CA2537669A1 - Inhibitors of nucleoside phosphorylases and nucleosidases for treating cancer - Google Patents
Inhibitors of nucleoside phosphorylases and nucleosidases for treating cancer Download PDFInfo
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- CA2537669A1 CA2537669A1 CA002537669A CA2537669A CA2537669A1 CA 2537669 A1 CA2537669 A1 CA 2537669A1 CA 002537669 A CA002537669 A CA 002537669A CA 2537669 A CA2537669 A CA 2537669A CA 2537669 A1 CA2537669 A1 CA 2537669A1
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- hydroxy
- methyl
- halogen
- deazaadenin
- pyrrolidine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
Use for treating cancer of a compound of the formula (I):
(see formula I)
(see formula I)
Description
METHODS OF TREATING CANCER
STATEMENT OF GOVERNMENT SUPPORT
The invention disclosed herein was made with U.S. Government support under National Institutes of Health (NIH) grant number number GM41916. Accordingly, the U.S. Government has certain rights in this invention.
TECHNICAL FIELD
The present invention relates to methods of treating cancer by administering to a patient in need thereof one or more inhibitors of 5'-methylthioadenosine phosphorylase (MTAP). In particular, the invention relates to methods of treating prostate cancer or head and neck cancer.
BACKGROUND
Certain nucleoside analogues have been identified as potent inhibitors of 5'-methylthioadenosine phosphorylase (MTAP) and 5'-methylthioadenosine nucleosidase (MTAN). These are the subject of WO 03/080620.
Compounds where the location of the nitrogen atom in the sugar ring is varied or where two nitrogen atoms form part of the sugar ring, have also been identified as inhibitors of MTAP and MTAN. These compounds are described in WO
2004/018496.
MTAP and MTAN function in the polyamine biosynthesis pathway, in purine salvage in mammals, and in the quorum sensing pathways in bacteria. MTAP catalyses the reversible phosphorolysis of methylthioadenosine (MTA) to adenine and 5-methyfthio-a-D-ribose-1-phosphate (MTR-1 P). MTAN catalyses the reversible hydrolysis of MTA
to adenine and 5-methylthio-a-D-ribose and of S-adenosyl-L-homocysteine (SAH) to adenine and S-ribosyl-homocysteine (SRH). The adenine formed is subsequently recycled and converted into nucleotides. Essentially, the only source of free adenine in the human cell is a result of the action of these enzymes. The MTR-1 P is subsequently converted into methionine by successive enzymatic actions.
STATEMENT OF GOVERNMENT SUPPORT
The invention disclosed herein was made with U.S. Government support under National Institutes of Health (NIH) grant number number GM41916. Accordingly, the U.S. Government has certain rights in this invention.
TECHNICAL FIELD
The present invention relates to methods of treating cancer by administering to a patient in need thereof one or more inhibitors of 5'-methylthioadenosine phosphorylase (MTAP). In particular, the invention relates to methods of treating prostate cancer or head and neck cancer.
BACKGROUND
Certain nucleoside analogues have been identified as potent inhibitors of 5'-methylthioadenosine phosphorylase (MTAP) and 5'-methylthioadenosine nucleosidase (MTAN). These are the subject of WO 03/080620.
Compounds where the location of the nitrogen atom in the sugar ring is varied or where two nitrogen atoms form part of the sugar ring, have also been identified as inhibitors of MTAP and MTAN. These compounds are described in WO
2004/018496.
MTAP and MTAN function in the polyamine biosynthesis pathway, in purine salvage in mammals, and in the quorum sensing pathways in bacteria. MTAP catalyses the reversible phosphorolysis of methylthioadenosine (MTA) to adenine and 5-methyfthio-a-D-ribose-1-phosphate (MTR-1 P). MTAN catalyses the reversible hydrolysis of MTA
to adenine and 5-methylthio-a-D-ribose and of S-adenosyl-L-homocysteine (SAH) to adenine and S-ribosyl-homocysteine (SRH). The adenine formed is subsequently recycled and converted into nucleotides. Essentially, the only source of free adenine in the human cell is a result of the action of these enzymes. The MTR-1 P is subsequently converted into methionine by successive enzymatic actions.
MTA is a by-product of the reaction involving the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine to putrescine during the formation of spermidine. The reaction is catalyzed by spermidine synthase. Likewise, spermine synthase catalyses the conversion of spermidine to spermine, with concomitant production of MTA as a by-product. The spermidine synthase is very sensitive to product inhibition by accumulation of MTA. Therefore, inhibition of MTAP or MTAN
severely limits the polyamine biosynthesis and the salvage pathway for adenine in the cells.
Although MTAP is abundantly expressed in normal cells and tissues, MTAP
deficiency due to a genetic deletion has been reported with many malignancies.
