 8.8 ml of anhydrous
THF are added 4.4 mg (190 umol) of sodium. The mixture is heated at 40°C for 2 hours to fully
dissolve it; after cooling to RT, 740 pi (5.09 mmol) of fe/t-butyl acrylate are added to the mixture.
Swing is continued for 15 hours at RT and the mixture is then concentrated under reduced
pressure and the crude product is purified by chromatography on silica gel, using a 98/2 to 90/10
DCM/methanol mixture as eluent. Compound 64 is obtained in the form of a colourless oil
(944 mg, 77%). 1H NMR (400 MHz, DMSO-of6): 1.44 (s, 9 H); 2.41 (t, J = 6.2 Hz, 2 H); 3.38 (t, J =
2.4 Hz, 1 H); 3.46 to 3.62 (m, 16 H); 3.59 (t, J = 6.2 Hz, 2 H); 4.14 (d, J = 2.4 Hz, 2 H). LCMS
(A1): ES m/z = 361 [M + H]+; m/z = 383 [M + Na]+; tR = 3.79 min.
Compound 65 : 3-(2-{2-[2-(2-prop-2-ynyloxyethoxy)ethoxy]ethoxy}ethoxy)propanoic acid
To a solution of 944 mg (2.62 mmol) of compound 64 are added 2 ml (52.4 mmol) of TFA. The
mixture is stirred for 6 hours at RT and then concentrated to dryness, taken up in a minimum
amount of DCM and entrained several times with toluene. Compound 65 is obtained in the form
of a colourless oil (722 mg, 91%). 1H NMR (400 MHz, DMSO-d6): 2.44 (t, J = 6.4 Hz, 2 H); 3.39 (t,
J = 2.4 Hz, 1 H); 3.46 to 3.56 (m, 16 H); 3.60 (t, J = 6.4 Hz, 2 H); 4.14 (d, J = 2.4 Hz, 2 H); 7.44 to
9.73 (very broad m, 1 H). LCMS (A2): ES m/z = 305 [M + H]+; m/z = 303 [M - H]"; tR= 0.48 min.
Example 20: 2,5-Dioxapyrrolidin-1 -yl 3-(2-{2-[2-(2-{1 -[1 -(4-{(2R,3R)-3-[(S)-1 -((EH 10R, 16S)-1O-(3-
chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclo-
I iexadec-13-en-16-yl)ethyl]oxiranyl}benzyl)-1,2,3-triazol-1-yl]methoxy}ethoxy)ethoxy]ethoxy}-
ethoxy)propanoate
Example 20 may be obtained from compounds 60 and 65 according to the method described for
compound 61, followed by activating the acid according to the method described for Example 19.
Example 21: 2,5-Dioxopyrrolidin-1-yl (4-{1-[(4-{(2R,3R)-3-[(S)-1-((EH3S,10RI16S)-1O-{3-
chloro-4-methoxybenzyl}-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diaza-
cyclohexadec-13-en-16-yl)ethyl]oxiranyl}benzyl)methylamino]methyl}-1,2,3-triazol-1-yl)-
butanoate
Compound 66: (E)-(3S,1 OR, 16S)-1O-(3-Chioro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-16-[(S)-
1-((2R,3R)-3-{4-[(methylprop-2-ynylamino)methyl]phenyl}oxiranyl)ethyl]-1,4-ciioxa-8,11-cliaza-
cyclohexadec-13-ene-2,5,9,12-tetraone
To a solution, purged with argon, of 40 mg (55.7 pmol) of compound 2 in 5 ml of anhydrous
acetonitrile are successively added 48.5 pi (279 umol) of DIPEA and 13.9 pi (167 pmol) of
N-methylpropargylamine. The mixture is heated at 40°C for 15 hours. Some starting compound 2
remains: a further 48.5 pi (279 pmol) of DIPEA and 13.9 pi (167 pmol) of
N-methylpropargylamine are added. After stirring for 24 hours at 40°C, the mixture is diluted with
5 ml of EtOAc and then washed with 5 ml of water. The aqueous phase is extracted with 3x5 ml
of EtOAc and the organic phases are combined, washed with saturated NaHCO3 solution (10 ml)
and with saturated NaCI solution (10 ml) and dried over MgSO4. After filtering and concentrating
under reduced pressure, the crude product is purified by chromatography on silica gel, using a
98/2 to 90/10 DCM/methanol mixture as eluent. Compound 66 is obtained in the form of a white
solid (45 mg, quant.). 1H NMR (400 MHz, DMSO-cfe): 0.78 (m, 6 H); 1.00 (s, 3 H); 1.05 (d, J = 6.8
Hz, 3 H); 1.12 (s, 3 H); 1.30 (m, 1 H); 1.49 to 1.61 (m, 2 H); 1.81 (m, 1 H); 2.20 (s, 3 H); 2.28 (m,
1 H); 2.63 to 2.76 (m, 2 H); 2.92 to 3.07 (m, 3 H); 3.17 (t, J = 2.4 Hz, 1 H); 3.27 (d, J = 2.4 Hz, 2
H); 3.33 (partially masked m, 1 H); 3.50 (s, 2 H); 3.81 (s, 3 H); 3.88 (d, J = 1.7 Hz, 1 H); 4.25
(ddd, J = 3.4 and 7.8 and 11.4 Hz, 1 H); 4.92 (dd, J = 3.5 and 9.7 Hz, 1 H); 5.11 (ddd, J = 1.5 and
5.5 and 11.2 Hz, 1 H); 5.80 (dd, J = 1.5 and 15.0 Hz, 1 H); 6.47 (ddd, J = 3.9 and 11.2 and 15.0
Hz, 1 H); 7.05 (d, J = 8.6 Hz, 1 H); 7.17 (dd, J = 2.1 and 8.6 Hz, 1 H); 7.22 (dd, J = 2.1 and 9.4
Hz, 1 H); 7.25 to 7.33 (m, 5 H); 8.33 (d, J = 7.8 Hz, 1 H). LCMS (A2): ES m/z = 750 [M + H]+; ES
m/z = 375.5 [M + 2H]2+ base peak; ES m/z = 748 [M - H]"; m/z = 794 [M - H + HCO2H]-; tR = 0.91
min.
Compound 67: Ethyl 4-azidobutyrate
2.86 ml (20 mmol) of ethyl 4-bromobutanoate and 2.6 g (40 mmol) of NaN3 are dissolved in 30 ml
of a 2/1 acetone/water mixture. The mixture is refluxed for 7 hours. After cooling to RT and
concentrating under reduced pressure, the residue is taken up in 50 ml of water. The aqueous
phase is extracted with 3x30 ml of DCM. The organic phases are combined, dried over MgSO4,
filtered and concentrated under reduced pressure to give compound 67 in the form of a
colourless oil (3 g, 95%). 1H NMR (400 MHz, chloroform-of): 1.27 (t, J = 7.2 Hz, 3 H); 1.92 (m, 2
H); 2.41 (t, J = 7.2 Hz, 2 H); 3.36 (t, J = 6.7 Hz, 2 H); 4.15 (q, J = 7.2 Hz, 2 H).
