Derivatives of dolastatin 10 and auristatins
The subject of the present invention concerns novel derivatives of dolastatin 10
and auristatins, their methods of production, pharmaceutical compositions containing
the same and the use thereof as medicinal product in particular in the treatment of
cancer.
Dolastatin 10 (D10) is a cytotoxic peptide derivative isolated from a marine
mollusc {Dolabella auricularia) whose absolute configuration was determined and later
confirmed after total synthesis of the product (Pettit G. R. J. Am. Chem. Soc. 1987, 109,
6883; Pettit G. R. J. Am. Chem. Soc. 1987, 109, 7581; Pettit, G. R Heterocycles 1989,
28, 553; Pettit, G. R. J. Am. Chem. Soc. 1989,1 11, 5015; Pettit G. R. J. Am. Chem. Soc.
1991, 113, 6692). D10 is formed of 5 units called dolavaline (Dov), valine (Val),
dolaiso leucine (Dil), dolaproine (Dap) and dolaphenine (Doe). A certain number of
analogues of this compound have been synthesised by modifying the nature of its
component amino acids (Pettit G. R. J. Med. Chem. 1990, 33, 3133; Miyazaki K.
Peptide Chemistry 1993, 31, 85; Miyazaki K. Chem. Pham. Bull. 1995, 43, 1706).
Modifications of the C-terminal part (right end) have also been performed and have led
to numerous derivatives which include auristatin E or F (Pettit G. R. Anticancer Drug
Design, 1998, 13, 243; Pettit G. R. Antimicrobial Agents And Chemotherapy, 1998,
2961).
Dolastatin 10
Auristatin F
The present invention has focused on modification of the N-terminal part (left
end) of derivatives of dolastatin 10 and auristatins E and F. The few examples published
in the literature on modifications made at this position have led to losses of activity
(Miyazaki K. Chem. Pham. Bull. 1995, 43, 1706). The compounds described in the
present invention differ from the prior art through their original chemical structures and
also through their remarkable biological property that is fully unexpected having regard
to the elements published in the literature. These remarkable activities result in making
these compounds suitable for use in the treatment of cancer.
In addition, these compounds have the advantage of being both active as
cytotoxic agents and more soluble than the parent compounds.
The subject of the present invention is thus a compound of following formula
(I):
where:
- Ri is H or OH,
- R2 is a group: (Ci-C 6)alkyl (e.g. methyl), COOH, COO-((Ci-C 6)alkyl) (such as
COOMe) or thiazolyl (such as thiazol-2-yl),
- ¾ is H or a (Ci-C )alkyl group (such as methyl), in particular a (Ci-C6)alkyl
group, and
- R4 is an aryl-(Ci-Cs)alkyl group substituted by one or more groups (in particular
one, preferably on the aryl part) chosen from among OH and NR9R10 groups with
R and Rio each independently of one another representing H or a (Ci-Ce)alkyl
group (such as methyl),
or a pharmaceutically acceptable salt, hydrate or solvate thereof.
The radicals R2 to R4, and in particular R4, may be chiral groups and may be in
the form of their different stereoisomers and optionally in the form of a mixture of
stereoisomers.
By « stereoisomer », in the meaning of the present invention is meant a
geometric isomer or an optical isomer.
Geometrical isomers result from the different position of the substituents on a
double bond which may therefore have a Z or E configuration.
Optical isomers result in particular from the different position in space of the
substituents on a carbon atom comprising 4 different substituents. This carbon atom
then forms a chiral or asymmetric centre. Optical isomers comprise diastereoisomers
and enantiomers. Optical isomers which are images of one another in a mirror but which
cannot be superimposed are called « enantiomers ». Optical isomers which are not
superimposable images of one another in a mirror are called « diastereoisomers ».
A mixture containing equal quantities of two individual enantiomer forms of
opposite chirality is called a « racemic mixture ».
In the present invention by « pharmaceutically acceptable » is meant that which
can be used in the preparation of a pharmaceutical composition which is generally, safe
non-toxic and neither biologically nor otherwise undesirable, and which is acceptable
for veterinary use as well as for human pharmaceutical use.
By « pharmaceutically acceptable salt, hydrate or solvate » of a compound is
meant a salt, hydrate or solvate which is pharmaceutically acceptable as defined herein
and which has the desired pharmacological activity of the parent compound.
Pharmaceutically acceptable salts notably comprise:
(1) the addition salts of a pharmaceutically acceptable acid formed with
pharmaceutically acceptable inorganic acids such as hydrochloric, hydrobromic,
phosphoric, sulfuric and similar acids; or formed with pharmaceutically acceptable
organic acids such as acetic, trifluoroacetic, propionic, succinic, fumaric, malic, tartaric,
citric, ascorbic, maleic, glutamic, benzoic, salicylic, toluenesulfonic, methanesulfonic,
stearic, lactic and similar acids; and
(2) the addition salts of a pharmaceutically acceptable base formed when an acid
proton present in the parent compound is either replaced by a metallic ion e.g. an
alkaline metal ion, an alkaline-earth metal ion or an aluminium ion; or coordinated with
a pharmaceutically acceptable organic base such as lysine, arginine and similar; or with
a pharmaceutically acceptable inorganic base such as sodium hydroxide, potash,
calcium hydroxide and similar.
These salts can be prepared from the compounds of the invention containing a
base or acid function, and the corresponding acids or bases using conventional chemical
methods.
The formula (I) compounds of the invention are preferably in salt form, and in
particular a pharmaceutically acceptable acid addition salt.
Preferably, the compounds of formula (I) according to the present invention are
in the form of a pharmaceutically acceptable acid addition salt, the acid possibly being
trifluoroacetic acid, acetic acid or hydrochloric acid for example, and in particular
trifluoroacetic acid.
The solvates comprise the conventional solvates obtained at the last preparation
step of the compounds of the invention due to the presence of solvent, the solvent
possibly being ethanol for example.
By « alkyl » in the present invention is meant a straight-chain or branched,
saturated hydrocarbon chain. For example, mention can be made of methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl groups.
By « (Cx- Cy)alkyl » in the meaning of the present invention is meant an alkyl
chain such as defined above comprising x to y carbon atoms. Therefore, a (Ci-C 6)alkyl
group is an alkyl chain having 1 to 6 carbon atoms.
By « aryl » in the meaning of present invention is meant an aromatic
hydrocarbon group preferably having 6 to 10 carbon atoms and able to comprise one or
two fused rings. For example a phenyl or a naphthyl can be cited. Advantageously it is a
phenyl.
By « aryl-(Ci-C8)alkyl » in the meaning of the present invention is meant an aryl
group such as defined above linked to the remainder of the molecule via an alkyl group
such as defined above and comprising 1 to 8, in particular 1 to 6, advantageously 1 to 4,
preferably 1 or 2 carbon atoms. The aryl moiety is preferably a phenyl moiety. The (Ci-
Cs)alkyl moiety is advantageously a (Ci-C4)alkyl, peferably a (Ci-C 2)alkyl. In
particular, the aryl-(Ci-C8)alkyl group is a benzyl or phenethyl group.
Among the compounds of the invention, one particularly appreciated class of
compounds corresponds to the formula (I) compounds in which Ri is OH and R2
represents a (Ci-C 6)alkyl group, such as methyl.
Another particularly appreciated class of compounds corresponds to the formula
(I) compounds in which Ri is a hydrogen and R2 is a thiazole (in particular a thiazol-2-
yl group).
Another class of particularly appreciated compounds corresponds to the formula
(I) compounds in which Ri is a hydrogen and R2 is a COO(Ci-Ce)alkyl group such as
COOMe.
Another class of particularly appreciated compounds corresponds to the formula
(I) compounds in which Ri is a hydrogen and R2 is a COOH group.
Therefore the compounds of the invention are advantageously formula (I)
compounds in which:
- Ri=OH and R2=Me (methyl), or
- Ri=H and R2=COOH, COOMe or thiazol-2-yl.
According to one particular embodiment of the present invention, R2 is more
particularly a methyl, COOH, COOMe or thiazol-2-yl group.
Preferably, Ri is H and R2 is COOH or COO(Ci-C 6)alkyl, notably COOH or
COOMe.
According to a first preferred embodiment, Ri is H and R2 is COOH.
According to a second preferred embodiment, Ri is H and R2 is COOMe.
R3 particularly represents H or a methyl group, advantageously a methyl group.
R4 represents advantageously an aryl-(Ci-Cs)alkyl group, notably an aryl-(Ci-
C4)alkyl group, such as an aryl-(Ci-C 2)alkyl group, substituted by one group chosen
from among OH and NR9R10, and notably being NR9R10.
R4 represents advantageously an aryl-(Ci-Cs)alkyl group, notably an aryl-(Ci-
C4)alkyl group, such as an aryl-(Ci-C 2)alkyl group, substituted by one group on the
aryl moiety chosen from among OH and NR9R10, and notably being NR9R10.
The aryl group is advantageously a phenyl group.
Thus R4 can represent in particular a phenyl-(Ci-C 2)alkyl substituted by one
group (preferably on the phenyl moiety) chosen from among OH and NR9R10, and
notably being NR9R10.
s have the following formula:
wherein X0 represents OH or NR9R10, n particular NR9R10, and m represents an integer
comprised between 1 and 8, notably between 1 and 4, and advantageously is 1 or 2.
According to a preferred embodiment, R4 has the following formula:
with Xo and m as defined previously, and in particular with and m=l or 2.
R 4 may in particular be chosen from among:
Advantageously, the formula (I) compound is chosen from among the
compounds 11-15, 19-20, 23-24, 27-29, 49-51 and 61-64 described in the examples
below.
A further subject of the present invention is a formal (I) compound such as
defined above for use as medicinal product, in particular for the treatment or prevention
of cancer or benign proliferative disorders.
The present invention also concerns the use of a formula (I) compound such as
defined above for producing a medicinal product, particularly intended for the treatment
or prevention of cancer or benign proliferative disorders.
The present invention also concerns a method for treating or preventing cancer
or benign proliferative disorders comprising the administration to a person in need
thereof of an effective amount of a formula (I) compound such as defined above.
The cancer to be treated or prevented is more particularly cancer of the lung,
pancreas, skin, head, neck, uterus, ovaries, anus, stomach, colon, breast, oesophagus,
small intestine, thyroid gland, lymphatic system, prostate, kidney, or bladder, or an
acute or chronic leukaemia, or a combination of two or more of these cancers.
By benign proliferative disorders is meant proliferating disorders which cannot
give rise to metastases or which have not yet progressed towards a cancer (pre
cancerous tumours).
A further subject of the present invention is a pharmaceutical composition
comprising a formula (I) compound such as defined above and at least one
pharmaceutically acceptable excipient.
The active ingredient can be administered in unit forms of administration, in a
mixture with conventional pharmaceutical carriers, to animals or to human beings.
Suitable unit forms of administration comprise forms via oral route, forms for
sublingual or buccal administration, forms for administration via parenteral route
(subcutaneous, intradermal, intramuscular or intravenous), forms for topical
administration (on the skin and mucosa, including intranasal and intraocular
administration) and forms for rectal administration.
Such compositions may be in the form of a solid, liquid, emulsion, lotion or
cream.
As solid compositions, for oral administration, use can be made of tablets, pills,
powders (hard or soft gelatine capsules) or granules. In these compositions, the active
ingredient of the invention is mixed with one or more inert diluents such as starch,
cellulose, sucrose, lactose or silica, in a stream of argon. These compositions may also
comprise substances other than diluents, for example one or more lubricants such as
magnesium stearate or talc, a colouring agent, a coating (coated tablets) or a varnish.
As liquid compositions for oral administration, use can be made of solutions,
suspensions, emulsions, syrups and elixirs that are pharmaceutically acceptable and
contain inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil.
These compositions may comprise substances other than diluents; for example wetting,
sweetening, thickening, flavouring or stabilising products.
The sterile compositions for parenteral administration may preferably be
aqueous or non-aqueous solutions, suspensions or emulsions. As solvent or vehicle, use
can be made of water, propyleneglycol, a polyethyleneglycol, vegetable oils, in
particular olive oil, injectable organic esters e.g. ethyl oleate or other suitable organic
solvents. These compositions may also contain adjuvants, in particular wetting, isotonic,
emulsifying, dispersing and stabilising agents. Sterilisation can be performed in several
manners, for example by sanitising filtration, by incorporating sterilising agents into the
composition, by radiation or by heating. They can also be prepared in the form of solid
sterile compositions which can be dissolved at the time of use in sterile water or any
other injectable sterile medium.
The compositions for rectal administration are suppositories or rectal capsules
which, in addition to the active ingredient, contain excipients such as cocoa butter,
semi-synthetic glycerides or polyethyleneglycols.
The compositions for topical administration may for example be creams, lotions,
eye drops, mouthwash, nasal drops or sprays.
The doses are dependent on the desired effect, on the length of treatment and the
route of administration used. In general the physician will determine the suitable dosage
in relation to the age, weight and all other factors particular to the subject to be treated.
Another active ingredient may be contained in the pharmaceutical compositions
according to the present invention. In particular, it may be an anticancer agent, and in
particular a cytotoxic anticancer agent such as navelbine, vinflunine, taxol, taxotere, 5-
fluorouracil, methotrexate, doxorabicin, camptothecin, gemcitabin, etoposide, cis-platin
or carmustine (also called BCNU); or a hormonal anticancer agent such as tamoxifen or
medroxyprogesterone.
Radiation treatment (X-ray or gamma ray) may also be associated with the
administering of a compound of the present invention. Such radiation can be given
using an external source or by implanting minute internal radioactive sources.
The present invention also concerns the preparation of the formula (I)
compounds according to the invention using the general methods described in the
following synthesis schemes, optionally supplemented by any standard operation when
needed that is described in the literature or well known to persons skilled in the art, or
described in the examples in the experimental part hereof.
Scheme 1
Scheme 1 illustrates the first general method which can be used to prepare
formula (I) compounds. In the above general formulas, R R2 and R3 are such as
previously defined, R a represents a R4 group such as previously defined optionally in
protected form and G is a protective group.
The first step consists of the condensing of compound (II), protected on its
amine function by a protective group G, with compound (III). X may represent a leaving
group such as a chlorine. In this case the first step consists of the reaction between an
acid chloride and an amine. This reaction can be conducted using methods and
techniques well known to those skilled in the art. In one particularly appreciated
method, the two entities are caused to react in the presence of an organic or inorganic
base e.g. Et3N, iPr NEt, pyridine, NaH, Cs C0 3, K2C0 in a solvent such as THF,
dichloromethane, DMF, DMSO, at a temperature notably between -20°C and 100°C. X
may also be a hydroxyl (OH). In this case, the first step is a condensation reaction
between the carboxylic acid (II) and the amine (III). This reaction can be performed
following methods and techniques well known to skilled persons. In one particularly
appreciated method, these two entities are caused to react in the presence of a coupling
agent such as l-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC), 3-hydroxyl,
2,3-benzotriazin-4(3H)-one, a tertiary amine such as diisopropylethylamine, in a polar
aprotic solvent such as dichloromethane or DMF, at a temperature notably between -
15°C and 40°C. In another particularly appreciated method, these two entities are
caused to react in the presence of diethyl phosphorocyanidate (DEPC), a tertiary amine
such as triethylamine, in a polar aprotic solvent such as dichloromethane or DMF, at a
temperature of between -15°C and 40°C. Another particularly appreciated method
consists of causing these two entities to react in the presence of 0-(7-azabenzotriazoll-
yl)-l,l,3,3-tetramethyl-uroniumhexafluorophosphate (HATU), a tertiary amine such
as diisopropylethylamine, in a polar aprotic solvent such as dichloromethane or DMF, at
a temperature of between -15°C and 100°C.
After deprotection of the intermediate using techniques well known to those
skilled in the art (« Protective Groups in Organic Synthesis », T.W. Greene, John Wiley
& Sons, 2006 and « Protecting Groups », P.J. Kocienski, Thieme Verlag, 1994),
compound (IV) can be condensed with compound (V) following the methods and
techniques described above to lead to compound (VI) after a deprotection step. This
compound can then, after condensation with the intermediate (VII) and optional
deprotection, lead to the formation of the formula (I) compounds. Compound (VI) can
also be coupled with a compound (VIF) in which R' 3 is a precursor of R , in particular
an R3 group protected by a protective group. Coupling followed by deprotection of
group R'3 to lead to R3 can be carried out following the same procedures as described
previously.
Scheme 2
Scheme 2 illustrates the second general method which can be used to prepare
formula (I) compounds. In the above general formulas Ri, R2, and R3 are such as
previously defined, R4a represents an R4 group such as previously defined optionally in
protected form, R4b is a precursor of an R4 group and G is a protective group.
At the first step, compound (IX) protected on its amine function by a protective
group G is condensed with compound (VI). X may represent a leaving group e.g. a
chlorine. In this case, the first step consists of the reaction between an acid chloride and
an amine. This reaction can be performed using methods and techniques well known to
persons skilled in the art. In one particularly appreciated method the two entities are
caused to react in the presence of an organic or inorganic base such as Et3N, iPr2NEt,
pyridine, NaH, Cs2C0 3, K2C0 3 in a solvent such as THF, dichloromethane, DMF,
DMSO at a temperature notably between -20° and 100°C. X may also represent a
hydroxyl. In this case, the first step is a condensation reaction between the carboxylic
acid (IX) and the amine (VI). This reaction can be conducted following methods and
techniques well known to skilled persons. In one particularly appreciated method, the
two entities are caused to react in the presence of l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDC), 3-hydroxy-l,2,3-benzotriazin-4(3H)-one, a tertiary amine such as
diisopropylethylamine, in a polar aprotic solvent such as dichloromethane or DMF, at a
temperature notably between -15°C and 40°C. In another particularly appreciated
method, these two entities are caused to react in the presence of diethyl
phosphorocyanidate (DEPC), a tertiary amine such as triethylamine, in a polar aprotic
solvent such as dichloromethane or DMF, at a temperature notably between -15°C and
40°C.
