WO 2007/026027 PCT/EP2006/065941
NOVEL ANSAMYCIN DERIVATIVES
Introduction
The present invention relates to derivatives of ansamycin compounds that are useful,
e.g. in the treatment of cancer or B-cell malignancies, in particular the derivatives are pro-drugs
of ansamycin compounds. The present invention also provides methods for the production of
these compounds and their use in medicine, in particular in the treatment and / or prophylaxis of
cancer or B-cell malignancies.
Background of the invention
The development of highly specific anticancer drugs with low toxicity and favourable
pharmacokinetic characteristics comprises a major challenge in anticancer therapy.
The 90 kDa heat shock protein (Hsp90) is an abundant molecular chaperone involved in
the folding and assembly of proteins, many of which are involved in signal transduction
pathways (for reviews see Neckers, 2002; Sreedhar ef ai, 2004a; Wegele ef ai, 2004 and
references therein). So far nearly 50 of these so-called client proteins have been identified and
include steroid receptors, non-receptor tyrosine kinases e.g. src family, cyclin-dependent
kinases e.g. cdk4 and cdk6, the cystic transmembrane regulator, nitric oxide synthase and
others (Donze and Picard, 1999; McLaughlin et ai, 2002; Chiosis et ai, 2004; Wegele ef ai,
2004; http://www.picard.ch/downloads/Hsp90interactors.pdf). Furthermore, Hsp90 plays a key
role in stress response and protection of the cell against the effects of mutation (Bagatell and
Whitesell, 2004; Chiosis ef ai, 2004). The function of Hsp90 is complicated and it involves the
formation of dynamic multi-enzyme complexes (Bohen, 1998; Liu et ai, 1999; Young ef ai,
2001; Takahashi et al., 2003; Sreedhar et al., 2004; Wegele et al., 2004). Hsp90 is a target for
inhibitors (Fang et al., 1998; Liu et al., 1999; Blagosklonny, 2002; Neckers, 2003; Takahashi ef
ai, 2003; Beliakoff and Whitesell, 2004; Wegele et al., 2004) resulting in degradation of client
proteins, cell cycle dysregulation and apoptosis. More recently, Hsp90 has been identified as an
important extracellular mediator for tumour invasion (Eustace ef ai, 2004). Hsp90 was identified
as a new major therapeutic target for cancer therapy which is mirrored in the intense and
detailed research about Hsp90 function (Blagosklonny et ai, 1996; Neckers, 2002; Workman
and Kaye, 2002; Beliakoff and Whitesell, 2004; Harris et al., 2004; Jez et al., 2003; Lee et al.,
2004) and the development of high-throughput screening assays (Carreras et al., 2003;
Rowlands ef ai, 2004). Hsp90 inhibitors include compound classes such as ansamycins,
macrolides, purines, pyrazoles, coumarin antibiotics and others (for review see Bagatell and
Whitesell, 2004; Chiosis et al., 2004 and references therein).
The benzenoid ansamycins are a broad class of chemical structures characterised by an
aliphatic ring of varying length joined either side of an aromatic ring structure. Naturally

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occurring ansamycins include: macbecin and 18,21-dihydromacbecin (also known as macbecin
I and macbecin II respectively) (1 & 2; Tanida et al., 1980), geldanamycin (3; DeBoer et al.,
1970; DeBoer and Dietz, 1976; WO 03/106653 and references therein), and the herbimycin
family (4; 5, 6, Omura et al., 1979, Iwai et al., 1980 and Shibata et al., 1986a, WO 03/106653
and references therein).

Ansamycins were originally identified for their antibacterial and antiviral activity,
however, recently their potential utility as anticancer agents has become of greater interest
(Beliakoff and Whitesell, 2004). Many Hsp90 inhibitors are currently being assessed in clinical
trials (Csermely and Soti, 2003; Workman, 2003). In particular, geldanamycin has nanomolar
potency and apparent specificity for aberrant protein kinase dependent tumour cells (Chiosis et
al., 2003; Workman, 2003).
It has been shown that treatment with Hsp90 inhibitors enhances the induction of tumour
cell death by radiation and increased cell killing abilities (e.g. breast cancer, chronic myeloid
leukaemia and non-small cell lung cancer) by combination of Hsp90 inhibitors with cytotoxic
agents has also been demonstrated (Neckers, 2002; Beliakoff and Whitesell, 2004). The
potential for anti-angiogenic activity is also of interest: the Hsp90 client protein HIF-1á plays a

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key role in the progression of solid tumours (Hur et al., 2002; Workman and Kaye, 2002; Kaur et
al., 2004),
Hsp90 inhibitors also function as immunosuppressants and are involved in the
complement-induced lysis of several types of tumour cells after Hsp90 inhibition (Sreedhar et
al., 2004). Treatment with Hsp90 inhibitors can also result in induced superoxide production
(Sreedhar et al., 2004a) associated with immune cell-mediated lysis (Sreedhar et al., 2004).
The use of Hsp90 inhibitors as potential anti-malaria drugs has also been discussed (Kumar et
al., 2003). Furthermore, it has been shown that geldanamycin interferes with the formation of
complex glycosylated mammalian prion protein PrPc (Winklhofer et al., 2003).
As described above, ansamycins are of interest as potential anticancer and anti-B-cell
malignancy compounds, however the currently available ansamycins exhibit poor
pharmacological or pharmaceutical properties, for example they show poor water solubility, poor
metabolic stability, poor bioavailability or poor formulation ability (Goetz et al., 2003; Workman
2003; Chiosis 2004). Both herbimycin A and geldanamycin were identified as poor candidates
for clinical trials due to their strong hepatotoxicity (review Workman, 2003) and geldanamycin
was withdrawn from Phase I clinical trials due to hepatotoxicity (Supko et al., 1995, WO
03/106653)
Geldanamycin was isolated from culture filtrates of Streptomyces hygroscopicus and
shows strong activity in vitro against protozoa and weak activity against bacteria and fungi. In
1994 the association of geldanamycin with Hsp90 was shown (Whitesell et al., 1994). The
biosynthetic gene cluster for geldanamycin was cloned and sequenced (Allen and Ritchie, 1994;
Rascher et al., 2003; WO 03/106653). The DNA sequence is available under the NCBI
accession number AY179507. The isolation of genetically engineered geldanamycin producer
strains derived from S. hygroscopicus subsp. duamyceticus JCM4427 and the isolation of 4,5-
dihydro-7-O-descarbamoyl-7-hydroxygeldanamycin and 4,5-dihydro-7-0-descarbamoyi-7-
hydroxy-17-O-demethylgeldanamycin were described recently (Hong et al., 2004). By feeding
geldanamycin to the herbimycin producing strain Streptomyces hygroscopicus AM-3672 the
compounds 15-hydroxygeidanamycin, the tricyclic geldanamycin analogue KOSN-1633 and
methyl-geldanamycinate were isolated (Hu et al., 2004). The two compounds 17-formyl-17-
demethoxy-18-0-21-O-dihydrogeldanamycin and 17-hydroxymethyl-17-
demethoxygeldanamycin were isolated from S. hygroscopicus NRRL 3602 containing plasmid
pKOS279-78 with various genes from the herbimycin producing strain Streptomyces
hygroscopicus AM-3672 (Hu et al., 2004).
In 1979 the ansamycin antibiotic herbimycin A was isolated from the fermentation broth
of Streptomyces hygroscopicus strain No. AM-3672 and named according to its potent
herbicidal activity. The antitumour activity was established by using cells of a rat kidney line
infected with a temperature sensitive mutant of Rous sarcoma virus (RSV) for screening for

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drugs that reverted the transformed morphology of the these cells (for review see Uehara,
2003). Herbimycin A was postulated as acting primarily through the binding to Hsp90 chaperone
proteins but the direct binding to the conserved cysteine residues and subsequent inactivation
of kinases was also discussed (Uehara, 2003).
Chemical derivatives have been isolated and compounds with altered substituents at
C19 of the benzoquinone nucleus and halogenated compounds in the ansa chain showed less
toxicity and higher antitumour activities than herbimycin A (Omura et al., 1984; Shibata et al.,
1986b). The sequence of the herbimycin biosynthetic gene cluster was identified in WO
03/106653 and in a recent paper (Rascher et al, 2005).
The ansamycin antibiotics macbecin (1) and 18,21-dihydromacbecin (2) (C-14919E-1
and C-14919E-1), identified by their antifungal and antiprotozoal activity, were isolated from the
culture supernatants of Nocardia sp No. C-14919 (Actinosynnema pretiosum subsp pretiosum
ATCC 31280) (Tanida et al., 1980; Muroi et al., 1980; US 4,315,989 and US 4,187,292). 18,21-
Dihydromacbecin is characterized by containing the hydroquinone form of the nucleus. Both
macbecin and 18,21-dihydromacbecin were shown to possess similar antibacterial and
antitumour activities against cancer cell lines such as the murine leukaemia P388 cell line (Ono
et al., 1982). Reverse transcriptase and terminal deoxynucleotidyl transferase activities were
not inhibited by macbecin (Ono et al., 1982). The Hsp90 inhibitory function of macbecin has
been reported in the literature (Bohen, 1998; Liu etal., 1999). The conversion of macbecin and
18,21-dihydromacbecin after adding to a microbial culture broth into a compound with a hydroxy
group instead of a methoxy group at a certain position or positions is described in patents US
4,421,687 and US 4,512,975.
During a screen of a large variety of soil microorganisms, the antibiotics TAN-420A to E
were identified from producer strains belonging to the genus Streptomyces (7-11, EP 0 110
710).

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In 2000, the isolation of the geldanamycin related, non-benzoquinone ansamycin
metabolite reblastatin from cell cultures of Streptomyces sp. S6699 and its potential therapeutic
value in the treatment of rheumatoid arthritis was described (Stead et al., 2000).
A further Hsp90 inhibitor, distinct from the chemically unrelated benzoquinone
ansamycins is Radicicol (monorden) which was originally discovered for its antifungal activity
from the fungus Monosporium bonorden (for review see Uehara, 2003) and the structure was
found to be identical to the 14-membered macrolide isolated from Nectria radicicola. In addition
to its antifungal, antibacterial, anti-protozoan and cytotoxic activity it was subsequently identified
as an inhibitor of Hsp90 chaperone proteins (for review see Uehara, 2003; Schulte et al., 1999).
The anti-angiogenic activity of radicicol (Hur et al., 2002) and semi-synthetic derivates thereof
(Kurebayashi et al., 2001) has also been described.
Recent interest has focussed on 17-amino derivatives of geldanamycin as a new
generation of ansamycin anticancer compounds (Bagatell and Whitesell, 2004), for example 17-
(allylamino)-17-desmethoxy geldanamycin (17-AAG, 12) (Hostein et al., 2001; Neckers, 2002;
Nimmanapalli et al., 2003; Vasilevskaya et al., 2003; Smith-Jones et al., 2004)and 17-
desmethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG, 13)(Egorin et al.,
2002; Jez et al., 2003). More recently geldanamycin was derivatised on the 17-position to
create 17-geldanmycin amides, carbamates, ureas and 17-arylgeldanamycin (Le Brazidec et
al., 2003). A library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogues has
been reported and tested for their affinity for Hsp90 and water solubility (Tian et al., 2004). A
further approach to reduce the toxicity of geldanamycin is the selective targeting and delivering
of an active geldanamycin compound into malignant cells by conjugation to a tumour-targeting
monoclonal antibody (Mandler et al., 2000).