Compound 68:4-Azidobutanoic acid
To a solution of 3 g (19.9 mmol) of compound 67 in 47 ml of methanol and 37 ml of water are
added portionwise 5.58 g (99.5 mmol) of potassium hydroxide. The mixture is stirred at RT for 6
hours and then concentrated under reduced pressure to about 28 ml. The residue is diluted with
25 ml of water and extracted with 2x20 ml of DCM. The aqueous phase is acidified to pH=1 by
adding concentrated HCI and is then extracted with 3x25 ml of diethyl ether. The organic phases
are combined, washed with saturated NaCI solution (25 ml) and dried over MgSO4. After filtering
and concentrating to dryness under reduced pressure, compound 68 is obtained in the form of a
colourless oil (2.05 g, 80%). 1H NMR (400 MHz, chloroform-of): 1.93 (m, 2 H); 2.49 (t, J = 7.1 Hz,
2 H); 3.39 (t, J = 6.7 Hz, 2 H); 11.24 (broad m, 1 H).
Compound 69: (4-{1-[(4-{(2R,3R)-3-[(S)-1-((E)-(3S,10R,16S)-1O-{3-Chloro^-methoxybenzyl}-3-
isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11 -diazacyclohexadec-13-en-16-yl)ethyl]-
oxiranyl}benzyl)methylamino]methyl}-1,2,3-triazol-1 -yl)butanoic acid
To a solution of 24 mg (32 umol) of compound 66 in 545 pi of THF are successively added
8.3 mg (13 umol) of compound 68, 545 pi of water, 128 pi of an aqueous 0.1M solution of copper
sulfate and 128 pi of aqueous 0.2M sodium ascorbate solution. The mixture is stirred for 45
minutes at RT and then diluted with 2 ml of water. The aqueous phase is extracted with 3x2 ml of
EtOAc. The organic phases are combined, washed with saturated NaCI solution (2 ml) and
filtered through MgSO4. After filtering and concentrating under reduced pressure, the crude
product is purified by chromatography on diol-grafted silica gel, using a 98/2 to 90/10
DCM/methanol mixture as eluent. Compound 69 is obtained in the form of a white solid (22.6 mg,
80%). 1H NMR (500 MHz, DMSO-d6): 0.76 (d, J = 6.6 Hz, 3 H); 0.78 (d, J = 6.6 Hz, 3 H); 1.00 (s,
3 H); 1.05 (d, J = 6.9 Hz, 3 H); 1.11 (s, 3 H); 1.30 (m, 1 H); 1.50 to 1.62 (m, 2 H); 1.81 (m, 1 H);
2.03 (m, 2 H); 2.10 (s, 3 H); 2.20 (t, J = 7.0 Hz, 2 H); 2.30 (m, 1 H); 2.64 to 2.74 (m, 2 H); 2.94 to
3.03 (m, 3 H); 3.35 (partially masked m, 1 H); 3.48 (s, 2 H); 3.61 (s, 2 H); 3.81 (s, 3 H); 3.87 (d, J
= 1.6 Hz, 1 H); 4.25 (ddd, J = 3.7 and 7.7 and 11.5 Hz, 1 H); 4.37 (t, J = 7.0 Hz, 2 H); 4.91 (dd, J
= 3.6 and 9.6 Hz, 1 H); 5.11 (ddd, J = 1.5 and 5.5 and 11.3 Hz, 1 H); 5.80 (dd, J = 1.5 and 15.1
Hz, 1 H); 6.47 (ddd, J = 3.7 and 11.3 and 15.1 Hz, 1 H); 7.05 (d, J = 8.5 Hz, 1 H); 7.17 (dd, J =
2.2 and 8.5 Hz, 1 H); 7.23 (dd, J = 2.5 and 9.8 Hz, 1 H); 7.26 (d, J = 8.5 Hz, 2 H); 7.28 (d, J = 2.2
Hz, 1 H); 7.34 (d, J = 8.5 Hz, 2 H); 8.02 (s, 1 H); 8.39 (broad d, J = 7.7 Hz, 1 H); 12.14 (broad m,
1 H). LCMS (A2): ES m/z = 879 [M + H]+; m/z = 877 [M - H]"; tR= 0.86 min.
Compound 70: Methyl (4-{1 -[(4-{(2R,3R)-3-[(S)-1 -((E)-(3S, 10R, 16S)-1O-{3-chloro-4-methoxy-
benzyl}-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11 -diazacyclohexadec-13-en-16-
yl)ethyl]oxiranyl}benzyl)methylamino]methyl}-1,2,3-triazol-1-yl)butanoate
To a solution, purged with argon, of 5.5 mg (6.2 umol) of compound 69 in 0.5 ml of DCM and
0.2 ml of methanol are added 4.7 ul (9.3 umol) of trimethylsilyldiazomethane. The mixture is
stirred for 45 minutes at RT and then concentrated to dryness. The crude product is purified by
chromatography on silica gel, using a 98/2 to 95/5 DCM/methanol mixture as eluent. Compound
70 is obtained in the form of a white solid (3.4 mg, 62%). 1H NMR (500 MHz, DMSO-cfe): 0.75 (d,
J = 6.6 Hz, 3 H); 0.77 (d, J = 6.6 Hz, 3 H); 1.00 (s, 3 H); 1.05 (d, J = 6.9 Hz, 3 H); 1.11 (s, 3 H);
1.28 (m, 1 H); 1.49 to 1.61 (m, 2 H); 1.80 (m, 1 H); 2.07 (m, 2 H); 2.10 (m, 3 H); 2.26 (m, 1 H);
2.31 (t, J = 7.0 Hz, 2 H); 2.62 to 2.74 (m, 2 H); 2.93 to 3.04 (m, 3 H); 3.27 to 3.37 (partially
masked m, 1 H); 3.48 (s, 2 H); 3.58 (s, 3 H); 3.61 (s, 2 H); 3.81 (s, 3 H); 3.87 (d, J = 2.0 Hz, 1 H);
4.25 (ddd, J = 3.7 and 8.2 and 11.6 Hz, 1 H); 4.38 (t, J = 7.0 Hz, 2 H); 4.91 (dd, J = 3.9 and 9.8
Hz, 1 H); 5.11 (ddd, J = 1.5 and 5.3 and 11.2 Hz, 1 H); 5.79 (dd, J --1.5 and 15.2 Hz, 1 H); 6.47
(ddd, J = 3.9 and 11.2 and 15.2 Hz, 1 H); 7,05 (d, J = 8.6 Hz, 1 H); 7.16 (dd, J = 2.2 and 8.6 Hz, 1
H); 7.22 (dd, J = 2.4 and 9.8 Hz, 1 H); 7.26 (d, J = 8.6 Hz, 2 H); 7.28 (d, J = 2.2 Hz, 1 H); 7.33 (d,
J = 8.6 Hz, 2 H); 8.03 (s, 1 H); 8.34 (d, J = 8.2 Hz, 1 H). LCMS (A2): ES m/z = 893 [M + H]+; m/z
= 447 [M + 2H]2+ base peak; m/z = 891 [M - H]"; m/z = 937 [M -H + HCO2H]" base peak; tR= 0.90
min.
Example 21: 2,5-Dioxopyrrolidin-1 -yl (4-{1 -[(4-{(2R,3R)-3-[(S)-1 -((E)-(3S, 10R, 16S)-1O-{3-chloro-
4-methoxybenzyl}-3-isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-
13-en-16-yl)ethyl]oxiranyl}benzyl)methylamino]methyl}-1,2,3-triazol-1 -yl)butanoate
Example 21 may be obtained by activating the acid 69 according to the method described for
Example 19.
Example 22
Compound 71: (EW3S.10R.16S)-16-{(S)-1-r(2S,3S)-3-(4-Azidomethvlphenvl)oxiranvnethvl>-1O-
(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11 -diazacyclohexadec-13-ene-
2,5,9,12-tetraone
The alcohol 57 (36 mg; 51.48 umol, prepared according to Al-awar R.S., et al., J.Med.Chem.
2003, 46, 2985-3007) is dissolved in anhydrous THF (2 ml). The solution is purged with argon
and cooled in a bath of ice-cold water, followed by addition of DPPA (74 umol) and then DBU
(80 umol). The mixture is allowed to warm to RT and stirring is continued overnight. The next day,
the solution is again cooled in a bath of ice-cold water, followed by addition of a further 74 umol of
DPPA and 80 umol of DBU. After reaction for 2 hours at 0°C and 2 hours at RT, the mixture is
hydrolyzed with 5 ml of water and then extracted 3 times with DCM. The combined organic
phases are dried over is^SCU, filtered and evaporated under reduced pressure. The crude
product is then purified by chromatography on silica gel, eluting with a 100/0 to 95/5
DCM/methanol mixture. Compound 57 is obtained in the form of a colourless solid (24 mg; 64%).