The loss of MTAP enzyme function in these cells is known to be due to homozygous deletions on chromosome 9 of the closely linked MTAP and p161MTS1 tumour suppressor gene. As absence of p16/MTS1 is probably responsible for the tumour, the lack of MTAP activity is a consequence of the genetic deletion and is not causative for the cancer. However, the absence of MTAP alters the purine metabolism in these cells so that they are mainly dependent on the de novo pathway for their supply of purines.
MTA has been shown to induce apoptosis in dividing cancer cells, but to have the opposite, anti-apoptotic effect on dividing normal cells such as hepatocytes (E.
Ansorena et al., Hepatology, 2002, 35: 274-280).
MTAP inhibitors may therefore be used in the treatment of cancer. Such treatments are described in WO 03/080620 and WO 2004/018496.
The need for new cancer therapies remains ongoing. For some prevalent cancers the treatment options are still limited. Prostate cancer, for example, is the most commonly diagnosed non-skin cancer in the United States. Current treatment options include radical prostatectomy, radiation therapy, hormonal therapy, and watchful waiting.
Although the therapies may offer successful treatment of an individual's condition, the pitfalls are quite unfavorable and lead to a decrease in a man's overall quality of life.
Surgery may inevitably result in impotence, sterility, and urinary incontinence. Side effects associated with radiation therapy include damage to the bladder and rectum as well as slow-onset impotence. Hormonal therapy will not cure the cancer and eventually most cancers develop a resistant to this type of therapy. The major risk associated with watchful waiting is that it may result in tumour growth, cancer progression and metastasis. It is therefore desirable that a better treatment option is made available to patients diagnosed with prostate cancer.
It is an object of the invention to provide a method of treating cancer, particularly prostate or head and neck cancer, or at least to provide a useful choice.
STATEMENTS OF INVENTION
Ina first aspect, the invention provides a method of treating cancer comprising administering to a patient in need thereof a compound of the formula (I):
V
W
X
Y
OH
(I) wherein:
V is selected from CH2 and NH, and W is selected from NR' and NR2; or V
is selected from NR' and NR2, and W is selected from CH2 and NH;
X is selected from CH2 and CHOH in the R or S-configuration;
Y is selected from hydrogen, halogen and hydroxy, except where V is selected from NH, NR' and NR2 then Y is hydrogen;
Z is selected from hydrogen, halogen, hydroxy, SQ, OQ and Q, where Q
is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
R' is a radical of the formula (II) B
H
N N
A' I
D
~-- G
(II) R2 is a radical of the formula (III) B
N N
A I
/ N E D
~-- G
(III) A is selected from N, CH and CR3, where R3 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen; or R3 is hydroxyl, halogen, NH2, NHR4, NR4R5;
or SR6, where R , R5 and R6 are alkyl, aralkyl or aryl groups, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
B is selected from NH2 and NHR7, where R7 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
D is selected from hydroxy, NH2, NHR8, hydrogen, halogen and SCH3, where R8 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
E is selected from N and CH;
G is selected from CH2 and NH, or G is absent, provided that where W is NR' or NR2 and G is NH then V is CH2, and provided that where V is NR' or NR2 and G is NH then W is CHZ;
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.
Preferably the cancer is prostate cancer or head and neck cancer.
In another aspect, the invention provides a method of treating cancer comprising administering to a patient in need thereof a compound of the formula (IV):
W
X
Y
OH
(IV) wherein:
V is selected from CH2 and NH, and W is selected from NR' and NR2; or V
is selected from NR' and NRZ, and W is selected from CH2 and NH;
X is selected from CH2 and CHOH in the R or S-configuration;
Y is selected from hydrogen, halogen and hydroxy, except where V is selected from NH, NR' and NR2 then Y is hydrogen;
Z is selected from hydrogen, halogen, hydroxy, SQ, OQ and Q, where Q
is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
R' is a radical of the formula (V) B
H
p N N
A I
D
~-- G
(V) R2 is a radical of the formula (VI) B
~ \''N
A I
N
~ E D
goG
(\A) A is selected from N, CH and CR3, where R3 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen; or R3 is hydroxyl, halogen, NH2, NHR4, NR4R5;
or SRs, where R4, R5 and R 6 are alkyl, aralkyl or aryl groups, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
B is selected from NH2 and NHR7, where R7 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
D is selected from hydroxy, NH2, NHR8, hydrogen, halogen and SCH3, where RB is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
E is selected from N and CH;
G is selected from CH2 and NH, or G is absent, provided that where W is NR' or NR2 and G is NH then V is CH2, and provided that where V is NR' or NR2 and G is NH then W is CH2;
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof;
provided that the compound (3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine is excluded.
Preferably Z is SQ.
Preferably Q is an alkyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy. It is further preferred that Q is a Cj-Ce alkyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy. Most preferably Q
is a methyl group Alternatively it is preferred that Z is not methylthio.
It is also preferred that Q is an aryl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy. More preferably Q is a phenyl or benzyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy.
Preferably G is CH2. It is also preferred that V is CH2 and W is NR1. It is further preferred that B is NHZ. It is also preferred that D is H, and it is preferred that A is CH.