Whilst these derivatives exhibit reduced hepatotoxicity they still have only limited water
solubility and require the use of a solubilising carrier (e.g. Cremophore®, DMSO-egg lecithin),
which itself may result in side-effects in some patients.
Therefore, there remains a need to identify novel ansamycins with improved water
solubility which will have an improved pharmacological profile and reduced side-effect profile for

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administration. The present invention discloses novel ansamycin derivatives which are pro-
drugs and may be cleaved, chemically or enzymatically, to the parent molecule and which
generally have improved pharmaceutical properties compared with the presently available
ansamycins; in particular they show improvements in respect of one or more of the following
properties: water solubility, bioavailability and formulation ability.
Summary of the invention
The present invention provides derivatives of ansamycins, methods for the preparation
of these compounds, intermediates thereto and methods for the use of these compounds in
medicine. In particular the derivatives of ansamycins are pro-drugs.
In its broadest aspect the present invention provides derivatives of benzenoid
ansamycins containing self-cleaving and water solubilizing derivatising group(s) at positions 18
and/or 21 of the parent molecule. These groups are designed to be chemically cleaved to
produce the bioactive parent molecule, alternatively cleavage may occur via enzymatic means.
Thus the invention relates to derivatives of a benzenoid ansamycin which contain a 1,4-
dihydroxyphenyl moiety bearing at position 6 an amino carboxy substituent, in which position 2
and the carboxy substituent at position 6 are connected by an aliphatic chain of varying length
characterised in that one or both of the 1 -hydroxy and the 4-hydroxy position(s) of the phenyl
ring are independently derivatised by an aminoalkyleneaminocarbonyl group, which alkylene
group (which may optionally be substituted by alkyl eg methyl groups) has a chain length of 2 or
3 carbons and which derivatising group(s) increase the water solubility and/or the bioavailability
of the parent molecule but which are capable of being removed in-vivo eg by self-cleavage.
in this context the "parent molecule" means the corresponding molecule bearing an
underivatised hydroxyl group at positions 1 and 4 of the phenyl ring (i.e. hydroquinone) or the
benzoquinone form thereof.
In a more specific aspect the present invention provides derivatives of ansamycins
according to the formulas (IA-IC) below, or a pharmaceutically acceptable salt thereof:


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wherein:
R1 represents H, OH, OMe, -NHCH2CH=CH2 or-NHCH2CH2N(CH3)2;
R2 represents OH, or keto;
R3 represents OH or OMe;
R5 represents H or
wherein:
n represents 0 or 1;
R6 represents H, Me, Et or iso-propyl;
R7, R8 and R9 each independently represent H or a C1-C4 branched or linear chain alkyl group;
or R7 and R8, or R8 and R9, may be connected so as to form a 6-membered carbocyclic ring;
R10 represents H or a C1-C4 branched or linear chain alkyl group;
provided however that the R5 moieties are not both H and that when neither R5 moiety
represents H then the two R5 moieties are the same.
The above structure shows a representative tautomer and the invention embraces all
tautomers of the compounds of formula (IA), (IB) and (IC) for example keto compounds where
enol compounds are illustrated and vice versa.
Compounds of formula (IA), (IB) and (IC) are referred to collectively in the foregoing as
compounds of formula (I).
In a further aspect, the present invention provides ansamycin derivatives such as
compounds of formula (I) or a pharmaceutically acceptable salt thereof, for use as a
pharmaceutical.

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Definitions
The articles "a" and "an" are used herein to refer to one or to more than one (i.e. at least
one) of the grammatical objects of the article. By way of example "an analogue" means one
analogue or more than one analogue.
As used herein the term "analogue(s)" refers to chemical compounds that are
structurally similar to another but which differ slightly in composition (as in the replacement of
one atom by another or in the presence or absence of a particular functional group).
As used herein, the term "cancer" refers to a malignant new growth that arises from
epithelium, found in skin or, more commonly, the lining of body organs, for example, breast,
prostate, lung, kidney, pancreas, stomach or bowel. A cancer tends to infiltrate into adjacent
tissue and spread (metastasise) to distant organs, for example to bone, liver, lung or the brain.
As used herein the term cancer includes both metastatic tumour cell types, such as but not
limited to, melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and
mastocytoma and types of tissue carcinoma, such as but not limited to, colorectal cancer,
prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer,
pancreatic cancer, bladder cancer, renal cancer, gastric cancer, gliobastoma, primary liver
cancer and ovarian cancer.
As used herein, the term "bioavailability" refers to the degree to which or rate at which
a drug or other substance is absorbed or becomes available at the site of biological activity after
administration. This property is dependent upon a number of factors including the solubility of
the compound, rate of absorption in the gut, the extent of protein binding and metabolism etc.
Various tests for bioavailability that would be familiar to a person of skill in the art are described
herein (see also Egorin et al. (2002).
As used herein the term "B-cell malignancies" includes a group of disorders that
include chronic lymphocytic leukaemia (CLL), multiple myeloma, and non-Hodgkin's lymphoma
(NHL). They are neoplastic diseases of the blood and blood forming organs. They cause bone
marrow and immune system dysfunction, which renders the host highly susceptible to.infection
and bleeding.
The term "pro-drug" as used in this application refers to a precursor or derivative form of a
pharmaceutically active substance that has an improved formulation profile compared to the parent
drug, e.g. it may be less cytotoxic or more soluble compared to the parent drug, and it is capable of
being activated (e.g. cleaved chemically or enzymatically) or otherwise converted into the more
active parent form (see, for example, Wilman D.E.V. (1986) "Pro-drugs in Cancer Chemotherapy"
Biochemical Society Transactions 14, 375-382 (615th Meeting, Belfast) and Stella V.J. et a! (1985)
"Pro-drugs: A Chemical Approach to Targeted Drug Delivery" Directed Drug Delivery R. Borchardt
et al (ed.) pages 247-267 (Humana Press).
The term "water solubility" as used in this application refers to solubility in aqueous media,

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e.g. phosphate buffered saline (PBS) at pH 7.4, or in 5% glucose solution. Tests for water
solubility are given below in the Examples as "water solubility assay".
As used herein, the term "ansamycin derivative" refers to a benzenoid ansamycin
derivative referred to above as representing the invention in its broadest aspect, for example a
compound according to formula (I) above, or a pharmaceutically acceptable salt thereof. These
compounds are also referred to as "compounds of the invention" or "derivatives of
ansamycins" and these terms are used interchangeably in the present application.
The pharmaceutically acceptable salts of compounds of the invention such as the
compounds of formula (I) include conventional salts formed from pharmaceutically acceptable
inorganic or organic acids or bases as well as quaternary ammonium acid addition salts. More
specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric,
nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric,
citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric,
toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic,
hydroiodic, malic, steroic, tannic and the like. Hydrochloric acid salts are of particular interest.
Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful
in the preparation of salts useful as intermediates in obtaining the compounds of the invention
and their pharmaceutically acceptable salts. More specific examples of suitable basic salts
include sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, N,N'-
dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-
methylglucamine and procaine salts. References hereinafter to a compound according to the
invention include both compounds of formula (I) and their pharmaceutically acceptable salts.
Alkyl, alkenyl and alkynyl groups may be straight chain or branched.
Examples of alkyl eg C1-C4 alkyl groups include methyl, ethyl, n-propyl, i-propyl and n-
butyl.
As used herein the terms "18,21-dihydromacbecin" and "macbecin II" (the hydroquinone
of macbecin I) are used interchangeably.
Figure Legend
Figure 1 Graph showing the decay of the parent compound and the relative amounts of
released compounds in a cleavage assay.
Figure 2 Graph showing group median relative tumour volumes over time in PRXF DU-145
xenografts.

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Description of the Invention
Strategies to improve physical properties of drug candidates e.g. bioavailability typically
employ a pro-drug precursor which relies upon environmental influence such as enzymatic
hydrolysis to release active parent drug. However, due to variation between individuals, release
of active drug at a required rate in vivo may not be achievable.
The present invention provides derivatives of ansamycins with an alternative method of
rate controlled active drug release. This approach utilises self-cleaving of an incorporated
amino side chain triggered at physiological pH via an intramolecular cyclisation-elimination
reaction. Such intramolecular attack by a terminal amino group upon a carbamate functionality
generates a cyclic urea fragment and leads to parent drug release.
The rate of drug release is governed by chemical cyclisation rate constants and
associated substituents rather than by external influence.
Whilst it is intended that the compounds of the invention are capable of chemically
mediated self-cleavage, it is also possible that they are substrates for enzymatic cleavage and
this is also encompassed within the scope of the present invention.
Thus, the present invention provides derivatives of ansamycins, as set out above,
methods for the preparation of these compounds, intermediates thereto and methods for the
use of these compounds in medicine.
In one example set of compounds of formula IA-IC, R6 represents H, Me or Et, In a
further example set of compounds of formula IA-IC, R10 represents a C1-4 branched or linear
alkyl group. In a further example set of compounds of formula IA-IC, R6 represents H, Me or Et
and R10 represents a C1-4 branched or linear alkyl group.
Each of the R5 groups will be the same or one will be H whilst the other is substituted as
described herein. In one example aspect one of the R5 groups is H; in an alternative example
aspect neither R5 group is H. Preferably the C21 R5 group is H.
Preferably R6 represents Me. Alternatively, preferably R6 represents Et.
Preferably R10 represents Me. Alternatively, preferably R10 represents Et.
Preferably R7 represents H. Preferably R8 represents H. Preferably R9 represents H.
When R7 and R8 or R8 and R9 are connected to form a six-membered carbocyclic ring
that ring may suitably be a cyclohexyl ring.
Preferably n = 0.
The stereochemistry of side chains relative to the ansamycin ring preferably follows that
of the corresponding parent compounds (i.e. macbecin, geldanamycin, herbimycin A, see eg
structure shown above Table 4).
The compound of formula (I) may, for example, represent a derivative of the following
compounds:

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• macbecin (formula (IA));
• geldanamycin (formula (IB) in which R1 represents OMe, particularly when R2 represents
OH);
• herbimycin B (formula (IB) in which R1 represents H, particularly when R2 represents
OH);
• 17-AAG (formula (IB) in which R1 represents NHCH2CH=CH2, particularly when R2
represents OH);
• 17-DMAG (formula (IB) in which R1 represents NHCH2CH2NMe2, particularly when R2
represents OH);
• herbimycin A (formula (IC) in which R2 represents OMe and R3 represents OMe); or
• herbimycin C (formula (IC) in which R2 represents OH and R3 represents OMe)
In general, the compounds of the invention are prepared by semi-synthetic derivatisation
of the ansamycin family of compounds.
Thus a process for preparing a compound of formula (I) or a pharmaceutically
acceptable salt thereof comprises:
(a) preparing a compound of formula (I) in which neither R5 moiety is H by reacting a
compound of formula (IIA), (MB) or (IIC):


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wherein L is a leaving group
or a protected derivative thereof, with a compound of formula (H)

wherein P represents a protecting group; or
(b) preparing a compound of formula (I) in which the C21 R5 moiety is H by reacting a
compound of formula (IID), (IIE) or (IIF):

wherein L is a leaving group
or a protected derivative thereof, with a compound of formula (H)

wherein P represents a protecting group; or
(c) converting a compound of formula (I) or a salt thereof to another compound of
formula (I) or another pharmaceutically acceptable salt thereof; or

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(d) deprotecting a protected compound of formula (I).
In the foregoing text the compounds of formula (IIA), (IIB), (IIC), (IID), (IIE) and (IIF) are
referred to collectively as compounds of formula (II).
In processes (a) and (b), exemplary leaving groups L include halogen (eg chlorine,
bromine), alkoxy (eg C1-4alkoxy), aryl (eg phenoxy or substituted phenoxy such as 4-
nitrophenoxy) or alkylaryl (eg C1-4alkyiaryl eg benzyloxy). Preferably L represents 4-
nitrophenoxy. Exemplary protecting groups P include t-butyloxycarbonyl ("Boc") and the trityl
group.
The reaction of compounds of formula (II) with compound of formula (H) may be
performed under conventional conditions known per se for carbamate formation eg reflux of the
ingredients in an inert solvent such as dichloromethane with water work-up.
Compounds of formula (II), or a protected derivative thereof, may be prepared by
reacting a compound of formula IIIA-IIIC:

or a protected derivative thereof, with a compound of formula (J):
L'-CO-L (J)

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wherein L' represents a leaving group, preferably one which is more labile than L.
Exemplary L' groups are as described for L, above. A preferred compound of formula J is 4-
nitrophenylchloroformate.
The reaction of compounds of formula (III) with compound of formula (J) may be
performed under conventional conditions known per se eg reflux of the ingredients in an inert
solvent such as dichloromethane.
Since the C18 OH group is more reactive than the C21 OH group, compounds of
formula (IID)-(IIF) may be obtained by reacting the corresponding compound of formula (IIIA)-
(IIIC) with a slight excess of the compound of formula J. Compounds of formula (IIA)-(IIC) may
be obtained by reacting the corresponding compound of formula (IIIA)-(IIIC) with a greater than
two times excess of the compound of formula J.
Compounds of formula IIIA-IIIC (hereinafter "compounds of formula (III)") and protected
derivatives thereof may be prepared as follows:
Firstly, the naturally occurring ansamycins for use as templates may be obtained via
direct fermentation of strains which produce the desired compound. A person of skill in the art
will be able to culture a producer strain under suitable conditions for the production and isolation
of the natural product template. The strains listed in Table 1 are examples of producer strains
for the natural product templates, but a person of skill in the art will appreciate that there may be
alternative strains available that will produce the same compound under appropriate conditions.