1H NMR (500 MHz, DMSO-d6) 5 (ppm): 0.84 (d, J = 6.3 Hz, 3 H); 0.86 (d, J = 6.3 Hz, 3 H);0.98
(d, J = 7.1 Hz, 3 H); 1.03 (s, 3 H); 1.15 (s, 3 H); 1.48 to 1.67 (m, 3 H); 1.88 (m, 1 H); 2.45 (m, 1
H);2.63 (m, 1 H); 2.71 (dd, J = 11.5 and 14.0 Hz, 1 H); 2.97 to 3.08 (m, 3 H); 3.36 (partially
masked m, 1 H); 3.82 (broad s, 4 H); 4.28 (ddd, J = 3.6 and 8.0 and 11.5 Hz, 1 H); 4.44 (s, 2 H);
4.99 (dd, J = 3.7 and 9.5 Hz, 1H); 5.12 (ddd, J = 1.4 and 5.7 and 11.4 Hz, 1 H); 5.88 (dd, J = 1.4
and 15.2 Hz, 1 H); 6.49 (ddd, J = 3.8 and 11.4 and 15.2 Hz, 1 H); 7.05 (d, J = 8.5 Hz, 1 H); 7.18
(dd, J = 2.2 and 8.5 Hz, 1 H); 7.26 to 7.32 (m, 4 H); 7.36 (d,J = 8.5 Hz, 2 H); 8.40 (d, J = 8.0 Hz, 1
H). LCMS (A2): ES m/z = 722 [M - H]"; m/z = 724 [M + H]+; m/z = 768 [M + HCO2H - H]-; tR = 1.19
min.
Compound 72: (EH3S.10R.16S)-16-((S)-1-r(2S.3SV3-(4-Aminomethvlphenynoxiranyl]ethyl}-1O-
(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11 -diazacyclohexadec-13-ene-
2,5,9,12-tetraone
Compound 71 (22 mg, 30.38 umol) is dissolved in a mixture of methanol (1 ml)/water (0.2 ml),
followed by addition of TCEP (34.89 umol). The solution obtained is stirred overnight at RT and
then concentrated under reduced pressure. The residue is then taken up in saturated aqueous
NaHCO3 solution and extracted 3 times with DCM. The combined organic phases are dried over
Na2SO4, filtered and concentrated under reduced pressure. The intermediate 72 is thus obtained
in the form of a colourless solid (21 mg, 99%), which is used in crude form in the following step.
LCMS (A5): ES m/z = 696 [M - H]'; m/z = 698 [M + H]+; m/z = 742 [M + HCO2H - H]"; tR = 3.19
min.
Compound 74: FmocVal-Cit-PABAC-a-aminocryptophycin 72
Compound 72 (21 mg, 30.08 umol) is dissolved in a mixture of acetonitrile (2 ml)/anhydrous DMF
(0.5 m!), followed by addition of a solution of compound 73 (23 mg, 30.1 umol; prepared
according to WO 2006/110 476) in acetonitrile (2 ml). The mixture is stirred for 22 hours at RT
end then concentrated under reduced pressure. The residue is taken up in DCM and washed with
saturated aqueous NaHCO3 solution and then dried over Na2SO4, filtered and concentrated
under reduced pressure. The crude product is then purified by chromatography on silica gel,
eluting with a 100/0 to 90/10 DCM/methanol mixture. Compound 74 is obtained in the form of a
white solid (22 mg), which is used in crude form in the following step. LCMS (A5): ES m/z = 1325
[M + H]+; m/z = 1369 [M + HCO2H - H]~; tR = 4.30 min.
Compound 75: Val-Cit-PABAC-oc-aminocryptophycin 72
Compound 74 (22 mg, 16.59 umol) is dissolved in DMF (3 ml), followed by addition of piperidine
(150 ul, 1.51 mmol) and the mixture is stirred for 30 minutes at RT. The mixture is concentrated
under reduced pressure; the residue is dissolved in a minimum amount of methanol and
precipitated from ether. Compound 75 is thus obtained in the form of a white solid (15 mg, 82%).
LCMS (A5): ES m/z = 1103 [M + H]+; m/z = 1101 [M - H]"; m/z = 1148 [M + HCO2H - H]~; tR =
3.38 min.
Compound 76: Glutaric acid-Val-Cit-PABAC-a-aminocryptophycin 72
A solution of glutaric anhydride (8 mg, 70 umol) in anhydrous DMF (200 u!) is added to
compound 75 (15 mg, 13.59 umol) conditioned under argon. The solution obtained is stirred
overnight at RT, followed by addition of saturated aqueous NH4CI solution and extraction 3 times
with DCM. The combined organic phases are dried over Na2SO4, filtered and concentrated under
reduced pressure. The residue obtained is taken up in DCM and precipitated with ether. After
filtering through a sinter funnel and washing with ether, compound 76 is obtained in the form of a
beige-coloured solid (8 mg, 48%). 1H NMR (500 MHz, DMSO-cfe) 5 (ppm): 0.80 to 0.92 (m, 12 H);
0.96 (d, J = 7.1 Hz, 3H); 1.02 (s, 3 H); 1.15 (s, 3 H); 1.30 to 1.72 (m, 9 H); 1.86 (m, 1 H); 2.00 (m,
1 H); 2.11 to 2.26 (m, 4 H); 2.42 (m, 1 H); 2.60 to 2.74 (m, 2 H); 2.91 to 3.11 (m, 5 H); 3.38
(partially masked m, 1 H); 3.76 (d,J = 1.8 Hz, 1 H); 3.81 (s, 3 H); 4.17 (m, 3 H); 4.27 (m, 1 H);
4.37 (m, 1 H); 4.90 to 5.04 (m, 3 H); 5.11(m, 1 H); 5.50 (broad m, 2 H); 5.88 (broad d, J = 15.2
Hz, 1 H); 6.20 (broad m, 1 H); 6.48 (m, 1 H); 7.06 (d, J = 8.3 Hz, 1 H); 7.11 to 7.32 (m, 9 H); 7.60
(m, 2 H); 7.75 (m, 1 H); 7.89 (m, 1 H); 8.28 (broad m, 1 H); 8.41 (d, J = 7.8 Hz, 1 H); 9.92 (broad
s, 1 H); 12.06 (broad m, 1 H). LCMS (A2): ES m/z = 1217 [M + H]+; m/z = 1215 [M - H]"; tR = 1.05
min.
Example 22
Compound 76 (6 mg, 4.93 umol) is dissolved in a mixture of DCM (0.5 ml) and DMF (0.1 ml),
followed by successive addition of N,N'-disuccinimidyl carbonate (6 mg, 23.42 umol) and DIPEA
(4 pi, 22.96 umol). After reaction for 3 hours at RT, saturated aqueous NH4CI solution is added
and the mixture is extracted 3 times with DCM. The combined organic phases are dried over
Na2SO4, filtered and concentrated under reduced pressure. The crude product is purified by
chromatography on silica gel, eluting with a 100/0 to 90/10 DCM/methanol mixture. Compound
Ex. 22 is obtained in the form of a white solid (4 mg; 61%). 1H NMR (500 MHz, DMSO-d6) 8
(ppm): 0.80 to 0.90 (m, 12 H); 0.96 (d, J = 7.3 Hz, 3 H); 1.03 (s, 3 H); 1.15 (s, 3 H); 1.32 to 1.76
(m, 7 H); 1.80 to 1.89 (m, 3 H); 1.97 (m, 1 H); 2.29 (m, 2 H); 2.44 (m, 1 H); 2.60 to 2.75 (m, 4 H);
2.81 (s, 4 H); 2.89 to 3.08 (m, 5 H); 3.41 (partially masked m, 1 H); 3.77 (d, J = 2.0 Hz, 1 H); 3.81
(s, 3 H); 4.14 to 4.24 (m, 3 H); 4.27 (ddd, J = 3.9 and 8.3 and 11.5 Hz, 1 H); 4.38 (m, 1 H); 4.93 to
5.03 (m, 3 H); 5.11 (m, 1 H); 5.39 (broad s, 2 H); 5.88 (broad d, J = 15.2 Hz, 1 H); 5.96 (t, J = 5.9
Hz, 1 H); 6.48 (ddd, J = 3.7 and 11.1 and 15.2 Hz, 1 H); 7.05 (d, J = 8.3 Hz, 1 H); 7.16 to 7.36 (m,
9 H); 7.59 (d, J = 7.8 Hz, 2 H); 7.76 (t, J = 6.1 Hz, 1 H); 7.88 (d, J = 8.8 Hz, 1 H); 8.10 (d, J = 7.3
Hz, 1 H); 8.41 (d, J = 8.3 Hz, 1 H); 9.97 (broad s, 1 H). LCMS (A2): ES m/z = 1314 [M + H]+; m/z
= 1358 [M + HCO2H - Hf; tR = 1.06 min.