After deprotection of the intermediate, using techniques well known to skilled
persons, the obtained compound (VIII) can lead to the formula (I) compounds after
reaction with R4Y. In this case, Y is a leaving group such as CI, Br, I, OS0 2CH3,
OS0 2CF3 or O-Tosyl. The reaction is conducted in the presence of an organic or
inorganic base such as Et3N, iPr2NEt, NaH, Cs2C0 3, K2C0 3, in a polar anhydrous
solvent such as dichloromethane, THF, DMF, DMSO at a temperature notably between
-20° and 100°C. In another particularly appreciated method, compound (VIII) is caused
to react with an aldehyde of formula R4b-CHO where R4b corresponds to a precursor of
R4. In this case, the reaction is a reductive amination in the presence of a reducing agent
such as NaBH4, NaBH3CN, NaBH(OAc) 3, in a polar solvent such as 1,2-
dichloroethane, dichloromethane, THF, DMF, MeOH, in the optional presence of
titanium isopropoxide (IV), at a pH which can be controlled by the addition of an acid
such as acetic acid at a temperature notably between -20°C and 100°C.
In the foregoing synthesis schemes, a formula (I) compound may lead to another
formula (I) compound after an additional reaction step such as saponification for
example using methods well known to skilled persons whereby an R2 group
representing an ester, preferably a methyl ester, is changed to an R2 group representing a
carboxylic acid.
If it is desired to isolate a formula (I) compound containing at least one base
function in the state of an acid addition salt, this is possible by treating the free base of
the formula (I) compound (containing at least one base function) with a suitable acid,
preferably in equivalent quantity. The suitable acid may in particular be trifluoroacetic
acid.
A further subject of the present invention is therefore a first method for
preparing a formula (I) compound, comprising a condensation reaction between a
compound of following formula (VI):
where Ri and R2 are such as defined previously, and
a compound of following formul
where R3 is such as previously defined, R4a corresponds to a R4 group such as
previously defined optionally in protected form, and X is OH or CI.
When X = OH, the coupling reaction can be performed under peptide coupling
conditions well known to persons skilled in the art.
Said peptide coupling can be performed in the presence of a coupling agent such
as diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), l-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), carbonyldiimidazole
(CDI), 2-(1H-benzotriazole- 1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU), 2-(lH-benzotriazole- 1-yl)- 1,1,3,3-tetramethyluronium tetrafluoroborate
(TBTU), 0-(7-azobenzotriazol- 1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU), diethyl phosphorocyanidate (DEPC) or (benzotriazol-l-yloxy)
tripyrrolodinophosphonium hexafluorophosphate (PyBOP), optionally associated with a
coupling auxiliary such as N-hydroxy succinimide (NHS), N-hydroxy benzotriazole
(HOBt), 3,4-dihydro-3-hydroxy-4-oxo-l,2,3-benzotriazole (HOOBt), l-hydroxy-7-
azabenzotriazole (HAt), N-hydroxysylfosuccinimide (sulfo NHS) or dimethyl
aminopyridine (DMAP). Preferably the coupling agent is HATU or DEPC.
The reaction can also be performed in the presence of a base such as DIEA
(diisopropylethylamine).
In particular, the peptide coupling is performed in the presence of HATU or
DEPC and DIEA.
Said reaction can be carried out in a polar aprotic solvent such as
dichloromethane (DCM) or dimethylformamide (DMF), in particular at a temperature of
between -15°C and 40°C.
When X = CI, the condensation reaction will be conducted in the presence of a
base which may be organic or inorganic, such as Et3N, iPr2NEt, pyridine, NaH, Cs2C0 ,
or K2C0 3.
The reaction can be carried out in a solvent such as tetrahydrofuran (THF),
dichloromethane (DCM), dimethylformamide (DMF), or dimethylsulfoxide (DMSO), in
particular at a temperature of between -20° and 100°C.
The compounds of formulas (VI) and (VII) can be prepared following synthesis
protocols described in the experimental part below or following techniques known to
those skilled in the art.
A further subject of the present invention is a second method for preparing a
formula (I) compound comprising a substitution reaction between a compound of
following formula (VIII):
(VIII)
where Ri, R2 an R3 are such has previously defined, and
a compound of following formula (X):
where R4a is an R4 group such as previously defined optionally in protected form, and Y
is a leaving group such as CI, Br, I, OS0 2CH3, OS0 2CF3 or O-Tosyl.
The substitution reaction will be notably conducted in the presence of a base
which may be organic or inorganic such as Et3N, iPr2NEt, NaH, Cs2C0 3, or K2C0 3.
This reaction can be implemented in a polar solvent, preferably anhydrous, such
as DCM, THF, DMF or DMSO, in particular at a temperature of between -20° and
100°C.
The compounds of formulas (VIII) and (X) can be prepared following the
synthesis protocols described in the experimental part below or using techniques known
to those skilled in the art.
A further subject of the present invention is a third method for preparing a
formula (I) compound in which R4 is a -CH 2R4b group with R4b representing an aryl or
aryl-(Ci-Cv)alkyl group substituted by one or more groups (in particular one,
preferably on the aryl moiety) chosen from among OH and NR9R10 groups,
comprising a reductive amination reaction between a compound of following formula
(VIII):
where Ri, R2 and R3 are such as previously defined, and
a compound of following formula (XI):
where R4b is such as defined above.
The reductive amination reaction can be carried out in the presence of a reducing
agent such as NaBH4, NaBH3CN or NaBH(OAc)3 and optionally titanium isopropoxide
(IV).
The pH can be controlled by adding an acid such as acetic acid, in particular to
reach a pH of between 4 and 5.
This reaction can be implemented in a polar solvent such as DCE (1,2-
dichloroethane), DCM, THF, DMF or methanol, in particular at a temperature of
between -20° and 100°C.
The compounds of formulas (VIII) and (XI) can be prepared following synthesis
protocols described in the experimental part below or using techniques known to those
skilled in the art.
The compound obtained after the condensation/substitution/reductive amination
step of one of the three above methods can be subjected to additional deprotection steps
particularly concerning the substituents R2 and R4 and optionally additional
functionalization steps using methods well known to skilled persons.
When R2 represents a COOH group, the condensation/substitution/reductive
amination step mentioned above can be performed from a compound of formula (VI)
with an R2 group representing a COO-((Ci-C )alkyl) ester function, this ester function
then possibly being saponified to yield a formula (I) compound with R2 = COOH.
When the R 4 group comprises a NH function, this can be protected before
performing the condensation/substitution/reductive amination reaction by substituting
the nitrogen atom by an N-protective group such as a Boc or Fmoc group.
By « protective group » in the present invention is meant a group which
selectively blocks a reactive site in a multifunctional compound such that a chemical
reaction can selectively be carried out at another non-protected reactive site in the
meaning conventionally associated therewith in chemical synthesis.
By « N-protective group » in the present invention is meant any substituent
which protects the NH group against undesirable reactions such as the N-protective
groups described in Greene, « Protective Groups In Organic synthesis », (John Wiley &
Sons, New York (1981)) and Harrison et al. « Compendium of Synthetic Organic
Methods », Vols. 1 to 8 (J. Wiley & sons, 1971 to 1996). The N-protective groups
comprise carbamates, amides, N-alkylated derivatives, amino acetal derivatives, Nbenzyl
derivatives, imine derivatives, enamine derivatives and N-heteroatom
derivatives. The N-protecting groups can be formyl; an aryl, such as a phenyl,
optionally substituted with one or several methoxy groups such as /?-methoxyphenyl
(PMP); an aryl-(Ci-C6)alkyl, such as a benzyl, the aryl moiety being optionally
substituted with one or several methoxy groups, such as benzyl (Bn), /?-methoxybenzyl
(PMB) or 3,4-dimethoxybenzyl (DMPM); -CO-R Gpi such as acetyl (Ac), pivaloyl (Piv
or Pv), benzoyl (Bz) or /?-methoxybenzylcarbonyl (Moz); -C0 2-RGPI such as
tbutyloxycarbonyl (Boc), trichloroethoxycarbonyl (TROC), allyloxycarbonyl (Alloc),
benzyloxycarbonyl (Cbz or Z) or 9-fluorenylmethyloxycarbonyl (Fmoc); -S0 2-RGPI
such as phenylsulfonyl, tosyl (Ts or Tos) or 2-nitrobenzenesulfonyl (also called nosyl -
Nos or Ns); and the like, with RGPI representing a (Ci-C )alkyl optionally substituted
with one or several halogen atoms such as F or CI; a (C2-Ce)alkenyl such as an allyl; an
aryl, such as a phenyl, optionally substituted with one or several groups chosen among
OMe (methoxy) and N0 2 (nitro); an aryl-(Ci-C )alkyl, such as a benzyl, the aryl moiety
being optionally substituted with one or several methoxy groups; or a 9-fluorenylmethyl
group.
In particular, the N-protective group comprises formyl, acetyl, benzoyl,
pivaloyl, phenylsulfonyl, benzyl (Bn), t-butyloxycarbonyl (Boc), benzyloxycarbonyl
(Cbz), p-methoxybenzyloxycarbonyl, p-nitrobenzyl-oxycarbonyl, trichloroethoxy
carbonyl (TROC), allyloxycarbonyle (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc),
trifluoro-acetyl, benzyl carbamates (substituted or not) and similar. It may in particular
be a Boc or Fmoc group.
The protection of the NH amine function by a Boc or Fmoc group and its
subsequent deprotection, after the condensation/substitution/reductive amination
reaction, are well known to persons skilled in the art and are described in particular in
the experimental part below.
The formula (I) compound obtained with one of the three methods mentioned
above can also be salified by adding a pharmaceutically acceptable base or acid, in
particular a pharmaceutically acceptable acid such as trifluoroacetic acid. Said step can
optionally be performed at the same time as another reaction step, in particular at the
same time as a deprotection step when this must be performed in an acid medium for
example.
The compound obtained with one of these three methods, optionally after
additional step(s) for deprotection, functionalization and/or salification, can be
separated from the reaction medium using methods well known to skilled persons, such
as by extraction, solvent evaporation or by precipitation and filtration.
The compound may also be purified if necessary using techniques well known to
skilled persons, e.g. by recrystallization if the compound is crystalline, by distillation,
by silica gel column chromatography or high performance liquid chromatography
(HPLC).
The following examples illustrate the invention without however limiting the
scope thereof.
EXAMPLES
I - Synthesis of the compounds of the invention
The following abbreviations are used in the following examples:
aq. Aqueous
ee enantiomeric excess
equiv equivalent
ESI Electrospray ionisation
LC/MS Liquid Chromatography coupled with Mass Spectrometry
HPLC High Performance Liquid Chromatography
NMR Nuclear Magnetic Resonance
sat. saturated
UV ultraviolet
Reference Example 1
(S)-2-((S)-2-((3-aminopropyl)(methyl)amino)-3-methylbutanamido)-7V-
((3R,4S,5S)-3-methoxy-l-((S)-2-((lR,2R)-l-methoxy-2-methyl-3-oxo-3-(((S)-
2-phenyl-l-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-l-yl)-5-methyl-loxoheptan-
4-yl)-7V,3-dimethylbutanamide, bis trifluoroacetic acid
Example 1A: (4R, 5S)-4-methyl-5-phenyl-3-propanoyl-l,3-oxazolidin-2-
one
(4R, 5S)-4-methyl-5-phenyl-l,3-oxazolidin-2-one (5.8 g, 32.7 mmol, 1.00
equiv) was dissolved in tetrahydrofuran (THF, 120 mL) in an inert atmosphere. The
mixture was cooled to -78°C and n-butyllithium (14.4 mL) was added drop-wise. After
agitation for 30 minutes at -78°C, propanoyl chloride (5.7 mL) was added. Agitation
was continued for 30 minutes at -78°C then overnight at ambient temperature. The
reaction mixture was concentrated then re-dissolved in 200 mL of water. The pH of the
solution was adjusted to 7 with sodium bicarbonate saturated aqueous solution. This
aqueous phase was extracted 3 times with 100 mL of ethyl acetate (EtOAc). The
organic phases were combined, dried over sodium sulfate, filtered and concentrated to
yield 6.8 g (89 %) of compound 1A in the form of a yellow oil.
Example IB: ter t-butyl (2S)-2-[(lR,2R)-l-hydroxy-2-methyl-3-[(4R,5S)-4-
methyl-2-oxo-5-phenyl- 1,3-oxazolidin-3yl]-3-oxopropyl]pyrrolidine- 1-carboxylate
Compound 1A (17.6 g, 75.45 mmol, 1.00 equiv) was dissolved in
dichloromethane (DCM, 286 mL) in an inert atmosphere. This solution was cooled with
an ice bath. Triethylamine (TEA, 12.1 mL, 1.15 equiv) and Bu2BOTf (78.3 mL, 1.04
equiv) were added drop-wise whilst holding the temperature of the reaction mixture
below 2°C. Agitation was continued at 0°C for 45 minutes, after which the reaction was
cooled to -78°C. A solution of tert-butyl (2S)-2-formylpyrrolidine-l- carboxylate (8.5
g, 42.66 mmol, 0.57 equiv) in DCM (42 mL) was added drop-wise. Agitation was
continued for 2 hours at -78°C, then for 1 hour at 0°C and finally 1 hour at ambient
temperature. The reaction was neutralised with 72 mL of phosphate buffer (pH = 7.2 -
7.4) and 214 mL methanol, and cooled to 0°C. A solution of 30 % hydrogen peroxide in
methanol (257 mL) was added drop-wise whilst maintaining the temperature below
10°C. Agitation was continued for 1 hour at 0°C. The reaction was neutralised with
142 mL of water, then concentrated under reduced pressure. The resulting aqueous
solution was extracted 3 times with 200 mL EtOAc. The organic phases were combined,
dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica
column with a mixture of EtOAc and petroleum ether (EtOAc:PE = 1:8) to yield 13.16
g (40 %) of compound IB in the form of a colourless oil.
Example 1C: (2R,3R)-3-[(2S)-l-[(ter t-butoxy)carbonyl]pyrrolidin-2-yl]-3-
hydroxy-2-methylpropanoic acid
Compound IB (13.16 g, 30.43 mmol, 1.00 equiv) was dissolved in THF
(460 mL) in the presence of hydrogen peroxide (30 % in water, 15.7 mL), then cooled
with an ice bath. An aqueous solution of lithium hydroxide (0.4 mol/L, 152.1 mL) was
added drop-wise whilst holding the reaction temperature below 4°C. The reaction
mixture was agitated 2.5 hours at 0°C. An aqueous solution of a2S0 3 ( 1 mol/L, 167.3
mL) was added drop-wise whist holding the temperature at 0°C. The reaction mixture
was agitated 14 hours at ambient temperature, then neutralised with 150 mL of cold
sodium bicarbonate saturated solution and washed 3 times with 50 mL of DCM. The pH
of the aqueous solution was adjusted to 2-3 with a 1M aqueous solution of KHS0 4.
This aqueous solution was extracted 3 times with 100 mL of EtOAc. The organic phases
were combined, washed once with saturated NaCl solution, dried over sodium sulfate,
filtered and concentrated to yield 7.31 g (88 %) of compound 1C in the form of a
colourless oil.
Example ID: (2R,3R)-3-[(2S)- l-[(ter t-butoxy)carbonyl]pyrrolidin-2-yl]-3-
methoxy-2-methylpropanoic acid
Compound 1C (7.31 g, 26.74 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in THF (135 mL) in the presence of iodomethane (25.3 mL). The reaction
medium was cooled with an ice bath after which NaH (60 % in oil, 4.28 g) was added in
portions. The reaction was left under agitation 3 days at 0°C and then neutralised with
100 mL of sodium bicarbonate saturated aqueous solution and washed 3 times with 50
mL ether. The pH of the aqueous solution was adjusted to 3 with 1M aqueous KHSO4
solution. This aqueous solution was extracted 3 times with 100 mL of EtOAc. The
organic phases were combined, washed once with 100 mL of Na2S20 3 (5 % in water),
once with NaCl-saturated solution, then dried over sodium sulfate, filtered and
concentrated to yield 5.5 g (72 %) of compound ID in the form of a colourless oil.
Example -methoxy - N-methyl-2-phenylacetamide
2-phenylacetic acid (16.2 g, 118.99 mmol, 1.00 equiv) was dissolved in
dimethylformamide (DMF, 130 mL) then cooled to -10°C. Diethyl phosphorocyanidate
(DEPC, 19.2 mL), methoxy(methyl)amine hydrochloride (12.92 g, 133.20 mmol,
1.12 equiv) and triethylamine (33.6 mL) were added. The reaction mixture was agitated
30 minutes at -10°C then 2.5 hours at ambient temperature. It was then extracted twice
with 1 litre of EtOAc. The organic phases were combined, washed twice with 500 mL
of NaHCC"3 (sat.), once with 400 mL of water, then dried over sodium sulfate, filtered
and concentrated. The residue was purified on a silica column with an EtOAc and PE
mixture (1:100 to 1:3) to yield 20.2 g (95 %) of compound IE in the form of a yellow
oil.