Alternatively, the compounds may be commercially available; Table 2 lists the
compounds that may be purchased together with their catalogue numbers:


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In addition to the specific methods and references provided herein a person of skill in the
art may also consult standard textbook references for synthetic methods, including, but not
limited to Vogel's Textbook of Practical Organic Chemistry (Furniss et al., 1989) and March's
Advanced Organic Chemistry (Smith and March, 2001).
The naturally occurring ansamycins exist and can be isolated predominantly in their
benzoquinone form. In some cases the hydroquinone form may be isolated from the
fermentation broth. If the benzoquinone form is isolated then it will need to be converted to the
corresponding compound of formula (III) (hydroquinones). It is well-known in the art that
benzoquinones can be chemically converted to hydroquinones (reduction) and vice versa
(oxidation). This can be applied to the ansamycin natural products, as described above, such
that where the benzoquinone form occurs naturally, the hydroquinone can be synthesised by a
variety of methods. As an example (but not by way of limitation) this may be achieved in
organic media with a source of hydride, such as but not limited to, LiAIH4 or SnCl2-HCl.
Alternatively this transformation may be mediated by dissolving the benzoquinone form of the
ansamycin in organic media and then washing with an aqueous solution of a reducing agent,
such as, but not limited to, sodium hydrosulfite (Na2S2O4 or sodium thionite). Preferably, this
transformation is carried out by dissolving macbecin orgeldanamycin in ethyl acetate and
mixing this solution vigorously with an aqueous solution of sodium hydrosulfite (Muroi et al.,
1980). The resultant organic solution can then be washed with water, dried and the solvent
removed under reduced pressure to yield an almost quantitative amount of 18,21-
dihydromacbecin or 18,21-dihydrogeldanamycin respectively.
Compounds of formula (IIB) and (IIC) such as those derived from geldanamycin may or
do contain a secondary hydroxyl group at the C-11 position. In order to derivatise these
compounds at the C-18 hydroxyl exclusively it may be necessary to first modify the C-11
hydroxyl. Additionally, compounds derivatised at the C-11 position in combination with
derivatisation at C-18 or C-21 are specifically contemplated as compounds of the invention in
their own right. Described below are methods for accomplishing this for geldanamycin, but a
person of skill in the art wili appreciate that these can equally be applied to other compounds of
formula (IIB) and (IIC), (IIE) and (IIF) for which the parent compound contains an OH group at
C-11.
For example, the C-11 hydroxyl could be oxidised to a ketone, by one of many standard
protocols for oxidising secondary alcohols to a ketone, such as but not limited to, Swern

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oxidation, Dess-Martin periodination, tetrapropylammonium peruruthenate (TPAP), Jones'
reagent, pyridinium chlorochromate (Corey's reagent) or pyridinium dichromate. With 11-
oxogeldanamycin in hand the benzoquinone may now be reduced to the hydroquinone, as
described above. It will be appreciated by a person of skill in the art that care must be taken to
not concomitantly reduce out the newly formed C-11 ketone, in other words the reducing agent
selected should be chemoselective for the benzoquinone system over the C-11 carbonyl, such
chemoselective agents are known to a person of skill in the art, an example of a suitable agent
(without limitation) is sodium hydrosulfite, unsuitable agents include, without limitation LiAIH4.
11-oxo-18,21-dihydrogeldanamycin can then be used as a template for further derivatisation
into a compound of Formula I, as described below.
Protecting groups may, if desired, be generally employed in the synthesis of compounds
of the invention and intermediate compounds as would be understood by a person skilled in the
art.
Hydroquinone ansamycins, as shown as Formula (III) converted if required from their
benzoquinone forms, may be used as templates for further semi-synthesis (i.e. process (a)).
Other compounds embraced by the invention may be prepared by methods described
herein and/or by methods known to a skilled person.
Compounds of formula (H) may be produced by methods known to a person of skill in
the art. One suitable method for the production of compounds of formula H is illustrated below,
but it will be appreciated that other, equally suitable, methods of production may be employed.


WO 2007/026027 PCT/EP2006/065941
17
Therefore, in one embodiment, a variety of self-cleaving and water solubilizing side-
chains (R5) can be introduced at the 18 and/or 21-position of 18,21-dihydromacbecin. This may
be achieved by treating a mono N-protected diamine of variable chain length and substitution
pattern (H) with an intermediate 4-nitrophenyl carbonate analogue of macbecin (e.g. a
compound of formula (II) above).
A primary amino alkyl benzyl ether (A), derived from protection group manipulation of an
amino alcohol, is reacted with benzaldehyde to give the Schiff base (B). The imine formed is
treated with an alkylating agent e.g. a trialkyloxonium tetrafluoroborate to introduce R10 and thus
generate an iminium salt (C). Hydride reduction to the benzylamine (D) and subsequent di-
debenzylation via catalytic hydrogenolysis provides secondary amines of type (E). N-Boc
protection to (F) is followed by hydroxyl activation to a suitable leaving group e.g. mesylate (G).
The Boc group of compounds (F), (G) and (H) in the diagram may be replaced by a different
protecting group if desired by treating compound (E) with an alternative protecting reagent. A
preferred alternative protecting group is trityl.
In process (c), salt formation and exchange may be performed by conventional methods
known to a person of skill in the art. Interconversions of compounds of formula (I) may be
performed by known processes for example hydroxy and keto groups may be interconverted by
oxidation/reduction as described elsewhere herein.
In processes (a), (b) and (d), examples of protecting groups and the means for their
removal can be found in T W Greene "Protective Groups in Organic Synthesis" (J Wiley and
Sons, 1991). Suitable hydroxyl protecting groups include alkyl (e.g. methyl), acetal (e.g.
acetonide) and acyl (e.g. acetyl or benzoyl) which may be removed by hydrolysis, and arylalkyl
(e.g. benzyl) which may be removed by catalytic hydrogenolysis, or silyl ether, which may be
removed by acidic hydrolysis or fluoride ion assisted cleavage. Suitable amine protecting
groups include sulphonyl (e.g. tosyl), acyi (e.g. benzyloxycarbonyl or t-butoxycarbonyl) and
arylalkyl (e.g. benzyl) which may be removed by hydrolysis or hydrogenolysis as appropriate.
Compound of formula (I) in which the C18 R5 is H may be prepared by deprotecting
compounds of formula (IVA)-(IVC) (collectively known as "compounds of formula (IV)"):


WO 2007/026027 PCT/EP2006/065941
18

wherein R1, R2, R3 and R5 are as defined above (save that R5 does not represent H) and
Pa represents a protecting group, or a further protected derivative thereof. Exemplary hydroxyl
protecting groups Pa include those mentioned above, especially silyl ethers. Deprotection by
removal of the silyl group may be achieved by hydrolysis under conventional conditions.
Compounds of formula (IV) or a further protected derivative thereof may be prepared by
reacting a compound of formula (VA)-(VC) (collectively known as "compounds of formula (V)"):

wherein L is a leaving group
or a protected derivative thereof, with a compound of formula (H).
Suitable conditions include those given above for processes (a) and (b).
Compounds of formula (V) or a protected derivative thereof may be prepared by reacting
a compound of formula (VIA)-(VIC) (collectively known as "compounds of formula (VI)"):

WO 2007/026027 PCT/EP2006/065941
19

or a protected derivative thereof, with a compound of formula (J).
Suitable conditions include those given above for the reaction of compounds of formula
(III) with compound of formula (J).
Compounds of formula (VI) or a protected derivative thereof may be prepared by
protecting compounds of formula (III). When Pa represent a silyl ether, protection may be
achieved by treating the compound of formula (III) with trialkylsilyl chloride or trialkylsilyl triflate
in the presence of base.
Other compounds of the invention may be prepared by methods known per se or by
methods analogous to those described above.
The compounds of the invention are useful directly, and as templates for further semi-
synthesis or bioconversion, to produce compounds useful as anticancer agents. Methods for
the semi-synthetic derivatisation of ansamycins such as geldanamycin and related compounds
are well known in the art and include (but are not limited to) those modifications described in
e.g. WO 03/013430, WO 02/079167, WO 03/066005.
In particular, the compounds of the invention have utility as pro-drugs, they may be
chemically cleaved to produce the active parent compound. Cleavage assays to assess the
rate of cleavage are known in the art and are
The above structures of intermediates may be subject to tautomerisation and where a
representative tautomer is illustrated it will be understood that and all tautomers for example
keto compounds where enoi compounds are illustrated and vice versa are intended to be

WO 2007/026027 PCT/EP2006/065941
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referred to.
Novel compounds of formula (II), (III), (IV), (V) and (VI) and protected derivatives thereof
are also claimed as an aspect of the invention.
The invention additionally provides for the use of a compound of the invention in the
treatment of cancer or B-cell malignancies. It also provides a compound of the invention for use
in the treatment of cancer or B-cell malignancies. It also provides a method of treatment of
cancer or B-cell malignancies which comprises administering to a patient an effective amount of
a compound of the invention. It also provides the use of a compound of the invention in the
preparation of a medicament for the treatment of cancer or B-cell malignancies.
Ansamycins are also known to have utilities in the treatment of other conditions,
including, but not limited to, the treatment of cardiac arrest and stroke (US 6,174,875, WO
99/51223), the treatment of fibrogenic disorders (WO 02/02123), the treatment or prevention of
restenosis (WO 03/079936), the treatment or prevention of diseases associated with protein
aggregation and amyloid function (WO 02/094259), the treatment of peripheral nerve damage
and the promotion of nerve regeneration (WO 01/03692, US 6,641,810, EP 1 024 806, US
2002/0086015, US 6,210,974, WO 99/21552, US 5,968,921) and the inhibition of angiogenesis
(WO 04/000307). The uses and methods involving the compounds of the invention also extend
to these other indications.
In a preferred embodiment, the present invention provides compounds with utility in the
treatment of cancer. One skilled in the art would be able by routine experimentation to
determine the ability of these compounds to inhibit tumour cell growth, (see Tian et al., 2004; Hu
et al. 2004; Dengler et al, 1995).
The present invention also provides a pharmaceutical composition comprising an
ansamycin derivative, or a pharmaceutically acceptable salt thereof, together with a
pharmaceutically acceptable carrier.
The existing ansamycin HSP90 inhibitors that are or have been in clinical trials, such as
geldanamycin, 17-AAG and 17-DMAG have poor pharmacological profiles, poor water solubility
and poor bioavailability. The present invention provides ansamycin derivatives which have
improved properties such as solubility and/or bioavailability. A person of skill in the art will be able
to readily determine the solubility of a given compound of the invention using standard methods. A
representative method is shown in the examples herein.
Additionally, a person of skill in the art will be able to determine the pharmacokinetics and
bioavailability of a compound of the invention using in vivo and in vitro methods known to a person
of skill in the art, including but not limited to those described below and in Egorin MJ et al., (2002).
The bioavailability of a compound is determined by a number of factors, (e.g. water solubility, rate
of absorption in the gut, the extent of protein binding and metabolism) each of which may be
determined by in vitro tests as described below, it will be appreciated by a person of skill in the art

WO 2007/026027 PCT/EP2006/065941
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that an improvement in one or more of these factors will lead to an improvement in the
bioavailability of a compound. Alternatively, the bioavailability of a compound may be measured
using in vivo methods as described in more detail below.
In vitro assays
a) Caco-2 permeation assay
Confluent Caco-2 cells (Li, A.P., 1992; Grass, G.M., et al., 1992, Volpe, D.A., et al.,
2001) in a 24 well Corning Costar Transwell format are used to establish the permeability and
efflux rate of compounds using methods as described herein, suitable formats include those
provided by In Vitro Technologies Inc. Baltimore, Maryland, USA. In a suitable format the apical
chamber contains 0.15 mL HBBS pH 7.4, 1% DMSO, 0.1 mM Lucifer Yellow and the basal
chamber contains 0.6 mL HBBS pH 7.4, 1% DMSO. Controls and test assays are incubated at
37 °C in a humidified incubator, shaken at 130 rpm. Lucifer Yellow is able to permeate via the
paracellular route only (i.e. between the tight junctions), a high Apparent Permeability (Papp)for
Lucifer Yellow indicates cellular damage during assay and all such wells are rejected. Suitable
reference controls in addition to the parent compound include propranolol, which has good
passive permeation with no known transporter effects and acebutalol, which has poor passive
permeation attenuated by active efflux by P-glycoprotein.
Compounds are tested in a uni- and bi-directional format by applying compound to the
apical or basal chamber (at 0.01 mM). Compounds in the apical or basal chambers are
analysed by LC-MS. Results are expressed as Apparent Permeability, Papp, (nm/s) and as the
Flux Ratio (A to B versus B to A).