Preferably any halogen is chlorine or fluorine.
In another aspect, the invention provides a method of treating cancer comprising administering to a patient in need thereof a compound of the formula (VII):
N
J I
~S N
\N
N
HO
(VI l) where J is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy.
Preferably the cancer is prostate cancer or head and neck cancer.
Preferably J is C1-C7 alkyl. More preferably J is methyl, ethyl, n-propyl, i-propyl, n-butyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl or cycloheptyl.
It is also preferred that J is phenyl, optionally substituted with one or more halogen substituents. More preferably J is phenyl, p-chlorophenyl, p-fluorophenyl or m-chlorophenyl.
It is also preferred that J is heteroaryl. More preferably J is 4-pyridyl.
It is also preferred that J is aralkyl. More preferably J is benzylthio.
Preferably J is -CH2CH2(NH2)COOH.
In another aspect, the invention provides a method of treating cancer comprising administering to a patient in need thereof a compound of the formula (VIII):
N
T S N
N N
HO OH
(VIII) where T is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, carboxy or straight- or branched-chain Cl-Cg alkyl.
Preferably the cancer is prostate cancer or head and neck cancer.
Preferably T is C1-C6 alkyl, optionally substituted with one or more substituents selected from halogen and hydroxy. More preferably T is methyl, ethyl, 2-fluoroethyl, or 2-hydroxyethyl. Most preferably T is methyl.
It is also preferred that T is aryl, optionally substituted with one or more substituents selected from halogen or straight-chain Cl-Ce alkyl. More preferably T is phenyl, naphthyl, p-tolyl, m-tolyl, p-chlorophenyl, m-chlorophenyl or p-fluorophenyl.
It is also preferred that T is aralkyl. More preferably T is benzyl.
Preferably the compound of formula (I) is:
(3R,4R)-1-[(8-aza-9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl) methyl]-3-hydroxy-4-(2-phenylethyl)pyrrolidine;
(3R,4S)-1 -[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzyFthiomethyl)pyrrolidine;
(3R,4S)-1-[(8-aza-9-deazaaden in-9-yl)methyl]-3-hydroxy-4-(benzylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-chlorophenylthiomethyl)pyrrolidine;
(3 R,4R)-1-[(9-deazaadenin-9-yl)methyl]-3-acetoxy-4-(acetoxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(n-butylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-fluorophenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yi)methyl]-3-hydroxy-4-(n-propylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaaden in-9-yl)methyl]-3-hydroxy-4-(cyclohexylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(3-chlorophenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(ethylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(phenylthiomethyl)pyrrolidine;
(3 R,4 S)-1-[(9-deazaaden in-9-yl) methy I]-3-hyd roxy-4-(4-pyridylthiomethyl)pyrrolidine;
(3 R,4S)-1-[(9-deazaadenin-9-yl) methyl]-3-hydroxy-4-n-propylpyrrolidine;
(3R,4S)-1-[(9-deazaaden in-9-yl)methyl]-3-hydroxy-4-(homocysteinylmethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzyloxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaaden in-9-yi)methyl]-3-hydroxy-4-(i-propylthiomethyl)pyrrolidine;
(3 R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methoxymethyl)pyrrolidine;
(3 R,4S)-1-[(9-deazaadenin-9-yl) methyl]-3-hydroxy-4-(cyclohexylmethylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cycloheptylthiomethyl)pyrrolid ine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclopentylthiomethyl)pyrrolid ine;
(3R,4S)-1-[(9-deazaaden in-9-yl)methyl]-3-hydroxy-4-(cyclobutylthiomethyl)pyrrolidine.
The compounds defined above may be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally or via an implanted reservoir. Preferably the compounds are administered orally.
BRIEF DESCRIPTION OF THE FIGURES
Figure la shows the survival of mouse prostate cancer cells (RM1) against increasing concentrations of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methyithiomethyl)pyrrolidine], either in the presence or absence of MTA.
Figure lb shows the survival of human prostate cancer cells (PC3) against increasing concentrations of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine], either in the presence or absence of MTA.
Figure 2 is a time dependent proliferation curve, showing the effect of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine]
and MTA on human prostate cancer cells (PC3).
Figure 3 is a time dependent proliferation curve, showing the effect of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine]
and MTA on SCC25 cells.
Figure 4 is a time dependent proliferation curve, showing the effect of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy4-(methylthiomethyl)pyrrolidine]
and MTA on FaDu cells.
Figure 5 shows phase contrast photographs of FaDu cells after 5 days of treatment with compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine] and MTA.
Figure 6 shows a cell cycle and apoptosis analysis of FaDu cells after 6 days of treatment with compound (2) ((3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-(methylthiomethyl)pyrrolidine] and MTA; (1) untreated resufts: G1 83.66%, S
8.08%, G2 8.26%, Apoptosis 6.06%; (2) treated with MTA results: G1 79.67%, S 10.42%, 9.91%, Apoptosis 6.66%; (3) treated with compound (3) results GI 72.06%, S
17.98%, G29.96%, Apoptosis 7.89%; (4) treated with MTA + compound (3) results GI
8.26%, S 31.25%, G2 60.49%, Apoptosis 29.41 %.