Example 23
Compound 77: (E)-(3S, 10R, 16S)-16-{(S)-1 -[(2R,3R)-3-(4-Aminomethylphenyl)oxiranyl]ethyl}-1O-
(3-chloro-4-methoxybenzyl)-3-isobutyl-6,6-dimethyl-1,4-dioxa-8,11 -diazacyclohexadec-13-ene-
2,5,9,12-tetraone
Compound 60 (23 mg, 31.76 umol) is dissolved in a mixture of methanol (1 ml) and water
(0.2 ml), followed by addition of TCEP (34.90 umol) and DCM (an amount sufficient to dissolve it).
The solution obtained is stirred overnight at RT and is then concentrated under reduced pressure.
The residue is then taken up in saturated aqueous NaHCO3 solution and extracted 3 times with
DCM. The combined organic phases are dried over Na2SO4, filtered and concentrated under
reduced pressure. The crude product is finally purified by chromatography on silica gel, eluting
with a 100/0 to 90/10 DCM/methanol mixture. Compound 77 is thus obtained in the form of a
white solid (12 mg, 59%). 1H NMR (500 MHz, DMSO-cfe) 8 (ppm): 0.78 (d, J = 6.4 Hz, 6 H); 1.00
(s, 3 H); 1.05 (d, J = 6.9 Hz, 3 H); 1.12 (s, 3 H); 1.30 (m, 1 H); 1.47 to 1.62 (m, 2 H); 1.79 (m, 1
H); 2.22 to 2.32 (m, 3 H); 2.63 to 2.73 (m, 2 H); 2.92 to 3.05 (m, 3 H); 3.35 (partially masked m, 1
H); 3.71 (s, 2 H); 3.81 (s, 3 H); 3.86 (d, J = 1.6 Hz, 1 H); 4.25 (ddd, J = 3.6 and 8.0 and 11.3 Hz, 1
H); 4.90 (dd, J = 3.6 and 9.6 Hz, 1 H); 5.11 (ddd, J = 1.5 and 5.2 and 11.3 Hz, 1 H); 5.79 (dd, J =
1.5 and 15.0 Hz, 1 H); 6.47 (ddd, J = 3.6 and 11.3 and 15.0 Hz, 1 H); 7.05 (d, J = 8.5 Hz, 1 H);
7.17 (dd, J = 1.9 and 8.5 Hz, 1 H); 7.19 to 7.26 (m, 3 H); 7.28 (d, J = 1.9 Hz, 1 H); 7.34 (d, J = 8.0
Hz, 2 H); 8.34 (d, J = 8.0 Hz, 1 H). LCMS (A2): ES m/z = 698 [M + H]+; m/z = 696 [M - H]"; m/z =
742 [M + HCO2H - Hf; tR = 0.87 min.
Compound 78: FmocVal-Cit-PABAC-oc-aminocryptophycin 77
Compound 77 (10 mg, 14.32 umol) is dissolved in anhydrous DMF (0.1 ml), followed by addition
of a solution of the intermediate 73 (11 mg, 14.35 umol; prepared according to WO
2006/110 476) in a mixture of acetonitrile (1 ml) and DMF (0.1 ml). The mixture is stirred for 3
hours at RT and then concentrated under reduced pressure. The residue is then purified by
chromatography on silica gel, eluting with a 100/0 to 90/10 DCM/methanol mixture. Compound 78
is obtained in the form of a white solid (18 mg, 95%). LCMS (A5): ES m/z = 1325 [M + H]+; m/z =
1369 [M + HCO2H - H]-; tR = 4.32 min.
Compound 79: Val-Cit-PABAC-oc-aminocryptophycin 77
Compound 78 (42 mg, 31.68 umol) is dissolved in DMF (5 ml), followed by addition of piperidine
(150 ul, 1.51 mmol) and the mixture is stirred for 30 minutes at RT. The mixture is concentrated
under reduced pressure and the residue is dissolved in a minimum amount of methanol and
precipitated from ether. Compound 79 is thus obtained in the form of a white solid (21 mg, 60%).
LCMS (A4): ES m/z = 1103 [M + H]+; m/z = 1101 [M - H]'; m/z = 1147 [M + HCO2H - H]_; tR =
3.67 min.
Compound 80: Glutaric acid-Val-Cit-PABAC-a-aminocryptophycin 77
Compound 79 (20 mg, 18.12 umol) is dissolved in a mixture of DMF (100 pi) and anhydrous DCM
(500 pi), followed by addition of glutaric anhydride (4 mg, 35.06 umol). The solution obtained is
stirred overnight at RT and then concentrated under reduced pressure. The residue is taken up in
a minimum amount of DCM and methanol, precipitated from a mixture of ether and pentane, and
filtered on a sinter funnel. Compound 80 is thus obtained in the form of a white solid (23 mg,
104% crude), which is used in crude form in the following step. 1H NMR (500 MHz, DMSO-cf6):
0.78 (d, J = 6.3 Hz, 6 H); 0.84 (d, J = 6.6 Hz, 3 H); 0.86 (d, J = 6.6 Hz, 3 H); 1.00 (s, 3 H); 1.04 (d,
J = 6.9 Hz, 3 H); 1.12 (s, 3 H); 1.22 to 1.47 (m, 3 H); 1.51 to 1.64 (m, 3 H); 1.72 (m, 3 H); 1.80 (m,
1 H); 1.99 (m, 1 H); 2.20 (m, 4 H); 2.28 (m, 1 H); 2.59 to 2.73 (m, 2 H); 2.90 to 3.07 (m, 5 H); 3.32
(partially masked m, 1 H); 3.81 (s, 3 H); 3.87 (broad s, 1 H); 4.19 (m, 3 H); 4.24 (m, 1 H); 4.38 (m,
1 H); 4.91 (dd, J = 3.3 and 9.6 Hz, 1 H); 4.97 (broad s, 2 H); 5.10 (dd, J = 4.4 and 10.7 Hz, 1 H);
5.40 (broad s, 2 H); 5.79 (broad d, J = 15.1 Hz, 1 H); 5.99 (broad m, 1 H); 6.47 (ddd, J = 3.6 and
11.3 and 15.1 Hz, 1 H); 7.05 (d, J = 8.5 Hz, 1 H); 7.17 (broad d, J = 8.5 Hz, 1 H); 7.20 to 7.34 (m,
8 H); 7.60 (broad d, J = 8.2 Hz, 2 H); 7.76 (broad t, J = 5.4 Hz, 1 H); 7.83 (d, J = 8.5 Hz, 1 H);
8.09 (broad d, J = 7.1 Hz, 1 H); 8.34 (d, J = 8.0 Hz, 1 H); 9.95 (broad s, 1 H); 11.98 (broad m, 1
H). LCMS (A2): ES m/z = 1217 [M + H]+; m/z = 1215 [M - H]'; tR = 1.03 min.
Example 23
Example 23 may be obtained by activating the acid 80 according to the method described for
Example 22.
Example 24: 4-((2S,3S)-3-{(S)-1-[(E)-(3S,10R,16S)-1O-(3-Chloro-4-methoxybenzyl)-3-
isobutyl-6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-
yl]ethyl}oxiranyl)benzyl [2-methyl-2-(pyridin-2-yldisulfanyl)propyl]methylcarbamate
Compound 81: Methyl[2-methyl-2-(pyridin-2-yldisulfanyl)propyl]amine
To a solution of the amine 15 (478 mg, 2.90 mmol) in MeOH (35 ml) is added TCEP (831 mg,
2.9 mmol). After reaction for 2 hours at RT, the solution obtained is added dropwise, under an
argon atmosphere, to a solution of 2,2'-dipyridyl disulfide (960 mg, 4.36 mmol) in ethanol (70 ml).