A positive value for the Flux Ratio indicates active efflux from the apical surface of the
cells. Therefore, improved bioavailability is shown in the above assay by an increased Papp
and/or a decreased flux ratio for the compound of the invention relative to its parent molecule.
b) Human Liver Microsomal (HLM) stability assay
Increased metabolic stability is also associated with improved bioavailability, this may be
determined using a HLM assay for example as described below. Liver homogenates provide a

WO 2007/026027 PCT/EP2006/065941
22
measure of a compounds inherent vulnerability to Phase I (oxidative) enzymes, including
CYP450S (e.g. CYP2C8, CYP2D6, CYP1A, CYP3A4, CYP2E1), esterases, amidases and flavin
monooxygenases (FMOs).
The half life (T1/2) of compounds can be determined, on exposure to Human Liver
Microsomes, by monitoring their disappearance over time by LC-MS. Compounds at 0.001 mM
are incubated at for 40 min at 37 °C, 0.1 M Tris-HCI, pH 7.4 with a human microsomal sub-
cellular fraction of liver at 0.25 mg/mL protein and saturating levels of NADPH as co-factor. At
timed intervals, acetonitrile is added to test samples to precipitate protein and stop metabolism.
Samples are centrifuged and analysed for parent compound.
Improved bioavailability is shown in the above assay by an increased T1/2 relative to the parent
compound.
In vivo assays
In vivo assays may also be used to measure the bioavailability of a compound.
Generally, a compound is administered to a test animal (e.g. mouse or rat) both intraperitoneally
(i.p.) or intravenously (i.v.) and orally (p.o.) and blood samples are taken at regular intervals to
examine how the plasma concentration of the drug varies over time. The time course of plasma
concentration overtime can be used to calculate the absolute bioavailability of the compound as
a percentage using standard models. An example of a typical protocol is described below.
Mice are dosed with 1, 10, or 75 mg/kg of the compound of the invention or the parent
compound i.p. i.v. or p.o.. Blood samples are taken at 5, 10, 15, 30, 45, 60, 90, 120, 180, 240,
360, 420 and 2880 minutes and the concentration of the compound of the invention or parent
compound in the sample is determined via HPLC. The time-course of plasma concentrations
can then be used to derive key parameters such as the area under the plasma concentration-
time curve (AUC - which is directly proportional to the total amount of unchanged drug that
reaches the systemic circulation), the maximum (peak) plasma drug concentration, the time at
which maximum plasma drug concentration occurs (peak time), additional factors which are
used in the accurate determination of bioavailability include: the compound's terminal half life,
total body clearance, steady-state volume of distribution and F%. These parameters are then
analysed by non-compartmental or compartmental methods to give a calculated percentage
bioavailability, for an example of this type of method see Egorin et al 2002, and references
therein.
The aforementioned compounds of the invention or a formulation thereof may be
administered by any conventional method for example but without limitation they may be
administered parenteraliy (including intravenous administration), orally, topically (including buccal,
sublingual or transdermal), via a medical device (e.g. a stent), by inhalation, or via injection

WO 2007/026027 PCT/EP2006/065941
23
(subcutaneous or intramuscular). The treatment may consist of a single dose or a plurality of
doses over a period of time.
Whilst it is possible for a compound of the invention to be administered alone, it is
preferable to present it as a pharmaceutical formulation, together with one or more acceptable
carriers. Thus there is provided a pharmaceutical composition comprising a compound of the
invention together with one or more pharmaceutically acceptable diluents or carriers. The
diluents(s) or carrier(s) must be "acceptable" in the sense of being compatible with the compound
of the invention and not deleterious to the recipients thereof. Examples of suitable carriers are
described in more detail below.
The compounds of the invention may be administered alone or in combination with other
therapeutic agents. Co-administration of two (or more) agents may allow for significantly lower
doses of each to be used, thereby reducing the side effects seen. There is also provided a
pharmaceutical composition comprising a compound of the invention and a further therapeutic
agent together with one or more pharmaceutically acceptable diluents or carriers.
In a further aspect, the present invention provides for the use of a compound of the
invention in combination therapy with a second agent for the treatment of cancer or B-cell
malignancies.
In one embodiment, a compound of the invention is co-administered with another
therapeutic agent for the treatment of cancer or B-cell malignancies preferred agents include, but
are not limited to, methotrexate, leukovorin, adriamycin, prenisone, bleomycin,
cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine,
doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2
monoclonal antibody (e.g. Herceptin™), capecitabine, raloxifene hydrochloride, EGFR inhibitors
(e.g. Iressa ®, Tarceva™, Erbitux™), VEGF inhibitors (e.g. Avastin™), proteasome inhibitors
(e.g. Velcade™) or Glivec® . Additionally, a compound of the invention may be administered in
combination with other therapies including, but not limited to, radiotherapy or surgery.
The formulations may conveniently be presented in unit dosage form and may be prepared
by any of the methods well known in the art of pharmacy. Such methods include the step of
bringing into association the active ingredient (compound of the invention) with the carrier which
constitutes one or more accessory ingredients. In general the formulations are prepared by
uniformly and intimately bringing into association the active ingredient with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the product.
The compounds of the invention will normally be administered orally or by any parenteral
route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in
the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically
acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the
route of administration, the compositions may be administered at varying doses.

WO 2007/026027 PCT/EP2006/065941
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For example, the compounds of the invention can be administered orally, buccally or
sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may
contain flavouring or colouring agents, for immediate-, delayed- or controlled-release
applications.
Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium
citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch
(preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and
certain complex silicates, and granulation binders such as polyvinylpyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and
acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl
behenate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high
molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds
of the invention may be combined with various sweetening or flavouring agents, colouring
matter or dyes, with emulsifying and/or suspending agents and with diluents such as water,
ethanol, propylene glycol and glycerin, and combinations thereof.
A tablet may be made by compression or moulding, optionally with one or more accessory
ingredients. Compressed tablets may be prepared by compressing in a suitable machine the
active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a
binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,
disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by
moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid
diluent. The tablets may optionally be coated or scored and may be formulated so as to provide
slow or controlled release of the active ingredient therein using, for example,
hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
Formulations in accordance with the present invention suitable for oral administration may
be presented as discrete units such as capsules, cachets or tablets, each containing a
predetermined amount of the active ingredient; as a powder or granules; as a solution or a
suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a
water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or
paste.
Formulations suitable for topical administration in the mouth include lozenges comprising
the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and
acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

WO 2007/026027 PCT/EP2006/065941
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It should be understood that in addition to the ingredients particularly mentioned above the
formulations of this invention may include other agents conventional in the art having regard to the
type of formulation in question, for example those suitable for oral administration may include
flavouring agents.
Pharmaceutical compositions adapted for topical administration may be formulated as
ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings,
sprays, aerosols or oils, transdermal devices, dusting powders, and the like. These compositions
may be prepared via conventional methods containing the active agent. Thus, they may also
comprise compatible conventional carriers and additives, such as preservatives, solvents to assist
drug penetration, emollient in creams or ointments and ethanol or oleyl alcohol for lotions. Such
carriers may be present as from about 1 % up to about 98% of the composition. More usually they
will form up to about 80% of the composition. As an illustration only, a cream or ointment is
prepared by mixing sufficient quantities of hydrophilic material and water, containing from about 5-
10% by weight of the compound, in sufficient quantities to produce a cream or ointment having the
desired consistency.
Pharmaceutical compositions adapted for transdermal administration may be presented as
discrete patches intended to remain in intimate contact with the epidermis of the recipient for a
prolonged period of time. For example, the active agent may be delivered from the patch by
iontophoresis.
For applications to external tissues, for example the mouth and skin, the compositions are
preferably applied as a topical ointment or cream. When formulated in an ointment, the active
agent may be employed with either a paraffinic or a water-miscible ointment base.
Alternatively, the active agent may be formulated in a cream with an oil-in-water cream
base or a water-in-oil base.
For parenteral administration, fluid unit dosage forms are prepared utilizing the active
ingredient and a sterile vehicle, for example but without limitation water, alcohols, polyols,
glycerine and vegetable oils, water being preferred. The active ingredient, depending on the
vehicle and concentration used, can be either suspended or dissolved in the vehicle. In
preparing solutions the active ingredient can be dissolved in water for injection and filter
sterilised before filling into a suitable vial or ampoule and sealing.
Advantageously, agents such as local anaesthetics, preservatives and buffering agents
can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after
filling into the vial and the water removed under vacuum. The dry lyophilized powder is then
sealed in the vial and an accompanying vial of water for injection may be supplied to
reconstitute the liquid prior to use.
Parenteral suspensions are prepared in substantially the same manner as solutions,
except that the active ingredient is suspended in the vehicle instead of being dissolved and

WO 2007/026027 PCT/EP2006/065941
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sterilization cannot be accomplished by filtration. The active ingredient can be sterilised by
exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a
surfactant or wetting agent is included in the composition to facilitate uniform distribution of the
active ingredient.
The compounds of the invention may also be administered using medical devices known in
the art. For example, in one embodiment, a pharmaceutical composition of the invention can be
administered with a needleless hypodermic injection device, such as the devices disclosed in U.S.
5,399,163; U.S. 5,383,851; U.S. 5,312,335; U.S. 5,064,413; U.S. 4,941,880; U.S. 4,790,824; or
U.S. 4,596,556. Examples of well-known implants and modules useful in the present invention
include : US 4,487,603, which discloses an implantable micro-infusion pump for dispensing
medication at a controlled rate; US 4,486,194, which discloses a therapeutic device for
administering medicaments through the skin; US 4,447,233, which discloses a medication infusion
pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable
flow implantable infusion apparatus for continuous drug delivery; US 4,439,196, which discloses an
osmotic drug delivery system having multi-chamber compartments; and US 4,475,196, which
discloses an osmotic drug delivery system. Many other such implants, delivery systems, and
modules are known to those skilled in the art.
The dosage to be administered of a compound of the invention will vary according to the
particular compound, the disease involved, the subject, and the nature and severity of the disease
and the physical condition of the subject, and the selected route of administration. The appropriate
dosage can be readily determined by a person skilled in the art.
The compositions may contain from 0.1% by weight, preferably from 5-60%, more
preferably from 10-30% by weight, of a compound of invention, depending on the method of
administration.
It will be recognized by one of skill in the art that the optimal quantity and spacing of
individual dosages of a compound of the invention will be determined by the nature and extent of
the condition being treated, the form, route and site of administration, and the age and condition of
the particular subject being treated, and that a physician will ultimately determine appropriate
dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop
the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal
clinical practice.
EXAMPLES
General Methods
Fermentation of cultures
Conditions used for growing the bacterial strains Actinosynnema pretiosum subsp.
pretiosum ATCC 31280 (US 4,315,989) and Actinosynnema mirum DSM 43827 (KCC A-0225,

WO 2007/026027 PCT/EP2006/065941
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Watanabe et al., 1982) are described in the patents US 4,315,989 and US 4,187,292. Both
strains may be grown on ISP2 agar (Shirling, E.B. and Gottlieb, D. (1966) International Journal
of Systematic Bacteriology 16:313-340) at 28 °C for 2-3 days and used to inoculate seed
medium (20 parts of glucose, 30 parts of soluble starch, 10 parts of corn steep liquor, 10 parts
of soybean flour, 5 parts of peptone, 3 parts of sodium chloride and 5 parts of calcium
carbonate/water 1000 parts by volume, pH 7.0, see US 4,315,989 and US 4,187,292). The
inoculated seed medium is incubated shaking at 28 °C for 48 h. For production of macbecin and
18,21-dihydromacbecin the fermentation medium (5% of glycerol, 2% of corn steep liquor, 2%
of yeast extract, 2% of KH2PO4, 0.5% of MgCI2 and 0.1% of CaCO3, pH6.5, see US 4,315,989
and US 4,187,292) is initially incubated at 28 °C for 24 h followed by an incubation period at 26
°C for four to six days. The culture is then harvested for extraction.
Extraction of culture broths for LCMS analysis
Culture broth (1 mL) and ethyl acetate (1 mL) is added and mixed for 15-30 min followed
by centrifugation for 10 min. 0.5 mL of the organic layer is collected, evaporated to dryness and
then re-dissolved in 0.25 mL of methanol.
LCMS analysis procedure for fermentation broth analysis and in vivo transformation studies
LCMS is performed on an integrated Agilent HP1100 HPLC system in combination with
a Bruker Daltonics Esquire 3000+ electrospray mass spectrometer operating in positive ion
mode. Chromatography was achieved over a Phenomenex Hyperclone column (C18 BDS, 3u,
150 x 4.6 mm) eluting over 11 min at a flow rate of 1 mL/min with a linear gradient from
acetonitrile / water (40/60) to acetonitrile / water (80/20).
Synthesis
All reactions are conducted under anhydrous conditions unless stated otherwise, in oven
dried giassware that is cooled under vacuum, using dried solvents. Reactions are monitored by
LC-UV-MS, on an Agilent 1100 HPLC coupled to a Bruker Daltonics Esquire3000 electrospray
mass spectrometer, switching between positive ion and negative ion modes for alternate scans.
Chromatography was achieved over a Phenomenex Hyperclone column, BDS C18 3u (150 x
4.6mm), with a linear gradient of acetonitrile:water (40:60 to 100) over 11 min at 1 mL/min.
Water Solubility Assay
Kinetic measurements:
Stock solutions of the compounds (10 mM) in DMSO were prepared. Aliquots (0.01 mL)
of each were made up to 0.5 mL with either PBS solution or DMSO. The resulting 0.2 mM
solutions were shaken for at room temperature on an IKA© vibrax VXR shaker.