Figures 7 to 19 show oral and IP availability of selected compounds that may be used in the methods of the invention.
Figure 20 shows the effects of compound 1[(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzylthiomethyl)pyrrolidine] on FaDu xenografts in NOD-SCID
mice.
Figure 21 shows representative tumours from each of the treatment cohorts for the above NOD-SCID mouse study.
Figure 22 shows MRI images of TRAMP mice (Panels A and B: Control TRAMP
(transgenic adenocarcinoma of mouse prostate) mice, Panels E and F: TRAMP mice treated with 1 mM Compound 1[(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-(benzylthiomethyl)pyrrolidine]) Figure 23 shows that compound (2) and MTA alter polyamine levels and induce cytostasis in PC3 cells (PUT=putrescine, SPD=spermidine, SPN=spermine). PC3 cells were cultured and treated in triplicate as follows: untreated control, substrate (MTA) alone, 1 M compound (2) alone, or a combination of both substrate and inhibitor. Both cells and spent media were harvested at 1, 6, and 12 days for polyamine analysis by HPLC fluorescence.
Figure 24 shows that compound (2) reduces tumour growth and metastasis in TRAMP mice, but does not alter polyamine levels in vivo. C56B1/6 mice were treated with 100 M compound (2) via their drinking water and sacrificed at 24, 48 hours, and 7 days. Livers were immediately removed for polyamine analysis. TRAMP mice were treated approximately 6-8 months with 100 [M compound (2) via their drinking water and control sacrificed. Livers were removed for polyamine analysis.
DETAILED DESCRIPTION
Derinitions The term "alkyl" is intended to include straight- and branched-chain alkyl groups, as well as cycloalkyl groups. The same terminology applies to the non-aromatic moiety of an aralkyl radical. Examples of alkyl groups include: methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, iso-butyl group, sec-butyl group, t-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-ethylpropyl group, n-hexyl group and 1-methyl-2-ethylpropyl group.
The term "aryl" means an aromatic radical having 6 to 18 carbon atoms and includes heteroaromatic radicals. Examples include monocyclic groups, as well as fused groups such as bicyclic groups and tricyclic groups. Some examples include phenyl group, indenyl group, 1-naphthyl group, 2-naphthyl group, azulenyl group, heptalenyl group, biphenyl group, indacenyl group, acenaphthyl group, fluorenyl group, phenalenyl group, phenanthrenyl group, anthracenyl group, cyclopentacyclooctenyl group, and benzocyclooctenyl group, pyridyl group, pyrrolyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, triazolyl group, tetrazolyl group, benzotriazolyl group, pyrazolyl group, imidazolyl group, benzimidazolyl group, indolyl group, isoindolyl group, indolizinyl group, purinyl group, indazolyl group, furyl group, pyranyl group, benzofuryl group, isobenzofuryl group, thienyl group, thiazolyl group, isothiazolyl group, benzothiazolyl group, oxazolyl group, and isoxazolyl group.
The term "halogen" includes fluorine, chlorine, bromine and iodine.
The compounds are useful for the treatment of certain diseases and disorders in humans and other animals. Thus, the term "patient" as used herein includes both human and other animal patients.
The term "prodrug" as used herein means a pharmacologically acceptable derivative of the compound of formula (I), (IV), (VII) or (VIII), such that an in vivo biotransformation of the derivative gives the compound as defined in formula (I), (IV), (VII) or (VIII). Prodrugs of compounds of formulae (I), (IV), (VII) or (VIII) may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to give the parent compound.
Prodrugs include compounds of formulae (I), (IV), (VII) and (VIII), tautomers thereof and/or pharmaceutically acceptable salts thereof, which include an ester functionality, or an ether functionality. It will be clear to the skilled person that the compounds of formulae (I), (IV), (VII) and (VIII) may be converted to corresponding ester or ether prodrugs using known chemical transformations.
Suitable prodrugs include those where the hydroxyl groups of the compounds of formula (I), (IV), (VII) or (VIII) are esterified to give, for example, a primary hydroxyl group ester of propanoic or butyric acid. Other suitable prodrugs are alkycarbonyoxymethyl ether derivatives on the hydroxyl groups of the compounds of formula (I), (IV), (VII) or (VIII) to give, for example, a primary hydroxyl group ether with a pivaloyloxymethyl or a propanoyloxymethyl group.
The term "pharmaceutically acceptable salts" is intended to apply to non-toxic salts derived from inorganic or organic acids, including, for example, the following acid salts: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, p-toluenesulfonate, salicylate, succinate, sulfate, tartrate, thiocyanate, and undecanoate.