After reaction for a further 2 hours at RT, the mixture is concentrated under reduced pressure.
The residue is taken up in DCM and washed with saturated aqueous NaHCO3 solution. The
organic phase is separated out and the aqueous phase is then extracted twice more with DCM.
The combined organic phases are dried over Na2SO4, filtered and concentrated under reduced
pressure. The crude product is purified by chromatography on silica gel, eluting with a 98/2 to
85/15 DCM/methanol mixture. Compound 81 is obtained in the form of a pale yellow oil (587 mg,
89%). 1H NMR (500 MHz, DMSO-cfe): 1.25 (s, 6 H); 1.80 (broad m, 1 H); 2.19 (s, 3 H); 2.45 (s, 2
H); 7.22 (ddd, J = 1.5 and 5.0 and 7.0 Hz, 1 H); 7.74 to 7.85 (m, 2 H); 8.42 (ddd, J = 1.1 and 1.5
and 5.0 Hz,1 H).
Example 24: 4-((2S.3S)-3-l(S)-1-f(EW3S.10R.16S)-1O-(3-Chloro-4-methoxvbenzvn-3-isobtjtyl-
6,6-dimethyl-2,5,9,12-tetraoxo-1,4-dioxa-8,11-diazacyclohexadec-13-en-16-yl]ethyl}oxiranyl)-
benzyl [2-methyl-2-(pyridin-2-yldisulfanyl)propyl]methylcarbamate
To a solution of the intermediate 58 (21 mg, 24.3 umol) in anhydrous DCM are successively
aaaea the amine 81 (10 mg, 43.79 pmol) and DIPEA (8 JJI, 45.43 pmol). After 24 hours at RT,
saturated aqueous NaHCO3 solution is added and the mixture is extracted 3 times with DCM. The
combined organic phases are dried over Na2SO4, filtered and concentrated under reduced
pressure. The crude product is purified by chromatography on silica gel, eluting with a 100/0 to
95/5 DCM/methanol mixture. Compound Ex. 24 is thus obtained in the form of a colourless solid
(7 mg, 30%). 1H NMR (500 MHz, DMSO-cfe): 0.82 (d, J = 6.0 Hz, 3 H); 0.84 (d, J = 6.0 Hz, 3 H);
0.97 (d, J = 6.8 Hz, 3 H); 1.03 (s, 3 H); 1.14 (s, 3 H); 1.25 (broad s, 6 H); 1.45 to 1.68 (m, 3 H);
1.88 (m, 1 H); 2.42 (partially masked m, 1 H); 2.58 to 2.75 (m, 2 H); 2.83 to 3.09 (m, 6 H); 3.34
(partially masked m, 1 H); 3.46 (broad s, 2 H); 3.81 (broad s, 4 H); 4.28 (m, 1 H); 4.91 to 5.17 (m,
4 H); 5.88 (broad d, J = 15.2 Hz, 1 H); 6.49 (m, 1 H); 7.05 (d, J = 8.6 Hz, 1 H); 7.15 to 7.38 (m, 8
H); 7.80 (broad m, 2 H); 8.39 (d, J = 7.8 Hz, 1 H); 8.43 (broad d, J = 4.6 Hz, 1 H). LCMS (A2): ES
m/z = 953 [M + H]+; m/z = 477 [M + 2H]2+; m/z = 997 [M + HCO2H - H]~; tR = 1.23 min.
Example 25: hu2H11-Ex17
According to the general method, 8.31 mg (0.057 pmol, 1.468 ml) of naked antibody hu2H11 at a
concentration of 5.66 mg/ml are treated with 5 eq. of compound Ex. 17 (0.442 mg, 0.340 pmol)
dissolved in 37.3 pi of DMA, such that the final antibody concentration is 3 mg/ml in the mixture.
After purifying on Superdex 200 pg and concentrating on Amicon Ultra-15 (Ultracel 50k
membrane, Millipore), 2.15 ml of conjugate are obtained at a concentration of 2.48 mg/ml with, on
average, 2.2 cytotoxic units per antibody and a monomer purity of 99.7% in an aqueous pH 6.5
buffer containing 0.01 M of phosphate, 0.14 M of NaCI and 20% by volume of NMP. The final
buffer change is performed in an aqueous pH 6.5 buffer containing 0.01 M of phosphate and
0.14 M of NaCI on 1 ml of conjugate. 1.5 ml of a solution of conjugate Ex. 25 at a concentration of
1.06 mg/ml with, on average, 2.8 cryptophycin derivatives (determined by UV) per antibody and a
monomer purity of 99.7% are thus obtained.
Example 26: hu2H11-Ex18
According to the general method, 7.8 mg (0.053 pmol, 1.458 ml) of naked antibody hu2H11 at a
concentration of 5.35 mg/ml are treated with 6 eq. of compound Ex. 18 (0.578 mg, 0.531 pmol,
purity 60%) dissolved in 272 pi of DMA, such that the final antibody concentration is 3 mg/ml in
the mixture. After purifying on Superdex 200 pg and concentrating on Amicon Ultra-15 (Ultracel
50k membrane, Millipore), 2.35 ml of conjugate are obtained at a concentration of 2.49 mg/ml
with, on average, 2.5 cryptophycin derivatives per antibody and a monomer purity of 100% in an
aqueous pH 6.5 buffer containing 0.01 M of phosphate, 0.14 M of NaCI and 20% by volume of
NMP. The final buffer change is performed in an aqueous pH 6.5 buffer containing 0.01 M of
phosphate and 0.14 M of sodium chloride on 1 ml of conjugate. 1.5 ml of a solution of conjugate
Ex. 26 at a concentration of 0.84 mg/ml with, on average, 2.22 cryptophycin derivatives
(determined by UV) per antibody and a monomer purity of 99.9% are thus obtained.
Example 27: hu2H11-Ex19
According to the general method, 12 mg (81.7 umol, 1.172 ml) of naked antibody hu2H11 at a
concentration of 10.24 mg/ml are treated with 2 * 5 eq. of compound Ex. 19 (0.382 mg,
0.41 umol) dissolved in 44.3 pi of DMA, such that the final antibody concentration is 3 mg/ml in
the reaction medium. After purifying on Superdex 200 pg in the presence of 20% NMP and
concentrating on Amicon Ultra-15 (Ultracel 50k membrane, Millipore), 1.04 ml of conjugates are
obtained in an aqueous pH 6.5 buffer containing 0.01 M of phosphate, 0.14 M of NaCI and 20%
by volume of NMP. The final buffer change is performed in an aqueous pH 6.5 buffer containing
0.01 M of histidine, 10% of sucrose (w/v) and 5% of NMP (v/v). 1.5 ml of a solution of conjugate
Ex. 27 at a concentration of 2.66 mg/ml with, on average, 1.4 cryptophycin derivatives (HRMS)
per antibody and a monomer purity of 99.8% are thus obtained.
The examples given above were prepared with a particular cryptophycin derivative (often the
derivative D-i), but may be applied to another derivative of the group DrD8 or to the cryptophycin
derivative of general formula (ii). Similarly, the examples of conjugates in Examples 10, 11, 12,
25, 26 and 27 may be applied to antibodies other than hu2H11.
Note: in said examples, -Lys- means that the attachment takes place on the e-amino groups of the lysines of the
antibody.