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After shaking the resulting solutions or suspensions were transferred to 2 mL Eppendorf
tubes and centrifuged for 30 minutes at 13200 rpm. Aliquots of the supernatant fluid were then
analysed by an Agilent 1100 HPLC coupled to a Bruker Daltonics Esquire3000 electrospray
mass spectrometer (for details see specific examples herein). Chromatography was achieved
over a Phenomenex Hyperclone column, BDS Ci8 3u (150 x 4.6 mm), with a linear gradient of
acetonitrile:water (40:60 to 100) over 11 min at 1 mL/min. UV absorbance was monitored at A =
258 and 280 nm.
All analyses were performed in triplicate and the solubilities of individual compounds
calculated by comparing their solubility in PBS with an assumed solubility of 100 % in DMSO at
0.2 mM.
Thermodvnamic measurements:
The appropriate amounts of compound to make solutions of the compounds at 2,5,10
and or 20 mM were mixed with the appropriate amounts of 5% glucose and with DMSO in
brown glass vials and shaken at room temperature on an IKA® vibrax VXR shaker. After 6
hours the resulting suspensions/solutions were centrifuged for 20 min at 13200 rpm.
Aliquots of the supernatant fluid are then analysed by an Agilent 1100 HPLC coupled to
a Bruker Daltonics Esquire3000 electrospray mass spectrometer (for details see specific
examples herein). Chromatography was achieved over a Phenomenex Hyperclone column,
BDS C18 3u (150 x 4.6mm), with a linear gradient of acetonitrile:water (40:60 to 100) over 11
min at 1 mL/min. UV absorbance was monitored at A = 258 and 280 nm.
All analyses were performed in triplicate and the solubilities of individual compounds
calculated by comparing their solubility in 5% glucose with an assumed solubility of 100 % in
DMSO.
Cleavage assays for pro-drugs of 18,21-dihydromacbecin
Assays to determine the rate of cleavage of the pro-drugs into their parent compounds
were performed as described herein. These were carried out in plasma, blood or buffer. Mixed
mouse, human or mixed rat plasma containing EDTA or heparin as an anti-coagulant was used
or whole defibrinated rabbit blood (mouse and rat plasma and rabbit blood were obtained from
Harlan-Sera Labs, human plasma was obtained from Biopredic International),
If using plasma the following protocol was used: The plasma was thawed at 37 °C in a
water bath. Plasma was dispensed into individual 20 mL tubes for each test compound allowing
0.5 mL plasma for each time point to be analysed. A tube containing plasma was also included
as a control. These tubes were incubated for 15 minutes at 37°C.
Each test compound was reconstituted in DMSO or other appropriate solvent. The
plasma was removed from the water bath and the compound was added to the plasma to give a
final concentration ot 0.001-0.010 mg/mL keeping the final concentration of solvent beiow 5%.

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In the case of the control, an equivalent volume of DMSO or other solvent was added.
Immediately after the addition of compound a 0.4 mL sample was taken from the tube,
transferred to a 2 mL microfuge tube. The plasma was promptly returned to the 37°C water
bath. 0.4 mL samples were taken at regular time points (e.g. after 30 min and then hourly) and
were immediately frozen at -80 °C.
The stability of the test compounds in phosphate-buffered saline (PBS) of specific pH
over the course of each experiment was also analysed using the same methodology.
After the final time point was sampled, the samples were extracted as follows.
For each time point sample an Oasis MCX (3cc/60mg) or Oasis HLB (333/60 mg) cartridge was
conditioned and equilibrated with 2 mL methanol followed by 2 mL water. 1.5 mL water and an
internal standard (IS) at 0.002 mg/mL were added to eac0.4 mL plasma sample. This was
loaded to the cartridge. The cartridge was washed with 2mL water and the analytes were eluted
in 2 ml MeOH. This extract was analysed without further treatment by LC/MS using the
conditions described below.
If using whole blood such as defibrinated whole rabbit blood the following protocol was
used: The blood was thawed at 37 °C in a water bath. Blood was dispensed into individual 20
mL tubes for each test compound allowing 0.5 mL for each time point to be analysed. A tube
containing blood was also included as a control. These tubes were incubated for 15 minutes at
37°C.
Each test compound was reconstituted in DMSO or other appropriate solvent. The blood
was removed from the water bath and the compound was added to give a final concentration of
0.001-0.010 mg/mL keeping the final concentration of solvent below 5%. In the case of the
control, an equivalent volume of DMSO or other solvent was added. Immediately after the
addition of compound a 0.4 mL sample was taken from the tube, transferred to a 2 mL
microfuge tube. The plasma was promptly returned to the 37°C water bath. 0.4 mL samples
were taken at regular time points (e.g. after 30 min and then hourly) and were immediately
frozen at -80 °C. To each 0.4 mL sample in its 2 mL microfuge tube was added 1.5 mL of 0.1 M
potassium dihydrogen phosphate, this was inverted to mix. A stable internal standard (IS) in
acetonitrile was added at 0.002mg/mL. This was agitated for 5 min on an IKA shaker at 1500
rpm. Centrifugation at 5000 rpm for 5 min lead to a dense pellet made up of red blood cells and
precipitated protein, and a pink/red supernatant. An Oasis HLB (333/60 mg) cartridge was
equilibrated with 2 mL of methanol followed by 2 mL of water. 1.5 mL of the supernatant was
transferred to the cartridge and allowed to flow through with application of mild positive pressure
when necessary. Each cartridge was dried by flashing it with a volume of air from a syringe and
analytes eluted with 1 mL of acetonitrile. 0.01 mL of 10 % formic acid was added to the eluant
which was then transferred to an HPLC vial for analysis.
The different LCMS analytical procedures used to measure the cleavage rates were:

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Method A
Injection volume: 0.03 ml_. HPLC was performed on a Phenomenex Hyperclone 3
micron BDS C18 column, 150 mm x 4.60 mm, running a mobile phase of:
Mobile phase A: 0.1% Formic acid in water
Mobile phase B: 0.1% Formic acid in acetonitrile
Flow rate: 1 mL/minute.
The HPLC conditions were: 30 % B for 2 min followed by a linear gradient to 100 %B
over a period of 14 min and an isocratic period of 4 min at 100 % B. The analytes were detected
by UV absorbance at 255 nm and mass spectrometry using a Bruker Daltonics Esquire 3000+
mass spectrometer coupled to the HPLC. The analytes were quantified based on the extracted
ion chromatogram. To ensure a reliable quantification, the linear response of the mass
spectrometer was checked for the concentration range of interest.
Method B
Injection volume: 0.030 mL. HPLC was performed on a Waters Symmetry C8 3.5 micron
column, 50 mm x 2.1 mm, running a mobile phase of:
Mobile phase A: 0.1% Formic acid in water
Mobile phase B: 0.1% Formic acid in acetonitrile
Flow rate: 1 mL/minute.
The HPLC conditions were: 10 % B for 1 min followed by a linear gradient to 100 %B
over a period of 7 min and an isocratic period of 2 min at 100 % B. The analytes were detected
by UV absorbance at 255 nm and mass spectrometry using a Bruker Daltonics Esquire 3000+
mass spectrometer coupled to the HPLC. The analytes were quantified based on the extracted
ion chromatogram. To ensure a reliable quantification, the linear response of the mass
spectrometer was checked for the concentration range of interest.
Method C
Injection volume: 0.02 mL. HPLC was performed on a Phenomenex Hyperclone 3
micron BDS C18 column, 150 mm x 4.60 mm, running a mobile phase of:
Mobile phase A: 0.1 % Formic acid in water
Mobile phase B: 0.1% Formic acid in acetonitrile
Flow rate: 1 mL/minute.
The HPLC conditions were: 10 % B for 1 min followed by a linear gradient to 100 %B
over a period of 7 min and an isocratic period of 2 min at 100 % B. The analytes were detected
by UV absorbance at 255 nm and mass spectrometry using a Bruker Daltonics Esquire 3000+
mass spectrometer coupled to the HPLC. The analytes were quantified based on the extracted
ion chromatogram. To ensure a reliable quantification, the linear response of the mass
spectrometer was checked for the concentration range of interest.

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In vitro bioassay for anticancer activity
In vitro evaluation of compounds for anticancer activity in a panel of 12 human tumour
cell lines in a monolayer proliferation assay may be carried out at the Oncotest Testing Facility,
Institute for Experimental Oncology, Oncotest GmbH, Freiburg. The characteristics of the 12
selected cell lines are summarised in Table 3.

The Oncotest celi lines were established from human tumor xenografts as described by
Roth et al., (1999). The origin of the donor xenografts is described by Fiebig et al., (1999).
Other cell lines were either obtained from the NCI (H460, SF-268, OVCAR-3, DU145, MDA-MB-
231, MDA-MB-468) or purchased from DSMZ, Braunschweig, Germany (LNCAP).
All cell lines, unless otherwise specified, were grown at 37 °C in a humidified
atmosphere (95 % air, 5 % CO2) in a 'ready-mix' medium containing RPMI 1640 medium, 10 %
fetal calf serum, and 0.1 mg/mL gentamicin (PAA, Colbe, Germany).
Monolayer assay - brief description of protocol:
A modified propidium iodide assay was used to assess the effects of the test
compound(s) on the growth of twelve human tumour cell lines (Dengler et al., (1995)).
Briefly, cells were harvested from exponential phase cultures by trypsinization, counter]
and plated in 96 well flat-bottomed microtitre plates at a cell density dependent on the cell line
(5 - 10.000 viable cells/well). After 24 h recovery to allow the cells to resume exponential

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growth, 0.010 ml_ of culture medium (6 control wells per plate) or culture medium containing
macbecin was added to the wells. Each concentration was plated in triplicate. Compounds were
applied in two concentrations (0.001 mM and 0.01 rnM). Following 4 days of continuous
exposure, cell culture medium with or without test compound is replaced by 0.2 ml_ of an
aqueous propidium iodide (PI) solution (7 mg/L). To measure the proportion of living cells, cells
are permeabilized by freezing the plates. After thawing the plates, fluorescence was measured
using the Cytofluor 4000 microplate reader (excitation 530 nm, emission 620 nm), giving a
direct relationship to the total number of viable cells.
Growth inhibition is expressed as treated/control x 100 (% T/C).
In vivo evaluation of antitumor efficacy - brief description of protocol:
PRXF DU-145 is a prostate cancer cell line initially isolated from a metastatic central
nervous system lesion of a 69-year-old man with prostate carcinoma. PRXF DU-145 cell
suspensions were injected subcutaneously into nude mice and the resulting xenografts were
passaged in nude mice until establishment of a stable growth pattern.
Tumour fragments were obtained from xenografts in serial passage in nude mice. After
removal of tumours from donor mice, they were cut into fragments (1-2 mm diameter) and
placed in RPMI 1640 culture medium (RPMI 1640 medium with 25 mM HEPES buffer with L-
Glutamine, Gibco, catalogue # 52400-025) until subcutaneous implantation. Recipient mice
were anaesthetized by inhalation of isoflurane. For bilateral implantation a small incision was
made in the skin of the back. Tumour fragments were transplanted with tweezers.
At randomization, tumour bearing animals were stratified into treatment and control
groups according to tumour volume, using "Lindner's Randomization Tables". Only animals
carrying a tumour of appropriate size (mean tumor diameter: 6-8 mm, minimum acceptable
tumor diameter: 5 mm) were considered for randomization. Mice were randomized when a
maximum number of mice qualified for randomization. The day of randomization was
designated as Day 0. Day 0 was also the first day of dosing. Tumour growth was then
compared between mice given different dosing regimes. Mice were monitored daily, with the
observation period lasting 42 Days.
The tumour volume was determined by two-dimensional measurement with a caliper on
the day of randomization (Day 0) and then twice weekly. Tumour volumes were calculated
according to the formula: (a x b2) x 0.5 where a represents the largest and b the perpendicular
tumor diameter,
Antitumour activity was evaluated as maximum tumour volume inhibition versus the
vehicle control group.