It will be appreciated that the representation of a compound of formula (I) or (IV) where B and/or D is a hydroxy group, is of the enol-type tautomeric form of a corresponding amide, and this will largely exist in the amide form. The use of the enol-type tautomeric representation is simply to allow fewer structural formulae to represent the compounds of the invention.
Similarly, it will be appreciated that the representation of a compound of formula (1) or (IV), where B and/or D is a thiol group, is of the thioenol-type tautomeric form of a corresponding thioamide, and this will largely exist in the thioamide form.
The use of the thioenol-type tautomeric representation is simply to allow fewer structural formulae to represent the compounds of the invention.
Detailed Description of the Invention The present invention relates to methods of treating cancer by administering to a patient in need thereof one or more inhibitors of 5'-methylthioadenosine phosphorylase (MTAP). In particular, the invention relates to methods of treating certain cancers, such as prostate cancer or head and neck cancer.
Suitable MTAP inhibitors which may be employed in the method of the present invention and the methods for preparing these inhibitors are described in WO
03/080620 and WO 2004/018496.
Certain MTAP inhibitor compounds are surprisingly effective for treating prostate and head and neck cancers. These are compounds of general formula (VII).
N
~N
NS N
HO
(VI l) This sub-class of MTAP inhibitors incorporates an adenine-like base moiety and a pyrrolidine moiety having an alkyl- aryl- or aralkylthiomethyl group at the 4-position.
Other MTAP inhibitor compounds are also surprisingly effective for treating prostate and head and neck cancers. These are compounds of general formula (VIII).
T~s N
N N
HO OH
(VIII) This sub-class of MTAP inhibitors also incorporates the adenine-like base moiety but has an iminoribitol moiety with an alkyl- aryl- or aralkylthiomethyl group at the 5'-position.
Examples of the first sub-class of inhibitors include compounds (1) and (2).
H NH= PN
NS N
HO HO
Compound (1) Compound (2) BT-DADMe-ImmA MT-DADMe-ImmA
The present studies show that compounds (1) and (2) are effective both in vitro and in vivo against a variety of cell lines (PC3, RM1, SCC25 and FaDu). These compounds are therefore particularly useful in the treatment of prostate and head and neck cancers.
The MTAP inhibitor compounds inhibit cell growth in vitro of the prostate cancer cell lines PC3 and RM1 and the head and neck cancer cell lines SCC25 and FaDu. An enhanced cell-killing effect is seen in vitro with combined administration of the MTAP
inhibitor compound plus MTA. Examples of this effect are shown in Figures 1 to 6.
Furthermore, the inhibitor compounds, when co-administered with MTA, exhibit a cytostatic effect on PC3 cells in vitro.
In order to determine whether the inhibition is selective for malignant cells, normal human fibroblast cells (GM02037) were also treated with compound (2) and MTA
for 3 weeks. No cytotoxicity was observed. Compound (2) is therefore cytotoxic for human HNSCC (human head and neck squamous cell carcinoma) cells at doses that exhibit minimal toxicity for normal cells. This selectivity is a further indication that the MTAP inhibitors described above are useful agents for the treatment of head and neck cancer.
The present in vivo studies further demonstrate the surprising efficacy of the compounds. In a NOD-SCID mouse model, compound (2) significantly delays the growth of established FaDu xenografts. The effect is seen either with or without co-administration of the inhibitor compound with MTA.
In addition, prostate cancer progression in the TRAMP mouse model is inhibited in mice treated with compound (1), either alone or in combination with MTA.
An example of the second sub-class of inhibitors is compound (3).
N
N
N
HO Compound (3) MT-ImmA
This compound also inhibits prostate cancer progression in the TRAMP mouse model, when administered either alone or in combination with MTA.
For the above in vivo models, the inhibitor compounds exhibit activity when administered with exogenous MTA and when administered alone. There is not a significant enhancement observed when the inhibitors are administered together with MTA. However, the in vitro results clearly demonstrate a surprising enhancement in activity when the inhibitors are administered in conjunction with MTA. Thus, the combined administration method provides a potential alternative treatment method for patients suffering from cancer, where the administration of an MTAP inhibitor is indicated.
The MTAP inhibitor compounds of formulae (I), (IV), (VII) and (VIII) (in particular the compounds of formulae (VII) and (VIII)) provide an effective alternative treatment option for cancer sufferers, especially for patients diagnosed with prostate and head and neck cancers.
General Aspects The MTAP inhibitor compounds are useful in both free base form and in the form of salts.
Figures 7, 9, 10, 12, 13, 15 and 16-19 show that the MTAP inhibitor compounds used in the methods of the present invention are orally available, and may therefore be formulated for oral administration. The compounds may also be administered by other routes. For example, the MTAP inhibitors may be administered to a patient orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally or via an implanted reservoir. The amount of compound to be administered will vary widely according to the nature of the patient and the nature and extent of the disorder to be treated. Typically the dosage for an adult human will be in the range less than 1 to 1000 milligrams, preferably 0.1 to 100 milligrams. The specific dosage required for any particular patient will depend upon a variety of factors, including the patient's age, body weight, general health, sex, etc.