Example 28: Evaluation of the inhibition of proliferation of MDA-MB-231, MDA-A1 and
HCT116 cell lines by the cytotoxic agents, study performed on the compounds of formula (II)
of the type SZa with Za = SMe or of the type -C(=O)-ZbRb with ZbRb = OMe or OCH2-CH=CH2
The MDA-MB-231, MDA-A1 or HCT116 cells in their exponential growth phase are trypsinized
and resuspended in their respective culture medium (DMEM/F12 Gibco #21331, 10% FCS Gibco
#1050O-056, 2 nM glutamine Gibco #25030 for the MDA cells; DMEM Gibco #11960, 10% FCS
Gibco #1050O-056, 2 mM glutamine Gibco #25030 for the HCT116 cells). The cell suspension is
seeded in Cytostar 96-well culture plates (GE Healthcare Europe, #RPNQ0163) in the whole
culture medium containing serum at a density of 5000 cells/well (MDA-MB-231, MDA-A1, '
HCT116). After incubation for 4 hours, successive dilutions of the cryptophycin derivatives are
added to the wells at concentrations decreasing from 10"7 to 1012 M (in triplicate for each
concentration). The cells are cultured for 3 days at 37°C under an atmosphere containing 5%
CO2 in the presence of the cytotoxic agents. On the 4th day, 10 ul of a 14C-thymidine solution
(0.1 uCi/well, Perkin Elmer #NEC56825000) are added to each well. The incorporation of 14C-
thymidine is measured 96 hours after the start of the experiment using a microbeta radioactivity
counter (Perkin Elmer). The data are expressed in the form of a percentage of survival by
determining the ratio between the reduced count obtained with the cells treated with cytotoxic
agent and the count obtained with the cells of the control wells (treated with the culture medium
alone).
Example 29: Evaluation of the inhibition of proliferation of the MDA-MB-231, MDA-A1 and
HCT116 cell lines by the antibody-cytotoxic agent conjugates
MDA-MB-231, MDA-A1 or HCT116 cells in their exponential growth phase are trypsinized and
resuspended in their respective culture medium (DMEM/F12 Gibco #21331, 10% FCS Gibco
#1050O-056, 2 nM glutamine Gibco #25030 for the MDA cells; DMEM Gibco #11960, 10% FCS
Gibco #1050O-056, 2 mM glutamine Gibco #25030 for the HCT116 cells). The cell suspension is
seeded in Cytostar 96-well culture plates (GE Healthcare Europe, #RPNQ0163) in the whole
culture medium containing serum at a density of 5000 cells/well (MDA-MB-231, MDA-A1,
HCT116). After incubation for 4 hours, successive dilutions of the cryptophycin derivatives are
added to the wells at concentrations decreasing from 10-7 to 10-12 M (in triplicate for each
concentration). The cells are cultured at 37°C in an atmosphere containing 5% CO2 in the
presence of the antibody-cytotoxic agent immunoconjugates for 3 days. On the 4th day, 10 pi of a
14C-thymidine solution (0.1 uCi/well, Perkin Elmer #NEC56825000) are added to each well. The
incorporation of 14C-thymidine is measured 96 hours after the start of the experiment with a
microbeta radioactivity counter (Perkin Elmer). The data are expressed in the form of a
percentage of survival by determining the ratio between the reduced count obtained with the cells
treated with the immunoconjugate and the count obtained with the cells of the control wells
(treated with the culture medium alone). In certain experiments, the naked antibody hu2H11 was
added to the wells at a concentration of 1 pM at the start of the experiment and the inhibition of
proliferation was measured as described previously.
CLAIMS
1. Cryptophycin derivative of formula (II):
in which:
■ R1 represents a halogen atom and R2 represents a group -OH, an acyl group derived
from an amino acid AA or a group (C1-C4)alkanoyloxy;
or alternatively R1 and R2 form an epoxide unit;
• AA denotes a natural or unnatural amino acid;
■ R3 represents a group (C1-C6)alkyl;
■ R4 and R5 both represent H or together form a double bond CH=CH between C13 and
C14;
» R6 and R7 represent, independently of each other, H or a group (C1-C6)alkyl;
■ R8 and R9 represent, independently of each other, H or a group (C1-C6)alkyl;
■ R10 represents at least one substituent of the phenyl nucleus chosen from: H, a group
OH, (C1-C4)alkoxy, a halogen atom or a group -NH2, -NH(C1-C6)alkyl or -N(C1-C6)alkyl2;
■ R-n represents at least one substituent of the phenyl nucleus chosen from H and a
group (C1-C4)alkyl;
■ L represents a linker in the ortho (o), meta (m) or para (p) position, preferably in the
para position, of the phenyl nucleus bearing the unit RCG1 chosen from:
-G' X (CR13R14),(OCH2CH2)y(CR15R16)u Q RCG1;
-G' X (CR13R14),(OCH2CH2)y-Y'-(CR15R16)u Q RCG1;
-G' X (CR13R14)t(CR17=CR18)(CR15R16)u(OCH2CH2)y Q RCG1;
-G' X (CR13R14)t(OCH2CH2)y(CR17=R18)(CR15R16)u Q RCG1;
-G' X (CR13R14)t-Phenyl-(CR15R16)u Y' Q RCG1; -G' X (CR13R14)t-furyl-(CR15R16)u Y Q
RCGl;
-G' X (CR13R14)t-oxazolyl-(CR15R16)u Y' Q RCG1; -G' X (CR13R14)t-thiazolyl-(CR15R16)u Y'
Q RCG; -G' X (CR13R14)rthienyl-(CR15R16)u Y' Q RCG1; -G' X (CR13R14)t-
imidazolyl-(CR15R16)u Y' Q RCG-,; -G' X (CR13R14)rpiperazinyl-CO(CR15R16)u Y' Q
RCG1; -G' X (CR13R14)t-piperidyl-methyl-NR12-CO(CR15R16)u Y' Q RCG1; -G' X
(CR13R14),-piperidyl-(CR15R16)u Y' Q RCG,; -G' X (CR13R14)t-piperidyl-NR12-(CR15R16)u Y'
Q RCG1; -G' X (CR13R14)t-triazolyKCR15R16)u Y' Q RCG1; -G' X (CR13R14)t-
triazolyl-(CR15R16)u Y' Q RCG1;
-G' X (CR13R14)t-phenyl-(CR15R16)u Q RCG1; -G' X (CR13R14)t-furyl-(CR15R16)u
QRCG1 -G' X (CR13R14)t-oxazolyl-(CR15R16)u Q RCG1; -G' X (CR13R14),-thiazolyl-
(CR15R16)U Q RCG1; -G' X (CR13R14)t-thienyl-(CR15R16)u Q RCG1; -G' X (CR13R14)t
imidazolyl-(CR15R16)u Q RCG1; -G' X (CR13R14)rPiperazinyl-(CR15R16)u Q RCG-,; -G' X
(CR13R14)t-piperidyl-(CR15R16)u Q RCG1; -G' X (CR13R14)t-piperidyl-rnethyl-NR12-
(CR15R16)u Q RCG1; -G' X (CR13R14)rpiperidyl-NR12-(CR15R16)u Q RCG1; -G' X (CR13R14)t-
triazolyl-(CR15R16)u Q RCG1;
or
-G" Y (CR13R14),(OCH2CH2)y(CR15R16)u Q RCG1;
-G" Y (CR13R14),(OCH2CH2)y-Y'-(CR15R16)u Q RCG1;
-G" Y (CR13R14)t(CR17=CR18)(CR15R16)u(OCH2CH2)y Q RCG1;
-G" Y (CR13R14)t(OCH2CH2)y(CR17=CR18)(CR15R16)u Q RCG1;
-G" Y (CR13R14)t-phenyl-(CR15R16)u Y' Q RCG,; -G" Y (CR13R14)t-furyl-(CR15R16)u Y' Q
RCG1; -G" Y (CR13R14)t-oxazolyl-(CR15R16)u Y' Q RCG1; -G" Y (CR13R14)t-
thiazolyl-(CR15R16)u Y' Q RCG1; -G" Y (CR13R14)t-thienyl-(CR15R16)u Y' Q RCG1; -G" Y
(CR13R14)t-imidazolyl-(CR15R16)u V Q RCG1; -G" Y (CR13R14)t-piperazinyl-CO(CR15R16)u
Y' Q RCG1; -G" Y (CR13R14)t-piperidyl-methyl-NR12-CO(CR15R16)u V Q RCG,; -G" Y
(CR13R l4)rpiperidyl-(CR15R16)u V Q RCG1; -G" Y (CR13R14)t-piperidyl-NR12-(CR15R16)u Y'
Q RCG1; -G" Y (CR13R14)rtriazolyl-(CR15R16)u V Q RCG1;
-G" Y (CK13R14)t-phenyl-(CR15R16)u Q RCG,; -G" Y (CR13R14)t-furyl-(CR15R16)u Q RCG,,
-G" Y (CR13R14)-oxazolyl-(CR15R16)u Q RCG1; -G" Y (CR13R14)tthiazolyl-(CR15R16)u Q
RCG,; -G" Y (CRi3R14)rthienyl-(CR15R16)u Q RCG,; -G" Y (CR13R14)t-imidazolyl-