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Rel. volumes of individual tumors (RTVs) were calculated by dividing the individual
tumor volume on Day X (Tx) by the individual tumor volume on Day 0 (TO) multiplied by 100%.

Group tumor volumes were expressed as the median RTV of all tumours in a group
(group median RTV). Group median RTV values were used for drawing growth curves and for
treatment evaluation.
For each group, tumour inhibition on a particular day (T/C in %) was calculated from the
ratio of the median RTV values of the respective test group and the vehicle control group,
multiplied by 100%.

The minimum (or optimum) T/C% value recorded for a particular test group during an
experiment represents the maximum antitumour activity for the respective treatment.
For the evaluation of the statistical significance of tumour inhibition, the U-test by Mann-
Whitney-Wilcoxon was performed. The test compares the ranking of individual tumours
according to relative volume in the vehicle control group on the one hand and in the test group
of interest on the other. By convention, p-values <0.05 indicate significance of tumour inhibition.
Intermediate 1: Production of macbecin using Actinosynnema mirum DSM 43827 &
Actinosynnema pretiosum subsp. pretiosum ATCC 31280 in falcon tubes
Falcon tubes containing 10 mL of seed medium were inoculated with an agar plug cut
from a dense A. mirum lawn grown on ISP2 agar and incubated as described in General
Methods. The seed culture (0.5 mL) was used for inoculation of 10 mL of production medium
followed by incubation and extraction as described in the General Methods section. Analysis by
LCMS indicated the presence of macbecin ([M+Na]', m/z = 581.3) which eluted at 9.5 min.
Intermediate 1 (alternative preparation): Production of macbecin using Actinosynnema
mirum DSM 43827 in a 15 litre fermenter
Five 2 L conical shaking flasks were prepared with 300 mL of seed medium each and
inoculated with 15 agar plugs per flask cut from a densely grown lawn of A. mirum. The

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cultures were shaken at 28 °C for 48 h. These seed cultures were used to inoculate (10 %
inoculum) a fermentation vessel containing 15 L of production medium. The fermentation was
carried out with an impeller tip speed of between 1.18 m/s and 2.75 m/s, an air flow of 1 vvm;
these were heated at 28 oC for 24 h then reduced to 24 "C. The pH was adjusted between pH
6.5 - 7 with 1 M H2SO4 and 1 M NaOH during the fermentation run. The baffles were tilted at 45
" and impeller tip speed was adjusted according to oxygen demand (minimum dissolved oxygen
was maintained at 30 %). Antifoam SAG471 was added at 0.2 % v/v prior to autoclaving and
then as and when required during the fermentation run. The fermenter was harvested after 230
h. Analysis by LCMS indicated the presence of macbecin ([M+Na]', m/z = 581.3) which eluted
after 9.5 min.
The fermentation broth was centrifuged for 30 min at 3500 rpm to separate the cells from
the supernatant. The supernatant was then partitioned three times with an equal volume of ethyl
acetate. The cell pellet was extracted twice with an equal volume of acetone. The organic
fractions were combined and the solvent removed in vacuo. The resulting aqueous slurry was
extracted three times with an equal volume of ethyl acetate and the combined organic fractions
concentrated to yield a crude extract.
Flash Silica gel was added to the crude extract previously dissolved in acetone and the
solvent then removed in vacuo. The impregnated silica was added onto an open column of flash
silica pre-conditioned with hexane. This was eluted with a step gradient of hexane:ethyl acetate
(100:0, 80:20, 75:25, 70:30, 50:50, 0:100) and finally ethyl acetate:methanol (50:50, 0:100).
Each step fraction volume was about one litre of solvent mixture. The fractions containing
macbecin were combined and evaporated to dryness. Further purification was realised by
preparative reversed-phase HPLC over a Phenomenex Luna column (C18,10 micron, 250 x 5u,
250 x 21.20 mm) eluting over 30 min with a gradient of eluant A (acetonitrile:water, 20:80) to
eluant B (acetonitrile) at a flow rate of 21 mL/min. Fractions containing macbecin were
combined and concentrated to yield to a yellow powder (45 mg). The structure of macbecin (in
CDCl3) was confirmed by multidimensional NMR spectroscopy using a Bruker Advance 500
MHz cryoprobe instrument, see Table 4. This was consistent with published data (Muroi et al.
1981).



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Example 1: Synthesis of 18O-(N,N'-dimethylethylenediamine-N'-carbamoyl)-18,21-
dihydromacbecin hydrochloride salt, 1 (Route 1)
Conversion of macbecin to 18,21 -dihydromacbecin
Macbecin (107.8 mg, 0.193 mmol) was dissolved in ethyl acetate (25 mL) and treated
with 96 mM sodium hydrosulfite solution (3x5 mL). On each occasion the phases were
vigorously mixed in a separating funnel and the aqueous drained off. The organic layer goes
from an intense yellow colour to virtually colourless. This organic layer was then washed with
water (3x10 mL), before being dried with anhydrous sodium sulfate, filtered and the solvent
removed under reduced pressure to yield 18,21-dihydromacbecin as an off-white glassy solid
(105.0 mg, 0.187 mmol, 97 % isolated yield). 18,21-dihydromacbecin was used without any
further purification
LCMS: macbecin, RT = 8.2 minutes ([M-H], m/z = 557.5, [M+Na]\ m/z = 581.2) UV Amax = 256
(sh) nm; 18,21-dihydromacbecin, RT = 3.5 minutes ([M-H]", m/z = 559.5, [M+Na]', m/z = 583.3)
UV Amax = 302 nm.
Preparation of N-tert-Butoxycarbonyl-N,N'-dimethylethylenediamine
N,N-dimethylethylenediamine (1.0 g, 11.3 mmol) was dissolved in anhydrous
dichioromethane (10 mL) and was treated with triethylamine (1.6 mL, 11.3 mmol). The mixture

WO 2007/026027 PCT/EP2006/065941
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was cooled to 0 °C for the addition of di-tert-butyl dicarbonate (2.5 g, 11.3 mmol). The reaction
stirred for 30 min at 0 °C then 2 hours at room temperature. The reaction mixture was then
washed with water (10 mL) and the aqueous layer extracted with further portions of
dichloromethane (2x10 mL). The combined organic phases were dried over Na2SO4 and the
solvent removed in vacuo. Purification by column chromatography (40:8:1,
dichloromethane:methanol:aqueous ammonia) yielded (508 mg, 24 %) of the desired N-tert-
butoxycarbonyl-N,N'-dimethylethylenediamine as a colourless oil.
O-Acylation of 18,21-dihydromacbecin with N-(tert-butoxycarbonyl)-N,N'-
dimethylethylenediamine to yield 18-O-[(N-tert-butoxycarbonyl)-N, N'-dimethylethylenediamine-
N '-carbamoyl]-18,21 -dihydromacbecin
18,21-dihydromacbecin (2.00 mg, 3.6 x 10-3 mmol) was dissolved in anhydrous
degassed dichloromethane (1 mL). 4-Nitrophenylchloroformate (0.86 mg, 4.3 x 10"3 mmol) was
added followed by 2,6-lutidine (2.48 x 10-3 mL, 21.4 x 10-3 mmol). The reaction mixture was
heated to reflux for 4 hrs to form the intermediate carbonate. N-tert-Butoxycarbonyl-N, N-
dimethylethylenediamine (2.70 mg, 14.2 x 10-3 mmol) was added and the reaction heated to
reflux for a further 2 h. The reaction mixture was washed with water (2 mL) and the crude
material analysed. The data corresponded to the that for the desired 18-O-[(N-tert-
butoxycarbonyl)-N, N -dimethylethylenediamine-N'-carbamoyl]-18,21 -dihydromacbecin.
LCMS: 18-O-[(N-tert-Butoxycarbonyl)-N,N'dimethylethylenediamine-N'-carbamoyl]-18,21-
dihydromacbecin, RT = 6.8 minutes ([M-H]-, m/z = 773.4), UV Amax = 266 nm.
Deprotection of 18-O-[(N-tert-butoxycarbonyl)-N,N'-dimethylethylenediamine-N'-carbamoyl]-
18,21 -dihydromacbecin to yield 18-O-(N,N'-dimethylethylenediamine-N'-carbamoyl)-18,21-
dihydromacbecin hydrochloride salt
18-O-[(N-tert-butoxycarbonyl)-N,N'-dimethylethylenediamine-N-carbamoyl]-18,21-
dihydromacbecin (20 mg, 25.8 umol) was treated with excess 2 M HCl in diethyl ether until all
the starting material was consumed. The residue was purified by tituration with diethyl ether to
yield 18-O-(N,N'-dimethylethylenediamine-N'-carbamoy!)-18,21 -dihydromacbecin hydrochloride
salt (11 mg, 60%) as a pale yellow solid.
Direct infusion MS: 18-O-(N,N'-dimethylethylenediamine-N'-carbamoyl)-18,21 -dihydromacbecin,
[M-H]\ m/z = 673.5, [M+Na]', m/z - 697.4, [M+NH]\ m/z - 675.4.
Example 2: Synthesis of 18-O-(N,N'-dimethylethylenediamine-N'-carbamoyl)-18,21-
dihydromacbecin hydrochloride salt, 1 (Route 2)

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Preparation of 18-O-(4-nitrophenylcarbonate)-18,21 -dihydromacbecin
Macbecin II (0.30 g, 0.54 mmol) was dissolved in anhydrous dichloromethane (72 ml).
To this solution was added 4-nitrophenylchloroformate as a solid (0.183 g, 0.91 mmol) followed
by 2,6-lutidine (0.217 ml, 1.87 mmol). The reaction mixture was heated at reflux under argon for
5 hours at 50°C (oil bath). The reaction was allowed to cool to ambient and washed
successively with equal volumes of 1N HCl and water, dried over Na2SO4 and filtered, and the
solvent removed under reduced pressure. The resulting material was purified over silica gel
eluting with a stepped gradient of acetone in hexane (5-40% acetone, increasing in 5%
increments) to yield the title compound. Isolated yield: 0.310 g (79%). NMR spectra acquired in
CDCl3 at 400 MHz were consistent with the title compound.
LCMS: RT = 7.2 min ([M-H]-, m/z = 724.0).
Preparation of N-trityl-N,N'-dimethylethylenediamine
N,N'-dimethylethylenediamine (5.0 g, 56.7 mmol) was dissolved in dichloromethane (20
ml) under argon and then treated with trimethylamine (5.74 g, 56.7 mmol). The stirring mixture
was cooled to 0°C prior to drop wise slow addition of a solution of tritylchloride (15.82 g, 56.7
mmol) in dichloromethane (20 mL). Following complete addition of this solution the reaction
mixture was stirred at 0 °C for a further 30 min, at which point the cooling bath was removed
and the reaction allowed to warm up to room temperature. The mixture was stirred under argon
at room temperature overnight. The resulting solution was partitioned between dichloromethane
and water, and a white precipitate of the HCl salt of the product was observed. This mixture was
the treated with a 10% aqueous solution of K2CO3 (100 mL) which resulted in dissolution of the
white solid and partitioning of the two phases. The two phases were separated and the aqueous
phase extracted with dichloromethane (2 x 200 mL). The combined organic extracts were dried
over MgSO4, and the solvent removed under reduced pressure. The residue was purified over
silica gel eluting with dichioromethane:methanol:aqueous ammonia (80:5:0.5). Pure fractions
were collected and the solvents removed under reduced pressure. Remaining water/ammonia
was removed by addition of isopropyl alcohol followed by removal under reduced pressure (3 x
300ml) and then under high vacuum to yield the title compound as a pale yellow solid. Isolated
yield, 6.30 g (34%). 1H NMR spectra acquired in CDCl3 at 400 MHz were consistent with the title
compound.
Preparation of 18-O-(N~trityl-N,N'-dimethylethylenediamine-N'-carbamoyl)-18-21~
dihydromacbecin
18-O-(4--nitrophenylcarbonate)-18,21-dihydromacbecin (0.89 g, 1.23 mmol) was
dissolved in dichloromethane (40 mL). N-trityl-N,N!-dimethylethylenediamine (1.21 g, 3.68