For oral administration the active compounds can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions and dispersions. Such preparations are well known in the art as are other oral dosage regimes not listed here. In the tablet form the compounds may be tableted with conventional tablet bases such as lactose, sucrose and corn starch, together with a binder, a disintegration agent and a lubricant. The binder may be, for example, corn starch or gelatin, the disintegrating agent may be potato starch or alginic acid, and the lubricant may be magnesium stearate. For oral administration in the form of capsules, diluents such as lactose and dried comstarch may be employed. Other components such as colourings, sweeteners or flavourings may be added.
When aqueous suspensions are required for oral use, the active ingredient may be combined with carriers such as water and ethanol, and emulsifying agents, suspending agents and/or surfactants may be used. Colourings, sweeteners or flavourings may also be added.
The compounds may also be administered by injection in a physiologically acceptable diluent such as water or saline. The diluent may comprise one or more other ingredients such as ethanol, propylene glycol, an oil or a pharmaceutically acceptable surfactant.
The compounds may also be administered topically. Carriers for topical administration of the compounds include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. The compounds may be present as ingredients in lotions or creams, for topical administration to skin or mucous membranes. Such creams may contain the active compounds suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyi alcohol and water.
The compounds may further be administered by means of sustained release systems.
For example, they may be incorporated into a slowly dissolving tablet or capsule.
Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).
EXAMPLES
Inhibitor Compounds Inhibitors of MTAP were synthesized as described earlier (Singh, V., Shi, W., Evans, G. B., Tyler, P. C., Furneaux, R H, Almo, S C, and Schramm, V L (2004) Biochemistry 43, 9-18; Evans G B, Furneaux R H, Lenz D H, et al., J Med Chem 2005:48, 4679-89). Solutions were standardized by the UV
absorbance of the 9-deazaadenine ring. Sterile solutions of inhibitors were prepared by filtration.
Example 1: Clonogenic Assays (Figure 1) PC3 cells were grown in equal (1:1) portions of Dulbecco's modified Eagle's medium and F12 containing 10% fetal bovine serum, 10 U/mL penicillin-G and 10 g/mL
streptomycin in monolayers to near confluency at 37 C. Cells were lysed in 50 mM
sodium phosphate pH 7.5, 10 mM KCI and 0.5% Triton X-100.
Example 2: Effect of Compound 2 and MTA on PC3 cells (Figure 2) PC3 cells were maintained in MEM Eagle's media supplemented with 10% fetal bovine serum, 100 units/mi penicillin, 100 g/mL streptomycin, 0.1 mM non essential amino acids and 1 mM sodium pyruvate.
Cell survival was evaluated using the WST-1 assay (Kicska G A, long Li, Horig H, et al. Proc Natl Acad Sci USA 2001;98:4593-98). Cells were seeded onto 96 well plates at a density of 104 cells per well, with either no additions, 1 M compound 2, MTA or 1 M compound 2 + 20 M MTA. IC50 was determined following the manufacturer's protocol (Roche Applied Science, IN). Cells were grown and measured in triplicate or quadruplicate and the error bars show the mean t SD
of the multiple samples.
Example 3: Effect of Compound 2 and MTA on SCC25 cells (Figure 3) SCC25 cells were maintained in MEM Eagle's media supplemented with 10% fetal bovine serum, 100 units/mI penicillin, 100 g/mL streptomycin, 0.1 mM non essential amino acids and 1 mM sodium pyruvate.
Cell survival was evaluated using the WST-1 assay (Kicska G A, long Li, Horig H, et al. Proc Naf/ Acad Sci USA 2001;98:4593-98). Cells were seeded onto 96 well plates at a density of 10 cells per well, with either no additions, 1 M MT-compound 2, 20 M MTA or 1 M compound 2 + 20 M MTA. IC50 was determined following the manufacturer's protocol (Roche Applied Science, IN). Cells were grown and measured in triplicate or quadruplicate and the error bars show the mean SD
of the multiple samples.
Example 4: Effect of MT-DADMe-ImmA (Compound 1) and MTA on FaDu cells (Figure 4) FaDu cells were maintained in MEM Eagle's media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 g/mL streptomycin, 0.1 mM non essential amino acids and 1 mM sodium pyruvate.
Cell survival was evaluated using the WST-1 assay (Kicska G A, long Li, Horig H, et al. Proc Natl Acad Sci USA 2001;98:4593-98). Cells were seeded onto 96 well plates at a density of 10d cells per well, with either no additions, 1 M compound 2, MTA or 1 M compound 2 + 20 M MTA. IC5o was determined following the manufacturer's protocol (Roche Applied Science, IN). Cells were grown and measured in triplicate or quadruplicate and the error bars show the mean SD
of the multiple samples.
Example 5: Phase Contrast Microscopy of FaDu Cells (Figure 5) FaDu cells were subjected to six days in culture using the same conditions described as for Example 4.