(CR15R16)u Q RCG,; -G" Y (CR13R14),-piperazinyl-(CR15R16)u Q RCG1; -G" Y (CR13R14)r
piperazinyl-(CR15R16)u Q CCR1; G" Y (CR13R14)rpiperidyl-(CR15R16)u Q RCG1; -G" Y
(CR13R14)t-piperidyl-methyl-NR12-(CR15R16)u Q RCG1; -G" Y (CR13R14)t-piperidyl-NR12-
(CR15R16)U Q RCG1; -G" Y (CR13R14)t-triazolyl-(CR15R16)u Q RCG1;;
in which formulae:
G' represents a group -CH=CH- or -(CH2)n-;
G" represents a group -(CH2)n-;
n represents an integer ranging from 1 to 6;
X represents a single bond or a group -CO-, -COO- or -CONR12-, the group CO being
attached to G';
Y represents a group -O-, -OCO-, -OCOO-, -OCONR12-, -NR12-, -NR12CO-, -NR12CONR'12-,
-NR12COO- or -S(O)q-, the atom O and the group NR12 being attached to G";
q represents an integer that may be 0,1 or 2;
Y' represents a group -O-, -OCO-, -OCOO-, -OCONR12-, -NR12-, -NR12CO, -NR12CONR'12-,
-NR12COO-, -S(O)q-, -CO-, -COO-, or -CONR12-;
R12, R12, R13, R14, R15, R16, R17 and R18 represent, independently of each other, H or a
group (C1-C6)alkyl;
t, u and y represent integers that may range from 0 to 20 and such that t+u+y is greater
than or equal to 1;
in the case of the linker of formula -G"Y(CR13R14)t(OCH2CH2)Y-Y'-(CR15R16)u Q RCG1' if y
is 0 and Q represents a single bond, then u cannot be 0;
Q represents a single bond, a group (C1-C10)alkylene or a group (OCH2CH2)i, i being an
integer ranging from 1 to 20, more particularly from 1 to 10, even more particularly from 1
to 8 or from 1 to 6, and even more particularly from 2 to 5;
■ RCG1 represents-SZa, -C(=O)-ZbRb, with R12 representing
H or (C1-C6)alkyl, more particularly a methyl group;
or L is chosen from:
in which formulae:
■ n represents an integer ranging from 1 to 6;
■ ALK represents a group (C1-C12)alkylene;
■ R12 and R'12 represent, independently of each other, H or a group (C1-C6)alkyl,
more particularly a methyl group;
■ i represents an integer ranging from 1 to 20, more particularly from 1 to 10, even
more particularly from 1 to 8, or from 1 to 6, and even more particularly from 2 to 5;
or alternatively L is a linker of formula (IV):
RbZb-CO-ALK
in which:
• (AA)W represents a sequence of w amino acids AA connected together via peptide
bonds;
• w represents an integer ranging from 1 to 12 and preferably from 1 to 6;
• n represents an integer ranging from 1 to 6;
• D represents one of the following units:
for which:
R12 represents H or a group (C1-C6)alkyl;
R19. R20, R21 and R22 represent, independently of each other, H, a halogen atom, -OH,
-CN or a group (C1-C4)alkyl;
T attached to (CH2)n represents NR12or O;
V1 represents O, S or NR12;
V2 represents CR22 or N;
V3, V4 and V5 are chosen, independently of each other, from CR22 and N;
■ Za represents H or the group -SRa Ra represents a group (C1-C6)alkyl, (C3-
C7)cycloalkyl, aryl, heteroaryl or (C3-C7)heterocycloalkyl;
■ Zb represents a single bond, -O- or -NH-, Rb representing H or a group (C1-C6)alkyl,
(C3-C7)cycloalkyl, aryl, heteroaryl or (C3-C7)heterocycloalkyl.
2. Cryptophycin derivative according to Claim 1, in which R10 represents at least one
substituent on the phenyl nucleus chosen from: H, a group OH, (C1-C4)alkoxy, a halogen
atom.
3. Cryptophycin derivative according to Claim 1 or 2, in which RCG1 represents -SZa
or-C(=O)-ZbRb.
4. Cryptophycin derivative according to Claims 1 to 3, in which
represents one of the following 7 groups:
5. Cryptophycin derivative according to Claims 1 to 4, in which n is 1.
6. Cryptophycin derivative according to Claim 1, in which L is chosen from:
■ R12 and R'12 represent, independently of each other, H or a group (C1-C6)alkyl,
more particularly a methyl group;
■ i represents an integer ranging from 1 to 20, more particularly from 1 to 10, even
more particularly from 1 to 8, or from 1 to 6, and even more particularly from 2 to 5;
• Za represents H or a group -SRa, Ra representing a group (C1-C6)alkyl, (C3-
C7)cycloalkyl, aryl, heteroaryl or (C3-C7)heterocycloalkyl;
■ Zb represents a single bond, -O- or -NH-, Rb representing H or a group (C1-C6)alkyl,
(C3-C7)cycloalkyl, aryl, heteroaryl or (C3-C7)heterocycloalkyl.
7. Cryptophycin derivative according to Claim 1, in which L is chosen from:
R12 representing H or a group (C1-C6)aikyl, more particularly a methyl group.
8. Cryptophycin derivative according to Claim 7, in which R12 represents H.
9. Cryptophycin derivative according to Claim 7, in which R12 represents (C1-C6)alkyl, more
particularly a methyl group.
10. Cryptophycin derivative according to Claims 1 to 9, in which Za represents H or
-S(C1-C6)alkyl, especially -SMe, ou -S-heteroaryl, especially or ZbRb
represents -0(C1-C6)alkyl, -OH, -OCH3, -OCH2CH=CH2, or
or the group in which Gl represents at least one
electroinductive group, or alternatively in which -C(=O)ZbRb represents
11. Cryptophycin derivative according to one of Claims 1 to 10 defined according to one of
the following formulae, L preferably being in the para position:
12. Cryptophycin derivative according to Claim 1, chosen from:
Za, Zb and Rb being as defined in Ciaim 1 or 10.
13. Cryptophycin derivative of formula (III):
in which the groups R1 to R11 have the same meanings as in Claim 1, 11 or 12 and G
represents a group -(CH2)nY, which is in an ortho (o), meta (m) or para (p) position on the
phenyl nucleus bearing the unit CR1, preferably in the para position,
n being an integer ranging from 1 to 6 and Y denoting -N3; -NR12-CH2-C=CH in which R12
represents H or a group (C1-C6)alkyl; -OMs; -OC(=O)-O-(4-nitrophenyl),
R12 representing H or (C1-C6)alkyl, more particularly a methyl group.
14. Cryptophycin derivative according to Claim 13, in which Y represents -N3; -NR12-CH2-
C=CH in which R12 represents H or a group (C1-C6)alkyl; -OMsor -OC(=O)-O-(4-
nitrophenyl).
15. Cryptophycin derivative according to one of Claims 1 to 12 or according to formula (III) of
Claim 13 with G= -(CH2)nY with Y= -CI, -N3, -OH, -NH2, maleimido, haloacetamido
intended to be conjugated to a binding agent.
16. Cryptophycin derivative according to Claim 15, in which the binding agent is a ligand, a
protein, an antibody, more particularly a monoclonal antibody, a protein or antibody
fragment, a peptide, an oligonucleotide or an oligosaccharide.
17. Process for preparing a binding agent to which is attached at least one cryptophycin
derivative, referred to as a conjugate, which consists in:
(i) placing in contact and leaving to react an aqueous solution of an optionally buffered
binding agent and a solution of the cryptophycin derivative as defined in one of Claims 1
to 16;
(ii) and then in optionally separating the conjugate formed in step (i) from the cryptophycin
derivative and/or the unreacted binding agent and/or any aggregates formed.