Example IF: 2-phenyl-l-(l ,3-thiazol-2-yl)ethan-l-one
Tetramethylethylenediamine (TMEDA, 27.2 mL) was dissolved in THF 300
mL) in an inert atmosphere, then cooled to -78°C before the drop-wise addition of n-
BuLi (67.6 mL, 2.5 M). 2-bromo-l,3-thiazole (15.2 mL) was added drop-wise and
agitation was continued 30 minutes at -78°C. Compound IE (25 g, 139.50 mmol, 1.00
equiv) dissolved in THF (100 mL) was added drop-wise. Agitation was continued for
30 minutes at -78°C then 2 hours at -10°C. The reaction was neutralised with 500 mL
of KHSO4 (sat.), then extracted 3 times with 1 litre of EtOAc. The organic phases were
combined, washed twice with 400 mL water and twice with 700 mL of NaCl (sat.), then
dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica
column with a mixture of EtOAc and PE (1:100 to 1:10) to yield 25 g (88 %) of
compound IF in the form of a yellow oil.
Example 1G: (lR)-2-phenyl-l-(l ,3-thiazol-2-yl)ethan-l-ol
In an inert atmosphere, a solution of compound IF (15 g, 73.8 mmol, 1.00
equiv.) in ether (300 mL) was added drop-wise to (+)-B-chloro
diisopinocampheylborane ((+)-Ipc 2BCl, 110.8 mL). The reaction mixture was agitated
24 hours at 0°C, then neutralised with 300 mL of a (1:1) mixture of NaOH (10 % in
water) and H20 2 (30 % in water), and finally extracted three times with 500 mL of
EtOAc. The organic phases were combined, washed twice with 300 mL of K2C0 3 (sat.)
and once with 500 mL of NaCl (sat.), then dried over sodium sulfate, filtered and
concentrated. The residue was purified on a silica column with a mixture of EtOAc and
PE ( 1:20 to 1:2) to yield 6.3 g (42 %) of compound 1G in the form of a white solid.
Example 1 - [(l S)-l-azido-2-phenylethyl]-l ,3-thiazole
Compound 1G (6 g, 29.23 mmol, 1.00 equiv.) was dissolved in an inert
atmosphere in THF (150 mL) in the presence of triphenylphosphine (13 g, 49.56 mmol,
1.70 equiv.), then cooled to 0°C. Diethylazodicarboxylate (DEAD, 7.6 mL) was added
drop-wise, followed by diphenylphosphorylazide (DPPA, 11 mL), the cold bath was
then removed and the solution was left under agitation 48 hours at ambient temperature.
The medium was concentrated under reduced pressure. The residue was purified on a
silica column with a mixture of EtOAc and PE (1:100 to 1:30) to yield 8 g of partly
purified compound 1H in the form of a yellow oil. Compound 1H was used as such in
the following step.
Example II: tert-butyl N -[(lS)-2-phenyl-l-(l,3-thiazol-2-yl)ethyl] carbamate.
Compound 1H (6.5 g, 28.2 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in THF (100 mL) in the presence of triphenylphosphine (6.5 g, 33.9 mmol,
1.20 equiv.), and heated to 50°C for 2 hours. Ammonia (70 mL) was then added and
heating was continued for 3 hours. The reaction was cooled, neutralised with 500 mL
water, then extracted 3 times with 500 mL of EtOAc. The organic phases were
combined and extracted twice with 500 mL of IN HC1. The aqueous phases were
combined, brought to pH 8-9 by adding a sodium hydroxide solution (10 % in water),
then extracted 3 times with 500 mL of DCM. The organic phases were combined, dried
over sodium sulfate, filtered and concentrated to yield 4.8 g (83 %) of (lS)-2-phenyll-(
l,3-thiazol-2-yl)ethan-l-amine in the form of a yellow oil. This compound was
then protected with a Boc group ((t rt-butoxy)carbonyl) so that it could be purified. It
was dissolved in an inert atmosphere in 1,4-dioxane (40 mL), then cooled to 0°C.
(Boc)20 (10.26 g, 47.01 mmol, 2.00 equiv) diluted in 20 mL of 1,4-dioxane was added
drop-wise. The cold bath was removed and the solution left under agitation overnight at
ambient temperature before being neutralised with 300 mL of water and extracted twice
with 500 mL of EtOAc. The organic phases were combined, dried over sodium sulfate,
filtered and concentrated. The residue was purified on a silica column with a mixture of
EtOAc and PE ( 1:100 to 1:20, ee = 93 %). It was then recrystallized in a hexane/acetone
mixture (~ 5-10 / 1, l g / 10 mL) to yield 6 g (84 %) of compound II in the form of a
white solid (ee > 99 %).
Example 1J: ter t-butyl (2S)-2-[(lR,2R)-l-methoxy-2-methyl-2-[[(lS)-2-
phen -1-(1,3-thiazol-2-yl)ethyl]carbamoyl]ethyl]pyrrolidine- 1-carboxylate
Compound II (3 g, 9.86 mmol, 1.00 equiv) was dissolved in an inert atmosphere
in 10 mL DCM. Trifluoroacetic acid (TFA, 10 mL) was added and the solution left
under agitation overnight at ambient temperature, then concentrated under reduced
pressure to yield 2.0 g (64 %) of (lS)-2-phenyl-l-(l,3-thiazol-2-yl)ethan-l-amine;
trifluoroacetic acid in the form of a yellow oil. This intermediate was re-dissolved in
20 mL of DCM after which compound ID (1.8 g, 6.26 mmol, 1.05 equiv), DEPC (1.1 g,
6.75 mmol, 1.13 equiv) and diisopropylethylamine (DIEA, 1.64 g, 12.71 mmol, 2.13
equiv) were added. The reaction mixture was left under agitation overnight at ambient
temperature, then concentrated under reduced pressure. The residue was purified on a
silica column with a mixture of EtOAc and PE (1:100 to 1:3) to yield 2.3 g (81 %) of
compound 1J in the form of a pale yellow solid.
Example IK: (2R,3R)-3-methoxy-2-methyl -N -[(lS)-2-phenyl-l-(l,3-thiazol-2-
yl)ethyl]-3- -pyrrolidin-2-yl]propanamide; trifluoroacetic acid
Compound 1J (2.25 g, 4.75 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in 10 mL of DCM. TFA (10 mL) was added and the solution left under
agitation overnight at ambient temperature, then concentrated under reduced pressure to
yield 2.18 g (94 %) of compound IK in the form of a yellow oil.
Example 1L: (2S,3S)-2-(benzylamino)-3-methylpentanoic acid
(2S,3S)-2-amino-3-methylpentanoic acid (98.4 g, 750 mmol, 1.00 equiv) was
added at ambient temperature and in portions to a 2N sodium hydroxide solution
(375 mL). Benzaldehyde (79.7 g, 751.02 mmol, 1.00 equiv) was quickly added and the
resulting solution was agitated 30 minutes. Sodium borohydride (10.9 g, 288.17 mmol,
0.38 equiv) was added in small portions, whilst holding the temperature at between 5
and 15°C. Agitation was continued for 4 hours at ambient temperature. The reaction
mixture was diluted with 200 mL of water, then washed twice with 200 mL of EtOAc.
The pH of the aqueous solution was adjusted to 7 with a 2N hydrochloric acid solution.
The formed precipitate was collected by filtering and gave 149.2 g (90 %) of compound
1L in the form of a white solid.
)- 2- [benzyl(methyl)amino]- 3- meth lpentanoic acid
Compound 1L (25 g, 112.97 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in formic acid (31.2 g) in the presence of formaldehyde (36.5 % in water,
22.3 g). The solution was agitated 3 hours at 90°C then concentrated under reduced
pressure. The residue was triturated in 250 mL of acetone, then concentrated. This
trituration/evaporation operation was repeated twice with 500 mL of acetone to yield
21.6 g (81 %) of compound 1M in the form of a white solid.
Exam le IN: (2S,3 S)-2-[benzyl(methyl)amino]-3-methylpentan- 1- o1
L1AIH4 (0.36 g) was suspended in 10 mL of THF in an inert atmosphere at 0°C.
Compound 1M (1.5 g, 6.37 mmol, 1.00 equiv) was added in small portions whilst
holding the temperature at between 0 and 10°C. The reaction mixture was agitated 2
hours at 65°C, then again cooled to 0°C before being neutralised with successive
additions of 360 of water, 1 mL of 15 % sodium hydroxide and 360 of water.
The aluminium salts which precipitated were removed by filtering. The filtrate was
dried over sodium sulfate, filtered and concentrated. The residue was purified on a silica
column with a mixture of EtOAc and PE (1:50) to yield 820 mg (58 %) of compound
IN in the form of a pale yellow oil.
Example IO: (2S,3S)-2-[benzyl(methyl)amino]-3-methylpentanal
Oxalyl chloride (0.4 mL) was dissolved in DCM (15 mL) in an inert atmosphere.
The solution was cooled to -70°C and a solution of dimethylsulfoxide (DMSO (0.5 mL)
in DCM (10 mL) was added drop-wise for 15 minutes. The reaction mixture was
agitated 30 minutes after which a solution of compound IN (820 mg, 3.70 mmol, 1.00
equiv) in DCM (10 mL) was added drop-wise for 15 minutes. The reaction mixture was
agitated a further 30 minutes at low temperature, then triethylamine (2.5 mL) was
slowly added. The reaction mixture was agitated 1 hour at -50°C, the cold bath was
then removed and the reaction neutralised with 25 mL of water whilst allowing the
temperature to return to normal. The solution was washed once with 30 mL of NaClsaturated
aqueous solution, then dried over sodium sulfate, filtered and concentrated.
The residue was purified on a silica column with a mixture of EtOAc and PE ( 1:200) to
yield 0.42 g (52 %) of compound lO in the form of a yellow oil.
Example IP: (2S,3S)-N - benz l- l ,l - dimethox - N ,3-dimethylpentan-2-amine
Compound lO (4.7 g, 21.43 mmol, 1.00 equiv) was dissolved in 20 mL of
methanol at 0°C. Concentrated sulfuric acid (4.3 mL) was added drop-wise and
agitation was continued for 30 minutes at 0°C. Trimethyl ortho formate (21.4 mL) was
added, the cold bath removed and the reaction medium left under agitation for 3 hours at
ambient temperature. The reaction medium was diluted with 200 mL of EtOAc,
successively washed with 100 mL of 10 % a2C0 3 and 200 mL of saturated NaCl, then
dried over sodium sulfate, filtered and concentrated under reduced pressure to yield 3.4
g (60 %) of compound IP in the form of a pale yellow oil.
Example 1 : [[l -(t rt-butoxy)ethenyl]oxy ](t rt-butyl)dimethylsilane
Diisopropylamine (20 g, 186.71 m mol, 1.08 equiv) was dissolved in 170 mL of
THF in an inert atmosphere and cooled to -78°C. nBuLi (2.4 M, 78.8 mL) was added
drop-wise and the solution agitated 30 minutes at low temperature (to give LDAlithium
diisopropylamide) before adding t rt-butyl acetate (20 g, 172.18 mmol, 1.00
equiv). The reaction mixture was agitated 20 minutes at -78°C before adding
hexamethylphosphoramide (HMPA, 25.8 mL) and a solution of
tertbutyldimethylchlorosilane (TBDMSCl, 28 g, 185.80 mmol, 1.08 equiv) in 35 mL of
THF. Agitation was continued for 20 additional minutes at low temperature, and the
cold bath was then removed. The solution was concentrated under reduced pressure.
The residue was re-dissolved in 100 mL of water and extracted 3 times with 100 mL of
PE. The organic phases were combined, washed once with 500 mL of NaCl-saturated
aqueous solution, dried over sodium sulfate, filtered and concentrated. The residue was
purified by distillation to yield 16.6 g (83 %) of compound 1Q in the form of a
colourless oil.
Example 1R: t r t-butyl (3R,4S,5S)-4-[benzyl(methyl)amino]-3-methoxy-5-
methyl he tanoate
Compound I P (2.0 g, 7.54 mmol, 1.00 equiv) and compound 1Q (2.6 g,
11.28 mmol, 1.50 equiv) were dissolved in 33 mL of DCM in an inert atmosphere. The
solution was cooled to 0°C. DMF ( 1.2 g) was added drop-wise together with a solution
of BF3 Et20 (2.1 g) in 7.5 mL of DCM. Agitation was continued for 24 hours at 0°C.
The reaction medium was washed once with 30 mL of sodium carbonate (10 %) and
twice with 50 mL of NaCl-saturated aqueous solution, then dried over sodium sulfate,
filtered and concentrated. The residue was purified on a silica column with a mixture of
EtOAc and PE (1:100) to yield 1.82 g (91 %) of compound 1R in the form of a yellow
oil.
Example I S: (3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoate
hydrochloride
Compound 1R (2.4 g, 6.87 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in 35 mL of ethanol in the presence of Pd/C (0.12 g) and concentrated
hydrochloric acid (0.63 mL). The nitrogen atmosphere was replaced by a hydrogen
atmosphere and the reaction medium was left under agitation 18 hours at ambient
temperature. The reaction medium was filtered and concentrated under reduced
pressure. The residue was triturated in 50 mL of hexane and the supernatant removed
which, after drying under reduced pressure, gave 1.66 g (82 %) of compound I S in the
form of a white solid.
Example IT: tert-butyl (3R,4S,5S)-4-[(2S)-2-[[(benzyloxy)carbonyl]amino]-
N,3-dimethylbutanamido] -3-methoxy-5 -methylheptanoate
(2S)-2-[[(benzyloxy)carbonyl]amino]-3-methylbutanoic acid (15 g, 0.40 mmol,
1.00 equiv) was dissolved in 300 mL of DCM in the presence of DIEA (38.3 mL) and
bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP, 32. 3g). The solution
was agitated 30 minutes at ambient temperature before adding compound IS (15.99g,
0.42 mmol, 1.07 equiv). The reaction medium was agitated 2 hours and then
concentrated. The residue was purified in reverse phase (CI 8) with a mixture of
acetonitrile (ACN) and water (30:70 to 100:0 in 40 minutes) to yield 17 g (58 %) of
compound IT in the form of a colourless oil.
Example 1U: tert-butyl (3R,4S,5S)-4-[(2S)-2-amino -N,3-dimethylbutanamido]-
3-methoxy-5-methylheptanoate
Compound IT (76 mg, 0.15 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in 10 mL of ethanol in the presence of Pd/C (0.05 g). The nitrogen
atmosphere was replaced by a hydrogen atmosphere and the reaction agitated 2 hours at
ambient temperature. The reaction medium was filtered and concentrated under reduced
pressure to yield 64 mg of compound 1U in the form of a colourless oil.
Example IV: (3R,4S,5S)-4-[(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]
amin - N,3-dimethylbutanamido]-3-methoxy-5-methylheptanoate
Compound 1U (18.19 g, 50.74 mmol, 1.00 equiv) was dissolved in 400 mL of a
1,4-dioxane/water mixture (1:1) in the presence of sodium bicarbonate (12.78 g, 152
mmol, 3.00 equiv) and 9H-fluoren-9-ylmethyl chloroformate (Fmoc-CI, 19.69 g, 76
mmol, 1.50 equiv), then agitated 2 hours at ambient temperature. The reaction medium
was then diluted with 500 mL of water and extracted 3 times with 200 mL of EtOAc.
The organic phases were combined, washed once with 200 mL of NaCl-saturated
aqueous solution, dried over sodium sulfate, filtered and concentrated to yield 40 g of
partly purified compound IV in the form of a pale yellow oil.
Example 1W: (3R,4S,5S)-4-[(2S)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]
amino]- ,3-dimethylbutanamido]-3-methoxy-5-methylheptanoic acid
Compound IV (40 g, 68.88 mmol, 1.00 equiv) was dissolved in a neutral
atmosphere in 600 mL of DCM. TFA (300 mL) was added. The solution was agitated 2
hours at ambient temperature, then concentrated under reduced pressure. The residue
was purified on a silica column with a mixture of methanol and DCM (1:10) to yield
23.6 g (65 %) of compound 1W in colourless oil form.
Example IX: 9H-fluoren-9-ylmethyl N-[(lS)-l-[[(3R,4S,5S)-3-methoxy-l-
[(2S)-2-[(lR,2R)-l-methoxy-2-methyl-2-[[(lS)-2-phenyl-l-(l,3-thiazol-2-
yl)ethyl]carbamoyl] ethyljpyrrolidin- 1-yl]-5-methyl- 1-oxoheptan-4-yl] (methyl)
carbamoyl]-2-methylpropyl]carbamate
Compound 1W (2.53 g, 4.82 mmol, 1.08 equiv) was dissolved in 20 mL of
DCM in the presence of compound IK (2.18 g, 4.47 mmol, 1.00 equiv), DEPC (875
mg, 5.37 mmol, 1.20 equiv) and DIEA (1.25 g, 9.67 mmol, 2.16 equiv). The reaction
mixture was left under agitation overnight at ambient temperature, then successively
washed with 50 mL of saturated KHS0 4 and 100 mL of water, dried over sodium
sulfate, filtered and concentrated. The residue was purified on a silica column with a
mixture of methanol and DCM (1:200 to 1:40) to yield 2.8 g (71 %) of compound IX in
the form of a pale yellow solid.