Example 7: Cell Cycle and Apoptosis Analysis of FaDu cells (Figure 6) FaDu cells were subjected to six days in culture using the same conditions described as for Example 4, before staining with propidium bromide and FACS cell sorting analysis.
Example 8: Oral Availability (Method for Compound (2)) Two groups of 3 C57BL6 mice received a single oral dose of compound (2) dissolved in sterile, deionized water, pippeted onto a crumb of food. Treated food was fed to each mouse individually under close observation at time zero. Two different single doses of inhibitor were administered: 50 g and 100 g. Mice were individually fed and closely observed for consumption of food. At specific time points, 4 L
blood samples were collected from the tail vein. The blood was mixed with 4 L 0.6%
Triton X-1 00 in PBS and stored at -80 C until time of analysis.
The amount of adenine produced was measured by the following MTAP activity assay:
Cells were harvested, washed three times with PBS and lysed with RIPA buffer.
The reaction mixture for MTAP activity assays contained the following: - 75 gg protein from cell lysates, 50 mM HEPES pH 7.4, 50 M MTA, and 20,000 dpm [2,8-3H]MTA.
Labeled MTA was synthesized from [2,8-3H]S-adenosylmethionine by a known method. Products of the MTAP reaction were resolved using TLC silica plates with 1 M ammonium acetate, pH 7.55, and 5% isopropanol. Adenine spots were excised and counted for label incorporation.
Example 9: FaDu Xenograft Studies (Figures 20 and 21) FaDu cells were injected into the dorsum of the foot of NOD-SCID mice. Groups were fed with 250 and 500 M of compound 2 p.o. or given i.p. injections of 100 p.l of compound 2, daily. Differences between treatment cohorts were determined using Student's t test. Compound 2 significantly delayed the growth of established FaDu xenografts.
Example 10: MRI Studies (Figure 22) MRI experiments were performed using a 9.4T 21 cm bore horizontal bore magnet (Magnex Scientific) Varian INOVA MRI system (Fremont, CA) equipped with a 28 mm inner diameter quadrature birdcage coil. Mice were anesthetized with isoflurane inhalation anesthesia (1-1.5% in 100% 02 administered via a nose cone) and positioned in the MRI coil. Body temperature was maintained (37-38 C) using a homeothermic warming system. After acquiring scout images, multi-slice spin-echo imaging with an echo time of 18 ms and a repetition time of 400ms ms was performed. A 40 mm field of view with a 256 x 256 matrix size was used. Nine to 15 slices along the transverse, sagittal, and coronal planes were acquired in each multi-slice experiment with a slice thickness of 1 mm and the gap between slices of 0.5 mm. MRI data were processed off-line with MATLAB-based MRI analysis software.
Example 11: Quantitation of Polyamines in Cells, Spent Media and Tissue Samples (Figure 23) Spent media and perchloric acid extracts of both PC3 cells and tissue samples were subjected to purification via cation exchange chromatography and dansyl-derivatized with minor changes. Disposable 10 ml BIO-RAD columns were centrifuged at 4,000 rpm for 3 minutes. Sodium carbonate used for derivatization was adjusted to pH
9.3 and the concentration of dansyl-chloride was adjusted to 100 mM. Dansyl-polyamines were quantitated by a Waters HPLC/ Fluorescence system. A
Phenomenex Luna 5 p, C18 column was used with a mobile phase of 30%
acetonitrile in a 50 mM ammonium acetate buffer at pH 6.8 (eluent A) and 100% acetonitrile (eluent B). Fluorescence detection was monitored by excitation at 338 nm and emission at 500 nm.
Example 12: Treatment of TRAMP Mice (Table 1, Figure 22) Short-Term: Mice were treated with sterile solutions of 100 M compound (2) (pH -6.4). Water bottles were autoclaved prior to filling with sterile inhibitor solutions.
Mice were sacrificed at 1, 2, and 7 days, with three mice in each group, with the control group sacrificed after 7 days. Livers were immediately removed upon sacrifice for polyamine analysis, conducted as described above.
Long-Term: Sterile solutions of 100 M compound (2) (pH -6.4). Water botties were autoclaved prior to filling with sterile inhibitor solutions. Water consumption was monitored every other day, with fresh inhibitor solution being administered to prevent bacterial growth. Mice were control-sacrificed and tissues (genitourinary system, liver, lungs) were collected for histology and polyamine analysis. Mass and dimensions of excised genitourinary system tumours were recorded. Sections of small intestine were also removed for toxicity analysis via H&E staining.
Discussion of the Examples Figure 1 a shows the effect of the addition of compound (2) to cultured mouse prostate cancer cells (RM1). Figure lb shows the effect of the addition of compound (2) to cultured human prostate cancer cells (PC3). Compound (2) was added either alone or in the presence of 20 ttM MTA. Figures 2, 3 and 4 show the effects of MTA
alone, compound (2) alone and MTA with compound (2) in time dependent cell proliferation experiments (PC3 cells, SCC25 cells and FaDu cells). The combination of compound (2) and MTA reduces cell proliferation. These data demonstrate that the compounds which are used in the methods of the present invention inhibit cell growth in vitro, when administered in combination with MTA.