18. Process according to Claim 17, in which:
> in the presence of a cryptophycin derivative comprising a reactive chemical group
RCG1 of the type -SZa, the binding agent comprises:
■ disulfide chemical groups in the case where RCG1 represents -SH;
■ thiol chemical groups in the case where RCG1 represents -SZa with ZaH;
■ maleimido or iodoacetamido chemical groups in the case where RCG1 represents
-SH;
> in the presence of a cryptophycin derivative comprising a reactive chemical group
RCG1 of the type -C(=O)-ZbRb, the cryptophycin derivative is reacted with the amino
functions of the binding agent, especially the e-amino groups borne by the side chains
of the lysine residues (Lys) of an antibody;
> in the presence of a cryptophycin derivative of formula (III) with G= -(CH2)nY, the
binding agent comprises groups -SH when Y= -CI or -maleimido, groups -C=CH
when Y= -N3 or carboxylic acid groups when Y= -OH or -NH2;
> in the presence of a cryptophycin derivative comprising a reactive chemical group
RCG1 of maleimido or haloacetamido type, the binding agent comprises thiol
chemical groups.
19. Process according to Claim 17 or 18, in which:
> in the presence of a cryptophycin derivative comprising a reactive chemical group
RCG1 of the type -SZa, the binding agent is modified with a modifying agent Ghosen
from a compound of formula: in which R represents a group (C1-
C6)alkyl, aryl, heteroaryl, (C3-C7)cycloalkyl, (C3-C7)heterocycloalkyl and ALK
represents a group (C1-C6)alkylene;
a pegylated analogue of formula:
or a sulfonic analogue of formula
in which X3, X4, X5 and X6 represent H or a group (C1-
C6)alkyl, X1 and X2 represent -H, -CONX8X9, -NO2, X8 and X9 representing H or a
group (C1-C6)alkyl, X7 represents -SO3"M+ or H or alternatively a quaternary
ammonium group and a denotes an integer ranging from 0 to 4 and b denotes an
integer ranging from 0 to 2000; in which:
- Hal represents a halogen atom;
- X10 represents a halogen atom or a group COOX14, nitro, unsubstituted or
halogenated (C1-C8)alkyl, unsubstituted or halogenated (C1-C8)alkoxy, unsubstituted
or halogenated (C2-C8)alkenyl, unsubstituted or halogenated (C2-C8)alkynyl,
unsubstituted (C3-C8)cycloalkyl, aryl that is unsubstituted or substituted with one to
three substituents selected from amino, halogen atom, unsubstituted or halogenated
(C1-C8)alkyl group, unsubstituted or halogenated (C1-C8)alkoxy;
- each of the X11, X12 and X13 independently represents a hydrogen atom or may
represent X3;
or X10 and X11 together form a ring (C2-C5)alkylene, which is unsubstituted or
substituted with one to five groups (C1-C4)alkyl;
or X10 and X11 form, together with X12, a ring (C1-C5)alkylene, which is unsubstituted
or substituted with one to five groups (C1-C4) alkyl;
and X14 is -H or a group (C1-C8)alkyl;
in the presence of a cryptophycin derivative comprising a reactive chemical group
RCG1 of the type -SH, the binding agent is modified with a modifying agent chosen
from succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate;
suifosuccinimidyi 4-(N-maleimidomethyl)cyclohexane-1-carboxylate;
* )
ALK denoting a group (C1-C12)alkylene and b being an integer
between 0 and 2000; succinimidyl-N-
bromoacetate; succinimidyl-3-(N-bromoacetamido)propionate;
b being an integer between 0 and 2000.
20. Conjugate that may be obtained via the process according to one of Claims 17 to 19.
21. Conjugate according to Claim 20, characterized by a DAR determined from the
deconvolution of the HRMS spectrum of greater than 1, preferably between 2 and 10 and
more particularly between 2 and 7.
22. Conjugate according to Claim 20, characterized by a DAR determined using a UV
spectrophotometer of greater than 0.5, preferably greater than 1, more particularly
between 1 and 10 and even more particularly between 2 and 7, the DAR being calculated
by the equation:
DAR = CD / CA
in which:
cD = [(ΕAΛ1 x Aλ2) - (εAλ2X Aλ1)] / [(εDλ2X εAλ1) - (ΕAΛ2X εDλ1)]
cA = [Aλ1-(CDxεDλ1)]/εAλ1
and
Aλ1 and Aλ2 denoting, respectively, the absorbances of the conjugate solution,
respectively, at the wavelengths λ1 and λ.2;
εD λ1 and εD λ2 denoting respectively, the molar absorption coefficients of the cryptophycin
derivative before conjugation at the wavelengths λ1 and λ2, these coefficients being
measured on the compounds of formula (II) of the type -SZa with Za = -SMe or of the type
-C(=O)-ZbRb with ZbRb = -OMe or -OCH2-CH=CH2;
εAλ1 and εAλ2 denoting, respectively, the molar absorption coefficients of the naked
antibody before conjugation at the two wavelengths A,1 and 12.
23. Conjugate according to Claim 22, in which λ1= 280 nm and λ2 is chosen within the
specific wavelength range 246 nm - 252 nm.
24. Conjugate solution that may be obtained via the process according to one of Claims 17 to
19 or comprising the conjugate as defined in one of Claims 20 to 23.
25. Use of a cryptophycin derivative according to one of Claims 1 to 13, for the preparation of
a binding agent to which is attached at least one of the said cryptophycin derivatives.
26. Use of a cryptophycin derivative of formula (III):
in which:
G represents a group -CH=CH2 or -(CH2)nY, which is in an ortho (o), meta (m) or
para (p) position on the phenyl nucleus bearing the unit CR1, preferably in the
para position;
Y represents -OH; -CI; -N3; -NH2; -SH; -COOH; -NR12-CH2-CECH in which R12
represents H or a group (C1-C6)alkyl, more particularly a methyl group; -OLG in
which LG represents a leaving group; -OC(=O)-O-(4-nitrophenyl); maleimido,
or of formula:
the groups R1 to R11 having the same meaning as in Claim 1, 11 or 12 and n being an
integer ranging from 1 to 6,
for the preparation of a binding agent to which is attached at least one of the said
cryptophycin derivatives.
27. Use of a cryptophycin derivative chosen from one of the following:
Za, Zb and Rb being as defined in Claim 1 or 10, for the preparation of a binding agent to
which is attached at least one cryptophycin derivative.
28. Cryptophycin derivative of formula:
the groups R1 to R11 having the same meanings as in Claim 1, 11 or 12 and n being an
integer ranging from 1 to 6.
29. Cryptophycin derivative according to one of Claims 1 to 15, for use as an anticancer
agent.
30. Conjugate according to Claims 20 to 23, for use as an anticancer agent.
31. Conjugate solution according to Claim 24, for use as an anticancer agent.
32. Use of a cryptophycin derivative according to one of Claims 1 to 15, for the preparation of
an anticancer medicament.'
33. Use of a conjugate according to Claims 20 to 23, for the preparation of an anticancer
medicament.
34. Use of a conjugate solution according to Claim 24, for the preparation of an anticancer
medicament.
ABSTRACT
The invention relates to a targeting agent, to which at least one cryptophycin is attached, of the formula (I), where:
R1 is a halogen atom and R2 is an OH group, an acyl group derived from an amino acid AA or a (C1-C4)alkanoyloxy group; or else
R1 and R2 together form an epoxy unit; AA denotes a natural or artificial amino acid; R3 is a (C1-C6)alkyi group; R4 and R5 are
both H or together form a CH=CH double bond between C13 and C14; R6 and R7 are, independently from one another, H or a
(C1-C6)alkyl group; R8 and R9 are, independently from one another, H or a (C1-C6)alkyl group; R10 is at least one substiruent of the
phenyl core selected from among: H, an OH group, (C1-C4)alkoxy, a halogen atom or a NH2, NH(C1-C6)alkyl, or N(C1-C6)alkyl
group; R11 is at least one substiruent of the phenyl core selected from among H or a (C1- C4)alkyl group; the targeting agent and
the cryptophycin derivative being covalently bonded, the bond being located at the ortho (O), meta (m), or para (p) position of the
phenyl core containing the CR1 unit.