Example 1Y: (2S)-2-amino -N -[(3R,5S)-3-methoxy-l-[(2S)-2-[(lR,2R)-lmethoxy-
2-methyl-2- [[(1S)-2-phenyl- 1-(1,3-thiazol-2-yl)ethyl] carbamoyl]ethyl]
pyrrolidin- 1-yl] -5-methyl- 1-oxoheptan-4-yl ]-N ,3-dimethylbutanamide
Compound IX (2.8 g, 3.18 mmol, 1.00 equiv) was dissolved in acetonitrile
(ACN, 12 mL) in the presence of piperidine (3 mL) and left under agitation 18 hours at
ambient temperature. The reaction was neutralised with 50 mL of water, then extracted
twice with 100 mL of DCM. The organic phases were combined, dried over sodium
sulfate, filtered and concentrated. The residue was purified on a silica column with a
mixture of methanol and DCM (1:100 to 1:40) to yield 1.2 g (57 %) of compound 1Y in
the form of a yellow solid.
Example IZA: (2S)-2-[ [(t r t-butoxy)carbonyl] (methyl)amino] -3-methyl
butanoic acid
(2S)-2 -[[(t r t-butoxy)carbonyl]amino]-3-methylbutanoic acid (63 g, 289.97
mmol, 1.00 equiv) was dissolved in an inert atmosphere in THF (1000 mL) in the
presence of iodomethane (181 mL). The solution was cooled to 0°C before adding
sodium hydride ( 116 g, 4.83 mol, 16.67 equiv) in small portions. The reaction mixture
was agitated for 1.5 hours at 0°C, the cold bath was then removed and agitation
continued for 18 hours. The reaction was neutralised with 200 mL of water and then
concentrated under reduced pressure. The residual aqueous phase was diluted with 4
litres of water, washed once with 200 mL of EtOAc and its pH adjusted to between 3
and 4 with a IN solution of hydrochloric acid. The mixture obtained was extracted 3
times with 1.2 L of EtOAc. The organic phases were combined, dried over sodium
sulfate, filtered and concentrated to yield 60 g (89 %) of compound IZA in the form of
a yellow oil.
Example 1ZB: benzyl (2S)-2- [[(t rt-butoxy)carbonyl] (methyl)amino] -3-methyl
butanoate
Compound 1ZA (47 g, 203.21 mmol, 1.00 equiv) was dissolved in DMF (600
mL) in the presence of L 12CO3 (15.8 g, 213.83 mmol, 1.05 equiv). The solution was
cooled to 0°C then benzyl bromide (BnBr 57.9 g, 338.53 mmol, 1.67 equiv) was added
drop-wise. The reaction mixture was left under agitation overnight before being
neutralised with 400 mL of water and filtered. The solution obtained was extracted
twice with 500 mL of EtOAc. The organic phases were combined, dried over sodium
sulfate, filtered and concentrated. The residue was purified on a silica column with a
mixture of EtOAc and PE (1:100 to 1:20) to yield 22.5 g (34 %) of compound 1ZB in
the form of a yellow oil.
Example 1 -3-methyl-2- methylamino)butanoate hydrochloride
Compound 1ZB (22.5 g, 70.00 mmol, 1.00 equiv) was dissolved in 150 mL of
DCM. Gaseous hydrochloric acid was bubbled. The reaction was agitated 1 hour at
ambient temperature and then concentrated under reduced pressure to yield 17 g (94 %)
of compound 1ZC in the form of a yellow solid.
Example 1ZD: tert-b t N-(3,3-diethoxypropyl)carbamate
3,3-diethoxypropan-l-amine (6 g, 40.76 mmol, 1.00 equiv) was dissolved in
1,4-dioxane (30 mL) in the presence of TEA (4.45 g, 43.98 mmol, 1.08 equiv), then
cooled to 0°C. (Boc)20 (9.6 g, 43.99 mmol, 1.08 equiv) diluted in 20 mL of 1,4-dioxane
was added drop-wise. The solution was agitated 2 hours at 0°C then overnight at
ambient temperature before being neutralised with 10 mL of water. The pH was
adjusted to 5 with HCI ( 1 %). The solution was extracted 3 times with 50 mL of EtOAc.
The organic phases were combined, dried over sodium sulfate, filtered and concentrated
to yield 8.21 g (81 %) of compound 1ZD in the form of a pale yellow oil.
Example 1Z: t rt-butyl N-(3-oxopropyl) carbamate
Compound 1ZD (8.20 g, 33.15 mmol, 1.00 equiv) was dissolved in 18.75 mL of
acetic acid and left under agitation overnight at ambient temperature. The reaction
medium was then extracted 3 times with 30 mL of EtOAc. The organic phases were
combined, washed 3 times with 30 mL of saturated NaCl solution, dried over sodium
sulfate, filtered and concentrated to yield 5 g (87 %) of compound 1ZE in the form of a
dark red oil.
Example 1ZF: (2S)-2- [(3 -[[(t rt-butoxy)carbonyl] amino]propyl)(methyl)
amino]-3-methylbutanoic ac
Compound 1ZE (2.4 g, 13.86 mmol, 1.00 equiv) was dissolved in 50 mL of THF
in the presence of compound 1ZC (3.56 g, 13.81 mmol, 1.00 equiv) and DIEA
(9.16 mL, 4.00 equiv). The reaction mixture was agitated 30 minutes at ambient
temperature before adding sodium triacetoxyborohydride (5.87 g, 27.70 mmol, 2.00
equiv). Agitation was continued overnight, then the reaction was neutralised with 100
mL of water and extracted 3 times with 50 mL of EtOAc. The organic phases were
combined, dried over sodium sulfate, filtered and concentrated. The residue was partly
purified on a silica column with a mixture of EtOAc and PE (1:4). The crude product
obtained was re-dissolved in 20 mL of methanol in the presence of Pd/C (1.2 g) and
hydrogenated for 20 minutes at normal temperature and pressure. The reaction medium
was filtered and concentrated under reduced pressure to yield 200 mg (5 %) of
compound 1ZF in the form of a white solid.
Example 1ZG: tert-butyl N-(3-[[(lS)-l-[[(lS)-l-[[(3R,4S,5S)-3-methoxy-l-
[(2S)-2-[(lR,2R)-l-methoxy-2-methyl-2-[[(lS)-2-phenyl-l-(l,3-thiazol-2-yl)ethyl]
carbamoyl]thyl]pyrrolidin-l-yl]-5-methyl-l-oxoheptan-4yl](rnethyl) carbamoyl]-2-
methylpropyl]carbamoyl]-2-methylpropyl](methyl)amino]propyl) carbamate
Compound 1Y (50 mg, 0.08 mmol, 1.00 equiv) was dissolved in 2 mL of DMF
in the presence of compound 1ZF (26.2 mg, 0.09 mmol, 1.20 equiv), DIEA (37.7 mL)
and 0-(7-azabenzotriazol-l-yl)-7V ,N,N',N, -tetramethyluronium hexafluorophosphate
(HATU, 43.3 mg, 0.1 1 mmol, 1.50 equiv). The reaction was left under agitation
overnight at ambient temperature, then diluted with 10 mL of water and extracted 3
times with 5 mL of EtOAc. The organic phases were combined, dried over sodium
sulfate, filtered and concentrated to yield 100 mg of compound 1ZG in the form of a
partly purified colourless oil.
Example 1: Compound 1ZG (90 mg, 0.10 mmol, 1.00 equiv) was dissolved in a
neutral atmosphere in 2 mL of DCM and the solution was cooled with an ice bath. TFA
( 1 mL) was added and the reaction agitated for 2 hours at ambient temperature, then
concentrated under reduced pressure. The residue was purified by preparative HPLC
(Pre-HPLC-001 SHIMADZU, SunFire Prep C 8 OBD column, 5 mh , 1 x 150 mm;
Eluting phase: water / ACN buffered with 0.05 % of TFA; Gradient of 18 % to 3 1 %
ACN in 7 minutes then 31 % to 100 % ACN in 2 minutes; Waters 2489 UV Detector at
254 nm and 220 nm). Compound 1 was obtained with a yield of 25 % (23 mg) in the
form of a white solid.
LC/MS/UV (Atlantis T3 column, 3 m , 4.6 x 100 mm; 35°C; 1 mL / min, 30 %
to 60 % ACN in water (20 mM ammonium acetate in 6 minutes); ESI (C44H 3N70 6S,
exact masse 827.53) m/z 829 (MH+), 5.84 min (93.7 %, 254 nm).
NMR (300MHz, CD3OD, ppm) d (Presence of rotamers) 7.85 - 7.80 (m,
1H); 7.69 - 7.66 ( , 1H), 7.40 - 7.10 (m, 5H), 5.80 - 5.63 (m, 1H), 4.80 - 4.65 ( , 2H),
4.22 -4.00 (m, 1H), 3.89 - 0.74 ( , 58H).
Reference Example 2
(S)-2-((S)-2-(((2-aminopyridin-4-yl)methyl)(methyl)amino)-3-
methylbutanamido)-7V-((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-lphenylpropan-
2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-
3-methoxy-5-methyl-l-oxoheptan-4-yl)-7V,3-dimethylbutanamide,
trifluoroacetic acid
Example 2A: rt-butyl (S)-2-((lR,2R)-3-(((l S,2R)- 1-hydroxy- 1-phenylpropan-
2-yl)amino)- 1-methoxy-2-methyl-3 -oxopropyl)pyrrolidine- 1-carboxylate
Compound ID (2.5 g, 8.70 mmol, 1.00 equiv) and (lS,2R)-2-amino-lphenylpropan-
l-ol (1.315 g, 8.70 mmol, 1.00 equiv) were dissolved in an inert
atmosphere in DMF (35 mL). The solution was cooled to 0 °C then DEPC (1.39 mL)
and TEA (1.82 mL) were added drop-wise. The reaction mixture was agitated 2 hours
at 0 °C then 4 hours at ambient temperature. The reaction mixture was diluted with 200
mL of water and extracted three times with 50 mL of EtOAc. The organic phases were
combined, washed once with 50 mL of KHS0 4 ( 1 mol/L), once with 50 mL of NaHC0 3
(sat.), once with 50 mL of NaCl (sat.), then dried over sodium sulfate, filtered and
concentrated under reduced pressure to yield 3.6 g (98 %) of compound 2A in the form
of a yellow solid.
Example 2B: (2R,3R)-N -((1 S,2R)- 1-hydroxy- 1-phenylpropan-2-yl)-3-methoxy-
2-methyl-3-((S)-pyrrolidin-2-yl)propanamide2,2,2-trifluoroacetate
Compound 2A (2.7 g, 6.42 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in DCM (40 mL) then cooled to 0 °C. TFA (25 mL) was added and the
solution agitated for 2 hours at 0 °C. The reaction mixture was concentrated under
reduced pressure to yield 4.4 g of compound 2B in the form of a yellow oil.
Example 2C: (9H-fluoren-9-yl)methyl ((S)- 1-(((3R,4S,5 S)- 1-((S)-2-((l R,2R)-3-
((( 1S,2R)- 1-hydroxy- 1-phenylpropan-2-yl)amino)- 1-methoxy-2-methyl-3 -oxopropyl)
pyrrolidin- 1-yl)-3-methoxy-5 -methyl- 1-oxoheptan-4-yl) (methyl)amino)-3 -methyl- 1-
oxobutan-2-yl)carbamate
Compounds 2B (4.4 g, 10.13 mmol, 1.00 equiv) and 1W (5.31 g, 10.12 mmol,
1.00 equiv) were dissolved in an inert atmosphere in DCM (45 mL). The solution was
cooled to 0°C then DEPC (1.62 mL) and DIEA (8.4 mL) were added drop-wise. The
reation mixture was agitated for 2 hours at 0 °C then at ambient temperature overnight.
The reaction mixture was diluted with 100 mL of water and extracted three times with
50 mL of DCM. The organic phases were combined, washed once with 50 mL of
KHSO4 ( 1 mol/L), once with 50 mL of NaHC0 3 (sat.), once with 50 mL of NaCl (sat.),
then dried over sodium sulfate, filtered and concentrated under pressure to yield 3.3 g
(39 %) of compound 2C in the form of a yellow solid.
Example 2D: (S)-2-amino -N -((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((lS,2R)-lhydroxy-
1-phenylpropan-2-yl)amino)- 1-methoxy-2-methyl-3 -oxopropyl)pyrrolidin- 1-
yl)-3 -methoxy-5 -methyl- 1-oxoheptan-4-yl )-N ,3-dimethylbutanamide
Compound 2C (300 mg, 0.36 mmol, 1.00 eq.) was dissolved in an inert
atmosphere in ACN (2 mL) and piperidine (0.5 mL). The solution was left under
agitation at ambient temperature overnight then evaporated to dryness under reduced
pressure. The residue was purified on a silica column with a mixture of DCM and
MeOH ( 1:100) to yield 150 mg (68 %) of compound 2D in the form of a white solid.
Example 2E: methyl 2-((t rt-butoxycarbonyl)amino)isonicotinate
Methyl 2-aminopyridine-4-carboxylate (2 g, 13.14 mmol, 1.00 equiv) was
dissolved in t r t-butanol (20 mL) after which di- t rt-butyl dicarbonate (4.02 g, 18.42
mmol, 1.40 equiv) was added. The reaction mixture was agitated at 60°C overnight then
the reaction was halted through the addition of an aqueous 1M NaHC0 3 solution (50
mL). The solid was recovered by filtration, washed with 50 mL of EtOH then dried in
vacuo to yield 2.5 g (75 %) of compound 2E in the form of a white solid.
Example 2F: t rt-butyl (4-(hydroxymethyl)pyridin-2-yl)carbamate
Compound 2E (2.5 g, 9.91 mmol, 1.00 equiv) and CaCl2 (1.65 g) were dissolved
in EtOH (30 mL). The solution was cooled to 0°C then NaBH4 (1.13 g, 29.87 mmol,
3.01 equiv) was gradually added. The solution was left under agitation overnight at
ambient temperature then the reaction was halted with the addition of water (50 mL).
The mixture was extracted three times with 20 mL of EtOAc. The organic phases were
combined, washed twice with 20 mL of NaCl (sat.) then dried over sodium sulfate,
filtered and concentrated under reduced pressure to yield 2.0 g (90 %) of compound 2F
in the form of a colourless solid.
Example 2G: t rt-butyl (4-formylpyridin-2-yl)carbamate
Compound 2F (2.5 g, 11.15 mmol, 1.00 equiv) was dissolved in DCE (25 mL)
then 19.4 g (223.14 mmol, 20.02 equiv) of Mn0 2 were added. The mixture was left
under agitation overnight at 70 °C then the solids were removed by filtering. The filtrate
was evaporated to dryness to yield 1.4 g (57 %) of compound 2G in the form of a white
solid.
Example 2H: benzyl (S)-2-(((2 -((t rt-butoxycarbonyl)amino)pyridin-4-yl)
methyl) (methyl)amino)-3-methylbutanoate
Compound 2G (2.3 g, 10.35 mmol, 1.00 equiv) was dissolved in 25 mL of
THF in the presence of compound 1ZC (2.93 g, 11.37 mmol, 1.10 equiv), DIEA
(5.39 g, 41.71 mmol, 4.03 equiv) and NaBH(OAc) 3 (4.39 g, 20.71 mmol, 2.00
equiv). The reaction mixture was agitated for 6 hours at ambient temperature then
neutralised with 60 mL of NaHC0 3 (sat.) and extracted 3 times with 20 mL of
AcOEt. The organic phases were combined, washed twice with 20 mL of NaCl (sat.),
dried over sodium sulfate, filtered and concentrated. The residue was purified on a
silica column with a mixture of EtOAc and PE (1:15) to yield 2.7 g (61 %) of
compound 2H in the form of a white solid.
Example 21: (S)-2-(((2 -((t rt-butoxycarbonyl)amino)pyridin-4-yl)methyl)
(methyl)amino)-3-methylbutanoic acid
Compound 2H (500 mg, 1.17 mmol, 1.00 equiv) was dissolved in 10 mL of
AcOEt and 2 mL of methanol in the presence of Pd/C (250 mg), and hydrogenated for 3
hours at ambient temperature and atmospheric pressure. The reaction medium was
filtered and concentrated under reduced pressure to yield 254 mg (64 %) of compound
21 in the form of a colourless solid.
Example 2J: tert-butyl (4-((3S,6S,9S,10R)-9-((S )- c-butyl)-10-(2-((S)-2-
((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-
oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-3,6-diisopropyl-2,8-dimethyl-4,7-dioxo-ll-oxa-
2,5,8-triazadodecyl)pyridin-2-yl) carbamate
Compound 2J was prepared in similar manner to compound 1ZG from the
amine 2D (85.2 mg, 0.14 mmol, 1.50 equiv), the acid 21 (31.7 mg, 0.09 mmol, 1.00
equiv), HATU (42.9 mg, 0.1 1 mmol, 1.20 equiv) and DIEA (36.7 mg, 0.28 mmol, 3.02
equiv) in DMF (3 mL). After evaporation to dryness, 100 mg of crude product were
obtained in the form of a white solid.
Example 2: Compound 2J (100 mg, 0.1 1 mmol, 1.00 equiv) was dissolved in 2
mL of DCM and 1 mL of TFA. The reaction was agitated for 1 hour at ambient
temperature, then concentrated under reduced pressure. The residue (80 mg) was
purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep CI 8 OBD
column, 5 m h, 19 x 150 mm; Eluting phase: water / ACN buffered with 0.05 % TFA;
Gradient of 20 % to 40 % ACN in 10 minutes then 40 % to 100 % ACN in 2 minutes;
Waters 2489 UV Detector at 254 nm and 220 nm). Compound 2 was obtained with a
yield of 6 % (6.3 mg) in the form of a white solid.