WO 2007/026027 PCT/EP2006/065941
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mmol) in dichloromethane (40 mL) was then added and the solution heated at reflux for 2 h, and
then overnight at room temperature. TLC showed the presence of starting material and the
mixture was refluxed for a further 2 h. This was then cooled to room temperature, washed with
water, dried over Na2SO4 and the solvent removed under reduced pressure. The resulting
material was purified by chromatography over silica gel eluting with hexane:acetone (5:3) to
yield a pale green solid. Isolated yield: 0.92 g (82%). NMR spectra acquired in CDCl3 at 400
MHz were consistent with the title compound.
LCMS: RT - 11.1 min ([M-H]', m/z = 915.6 (weak); [M-H-trityl]-, m/z = 673.4; [M+H-trityl]+ m/z =
675.5).
Preparation of 18-O-(N,N'-dimethylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin
hydrochloride salt, 1
18-O-(N-trityl-N,N'-dimethylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin (0.10
g, 0.11 mmol) was dissolved in anhydrous dichloromethane (40 mL) under argon and cooled to
-5 °C using an ice/salt bath. A solution of 2 M HCl in ether (0.104 mL, 0.21 mmol) was dissolved
in anhydrous dichloromethane (0.2 mL) and then added drop wise to the cooled substrate
solution. The reaction was stirred for 5 min at -5 °C and then 90 min at room temperature.
Hexane (40 mL) was added to precipitate the product salt and remaining starting material. The
precipitate was triturated and then washed twice with cold ether to remove any starting material
and other impurities. Isolated yield: 0.060 g (77%).
LCMS: RT = 3.1 min ([M-H]-, m/z = 673.5; [M+H]+, m/z = 675.4).
Example 3: Synthesis of 18-0-(N-methylethylenediamine-N'-carbamoyl)-18,21-
dihydromacbecin hydrochloride salt. 2
Preparation of N-Trityl-N-methylethylenediamine
N-Methylethylenediamine (5.96 g, 80.41 mmol) was dissolved in dichloromethane (100
mL) under argon. The stirring mixture was cooled to 0°C prior to drop wise slow addition of a
solution of tritylchloride (6.47 g, 23.21 mmol) in dichloromethane (40 ml). Following complete
addition of this solution the reaction mixture was stirred at 0°C for a further 30 min, at which
point the cooling bath was removed and the reaction allowed to warm up to room temperature.
The mixture was stirred under argon at room temperature overnight. The solvent was removed
from the resulting solution and ethyl acetate (200 mL) and saturated aqueous NaHCO3 (200
mL) were added. The resulting mixture was shaken, separated, and the aqueous extracted with
a further equal volume of ethyl acetate. The combined organics were dried over Na2SO4, filtered
and the solvent removed under reduced pressure. The residue was purified over silica gel

WO 2007/026027 PCT/EP2006/065941
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eluting with dichloromethane:methanol:aqueous ammonia (80:5:0.1). Two regioisomers were
observed and isolated separately. Pure fractions of the title compound (the more polar on silica)
were collected and the solvents removed under reduced pressure. Isolated yield, 1.66 g (22%).
1H NMR spectra acquired in DMSO at 400 MHz were consistent with the title compound and
allowed assignment of the correct regioisomer.
Preparation of 18-0-(N-trityl-N-methylethylenediamine-N'-carbamoyl)-18,21 -dihydromacbecin
18-0-(4-nitrophenylcarbonate)-18,21-dihydromacbecin (0.100 g, 0.14 mmol) was
dissolved in dichloromethane (20 ml). N-Trityl-N-methylethylenediamine (0.131 g, 0.41 mmol)
was then added and the solution heated at reflux for 2 hr, and then overnight at room
temperature. The organics were washed with water (20 ml_) and then dried over anhydrous
Na2SO4, filtered and the solvent removed under reduced pressure. The material was purified by
chromatography over the silica gel column eluting with hexane:acetone (7:3). Isolated yield:
0.093 g (74%). NMR spectra acquired in CDCl3 at 400 MHz were consistent with the title
compound.
LCMS: RT = 11.2 min ([M-H]-, m/z = 901.4).
Preparation of 18-O-(N-methylethylenediamine-N'-carbamoyl)-18,21 -dihydromacbecin
hydrochloride salt, 2
18-O-(N-trityl-N-methylethylenediamine-N'-carbamoyl)-18,21 -dihydromacbecin
(0.093 g, 0.10 mmol) was dissolved in anhydrous dichloromethane (19.5 mL) under argon and
cooled to -5 °C using an ice/salt bath. A solution of 2M HCl in ether (0.098 mL, 0.196 mmol)
was then added drop wise to the cooled substrate solution. The reaction was stirred for 5 min at
-5 °C and then 60 min at room temperature. Hexane (20 mL) was added to precipitate the
product salt and remaining starting material. The precipitate was triturated and then washed
twice with cold ether to remove any starting material and other impurities. The resulting solid
was recrystailized from dichloromethane:ether. Isolated yield: 0.021 g (29%).
LCMS: 9.7 min ([M-H]", 658.9).
Example 4: Synthesis of 18-O-(N,N'-diethy!ethylenediamine-N'-carbamoyl)-18,21-
dihydromacbecin hydrochloride salt, 3
Preparation of N-trityl-N.N'-diethylethylenediamine
N.N'-diethylethylenediamine (2.5 g, 21.5 mmol) was dissolved in dichloromethane (20
ml) under argon. The stirring mixture was cooled to 0 °C prior to drop wise slow addition of a
solution of tritylchloride (2.4 g, 8.6 mmol) in dichloromethane (20 mL). Following complete

WO 2007/026027 PCT/EP2006/065941
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addition of this solution the reaction mixture was stirred at 0 °C for a further 30 min, at which
point the cooling bath was removed and the reaction allowed to warm up to room temperature.
The mixture was stirred under argon at room temperature overnight. The solvent was removed
from the resulting solution and ethyl acetate (200 mL) and saturated aqueous NaHCO3 (200
mL) were added. The resulting mixture was shaken, separated, and the aqueous extracted with
a further equal volume of ethyl acetate. The combined organics were dried over Na2SO4, filtered
and the solvent removed under reduced pressure. The residue was purified over silica gel
eluting with dichloromethane:methanol:aqueous ammonia (80:5:0.5). Pure fractions were
collected and the solvents removed under reduced pressure. Remaining water/ammonia was
removed by addition of isopropyl alcohol and subsequent removal under reduced pressure (2 x
100 mL), and then under high vacuum to yield the title compound as a pale yellow solid.
Isolated yield: 2.60 g (84%). 1H NMR spectra acquired in CDCl3 at 400 MHz were consistent
with the title compound.
Preparation of 18-O-(N-trityl-N, N'-diethylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin
18-O-(4-nitrophenylcarbonate)-18,21-dihydromacbecin (0.296 g, 0.41 mmol)was
dissolved in dichloromethane (15 mL). N-trityl-N,N'-diethylethylenediamine (0.438 g, 1.22 mmol)
in dichloromethane (15 mL) was then added and the solution heated at reflux for 2 hr, and then
overnight at room temperature. The resulting solution was washed with water, dried over
Na2SO4 and the solvent removed under reduced pressure. The resulting material was purified
by chromatography over silica gel eluting with a stepwise gradient acetone in hexane (5 - 25 %
acetone) to yield a pale yellow solid. Isolated yield: 0.23 g (59 %). NMR spectra acquired in
CDCl3 at 400 MHz were consistent with the title compound.
LCMS: the material cleaved under mobile phase conditions, losing the trityl group to liberate the
secondary amine, RT = 4 min (broad) ([M-H]-, m/z = 701.6; [M+H]+, m/z = 703.6).
Preparation of 18-O-(N,N'-diethylethylenediamine-N'-carbamoyl)-18,21~dihydromacbecin
hydrochloride salt, 3
18-O-(N-trityl-N,N'--diethylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin (0.206
g, 0.22 mmol) was dissolved in anhydrous dichloromethane (40 mL) under argon and cooled to
-5 °C using an ice/salt bath. A solution of 2M HCl in ether (0.207 mL, 0.41 mmol) was dissolved
in anhydrous dichloromethane (1 mL) and then added drop wise to the cooled substrate
solution. The reaction was stirred for 5 min at -5 oC and then 90 min at room temperature.
Hexane (50 mL) was added to precipitate the product salt and remaining starting material. The
precipitate was triturated and then washed twice with cold ether to remove any starting material
and other impurities. Isolated yield: 0.090 g (55%).

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42
LCMS: RT = 3.4 min ([M-H]-, m/z= 701.6; [M+H]+, m/z = 703.6).
Example 5: Synthesis of 18-O-(N,N'-dimethyl-1,3-propanediamine-N'-carbarnoyl)-18,21-
dihydromacbecin hydrochloride salt, 4
Preparation of N-Trityl-N,N'-dimethyl-1,3-propanediamine
N,N'-Dimethyl-1,3-propanediamine (2.50 g, 24.5 mmol) was dissolved in
dichloromethane (20 mL) under argon. The stirring mixture was cooled to 0 °C prior to drop wise
slow addition of a solution of tritylchloride (2.73 g, 9.80 mmol) in dichloromethane (20 mL).
Following complete addition of this solution the reaction mixture was stirred at 0 °C for a further
30 min, at which point the cooling bath was removed and the reaction allowed to warm up to
room temperature. The mixture was stirred under argon at room temperature overnight. The
solvent was removed from the resulting solution and ethyl acetate (200 ml_) and saturated
aqueous NaHCO3 (200 mL) were added. The resulting mixture was shaken, separated, and the
aqueous extracted with a further equal volume of ethyl acetate. The combined organics were
dried over Na2SO4, filtered and the solvent removed under reduced pressure. The residue was
purified over silica gel eluting with dichloromethane:methanol:aqueous ammonia (80:5:0.5).
Pure fractions were collected and the solvents removed under reduced pressure. Remaining
water/ammonia was removed addition of ethanol (2 x 100 mL) and removal under reduced
pressure at 50°C, and then drying under high vacuum to yield the title compound. Isolated yield,
2.60 g (76 %). 1H NMR spectra acquired in CDCl3 at 400 MHz were consistent with the title
compound.
Preparation of 18-O-(N-trityl-N,N'-dimethyl-1,3-propanediamine-N'-carbamoyl)-18,21-
dihydromacbecin
18-O-(4-nitrophen.ylcarbonate)-18,21-dihydromacbecin (0.161 g, 0.22 mmol) was
dissolved in dichloromethane (15 mL). N-Trityl-N,N'-dimethyl-1,3-propanediamine (0.229 g, 0.67
mmol) in dichioromeihane (15 mL) was then added and the solution heated at reflux for 3.5 hr,
and then overnight at room temperature. The resulting solution was washed with water, dried
over Na2SO4 and the solvent removed under reduced pressure. The resulting material was
purified by chromatography over silica gel eluting with a stepwise gradient of acetone in hexane
(30 -- 50 % acetone) to yield a pale yellow solid. Isolated yield: 0.080 g (37 %). NMR spectra
acquired in CDCl3 at 400 MHz were consistent with the title compound.
LCMS: RT = 8.3 min ([M-H-trityl], m/z = 687.5; [M+H-trityl]', m/z = 689,5); the material also
cleaved under mobile phase conditions to liberate the free amine, RT = 3.2 min ([M-H-trityl]-,
687.5; [M+H-trifyl]+, 689.5).