LC/MS/UV (Ascentis Express CI 8 column, 2.7 mhi, 4.6 x 100 mm; 40°C;
1.8mL/min, from 10 % to 95 % ACN in water (0.05 % TFA) in 6 minutes); ESI
(C 5H7 N70 7, exact mass 823.56) m/z: 824.5 (MH+) and 412.9 (M.2H+/2, 100 %), 3.21
min (99.2 %, 210 nm).
H NMR (400MHZ, CD3OD, ppm): (Presence of rotamers) 7.81 - 7.79 (m,
1H); 7.39 - 7.29 (m, 5H); 6.61 - 6.59 (m, 2H); 4.84 - 4.52 (m, 1H); 4.32 - 4.02 ( , 1H);
3.90 - 2.98 (m, 10H); 2.90 - 2.78 (m, 1H); 2.55 - 0.81 (m, 39H).
Reference Example 3
methyl ((S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-7V,3-dimethyl-2-((S)-3-
methyl-2-(methyl(pyridin-4-ylmethyl)amino)butanamido)butanamido)-3-
methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-3-phenylpropanoate, trifluoroacetic acid
Compound ID (3 g, 10.44 mmol, 1.00 equiv) and methyl (S)-2-amino-3-
phenylpropanoate (2.25 g, 12.55 mmol, 1.20 equiv) were dissolved in an inert
atmosphere in DMF (40 mL). The solution was cooled to 0 °C then DEPC (1.67 mL,
1.05 equiv) and TEA (3.64 mL, 2.50 equiv) were added drop-wise. The reaction
mixture was agitated 2 hours at 0 °C then at ambient temperature overnight. The
reaction mixture was diluted with 100 mL of water and extracted three times with 50
mL EtOAc. The organic phases were combined, washed once with 100 mL of KHS0 4
( 1 mol/L), once with 100 mL of NaHC0 3 (sat.), once with 100 mL of NaCl (sat.), then
dried over sodium sulfate, filtered and concentrated under pressure to yield 4 g (85 %)
of compound 3A in the form of a colourless oil.
Example 3B: 2,2,2-trifluoroacetate of methyl (S)-2-((2R,3R)-3-methoxy-2-
methyl-3 -((S)-pyrrolidin-2-yl)propanamido)-3 -phenylpropanoate
Compound 3A (5 g, 11.15 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in DCM (40 mL). TFA (25 mL) was added and the solution agitated for 2
hours. The reaction mixture was concentrated under reduced pressure to yield 8 g of
compound 3B in the form of a yellow oil.
Example 3C: methyl (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((((9Hfluoren-
9-yl)methoxy)carbonyl)amino )-N ,3-dimethylbutanamido)-3-methoxy-5-methyl
heptanoyl)pyrro lidin-2-yl)-3 -methoxy-2-methylpropanamido)-3 -phenylpropanoate
Compounds 3B (8.03 g, 17.36 mmol, 1.00 equiv) and 1W (9.1 g, 17.34 mmol,
1.00 equiv) were dissolved in an inert atmosphere in DCM (80 mL). The solution was
cooled to 0 °C then DEPC (2.8 mL) and DIEA (12 mL) were added drop-wise. The
reaction mixture was agitated for 2 hours at 0 °C then at ambient temperature overnight.
The reaction mixture was diluted with 200 mL of water and extracted three times with
50 mL of DCM. The organic phases were combined, washed once with 50 mL of
KHSO4 ( 1 mol/L), once with 50 mL of NaHC0 3 (sat.), once with 50 mL of NaCl (sat.),
then dried over sodium sulfate, filtered and concentrated under reduced pressure to yield
5 g (34 %) of compound 3C in the form of a yellow solid.
Example 3D: methyl (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-amino -N ,3-
dimethylbutanamido)-3 -methoxy-5 -methylheptanoyl)pyrrolidin-2-yl)-3 -methoxy-2-
methylpropanamido)-3-phenylpropanoate
Compound 3C (5.5 g, 6.43 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in a solution of tetrabutylammonium fluoride (TBAF, 2.61 g, 9.98 mmol,
1.55 quiv) in DMF (100 mL). The solution was agitated at ambient temperature for 2
hours then diluted with 100 mL of water and extracted three times with 50 mL of
EtOAc. The organic phases were combined then dried over sodium sulfate, filtered and
concentrated under reduced pressure to yield 3.3 g (81 %) of compound 3D in the form
of a yellow solid.
Example 3E: benzyl (S)-3-methyl-2-(methyl(pyridin-4-ylmethyl)amino)
butanoate
Pyridine-4-carbaldehyde ( 1 g, 9.34 mmol, 1.00 equiv) was dissolved in 10 mL of
1,2-dichloroethane (DCE) in the presence of compound 1ZC (2.9 g, 11.25 mmol, 1.21
equiv) and titanium isopropoxide (IV) (4.19 mL, 1.40 equiv). The mixture was agitated
at ambient temperature for 30 minutes then 2.77 g of NaBH(OAc ) 3 (13.07 mmol, 1.40
equiv) were added. The reaction medium was left under agitation overnight then
neutralised with 100 mL of water and the mixture extracted 3 times with 50 mL of
AcOEt. The organic phases were combined and evaporated to dryness. The residue was
purified on a silica column with a mixture of EtOAc and PE ( 1:20) to yield 1.3 g (45 %)
of compound 3E in the form of a colourless oil.
Example 3F: (S)-3-methyl-2-(methyl(pyridin-4-ylmethyl)amino)butanoic acid
Compound 3E (800 mg, 2.56 mmol, 1.00 equiv) was dissolved in 30 mL of
AcOEt in the presence of Pd/C (300 mg) and hydrogenated for 3 hours at ambient
temperature and atmospheric pressure. The reaction medium was filtered and
concentrated under reduced pressure. The residue was purified on a silica column with a
mixture of DCM and MeOH (100:1 to 5:1) to yield 100 mg (18 %) of compound 3F in
the form of a white solid.
Example 3: Compounds 3D (50 mg, 0.08 mmol, 1.00 equiv) and 3F (26.34 mg,
0.12 mmol, 1.50 equiv) were dissolved in 3 mL of DCM. The solution was cooled to 0
°C then 0.018 mL of DEPC and 0.0392 mL of DIEA were added. The reaction was
agitated at 0°C for 2 hours then at ambient temperature overnight. The reaction medium
was concentrated under reduced pressure and the residue (70 mg) was purified by
preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep CI8 OBD column, 5
mih, 19 x 150 mm; Eluting phase: water / ACN buffered with 0.05 % of TFA; Gradient
of 20 % to 40 % ACN in 10 minutes then 40 % to 100 % ACN in 2 minutes; Waters
2545 UV Detector at 254 nm and 220 nm). Compound 3 was obtained with a yield of
27 % (20 mg) in the form of a white solid.
LC/MS/UV (Ascentis Express C18 column, 2.7 m h, 4.6 x 100 mm; 40°C;
1.5 mL/min, 10 % to 95 % ACN in water (0.05 % TFA) in 8 minutes);
ESI (C46H 2N6O , exact mass 836.5) m/z 837.5 (MH+) and 419.4 (M.2H+/2 (100 %)),
7.04 min (90.0 %, 210 nm).
1H NMR (400MHz, CD3OD, ppm) d (Presence of rotamers) 8.76 - 8.74 (m,
2H); 8.53 - 8.48 (m, 0.4H, NHCO incomplete exchange); 8.29 - 8.15 (m, 0.8H, NHCO
incomplete exchange); 8.01 (s, 2H), 7.31 - 7.22 (m, 5H), 4.88 - 4.68 (m, 3H); 4.31 -
4.07 (m, 2H); 3.94 - 2.90 (m, 18H); 2.55 - 0.86 (m, 38H).
Reference Example 4
(S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-7V,3-dimethyl-2-((S)-3-methyl-2-
(methyl(pyridin-4-ylmethyl)amino)butanamido)butanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-
phenylpropanoic acid, trifluoroacetic acid
Example 4: Compound 3 (100 mg, 0.1 1 mmol, 1.00 equiv) was dissolved in a
mixture of water (5 mL), ACN (5 mL) and piperidine (2.5 mL). The reaction mixture
was left under agitation overnight then concentrated under reduced pressure. The
residue was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep
CI8 OBD column, 5 mih, 19 x 150 mm; Eluting phase: water / ACN buffered with
0.05 % TFA; Gradient of 20 % to 40 % ACN in 10 minutes then 40 % to 100 % ACN in
2 minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to yield 20 mg (20 %) of
compound 4 in the form of a white solid.
LC/MS/UV (Ascentis Express C18 column, 2.7 mih, 4.6 x 100 mm; 40°C;
1.5 mL/min, 10 % to 95 % ACN in water (0.05 % TFA) in 8 minutes);
ESI (C45H 0N6O , exact mass 822.5) m/z 823.5 (MH+) and 412.4 (M.2H+/2, 100 %),
6.84 min (89.1 %, 210 nm).
1H NMR (400MHz, CD3OD, ppm): d (Presence of rotamers) 8.79 - 8.78 (m,
2H); 8.09 (m, 2H); 7.30 - 7.21 (m, 5H); 4.80 - 4.80 (m, 1H), 4.36 - 0.87 (m, 58H).
Reference Example 6
methyl (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-((3-aminopropyl)
(methyl)amino)-3-methylbutanamido)-7V,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-
phenylpropanoate, bis trifluoroacetic acid
Example 6A: methyl (2S)-2-[(2R)-2-[(R)-[(2S)-l-[(3R,4S,5S)-4-[(2S)-2-[(2S)-
2-[(3 -[[(t rt-butoxy)carbonyl] amino]propyl)(methyl)amino] -3-methyl butanamido] -N,3-
dimethylbutanamido]-3-methoxy-5-methylheptanoyl]pyrrolidin-2-yl](methoxy)methyl]
propanamido] -3-phenylpropanoate
Compound 3D (157.5 mg, 0.25 mmol, 1.00 equiv) was dissolved at 0°C in an inert
atmosphere in 3 mL of DCM in the presence of carboxylic acid 1ZF (78.7 mg, 0.27
mmol, 1.10 equiv), DEPC (46 mΐ ) and DIEA (124 mΐ ) . The reaction mixture was agitated
2 hours at low temperature and the cold bath was then removed and agitation continued
for 4 hours. It was then concentrated under reduced pressure to yield 200 mg of
compound 6A in the form of a crude yellow oil. It was used as such in the following
step.
Example 6: Compound 6A (200 mg, 0.22 mmol, 1.00 equiv) was dissolved in
an inert atmosphere at 0°C in 2 mL of DCM. TFA ( 1 mL) was added drop-wise and the
cold bath removed. The reaction mixture was agitated 1 hour at ambient temperature
then concentrated under reduced pressure. The residue was purified by preparative
HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep CI8 OBD column, 5 mh , 19 x 150
mm; Eluting phase: water / ACN buffered with 0.05 % TFA; Gradient of 20 % to 40 %
ACN in 10 minutes then 40 % to 100 % ACN in 2 minutes; Waters 2489 UV Detector
at 254 nm and 220 nm), to yield 60 mg (26 %, yield in 2 steps) of compound 6 in the
form of a white solid.
LC/MS/UV (Zorbax Eclipse Plus C8, 3.5 mh , 4.6 x 150 mm; 1 mL/min, 40°C,
30 to 80 % methanol in water (0.1 % H3P0 4) in 18 minutes); ESI (C43H74N60 , exact
mass 802.56) m/z 804 (MH+); 1 .50 min (91.5 %, 210 nm).
H NMR (300MHz, CD OD, ppm): d (Presence of rotamers) 8.52 (d, 0.3H,
NHCO incomplete exchange); 8.25 (d, 0.5H, NHCO incomplete exchange); 7.30-7.22
(m, 5H); 4.9^.6 (m, 3H); 4.2-4.0 (m, 1H); 4.0-0.86 (m, 6 H).
Reference Example 7
(S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-((3-aminopropyl)
(methyl)amino)-3-methylbutanamido)-7V,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl) pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-
lylpropanoic acid, bis trifluoroacetic acid
Example 7: Compound 6 (70 mg, 0.08 mmol, 1.00 equiv) was dissolved in a
mixture of water (5 mL), ACN (2.5 mL) and piperidine (5 mL). The reaction mixture
was left under agitation overnight at ambient temperature, then concentrated under
reduced pressure. The residue was purified by preparative HPLC (Pre-HPLC-00 1
SHIMADZU, SunFire Prep C18 OBD column, 5 mih, 19 x 150 mm; Eluting phase:
water / ACN buffered with 0.05 % TFA; Gradient of 20 % to 40 % ACN in 10 minutes
then 40 % to 100 % ACN in 2 minutes; UV Waters 2489 UV Detector at 254 nm and
220 nm), to yield 14.6 mg (21 %) of compound 7 in the form of a white solid.
LC/MS/UV (Ascentis Express C18, 2.7 m h, 4.6 x 100 mm; 1.5 mL/min, 40°C, 0
to 80 % methanol in water (0.05 % TFA) in 8 minutes); ESI (C42H 2N O , exact mass
788.54) m/z 790 (MH+), 5.71 min (96.83 %, 210 nm).
1H NMR (300MHz, CD3OD, ppm): d (Presence of rotamers) 8.42 (d, 0.3H,
NHCO incomplete exchange); 8.15 (d, 0.2H, NHCO incomplete exchange); 7.31-7.21
(m, 5H); 4.9-4.6 (m, 3H); 4.25-4.0 (m, 1H); 4.0-0.86 (m, 59H).
Example 11
(S)-7V-((3R,4S,5S)-3-methoxy-l-((S)-2-((lR,2R)-l-methoxy-2-methyl-3-oxo-3-
(((S)-2-phenyl-l-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-l-yl)-5-methyl-loxoheptan-
4-yl)-7V,3-dimethyl-2-((S)-3-methyl-2-(methyl(4-
(methylamino)phenethyl)amino) butanamido)butanamide, trifluoroacetic acid
Example 11A: t rt-butyl N-[4-(2-hydroxyethyl)phenyl] carbamate
Compound 11A was obtained with a yield of 75 % after reaction at ambient
temperature of 2-(4-aminophenyl)ethanol with BOC20 in THF.
Example 11B: tert-butyl N-[4-(2-oxoethyl)phenyl]carbamate
Compound 11A (2.5 g, 10.5 mmol, 1.00 equiv) was dissolved in 25 mL of DCM
then cooled to -78°C. A Dess-Martin Periodinane solution (DMP, 6.71 g, 15.8 mmol,
1.5 equiv) in DCM (10 mL) was added drop-wise. The cold bath was removed and
agitation continued for 1 hour at ambient temperature. The reaction was neutralised with
60 mL of a 50/50 mixture of sodium bicarbonate-saturated aqueous solution and
Na2S203-saturated aqueous solution. The resulting solution was extracted 3 times with
30 mL of EtOAc. The organic phases were combined, washed twice with NaClsaturated
aqueous solution, dried over anhydrous sodium sulfate, filtered and
concentrated under reduced pressure. The residue was purified on silica gel (EtOAc/PE
1/15) to yield 1.0 g (40 %) of compound 11B in the form of a pale yellow solid.
Example 11C: benzyl (2S)-2-[[2-(4 -[[(t rt-butoxy)carbonyl]amino]phenyl)
ethyl](methyl)amino]-3-methylbutanoate
Compound 1ZC (3.5 g, 13.6 mmol, 1.1 equiv) was dissolved in THF (30 mL) in
the presence of DIEA (6.4 g, 49.7 mmol, 4.0 equiv), aldehyde 11B (2.9 g, 12.3 mmol,
1.0 equiv) and sodium triacetoxyborohydride (5.23 g, 49.7 mmol, 2.0 equiv). The
reaction mixture was left under agitation overnight at ambient temperature, then
neutralised with 60 mL of sodium bicarbonate-saturated solution. The resulting solution
was extracted 3 times with 30 mL EtOAc. The organic phases were combined, washed
twice with NaCl-saturated aqueous solution, dried over anhydrous sodium sulfate,
filtered and concentrated under reduced pressure. The residue was purified on silica gel
(EtOAc/PE 1:20) to yield 3.7 g (68 %) of compound 11C in the form of a yellow oil.
Example IIP : (2S)-2-[[2-(4-[[(ter t-butoxy)carbonyl]amino]phenyl)ethyl]
(methyl)amino] -3-methylbutanoic acid
Compound 11C (2 g, 4.5 mmol, 1 equiv) was dissolved in 10 mL of methanol in
the presence of Pd/C (2 g) and hydrogenated for 2 hours at normal temperature and
pressure. The reaction medium was filtered and concentrated under reduced pressure to
yield 1.2 g (75 %) of compound 11D in the form of a yellow oil.
Example HE: (2S)-2-[[2-(4-[[(ter t-butoxy)carbonyl](methyl)amino]phenyl)
ethyl] (methyl) amino]-3-methylbutanoic acid
Compound 11D (1.2 g, 3.4 mmol, 1.00 equiv) was dissolved in an inert
atmosphere in THF (20 mL). The reaction medium was cooled with an ice bath after
which NaH (60 % in oil, 549 mg, 13.7 mmol, 4.0 equiv) was added in portions,
followed by iodomethane (4.9 g, 34 mmol, 10 equiv). The reaction was left under
agitation overnight at ambient teperature, then neutralised with water and washed with
100 mL of EtOAc. The pH of the aqueous solution was adjusted to 6-7 with IN HC1.
This aqueous solution was extracted 3 times with 100 mL of EtOAc. The organic phases
were combined, dried over sodium sulfate, filtered and concentrated to yield 800 mg
(64 %) of compound HE in the form of a yellow solid.
Example 11F: t rt-butyl N-[4-(2-[[(lS)-l-[[(lS)-l-[[(3R,4S,5S)-3-methoxy-l-
[(2S)-2- [(1R,2R)- 1-methoxy-2-methyl-2-[ [(1S)-2-phenyl- 1-(1,3-thiazol-2-yl)
ethyl]carbamoyl] ethyl]pyrrolidin- 1-yl] -5-methyl- 1-oxoheptan-4yl] (methyl)carbamoyl] -
2-methylpropyl] carbamoyl]-2-methylpropyl] (methyl)amino] ethyl)phenyl] -N-methyl
carbamate
Compound 11F was prepared in similar manner to compound 6A from the
amine 1Y (150 mg, 0.22 mmol, 1.2 equiv) and the acid HE (70 mg, 0.19 mmol, 1.0
equiv). After purification on silica gel (EtOAc/PE 1:1) 100 mg (52 %) of desired
product were obtained in the form of a pale yellow solid.
Example 11: Compound 11 was prepared in the same manner as for compound
1 from the intermediate 11F (100 mg, 0.1 mmol). The residue was purified by
preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep CI8 OBD column, 5
mh , 19 x 150 mm; Eluting phase: water / ACN buffered with 0.05 % TFA; Gradient of
20 % to 40 % ACN in 10 minutes then 40 % to 100 % ACN in 2 minutes; Waters 2489
UV Detector at 254 nm and 220 nm). Compound 11 was obtained with a yield of 39 %
(39.7 mg) in the form of a white solid.
LC/MS/UV (Eclipse Plus C8, 3.5 m, 4.6 x 150 mm; 1 mL/min, 40°C, 50 to
95 % methanol in water (0.05 % TFA) in 18 minutes); ESI (C oH7 N70 S, exact mass
903.57) m/z 904.5 (MH+), 7.53 min (93.68 %, 254 nm).
H NMR (300MHz, CD3OD, ppm): d (Presence of rotamers) 8.84 (d, 0.5H,
NHCO incomplete exchange); 8.7-8.5 (m, 0.9H, NHCO incomplete exchange); 7.76-
7.73 ( , 1H); 7.55 - 7.4 (m, 1H); 7.28-7.22 (m, 7H); 7.08-7.05 (m, 2H); 5.51-5.72 (m,
1H); 4.9^.80 (m, 2H); 4.3-0.7 (m, 60H).
Example 12
methyl (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-7V,3-dimethyl-2-((S)-3-
methyl-2-(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamido)-3-
methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-3- phenylpropanoate, trifluoroacetic acid
Example 12: In the same manner as for the final phases in the synthesis of
compound 1, compound 12 was prepared in two steps from the amine 3D ( 118 mg, 0.19
mmol) and the acid HE (82 mg, 0.22 mmol). The final residue was purified by
preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep CI8 OBD column, 5
mih, 19 x 150 mm; Eluting phase: water / ACN buffered with 0.05 % TFA; Gradient of
20 % to 40 % ACN in 10 minutes then 40 % to 100 % ACN in 2 minutes; Waters 2489
UV Detector at 254 nm and 220 nm). Compound 12 was obtained with a yield of 7 %
(13.7 mg) in the form of a white solid.
LC/MS/UV (Eclipse Plus C8, 3.5 m h, 4.6 x 150 mm; 1 mL/min, 40°C, 40 to 95
% methanol in water (0.05 % TFA) in 18 minutes); ESI (C4 H N O , exact mass
878.59) m/z 879.7 (MH+), 10.07 min (90.6 %, 254 nm).
1HNMR (300MHz, CD3OD, ppm) d (Presence of rotamers) 7.40 (se, 2H);
7.38-7.22 (m, 7H); 4.95-4.7 (m, 3H); 4.2-4.0 (m, 1H); 3.9-0.86 (m, 62H).
Example 13
(S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-7V,3-dimethyl-2-((S)-3-methyl-2-
(methyl(4-(methylamino)phenethyl)amino)butanamido)butanamido)-3-methoxy-
5-methyl heptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-
phenylpropanoic acid, trifluoroacetic acid
Example 13 : Compound 13 was prepared in the same manner as for compound
7 from compound 12 (100 mg, 0.10 mmol). The residue was purified by preparative
HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep CI8 OBD column, 5 m h, 19 x 150
mm; Eluting phase: water / ACN buffered with 0.05 % TFA; Gradient of 20 % to 40 %
ACN in 10 minutes then 40 % to 100 % ACN in 2 minutes; Waters 2489 UV Detector
at 254 nm and 220 nm). Compound 13 was obtained with a yield of 20 % (20 mg) in the
form of a white solid.
LC/MS/UV (Ascentis Express CI 8, 2.7 m h, 4.6 x 100 mm; 1.5 mL/min, 40°C,
10 to 95 % methanol in water (0.05 % TFA) in 8 minutes); ESI exact
mass 864.57) m/z 865.6 (MH+), 6.05 min (90.9 %, 210 nm).
1H NMR: (300MHz, CD3OD, ppm) d (Presence ofrotamers) 7.32-7.19 (m, 9H);
4.9-4.65 (m, 3H); 4.2-4.0 (m, 1H); 3.9-0.86 (m, 59H).
Example 14
(S)-2-((S)-2-((3-aminobenzyl)(methyl)amino)-3-methylbutanamido)-7V-
((3R,4S,5S)-3-methoxy-l-((S)-2-((lR,2R)-l-methoxy-2-methyl-3-oxo-3-(((S)-
2-phenyl-l-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-l-yl)-5-methyl-loxoheptan-
4-yl)-7V,3-dimethylbutanamide, trifluoroacetic acid
Example 14A: t rt-butyl (3-(hydroxymethyl)phenyl) carbamate
(3-aminophenyl)methanol (3 g, 24.36 mmol, 1.00 equiv) was dissolved in THF
(60 mL) after which di- t r t-butyl dicarbonate (6.38 g, 29.23 mmol, 1.20 equiv) was
then added. The reaction mixture was left under agitation overnight at ambient
temperature and the reaction was then diluted by adding 200 mL of water. The product
was extracted 3 times with 100 mL of AcOEt and the organic phases were then
recombined, dried over sodium sulfate, filtered and concentrated under reduced pressure
to yield the crude product (13.85 g of compound 14A) in the form of a yellow oil.
Exam le 14B rt- but l 3- form l hen l carbamate
Compound 14A (13.8 g, 61.81 mmol, 1.00 equiv) was dissolved in DCE (400
mL) and Mn0 2 (54 g, 621.14 mmol, 10.05 equiv) was then added. The mixture was left
under agitation at ambient temperature for 3 days after which the solids were removed
by filtering. The filtrate was evaporated to dryness and the residue was purified on a
silica column with a mixture of EtOAc and PE (1:30) to yield 3 g (22 %) of compound
14B in the form of a white solid.
Example 14C: benzyl (S)-2-((3 -((t rt-butoxycarbonyl)amino)benzyl) (methyl)
amino)-3-methylbutano
Compound 14B ( 1 g, 4.52 mmol, 1.00 equiv) was dissolved in 20 mL of THF in
the presence of compound 1ZC (1.16 g, 4.50 mmol, 1.00 equiv), DIEA (3 mL) and
NaBH(OAc) 3 (1.92 g, 9.06 mmol, 2.01 equiv). The reaction mixture was left under
agitation overnight at ambient temperature and then neutralised with 100 mL of water
and extracted 3 times with 50 mL of AcOEt. The organic phases were combined, dried
over sodium sulfate, filtered and concentrated. The residue was purified on a silica
column with a mixture of EtOAc and PE (1:50) to yield 1.9 g (99 %) of compound 14C
in the form of a white solid.
Exemple 14D: (S)-2-((3-((i er t-butoxycarbonyl)amino)benzyl)(methyl)amino)-
3-methylbutanoic acid
Compound 14C ( 1 g, 2.34 mmol, 1.00 equiv) was dissolved in 30 mL of AcOEt
and 4 mL of methanol in the presence of Pd/C (400 mg) and hydrogenated for 1 hour at
ambient temperature and atmospheric pressure. The reaction medium was filtered and
concentrated under reduced pressure to yield 680 mg (86 %) of compound 14D in the
form of a white solid.
Example 14E: t r t-butyl (3-((3S,6S,9S,10R)-9-((S)-sec-butyl)-3,6-diisopropyl-
10-(2-((S)-2-(( 1R,2R)- 1-methoxy-2-methyl-3 -oxo-3-(((S)-2-phenyl- 1-(thiazol-2-yl)
ethyl)amino)propyl)pyrrolidin-l-yl)-2-oxoethyl)-2,8-dimethyl-4,7-dioxo-llamine
1Y (100 mg, 0.15 mmol, 1.00 equiv), the acid 14D (102.27 mg, 0.30 mmol, 2.00
equiv), DEPC (0.053 mL) and DIEA (0.046 mL) in DCM (3 mL). The crude product
(80 mg) was purified on a silica column with a mixture of EtOAc and PE (1:1) to yield
100 mg (67 %) of compound 14E in the form of a pale yellow solid.
Example 14 : Compound 14 was synthesised in the same manner as for
compound 2 from the intermediate 14E (100 mg, 0.10 mmol, 1.00 equiv). The crude
product (80 mg) was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU,
SunFire Prep CI8 OBD column, 5 m , 19 x 150 mm; Eluting phase: water / ACN
buffered with 0.05 % TFA; Gradient of 20 % to 40 % ACN in 0 minutes then 40 % to
100 % ACN in 2 minutes; Waters 2545 UV Detecctor at 254 nm and 220 nm).
Compound 14 was obtained with a yield of 10 % (10 mg) in the form of a white solid.
LC/MS/UV (Eclipse plus C8 column, 3.5 mhi, 4.6 x 150 mm; 40°C; 1.0 mL /
min, 40 % to 95 % MeOH in water (0.05 % TFA) in 18 minutes); ESI (C48H7 N70 6S,
exact mass 875.5) m/z 876.5 (MH+) and 438.9 (M.2H+/2, 100 %), 11.35 min (95.6 %,
210 nm).
1H NMR (400MHz, CD3OD, ppm): d (Presence of rotamers) 8.92 - 8.86 ( ,
0.4H, NH incomplete exchange); 8.70 - 8.54 (m, 0.6H, NH incomplete exchange); 7.88
- 7.78 (m, 1H); 7.60 - 7.50 (m, 1H); 7.45 - 6.97 (m, 9H); 5.80 - 5.65 (m, 1H); 4.85 -
4.70 (m, 1H); 4.40 - 0.80 (m, 56H).
Example 15
methyl (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-((3-aminobenzyl)
(methyl)amino)-3-methylbutanamido )- iV,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-
phenylpropanoate, trifluoroacetic acid
Example 15A: methyl (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)^-((S)-2-((S)-
2-((3-((/eri-butoxycarbonyl)amino)benzyl)(methyl)amino)-3-methylbutanamido)-
N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-
methoxy-2-methylpropanamido)-3-phenylpropanoate
Compound 15A was synthesised in the same manner as for compound 3 from
the amine 3D (200 mg, 0.32 mmol, 1.00 equiv), the acid 14D (212.6 mg, 0.63 mmol,
2.00 equiv), DEPC (0.1 103 mL) and DIEA (0.157 mL, 3.00 equiv) in DCM (5 mL).
The crude product was purified on a silica column with a mixture of EtOAc and PE
( 1:1) to yield 200 mg (67 %) of compound 15A in the form of a yellow solid.
Example 15: Compound 15 was synthesised in the same manner as for
compound 2 from the intermediate 15A (200 mg, 0.21 mmol, 1.00 equiv). The crude
product was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU, SunFire Prep
CI 8 OBD column, 5 mih, 19 x 150 mm; Eluting phase: water / ACN buffered with 0.05
% TFA; Gradient of 20 % to 40 % ACN in 10 minutes then 40 % to 100 % ACN in 2
minutes; Waters UV Detector 2545 at 254 nm and 220 nm). Compound 15 was obtained
with a yield of 19 % (38.6 mg) in the form of a white solid.
LC/MS/UV (Ascentis Express C18 column, 2.7 mih, 4.6 x 100 mm; 40°C;
1.5 mL/min, 10 % to 95 % MeOH in water (0.05 % TFA) in 8 minutes);
ESI (C4 H 4N6O , exact mass 850.5) m/z 851.5 (MH+) and 426.4 (M.2H+/2, 100 %),
6.61 min (91.1 %, 210 nm).
1H NMR (400MHz, CD3OD, ppm) d (Presence of rotamers) 7.53 - 7.42 (m,
1H); 7.35 - 7.18 (m, 8H); 4.88 - 4.79 (m, 2H); 4.42 - 4.00 (m, 3H); 3.93 - 2.71 (m,
22H); 2.61 - 0.81 (m, 33H).
Examples 19 and 20
Compounds 19 and 20 were prepared in the same manner as for compound 1,
from the amines 1Y and 1ZC and corresponding aldehydes.
The tert- ty 4-formylphenyl carbonate involved in the preparation of
compound 19 was prepared in a single step as follows: 4-hydroxybenzaldehyde (2.5 g,
20.5 mmol, 1.0 equiv) was dissolved in an inert atmosphere in THF (20 mL) in the
presence of 18-crown-6 (0.25 g) and potassium carbonate (5 g). The reaction mixture
was cooled to 0°C and the di- t r t-butyl dicarbonate (5.8 g, 26.58 mmol, 1.30 equiv)
was then added. Agitation was continued for 1 hour at low temperature after which the
reaction was neutralised with 30 mL of water. The resulting solution was extracted three
times with 200 mL of EtOAc. The organic phases were combined, dried over anhydrous
sodium sulfate filtered and concentrated under reduced pressure. The residue was
purified on silica gel (EtOAc/PE 1:10) and yielded 4.2 g (92 %) of ter t-butyl 4-
formylphenyl carbonate in the form of a pale yellow solid.
The 4-nitrobenzaldehyde involved in the preparation of compound 20 was
commercial.
The synthesis of compound 20 was completed by reducing the nitro group. This
was performed as follows: (2S)-N -[(3R,4S,5S)-l-[(2S)-2-[(lR,2R)-2-[[(lS,2R)-lhydroxy-
1-phenylpropan-2-yl] carbamoyl]-1-methoxy-2-methylethylpyrrolidin- 1-yl] -3-
methoxy-5 -methyl- 1-oxoheptan-4-yl] -N ,3-dimethyl-2- [(2S)-3-methyl-2- [methyl[(4-
nitrophenyl)methyl]amino]butanamido]butanamide (40 mg, 0.05 mmol, 1.0 equiv) was
dissolved in 15 mL of ethanol. Dihydrated tin chloride (II) (317 mg, 1.4 mmol, 30
equiv) was added and the solution left under agitation for 3 days at ambient
temperature. The reaction was neutralised with 50 mL of water, then extracted three
times with 50 mL of EtOAc. The organic phases were combined, dried over anhydrous
sodium sulfate, filtered and concentrated under reduced pressure to yield compound 20
in the crude state.
Name X R Purity* Quantity
(S)-2-((S)-2-((4-hydroxybenzyl)
(methyl)amino)-3 -methyl
butanamido )-N -((3R,4S,5S)-3-
methoxy- 1-((S)-2-((l R,2R)- 1-
methoxy-2-methyl-3-oxo-3-(((S)-2- 1 93.2 % 21.6 mg
phenyl- 1-(thiazol-2-yl)ethyl)amino)
propyl)pyrrolidin- 1-yl)-5-methyl- 1-
oxoheptan-4-yl )-N ,3-dimethyl
butanamide, trifluoroacetic acid
Name X R Purity* Quantity
(S)-2-((S)-2-((4-aminobenzyl)
(methyl)amino)-3 -methyl
butanamido )-N-((3R,4S,5S)-3-
methoxy- 1-((S)-2-((l R,2R)- 1-
methoxy-2-methyl-3-oxo-3-(((S)-2- 1 96.7 % 21.1 mg
phenyl- 1-(thiazol-2-yl)ethyl)amino)
propyl)pyrrolidin- 1-yl)-5-methyl- 1-
oxoheptan-4-yl )-N,3-dimethyl
butanamide, trifluoroacetic acid
* The compounds were purified by preparative HPLC (Pre-HPLC-001
SHIMADZU, SunFire Prep C18 OBD column, 5 mih, 19 x 150 mm; Eluting phase:
water / ACN buffered with 0.05 % TFA; Gradient of 20 % to 40 % ACN in 10 minutes
then 40 % to 100 % ACN in 2 minutes; Waters 2489 UV Detector at 254 nm and 220
nm), to give the corresponding TFA salts in the form of white solids.