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Preparation of 18-O-(N,N'-dimethyl-1,3-propanediamine-N'-carbamoyl)-18,21-dihydromacbecin
hydrochloride salt, 4
18-0-(N-trityl-N,N'-dimethyl-1,3-propanediamine-N'-carbamoyl)-18,21-dihydromacbecin
(0.075 g, 0.08 mmol) was dissolved in anhydrous dichloromethane (8 mL) under argon and
cooled to -5oC using an ice/salt bath. A solution of 2M HCI in ether (0.079 mL, 0.16 mmol) was
dissolved in anhydrous dichloromethane (0.5 mL) and then added drop wise to the cooled
substrate solution. The reaction was stirred for 5 min at -5 °C and then 90 min at room
temperature. Hexane (20 mL) was added to precipitate the product salt and remaining starting
material. The precipitate was triturated and then washed twice with cold ether to remove any
starting material and other impurities. Isolated yield: 0.038 mg (65 %).
LCMS: RT = 3.3 min ([M-H]', m/z= 687.7; [M+H]+, 689.7).
Example 6: Synthesis of 18-O-(N,N'-diisopropylethylenediamine-N'-carbamoyl)-18,21-
dihydromacbecin hydrochloride salt, 5
Preparation of 18-O-(N-trityl-N,N'-diisopropylethylenediamine-N'-carbamoyl)-18,21'-
dihydromacbecin
18-O-(4-nitrophenylcarbonate)-18,21-dihydromacbecin (0.161 g, 0.22 mmol) was
dissolved in dichloromethane (15 mL). N-trityl-N,N'-diisopropylethylenediamine (0.257 g, 0.67
mmol) in dichloromethane (15 mL) was then added and the solution heated at reflux for 3.5 h,
and then overnight at room temperature. The resulting solution was washed with water, dried
over Na2SO4 and the solvent removed under reduced pressure. The resulting material was
purified by chromatography over silica gel eluting with a stepwise gradient acetone in hexane
(30 - 50 % acetone) to yield a pale yellow solid. Isolated yield: 0.060 g (28 %). NMR spectra
acquired in CDCl3 at 400 MHz were consistent with the title compound.
LCMS: RT = 7.8 min [M-H-trityi], m/z - 729.6; [M+H-trityl]+, m/z = 731.6.; the material also
cleaved under mobile phase conditions to liberate the secondary amine, RT = 5.7 min ([M-H]-,
m/z = 729.6; [M+H]+, m/z = 731.6).
Preparation of 18-O-(N,N'-diisopropylethylenediamine-N'-carbamoyl)-18,21 -dihydromacbecin
hydrochloride salt, 5
18-O-(N-trityl-N,N'-diisopropylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin
(0.055 g, 0.06 mmol) was dissolved in anhydrous dichloromethane (10 mL) under argon and
cooled to -5oC using an ice/salt bath. A solution of 2M HCl in ether (0.055 mL, 0.11 mmol) was
dissolved in anhydrous dichloromethane (1 mL) and then added drop wise to the cooled

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substrate solution. The reaction was stirred for 5 min at -5 °C and then 90 min at room
temperature. Hexane (20 mL) was added to precipitate the product salt and remaining starting
material. The precipitate was triturated and then washed twice with cold ether to remove any
starling material and other impurities. Isolated yield: 0.032 mg (70 %).
LCMS: RT = 5.5 min ([M-H]-, m/z = 729.9; [M+H]+, 731.9).
Example 7: Cleavage assay for compound 1 in human plasma
Compound 1 was incubated in human plasma as described in the General Methods-
Cleavage Assay above. An aliquot was sampled every 15 min and acidified by the addition of
0.01 ml_ phosphoric acid to stop the chemical triggered cleavage of the parent compound. The
samples were subsequently extracted as described above and analysed immediately using
Analysis Method B. The decay of the parent compound and the relative amounts of released
compounds are shown in Figure 1, where the open circles show the decay of 1 and the filled
squares and filled triangles indicate the accumulation of macbecin and 18,21-dihydromacbecin
respectively.
Example 8: Comparison of cleavage rates for compounds 1-5 in phosphate buffer and in
whole blood
Each compound was assessed for cleavage to 18,21-dihydromacbecin as follows.
Compounds 1-5 were incubated in whole defribrinated rabbit blood as described in the General
Methods - Cleavage Assay above. An aliquot was sampled at 2 min, 1 h, 2.25 h and 3.5 h. 1.5
mL of potassium dihydrogen phosphate was added and the internal standard (IS) to 0.002
mg/mL. The samples were applied to a cartridge described above and analysed immediately
using Analysis Method C. T1/2 values were calculated and are shown in Table 5.
The cleavage of compounds 1-3 was also measured in phosphate buffer as described in
the General Methods - Cleavage Assay above at pH 7.2 and pH 7.4 and T1/2 values calculated
as indicated in Table 5 below.


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45

NT, not tested in the run
*, the batch of compound 2 contained impurities which limited accuracy of the analysis
Example 9: Solubility measurements of compounds 1-3
The compounds 1-3 were tested for their thermodynamic solubility using the method
described in the General methods. The results are shown in Table 6 below.

Example 10: Biological data - In vitro evaluation of anticancer activity of macbecin
In vitro evaluation of macbecin for anticancer activity in a panel of 12 human tumor cell
lines in a monolayer proliferation assay was carried out as described in the general methods
using a modified propidium iodide assay.
The results are displayed in Table 7 below; each result represents the mean of duplicate
experiments.


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46

Example 11: Biological data - In vivo evaluation of 18-O-(N,N'-dimethylethylenediamine-
N'-carbamoyl)-18,21-dihydromacbecin hydrochloride salt (compound 1)
In vivo evaluation of 18-O-(N,N'-ciirnethylethylenediamine-N'-carbamoyl)-18,21-
dihydromacbecin hydrochloride 1 was carried out in nude mice bearing xenografts of the human
prostate carcinoma cell line DU-145, as described in the general methods. 18-O-(N,N'-
dimethylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin hydrochloride 1 was
administered intraperitoneally at a dose level of 60 mg/kg/d on days 0-4, 7-11, 14-18, 21, 24,
25, 28 and 29, in a vehicle of 5% glucose, and compared to a control group dosed with the
standard vehicle 10% DMSO, 0.05% Tween 80.
18-0-(N,N'-dimethylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin
hydrochloride 1 at 60 mg/kg/d i.p. on Days 0-4, 7-11, 14-18, 21, 24, 25, 28 and 29 significantly
(p<0.001; U-test by Mann-Whitney-Wilcoxon) reduced tumour growth rates (minimum T/C value:
24.3%, recorded on day 31). A graph showing the group median RTV value throughout the
course of the study is displayed as Figure 2.
Antitumour activity was determined as tumour volume inhibition relative to tumours in a
control group receiving the standard vehicle 10% DMSO, 0.05% Tween80 in PBS i.p. at 10
mL/kg/d on Days 0-4, 7-11, 14-18,21, 24, 25, 28 and 29. Group size was 6 mice for the vehicle
control group and 6 mice in the therapy groups implanted with two tumour fragments. The
experiment was terminated on Day 42.
Ail references including patent and patent applications referred to in this application are
incorporated herein by reference to the fullest extent possible.
Throughout the specification and the claims which follow, unless the context requires otherwise,
the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to
imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any
other integer or step or group of integers or steps.

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Workman, P. and Kaye, S.B. (2002) Translating basic cancer research into new cancer
therapeutics. Trends in Molecular Medicine 8:S1-S9.
Young, J.C.; Moarefi, I. and Hartl, U. (2001) Hsp90: a specialized but essential protein folding
tool. J, Cell. Bioi. 154:267-273.

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Claims
1. A derivative of a benzenoid ansamycin which contains a 1,4-dihydroxyphenyl moiety
bearing at position 6 an amino carboxy substituent, in which position 2 and the carboxy
substituent at position 6 are connected by an aliphatic chain of varying length characterised ii
that one or both of the 1-hydroxy and the 4-hydroxy position(s) of the phenyl ring are
independently derivatised by an aminoalkyleneaminocarbonyl group, which alkylene group,
which may optionally be substituted by alkyl groups, has a chain length of 2 or 3 carbons and
which derivatising group(s) increase the water solubility and/or the bioavailability of the paren
molecule but which are capable of being removed in-vivo.
2. A compound according to Formula (IA-IC) below:

wherein:
R1 represents H, OH, OMe, -NHCH2CH=CH2 or-NHCH2CH2N(CH3)2;
R2 represents OH, or keto;
R3 represents OH or OMe;

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R5 represents H or
wherein:
n represents 0 or 1;
R6 represents H, Me, Et or iso-propyl;
R7, R8 and R9 each independently represent H or a C1-C4 branched or linear
chain alkyl group; or R7 and R8, or R8 and R9, may be connected so as to form a
6-membered carbocyclic ring;
R10 represents H or a C1-C4 branched or linear chain alkyl group;
provided however that the R5 moieties are not both H and that when neither R5 moiety
represents H then the two R5 moieties are the same; or a pharmaceutically acceptable salt
thereof.
3. A compound according to claim 2 wherein R6 represents H, Me or Et.
4. A compound according to claim 2 wherein R10 represents a C1-C4 branched or linear
chain alkyl group.
5. A compound according to claim 2 wherein R6 represents H, Me or Et and
R10 represents a C1-C4 branched or linear chain alkyl group.
6. A compound according to any one of claims 2 to 5, wherein neither R5 represents H.
7. A compound according to any one of claims 2 to 5, wherein one R5 group represents H.
8. A compound according to claim 7 wherein the C21 R5 group is H.
9. A compound according to any one of claims 2 to 8 defined by structure (IA).
10. A compound according to any one of claims 2 to 8 defined by structure (IB).
11. A compound according to claim 10 wherein R1 represents -NHCH2CH=CH2.
12. A compound according to claim 10 wherein R1 represents -NHCH2CH2N(CH3)2.

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13. A compound according to claim 10 wherein R1 represents OMe.
14. A compound according to any one of claims 2 to 8 defined by structure (IC).
15. A compound according to any one of claims 2 to 14 wherein n is 0.
16. A compound according to any one of claims 2 to 15 wherein R6 represents Me.
17. A compound according to any one of claims 2 to 15 wherein R6 represents Et.
18. A compound according to any one of claims 2 to 17 wherein R10 represents Me.
19. A compound according to any one of claims 2 to 17 wherein R10 represents Et.
20. A compound according to any one of claims 2 to 19 wherein R7 represents H.
21. A compound according to any one of claims 2 to 20 wherein R8 and R9 represent H.
22. A compound according to claim 2 which is 18-O-(N,N'-dimethylethylenediamine -N'-
carbamoyl)-18,21-dihydromacbecin or a pharmaceutically acceptable salt thereof.
23. A compound according to claim 2 selected from:
18-O-(N-methylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin;
18-O-(N,N'-diethylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin;
18-O-(N,N'-dimethyl-1,3-propanediamine-N'-carbamoyl)-18,21-dihydromacbecin; and
18-(N.N'-diisopropylethylenediamine-N'-carbamoyl)-18,21-dihydromacbecin
or a pharmaceutically acceptable salt of any one thereof.
24. A compound according to any one of claims 1 to 23 in the form of a hydrochloride salt.
25. A compound according to any one of claims 1 to 24 for use as a pharmaceutical.
26. A compound according to any one of claims 1 to 24 for use a pharmaceutical in the
treatment of cancer or B-cell malignancies.
27. A pharmaceutical composition comprising a compound according to any one of claims 1
to 24 together with one or more pharmaceutically acceptable diluents or carriers.

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28. A method of treatment of cancer or B-cell malignancies which comprises administering
to a patient an effective amount of a compound according to any one of claims 1 to 24.
29. Use of a compound according to any one of claims 1 to 24 in the preparation of a
medicament for the treatment of cancer or B-cell malignancies.
30. A process for preparing a compound of formula (I) or a pharmaceutically acceptable salt
thereof which comprises:
(a) preparing a compound of formula (I) in which neither R5 moiety is H by reacting a
compound of formula (IIA), (IIB) or (IIC):

wherein R1, R2 and R3 are as defined in any one of claims 2-23 and L is a leaving group,
or a protected derivative thereof, with a compound of formula (H)


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wherein n, R6, R7, R8, R9 and R10 are as defined in any one of claims 2 to 23 and P
represents a protecting group; or
(b) preparing a compound of formula (I) in which the C21 R5 moiety is H by reacting a
compound of formula (IID), (IIE) or (IIF):

wherein R1, R2 and R3 are as defined in any one of claims 2-23 and L is a leaving group
or a protected derivative thereof, with a compound of formula (H)

wherein n, R6, R7, R8, R9 and R10 are as defined in any one of claims 2 to 23 and P
represents a protecting group; or
(c) converting a compound of formula (I) or a salt thereof to another compound of
formula (I) or another pharmaceutically acceptable salt thereof; or
(d) deprotecting a protected compound of formula (I).
31. A compound of formula (IIA), (IIB) or (IIC):

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wherein R1, R2 and R3 are as defined in any one of claims 2-23 and L is a leaving group, or a
protected derivative of any one thereof.
32. A compound of formula (IID), (IIE) or (IIF)


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wherein R1, R2 and R3 are as defined in any one of claims 2-23 and L is a leaving group
or a protected derivative of any one thereof.
33. A compound of formula (IVA), (IVB) or (IVC)

wherein R1, R2 R3 and R5 are as defined in any one of claims 2 to 23 and Pa is a protecting
group, or a protected derivative of any one thereof.
34. A compound of formula (VA), (VB) or (VC)

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wherein R1, R2 and R3 are as defined in any one of claims 2 to 23, L is a leaving group and Pa
is a protecting group, or a protected derivative of any one thereof.

There are provided inter alia derivatives of a benzenoid ansamycin which contain a 1,4-dihydroxyphenyl moiety bearing at position 6 an amino carboxy substituent, in which position 2 and the carboxy substituent at position 6 are connected by an aliphatic chain of varying length characterised in that one or both of the 1-hydroxy and the 4-hydroxy position(s) of the phenyl ring are independently derivatised by an aminoalkyleneaminocarbonyl group, which alkylene group, which may optionally be substituted
by alkyl groups, has a chain length of 2 or 3 carbons and which derivatising group(s) increase the water solubility and/or the bioavailability of the parent molecule but which are capable of being removed in-vivo. Such compounds are described for the treatment of cancer or B-cell malignancies.