Characterization of the end products: Compound 19 LC/MS/UV ESI:
(C4 H 2N6O7S, exact mass 876.52) m/z 877 (MH+), 439 [100 %, (M.2H+)/2]; UV: RT =
1.76 min (93.2 %, 220 nm). Compound 20 1H NMR: (400MHz, CD3OD, ppm): d
(Presence of rotamers) 7.85-7.80 (m, 1H); 7.6-7.5 (m, 1H); 7.4-7.15 (m, 5H); 7.1-7.05
(m, 2H); 6.73-6.70 (m, 2H); 5.8-5.55 (m, 1H); 5.0-4.7 (m, 2H); 4.25-4.05 (m, 1H);
4.0-0.8 (m, 54H). LC/MS/UV ESI: (C48H73N7O7S, exact mass 875.53) m/z 876 (MH+),
439 [75 %, (M.2H+)/2]; UV: RT = 4.83 min (96.8 %, 254 nm). 1H NMR (400MHz,
CD3OD, ppm): d (Presence of rotamers) 7.85-7.80 (m, 1H); 7.6-7.5 (m, 1H); 7.4-7.1
(m, 7H); 6.76-6.72 (m, 2H); 5.8-5.55 (m, 1H); 4.9-4.65 (m, 2H); 4.25-4.05 (m, 1H);
4.0-0.8 (m, 54H).
Examples 23 and 24
Compounds 23 and 24 were prepared in the same manner as for compounds 19
20, replacing the amine 1Y by the amine 2D.
* The compounds were purified by preparative HPLC (Pre-HPLC-001
SHIMADZU, SunFire Prep C18 OBD column, 5 mih, 19 x 150 mm; Eluting phase:
water / ACN buffered with 0.05 % TFA; Gradient of 20 % to 40 % ACN in 10 minutes
then 40 % to 100 % ACN in 2 minutes; Waters 2489 UV Detector at 254 nm and 220
nm), to give the corresponding TFA salts in the form of white solids.
Characterization of the end products: Compound 23 LC/MS/UV (ESI)
(C46H73N 50 , exact mass 823.55) m/z 824 (MH+), 846 (MNa+), 413 (100 %,
(M.2H+)/2); UV: 4.76 min (98.5 %, 215 nm). 1H NMR (400MHz, CDC13, ppm) d
(Presence of rotamers) 7.5-7.2 (m, 5H); 7.9-7.75 (m, 2H); 5.5-5.3 (m, 1H); 4.9-4.6
(m, 2H); 4.55-4.15 (m, 4H); 4.0-0.8 (m, 55H). Compound 24 LC/MS/UV (ESI)
(C46H 4N60 7, exact mass 822.56) m/z 823 (MH+), 845 (MNa+), 861 (MK+); UV: 3.68
min (99.15 %, 254 nm). 1H NMR (400MHz, CD3OD, ppm): d (Presence of rotamers)
8.0-7.7 (m, 0.5H, NHCO incomplete exchange); 7.5-7.0 (m, 7H); 6.75-6.65 (m, 2H);
4.85-4.5 (m, 2H); 4.4-4.05 (m, 2H); 4.0-0.8 (m, 56H).
Example 27
methyl (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-((4-
hydroxyphenethyl)(methyl)amino)-3-methylbutanamido)-/V,3-
dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-
methoxy-2-methylpropanamido)-3- phenylpropanoate, trifluoroacetic acid
Example 27: Compound 27 was prepared in the same manner as for compound
3 from the amine 3D (70 mg, 0.1 1 mmol, 1.00 equiv), the acid 49C (55.5 mg,
0.22 mmol, 2.00 equiv), DEPC (0.034 mL, 2.00 equiv) and DIEA (0.055 mL, 3.00
equiv) in DCM (3 mL). The crude product was purified by preparative HPLC (Pre-
HPLC-001 SHIMADZU, SunFire Prep C18 OBD column, 5 mih, 19 x 150 mm; Eluting
phase: water / ACN buffered with 0.05 % TFA; Gradient of 20 % to 45 % ACN in 10
minutes then 40 % to 100 % ACN in 2 minutes; Waters 2545 UV Detector at 254 nm
and 220 nm). Compound 27 was obtained with a yield of 3 % (2.9 mg) in the form of a
white solid.
LC/MS/UV (Eclipse Plus C8 column, 3.5 m, 4.6 x 150 mm; 40°C; 1.5 mL/min,
10 % to 95 % MeOH in water (0.05 % TFA) in 8 minutes); ESI (C4 H75N50 9, exact
mass 866.56) /z : 866.5 (MH+) and 433.9 (M.2H+/2, 100 %), 6.61 min (89.1 %,
210 ran).
1H NMR (400MHz, CD3OD, ) : d (Presence of rotamers) 8.70 - 8.49 (m, 0.9
H, NH/OH incomplete exchange); 8.30 - 8.22 (m, 0.3H, NH incomplete exchange); 7.36
- 7.02 (m, 7H); 6.86 - 6.62 (m, 2H); 4.82 - 4.69 (m, 2H); 4.20 - 4.03 (m, 1H); 3.91 -
3.33 (m, 12H); 3.30 - 2.90 (m, 17H); 2.55 - 0.80 (m, 35H).
Example 28
(S)-2-((S)-2--((3-aminobenzyl)(methyl)amino)-3-methylbutanamido)-7V-
((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-
yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-3-methoxy--5-
methyl—l-oxoheptan-4— yl)- N,3—dimethylbutanamide, trifluoroacetic acid
Example 28A: teri-butyl (3-((3S,6S,9S,10R)-9-((S)-5ec-butyl)-10-(2-((S)-
2-(( 1R,2R)-3-((( 1S,2R)-1 -hydroxy-1 -phenylpropan-2-yl)amino)-l -methoxy-2-
methyl-3-oxopropyl)pyrrolidin-l-yl)-2-oxoethyl)-3,6-diisopropyl-2,8-dimethyl-
4,7-dioxo-ll-oxa-2,5,8-triazadodecyl)phenyl)carbamate
amine 2D (100 mg, 0.17 mmol, 1.00 equiv), the acid 14D ( 111.25 mg, 0.33 mmol, 2.00
equiv), DEPC (0.058 mL) and DIEA (0.05 mL) in DCM (3 mL). The residue was
purified on a silica column with a mixture of EtOAc and hexane (1:1) to yield 100 mg
(66 %) of compound 28A in the form of a white solid.
Example 28: Compound 28 was synthesised in the same manner as for
compound 2 from the intermediate 28A (100 mg, 0.1 1 mmol, 1.00 equiv). The crude
product (80 mg) was purified by preparative HPLC (Pre-HPLC-001 SHIMADZU,
SunFire Prep C18 OBD column, 5 mih, 19 x 150 mm; Eluting phase: water / ACN
buffered with 0.05 % TFA; Gradient of 20 % to 40 % ACN in 10 minutes then 40 % to
100 % ACN in 2 minutes; Waters 2545 UV Detector at 254 nm and 220 nm).
Compound 28 was obtained with a yield of 20 % (20 mg) in the form of a white solid.
LC/MS/UV (Ascentis Express C18 column, 2.7 mih, 4.6 x 100 mm; 40°C;
1.5 mL/min, 10 % to 95 % MeOH in water (0.05 % TFA) in 8 minutes);
ESI (C46H 4N6O7, exact mass 822.56) m/z 823.5 (MH+) and 412.4 (M.2H+/2, 100 %),
12.45 min (87.2 %, 210 nm).
1H NMR: (400MHz, CD3OD, ppm) d (Presence of rotamers) 7.47 - 7.20 (m,
5H); 7.10 - 7.01 (m, 1H); 6.80 - 6.56 (m, 3H); 4.82 - 4.52 (m, 3H); 4.33 - 4.03 (m, 2H);
3.91 - 3.82 (m, 0.5H); 3.75 - 3.35 (m, 9.5H); 3.28 - 3.10 (m, 2H); 2.79 - 2.90 (m, 1H);
2.60 - 2.40 (m, 2H); 2.30 - 0.80 (m, 40H).
Example 29
(S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-((3-aminobenzyl)
(methyl)amino)-3-methylbutanamido)-7V,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-
phenylpropanoic acid, trifluoroacetic acid
Example 29: Compound 15 (100 mg, 0.10 mmol, 1.00 equiv) was dissolved in a
mixture of water (5 mL), ACN (5 mL) and piperidine (2.5 mL). The reaction mixture
was left under agitation overnight at ambient temperature and then concentrated under
reduced pressure. The residue was purified by preparative HPLC (Pre-HPLC-00 1
SHIMADZU, SunFire Prep C18 OBD column, 5 mih, 19 x 150 mm; Eluting phase:
water / ACN buffered with 0.05 % TFA; Gradient of 20 % to 40 % ACN in 10 minutes
then 40 % to 100 % ACN in 2 minutes; Waters 2545 UV Detector at 254 nm and 220
nm), to yield 20 mg (20 %) of compound 29 in the form of a white solid.
LC/MS/UV (Eclipse Plus C8 column, 3.5 m h, 4.6 x 150 mm; 40°C; 1.0 mL/min,
40 % to 95 % MeOH in water (0.05 % TFA) in 18 minutes); ESI (C46H72N6O , exact
mass 836.54) m/z 837.5 (MH+) and 419.4 (M.2H+/2, 100 %), 10.61 min (92.5 %,
210 nm).
1H NMR: (400MHz, CD3OD, ppm): d (Presence of rotamers) 7.38 - 7.15 (m, 6H);
7.00 - 6.99 (m, 3H); 4.85 - 4.68 (m, 2H); 4.37 - 3.38 (m, 11H); 3.31 - 2.70 (m, 8H); 2.60
- 0.82 (m, 35H).
Example 49
(S)-2-((S)-2-((4-hydroxyphenethyl)(mthyl)amino)-3-methylbutanamido)-7V-
((3R,4S,5S)-3-mthoxy-l-((S)-2-((lR,2R)-l-methoxy-2-methyl-3-oxo-3-(((S)-2-
phenyl-l-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-l-yl)-5-methyl-loxoheptan-
4-yl)-7V,3-dimethylbutanamide, trifluoroacetic acid
4-(2-hydroxyethyl)phenol (4 g, 28.95 mmol, 1.00 quiv) was dissolved in
DMSO (32 mL) and TEA (8.8 mL, 2.20 equiv) was then added dropwise. A solution of
S0 .Py (10 g, 2.20 equiv) in DMSO (36 mL) was added and the mixture was left under
agitation overnight at ambient temperature. The reaction mixture was neutralised with
250 mL of water and extracted 3 times with 100 mL of AcOEt. The organic phases were
combined, washed 5 times with water (100 mL) then twice with 150 mL of NaCl (sat.),
dried over sodium sulfate, filtered and concentrated. The residue was purified on silica
gel (EtOAc/PE (1:10) to yield 1 g (25 %) of compound 49A in the form of a colourless
oil.
Example 49B: benzyl (S)-2-((4-hydroxyphenethyl)(methyl)amino)-3 -methyl
butanoa
Compound 49B was synthesised in the same manner as for compound 14C from
the amine 1ZC (1.5 g, 5.82 mmol, 0.99 equiv), the aldehyde 49A (800 mg, 5.88 mmol,
1.00 equiv), NaBH(OAc) 3 (2.7 g, 12.74 mmol, 2.17 equiv) and DIEA (4.23 mL) in THF
(25 mL). The reaction mixture was neutralised with 50 mL of water and extracted 3
times with 50 mL of AcOEt. The organic phases were combined, dried over sodium
sulfate, filtered and concentrated. The residue was purified on silica gel (EtOAc/PE
(1:10) to yield 600 mg (37 %) of compound 49B in the form of a white solid.
Example 49C: (S)-2-((4-hydroxyphenethyl)(methyl)amino)-3-methylbutanoic
acid
Compound 49B (0.5 g, 1.46 mmol, 1.00 equiv) was dissolved in 40 mL of
MeOH in the presence of Pd/C (250 mg) and hydrogenated for 3 hours at ambient
temperature and atmospheric pressure. The reaction medium was filtered and
concentrated under reduced pressure to yield 0.4 g of compound 49C in the form of a
white solid.
Example 49: Compound 49 was synthesised in the same manner as for
compound 3 from the amine 1Y (53.4 mg, 0.08 mmol, 2.00 equiv), the acid 49C (70
mg, 0.28 mmol, 1.00 equiv), DEPC (0.032 mL, 2.00 equiv) and DIEA (0.053 mL, 3.00
equiv) in DCM (3 mL). The residue was purified by preparative HPLC (Pre-HPLC-00 1
SHIMADZU, Atlantis Prep OBD T3 column, 5 mih, 19 x 150 mm; Eluting phase: water
/ ACN buffered with 0.05 % TFA; Gradient of 20 % to 45 % ACN in 10 minutes then
45 % to 100 % ACN in 2 minutes; Waters 2545 UV Detector at 254 nm and 220 nm), to
yield 3 mg ( 1 %) of compound 49 in the form of a white solid.
LC/MS/UV (Ascentis Express C18 column, 2.7 m h, 4.6 x 100 mm; 40°C;
1.5 mL/min, 10 % to 95 % MeOH in water (0.05 % TFA) in 8 minutes);
ESI (C49H74N6O7S, exact mass 890.5) m/z 891.5 (MH+) and 446.4 (M.2H+/2, 100 %),
6.69 min (100 %, 210 nm).
1H NMR: (400MHz, CD3OD, ppm): d (Presence of rotamers) 8.92 - 8.87 (m, 0.5
H, NHCO, incomplete exchange), 8.70 - 8.63 (m, 0.4 H, NHCO, incomplete exchange),
8.85 - 8.77 (m, 1H), 7.59 - 7.51 (m, 1H), 7.35 - 7.03 (m, 7H), 6.82 - 6.71 (m, 2H), 5.77 -
5.58 (m, 1H), 5,81 - 5.70 (m, 1H), 4.21 - 0.80 (m, 58H).
Example 50
(S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-((4-
hydroxyphenethyl)(methyl)amino)-3-methylbutanamido)-7V,3-
dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-
methoxy-2-methylpropanamido)-3-phenylpropanoic acid, trifluoroacetic acid
Example 50: Compound 50 was prepared in the same manner as for compound
4, from compound 27 (100 mg, 0.10 mmol, 1.00 equiv). The residue was purified by
preparative HPLC (Pre-HPLC-001 SHIMADZU, Atlantis Prep OBD T3 column, 5 mih,
19 x 150 mm; Eluting phase: water / ACN buffered with 0.05 % TFA; Gradient of 20 %
to 40 % ACN in 10 minutes then 40 % to 100 % ACN in 2 minutes; Waters 2545 UV
Detector at 254 nm and 220 nm), to yield 10.7 mg ( 11 %) of compound 50 in the form
of a white solid.
LC/MS/UV (Ascentis Express C18 column, 2.7 m h, 4.6 x 100 mm; 40°C;
1.5 mL/min, 10 % to 95 % MeOH in water (0.05 % TFA) in 8 minutes);
ESI (C47H73N5O9, exact mass 851.5) m/z 852.5 (MH ) and 426.8 (M.2H72, 100 %),
6.46 min (91.7 %, 210 nm).
1H NMR: (400MHz, CD3OD, ppm) d (Presence of rotamers) 7.34 - 7.15 (m,
5H); 7.15 - 7.04 (se, 2H), 6.82 - 6.83 (m, 2H), 4.83 - 4.70 (m, 1H), 4.21 - 4.00 (m, 1H),
3.90 - 3.80 (m, 1H), 3.74 - 3.62 (m, 1H), 3.57 - 2.86 (m, 20H), 2.56 - 0.80 (m, 36H).
Example 51
methyl (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-((4-
hydroxybenzyl)(methyl)amino)-3-methylbutanamido)-7V,3-
dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-
methoxy-2-methylpropanamido)-3- phenylpropanoate, trifluoroacetic acid
Example 51A: t rt-butyl (4-formylphenyl)carbonate
4-hydroxybenzaldehyde (3.0 g, 24 mmol) was dissolved in 30 mL of DCM in
the presence of 4-DMAP (300 mg, 2.46 mmol, 0.1 equiv.) and di- t rt-butyl
dicarbonate (5.35 g, 24 mmol, 1.0 equiv.) and agitated 1 hour at ambient temperature.
The solution was then diluted with 200 mL of water and extracted 3 times with 100 mL
of DCM. The organic phases were combined, dried over sodium sulfate, filtered and
concentrated under reduced pressure to yield 5 g (92 %) of compound 51A in the form
of a white solid.
Example 51B: benzyl (S)-2((4 -((t rt-butoxycarbonyl)oxy)benzyl)(methyl)
amino)-3-methylbutanoate
NaBH(OAc)3 DIEA.THF
Compound 51A (220 mg, 0.99 mmol) was dissolved in 5 mL of THF in the
presence of compound IZC (255 mg, 0.99 mmol, 1.0 equiv.), NaBH(OAc)3 (420 mg, 2
mmol, 2.0 equiv.) and DIEA (654 mΐ ) and agitated overnight at ambient temperature.
5 The solution was then diluted with 100 mL of water and extracted 3 times with 50 mL
of EtOAc. The organic phases were combined, dried over sodium sulfate, filtered and
concentrated under reduced pressure. The residue was purified on a silica column with a
mixture of EtOAc and PE (1:100) to yield 200 mg (47 %) of compound 51B in the form
of a white solid.
0 Example 51C: (S)-2-((4-((ier t-butoxycarbonyl)oxy)benzyl)(methyl)amino)-3-
methyl butanoic acid
Compound 51C was prepared by hydrogenation of compound 5IB (200 mg),
following the protocol used for the preparation of compound 3F.
5 Example 51D: methyl (S)-2-((2R,3R)-3-((S)-l-((3R,4S,5S)^l-((S)-2-((S)-
2-((4 -((t ri-butoxycarbonyl)oxy)benzyl)(methyl)amino)-3-methylbutanamido )-N ,3-
dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-3-phenylpropanoate
0 Compound 51D was prepared by coupling compound 51C with amine 3D,
following the protocol used for the preparation of compound 3 to obtain the desired
product in the form of yellow oil with a yield of 60 %.
Example 51: Compound 51D (80 mg, 0.08 mmol) was dissolved in 1 mL of
DCM in the presence of 0.5 mL TFA, agitated 2 hours at ambient temperature and then
concentrated under reduced pressure. The residue was purified by preparative HPLC
(Pre-HPLC-010, SunFire Prep C18 OBD column, 5 mih, 19 x 150 mm; Eluting phase:
water / ACN buffered with 0.05 % TFA; Gradient of 23 % to 40 % ACN in 10 minutes
then 40 % to 95 % ACN in 2 minutes; Waters 2489 UV Detector at 254 nm and 220
nm). Compound 51 was obtained with a yield of 24 % (20 mg) in the form of a white
solid.
LC/MS/UV (Zorbax SB-Aq, 1.8 m h, 4.6 x 100 mm; 2 % MeOH in water
(0.05 % TFA) for 1 minute then 2 % to 95 % MeOH in 13 minutes); ESI
exact mass 851.54) m/z 874.5 (MNa+), 426.9 (M.2H+/2); 12.48 min (96 %, 210 nm).
1H NMR: (300MHz, CD3OD, ppm) d (Presence of rotamers) 8.1 - 8.6 (m, 0.9H,
NHCO incomplete exchange); 7.29 - 7.27 (m, 2H), 7.25 - 6.86 (m, 5H), 6.84 - 6.83 (m,
2H), 4.83 - 4.72 (m, 3H), 4.26 - 0.82 (m, 58H).
Example 61
(S)-2-((S)-2-((4-aminophenethyl)(methyl)amino)-3-methylbutanamido)-7V-
((3R,4S,5S)-3-methoxy-l-((S)-2-((lR,2R)-l-methoxy-2-methyl-3-oxo-3-(((S)-2-
phenyl-l-(thiazol-2-yl)ethyl)amino)propyl)pyrrolidin-l-yl)-5-methyl-l-oxoheptan-
4-yl)-7V,3-dimethylbutanamide
Example 61A: N-(4-aminophenethyl )-N-methyl-Z-valine dihydrochloride
Compound 11D (962 mg, 2.75 mmol) was dissolved in 10 ml of a commercially
available solution of HC1 in propan-2-ol (5 - 6 M), and stirred at room temperature for 2
hours. TLC analysis indicated complete consumption of starting material. The solvent
was evaporated under reduced pressure, and the resulting yellow solid triturated with
Et20 (2 x 10 ml). The product was dried under vacuum to furnish compound 61A as a
yellow solid (322 mg, 47 %).
Example 61: Carboxylic acid 61A (73 mg, 0.23 mmol, 1 eq.) and amine 1Y
(150 mg, 0.23 mmol, 1 eq.) were dissolved in dry DMF (2 ml). DIEA (158 mΐ ,
0.90 mmol, 4 eq.) and DECP (51 mΐ, 0.34 mmol, 1.5 eq.) were added and the reaction
stirred for 4 hours at room temperature. Analysis by LC-MS showed complete
consumption of the starting material. The solvent was evaporated under reduced
pressure, and the residue purified by flash chromatography on silica gel (DCM/MeOH)
to furnish compound 61 as a light yellow solid (83 mg, 40 %).
1H NMR: (500MHz, DMSO-d 6, pp ) d (Presence of rotamers), 8.86 (d, 0.5H,
NHCO); 8.65 (d, 0.5H, NHCO), 8.1 1-8.05 (m, 1H, NHCO), 7.80 (d, 0.5H, thiazole),
7.78 (d, 0.5H, thiazole), 7.65 (d, 0.5H, thiazole), 7.63 (d, 0.5H, thiazole), 7.32 - 7.12
(m, 5H), 6.83 (d, J=8.3 Hz, 2H), 6.45 (d, J=8.3 Hz, 2H), 5.56 - 5.49 (m, 0.5 H), 5.42 -
5.35 (m, 0.5H), 4.78 (s, 2H, NH2), 4.74 - 4.46 (m, 2H), 4.01 - 0.66 (m, 57H).
HPLC (Xbridge Shield C18, 3.5 m h, 4.6 x 50 mm ; 3.5 ml/min, 40°C, 0 to 95 %
MeCN in water (0.1 % TFA) in 2.25 minutes then 95 % MeCN for 0.5 minutes, Tr =
1.31 min (96.5 %, 220 nm).
m/z (Q-TOF ESI+) 890.5558 (2%, MH+, C4 H 6 7O6S requires 890.5572),
445.7834 (100 %, (MH2) +, C4 H 7 7O6S requires 445.7823).
Example 62
Methyl ((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-((4-
aminophenethyl)(methyl)amino)-3-methylbutanamido)-7V,3-dimethylbutanamido)-
3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-Lphenylalaninate
Example 62: Compound 62 was prepared in the same manner as for compound
61, using carboxylic acid 61A (69 mg, 0.21 mmol, 1 eq.), amine 3D (135 mg, 0.21
mmol, 1 eq.), DIEA (75 mΐ, 0.43 mmol, 2 eq.) and DECP (49 mΐ, 0.32 mmol, 1.5 eq.).
The crude product was purified by flash chromatography on silica gel (DCM/MeOH) to
furnish compound 62 as a yellowish solid (82 mg, 45 %).
1H NMR: (500MHz, OMSO-d6, ppm): d (Presence of rotamers), 8.50 (d, J=8.3,
0.5H, NHCO); 8.27 (d, J=8.0, 0.5H, NHCO), 8.15-8.04 (m, 1H, NHCO), 7.27 - 7.13
(m, 5H), 6.86 - 6.79 (m, 2H), 6.48 - 6.42 (m, 2H), 4.78 (s, 2H, NH2), 4.74 - 4.44 (m,
3H), 4.01 - 3.72 (m, 1.5H), 3.66 (s, 1.5H, C0 2Me), 3.63 (s, 1.5H, C0 2Me), 3.57 - 0.65
(m, 55.5H).
HPLC (Xbridge Shield C18, 3.5 m h, 4.6 x 50 mm ; 3.5 ml/min, 40°C, 0 to 95 %
MeCN in water (0.1 % TFA) in 2.25 minutes then 95 % MeCN for 0.5 minutes, Tr =
1.29 min (95 .3 %, 220 nm).
m/z (Q-TOF ESI+) 865.5800 (2%, MH+, C48H77N6O8 requires 865.5797),
433.2937 (100 %, (MH2) +, C4 H N 60 requires 433.2935).
Example 63
((2R,3R)-3-((S)-l-((3R,4S,5S)-4-((S)-2-((S)-2-((4-aminophenethyl)(methyl)amino)-
3-methylbutanamido)-7V,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine
2,2,2-trifluoroacetate
Example 63: Compound 62 (23 mg, 0.03 mmol) was dissolved in a mixture of
water ( 1 ml) and acetonitrile ( 1 ml). Piperidine (0.75 ml) was added and the mixture
stirred at room temperature for 5 hours. TLC analysis indicated complete consumption
of the starting material. The solvent was evaporated under reduced pressure, and the
residue purified by preparative HPLC (SunFire Prep column C18 OBD, 5 mih, 19 x 150
mm; Mobile phase: water/MeCN buffered with 0.1 % TFA; Gradient of 20 % to 40 %
MeCN in 10 minutes, then from 40 % to 100 % MeCN in 2 minutes; Detector UV
Waters 2545 at 254 nm et 220 nm). Compound 63 was obtained as a white solid (14 mg,
66 %).
1H NMR: (500MHz, DMSO-d 6, ppm): d (Presence ofrotamers), 12.7 (s(br), 1H,
C0 2H), 9.58 (m(br), 1H); 9.04 - 8.89 (m, 1H), 8.41 (d, 0.6H, NHCO), 8.15 (d, 0.4H,
NHCO), 7.27 - 7.13 (m, 5H), 7.13 - 6.99 (m(br), 2H), 6.90 - 6.64 (s(br), 2H), 4.77 -
3.40 (m, 10H), 3.34 - 2.75 (m, 20H), 2.34 - 1.94 (m, 4H), 1.90 - 0.7 (m, 25H).
HPLC (Xbridge Shield CI 8, 3.5 m h, 4.6 x 50 mm ; 3.5 ml/min, 40°C, 0 to 95 % MeCN
in water (0.1 % TFA) in 2.25 minutes then 95 % MeCN for 0.5 minutes, Tr = 1.24
min (100 %, 220 nm).
m/z (Q-TOF EST) 851.5641 (6%, MH+, C47H75N60 requires 851.5641),
426.2854 (100 %, (MH2) +, requires 426.2857).
Example 64
(S)-2-((S)-2-((4-aminophenethyl)(methyl)amino)-3-methylbutanamido)-7V-
((3R,4S,5S)-l-((S)-2-((lR,2R)-3-(((lS,2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-
l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-3-methoxy-5-methyl-loxoheptan-
4-yl)-7V,3-dimethylbutanamide
Compound 64 was prepared in the same manner as for compound 61, using
carboxylic acid 61A (93 mg, 0.29 mmol, 1 eq.), amine 2D (174 mg, 0.29 mmol, 1 eq.),
DIEA (100 mΐ, 0.58 mmol, 2 eq.) and DECP (66 mΐ, 0.43 mmol, 1.5 eq.). The crude
product was purified by flash chromatography on silica gel (DCM/MeOH) to furnish
compound 64 as an off-white solid (51 mg, 2 1 %).
1H NMR: (500MHz, DMSO-d6, ppm) d (Presence of rotamers), 9.61 (m(br),
1H); 9.05 - 8.89 (m, 1H), 7.93 (d, 0.6H, NHCO), 7.64 (d, 0.4H, NHCO), 7.36 - 6.98
(m, 7H), 6.92 - 6.70 (m(br), 2H), 5.45 (s(br), 1H), 4.80 - 4.41 (m, 3H), 4.06 - 3.44 (m,
4H), 3.37 - 2.79 (m, 18H), 2.45 - 2.21 (m, 3H), 2.17 - 0.70 (m, 35H).
HPLC (Xbridge Shield C18, 3.5 m h, 4.6 x 50 mm ; 3.5 ml/min, 40°C, 0 to 95 %
MeCN in water (0.1 % TFA) in 2.25 minutes then 95 % MeCN for 0.5 minutes, Tr =
1.20 min (100 %, 220 nm).
m/z (Q-TOF ESI+) 837.5826 (33%, MH+, C47H77N6O7 requires 837.5848),
419.2956 (100 %, (MH2) +, C47H76N6O8 requires 419.2961).
II - Biological activity of the compounds of the invention
Method:
Cell culture. A549 (Non Small Cell Lung Cancer - ATCC CCL- 185) and MDAMB-
231 (breast adenocarcinoma - ATCC HTB-26) cells were cultured in Minimum
Essential Medium Eagle (MEM) with 5% fetal calf serum (FCS) and Dulbecco's
modified Eagle Medium (DMEM) with 10% FCS respectively. MCF7 (breast ductal
carcinoma - ATCC HTB-22) and SN-12C (kidney carcinoma - ATCC) cells were
maintained in RPMI1640 medium (without phenol red for MCF7 cells) containing 10%
FCS. All the media were supplemented with fungizone (1.25 mg/mL) and penicillinstreptomycin
(100 U / 100 mg/mL). Cells were cultured under standard conditions in an
incubator at 37°C, 5%> C0 2 and 95%> atmospheric humidity.
Antiproliferative activity on 4 tumor cell lines. Compounds according to the
invention were investigated for their antiproliferative activity using an ATPlite
proliferation assay (Perkin Elmer, Villebon sur Yvette, France) on a comprehensive
panel of 4 cell lines. Cells were seeded in 96 well plates (10 cells/well for A549, 2.10
for MCF7, MDA-MB-23 1 and SN12C) at day 0 at a concentration to ensure cells
remained in logarithmic cell growth phase throughout the 72 h drug treatment period.
After a 24h incubation period, all the cells were treated with serial dilutions of the tested
compounds ( 11 of a 10X solution in 1% DMSO - 6 wells/ condition). To avoid
adherence of the compounds onto the tips, tips were changed between two consecutive
dilutions. Cells were then placed in 37°C, 5% C0 2 incubator. On day 4, cell viability
was evaluated by dosing the ATP released by viable cells. The number of viable cells
was analyzed in comparison with the number of solvent treated cells. The EC50 values
were determined with curve fitting analysis (non linear regression model with a
sigmoidal dose response, variable hill slope coefficient), performed with the algorithm
provided by the GraphPad Software (GraphPad Software Inc., CA, USA).
Results:
Various compounds:
Various compounds according to the invention were tested to determine their
antiproliferative activity on the MDA-MB-23 1 cell line following the above-described
method. The measured activities gave values of EC50 < 0.1 mM.
The few following examples chosen from among the compounds according to
the invention illustrate their fully remarkable antiproliferative properties:
Example 12: EC50 = 5.80xl0 10 M; Example 13: EC50 = 7.95xl0 8 M; Example 15: EC50
= 1.70xl0 10 M; Example 27: EC50 = 1.20xl0 10 M.
Various cell lines:
Compound 15 was tested on different cell lines (A549, MDA-MB-23 1, MCF-7,
SN12C) following the above-described method. The measured activities gave values of
ECso < 0.1 mM.
ECso (M) A549 MDA-MB-23 1 MCF-7 SN12C
Compound 15 1.45xlO 1.70xlO 7.15xlO 2.18c10
Comparative examples:
The substitution on the phenyl ring (amino/hydroxyl v. carboxyl) was studied in
the comparative examples below showing the improved antiproliferative activity of the
drugs according to the invention comprising an amino or hydroxyl substituent.

CLAIMS
1. A compound of following formula (I):
(I)
where:
- Ri is H or OH,
- R2 is a (Ci-C 6)alkyl, COOH, COO-((Ci-C 6)alkyl) or thiazolyl group,
- R is H or a (Ci-C )alkyl group, and
- R4 is an aryl-(Ci-C8)alkyl group substituted by one or more groups chosen from
among OH and NR9R10 groups with R and Rio each independently of one another
representing H or a (Ci-C )alkyl group,
or a pharmaceutically acceptable salt, hydrate or solvate thereof.
2. The compound according to claim 1, characterized in that:
- Ri=OH and R2 represents a (Ci-C )alkyl group, or
- Ri=H and R2 represents a COOH, COO-(Ci-Ce)alkyl or thiazole group.
3. The compound according to claim 1 or 2, characterized in that Ri
represents H and R2 represents COOH or COOMe.
4. The compound according to any one of claims 1 to 3, characterized in that
R 3 represents H or a methyl group.
5. The compound according to any one of claims 1 to 4, characterized in that
R4 represents an aryl-(Ci-C4)alkyl group substituted by one group on the aryl moiety
chosen from among OH and NR9R10, and notably being NR9R10.
6. The compound according to any one of claims 1 to 4, characterized in that
R 4 represents a phenyl-(Ci-C 2)alkyl group substituted by one group on the phenyl
moiety chosen from among OH and NR9R10 , and notably being NR9R10 .
7. The compound according to any one of claims 1 to 4, characterized in
llowing formula:
wherein X0 represents OH or NR9R10, in particular NR9R10, and m represents an integer
comprised between 1 and 8, advantageously m is 1 or 2.
8. The compound according to any one of claims 1 to 4, characterized in
that R4 has the following formula:
wherein X0 represents NR9R10 and m represents 1 or 2.

and the pharmaceutically acceptable salts thereof such as the salts formed with
trifluoroacetic acid.
10. A compound according to any one of claims 1 to 9 for use as a medicinal
product.
11. A compound according to any one of claims 1 to 9 for use as medicinal
product intended for the treatment of cancer or benign proliferative disorders.
12. A pharmaceutical composition comprising a formula (I) compound
according to any one of claims 1 to 9 and at least one pharmaceutically acceptable
excipient.
13. The pharmaceutical composition according to claim 12, further comprising
another active ingredient, advantageously chosen from among anticancer agents, in
particular comprising cytotoxic anticancer agents such as navelbine, vinflunine, taxol,
taxoter, 5-fluorouracil, methotrexate, doxorabicin, camptothecin, gemcitabin, etoposide,
cis-platin or carmustin; and hormonal anticancer agents such as tamoxifen or
medroxyprogesterone.
14. A method for preparing a formula (I) compound according to any one of
claims 1 to 9 comprising a condensation reaction between a compound of following
formula (VI):
where Ri and R2 are as defined in claim 1,
a compound of following formul
where R3 is as defined in claim 1, R4a represents an R4 group as defined in claim 1,
optionally in protected form, and X is OH or CI.
15. A method for preparing a formula (I) compound according to any one of
claims 1 to 9 comprising a substitution reaction between a compound of following
formula (VIII):
where Ri, R2 a,d R3 are as defined in claim 1, and
a compound of following formula (X):
where R4a represents an R4 group as defined in claim 1 optionally in protected form,
Y is a leaving group such as CI, Br, I, OS0 2CH3, OS0 2CF3 or O-Tosyl.
16. A method for preparing a formula (I) compound according to any one of
claims 1 to 9 where R4 represents a -CH 2R4b group with R4b representing an aryl or
aryl-(Ci-Cv)alkyl group substituted by one or more groups chosen from among OH and
NR9R10 groups,
comprising a reductive amination reaction between a compound of following formula
(VIII):
where Ri, R2 and R3 are as defined in claim 1, and
a compound of following formula (XI):
where R4b is as previously defined.