Figure 5 further demonstrates, showing phase contrast photographs of FaDu cells after 5 days of treatment with compound (2)/compound (2) + MTA, that the inhibitor compound + MTA is effective in inhibiting cell growth.
Thus, administration of MTA in circumstances where its degradation by MTAP is inhibited by an MTAP inhibitor leads to greater circulatory and tissue levels of MTA
and consequently an enhanced effect in the treatment of cancer.
Figure 6 shows that compound (2) in combination with MTA is also effective for stopping cell cycling (for FaDu cells) such that the cells become apoptotic.
Figures 20 and 21 show the results of in vivo studies. FaDu cells were injected into the dorsum of the foot of NOD-SCID mice. Groups were fed with 250 and 500 M
of compound (2) p.o. or given i.p. injections of 100 i of 4 mM compound (2), daily.
Figure 21 shows representative tumours from each of the treatment cohorts.
Differences between treatment cohorts were determined using Student's t test.
Figure 20 is a summary of the data for all treatment cohorts. The results show that compound (2) significantly delays the growth of established FaDu xenografts.
Longitudinal MRI provides a noninvasive means of monitoring prostate tumour growth in mice (Gupta S, Hastak K, Ahmad N, Lewin J S, Mukhtar H Proc Nat/ Acad Sci USA
2001 Aug 28;98(18):10350-5; Eng M H, Charles L G, Ross B D, Chrisp C E, Pienta K
J, Greenberg N M, Hsu C X, Sanda M G Urology 1999 Dec:54(6):1112-9; Song S K, Qu Z, Garabedian E M, Gordon J I, Milbrandt J, Ackerman J J Cancer Res. 2002 Mar 1:62(5):1555-8.).
MRI is used in the present case to evaluate prostate tumour growth and progression longitudinally in TRAMP mice (either untreated or treated with a compound that may be used according the methods of the invention). Mice were imaged approximately monthly from 12-33 weeks of age. Representative MRI images comparing untreated control TRAMP and treated TRAMP mice at approximately 30 weeks of age are shown in Figure 22.
Panels A and B show results from control mice. Panel A shows a coronal section through of a 30 week old TRAMP mouse with a large tumour (bright tissue) that weighed 8.76 g upon dissection at 34 weeks of age. The inset shows a more posterior coronal section. The bright tumour is smaller in this section but metastasis to the liver is observed (white arrow). Panel B shows a coronal section through the prostate region of a 30 week old TRAMP mouse. The seminal vesicles (SV) are enlarged. A large tumour (weighing 4.89 g upon dissection at 36 weeks of age) that spanned from the kidney to bladder (BL) is visible in the transverse section shown in the inset (white arrow).
Panels E and F show results for mice treated with 1 mM compound (1). Panel E
shows a coronal section through the prostate region of a 30 week old treated TRAMP
mouse. The tumour, weighing 0.41 g upon dissection at 34 weeks of age, was not observed during the imaging session. Panel F shows a similar section through a week old treated TRAMP mouse that exhibited a 0.64 g tumour upon dissection at weeks of age. The tumour is indicated by the white arrow in the MRI image shown in this panel.
Untreated TRAMP mice therefore demonstrate primary prostate tumour growth.
However, prostate cancer progression in the TRAMP mouse is inhibited in mice treated with compound (1), either alone or in combination with MTA.
Figure 23 shows that compound (2) and MTA, administered together, alter polyamine levels and induce cytostasis in PC3 cells. Combination treatment of PC3 cells with compound (2) and MTA for 1 day resulted in a significant 6-fold increase in intracellular PUT levels (3.03 x 10'3 t 2.86 x 10-2, combination treated cells vs. 5.04 X
10'2 f 1.08 x 10-2, control, p= 0.001, pmoles PUT/mg protein), a 2-fold increase in spent media PUT levels [1.19 x 10'3 2.04 x 10-1, combination treated media vs. 5.85 x 10'2 5.09 x 10"0, control media, p= 0.0001, pmoles PUT/mL spent media, as well as roughly a 2.5-fold increase in intracellular SPD levels (7.19 x 10'3 4.38 x 10"2, combination treated cells vs. 3.05 x 10'3 6.3 x 10'2, control, p=0.001 pmoles SPD/mg protein). SPN levels in combination treated spent media also slightly decreased (p=0.02). After 6 days of treatment, cellular SPN levels were decreased roughly 0.5-fold (4.0 x 10"3 7.38 x 10-2, combination treated cells vs. 6.87 x 10-3 9.68 x 10-2, control, p= 0.005, pmoles SPN/mg protein), with both PUT and SPD
elevated (p= 0.02 and p= 0.01, respectively in compa(son to controls). Most significantly, levels of PUT in spent media were almost double that of the control (2.41 x 10'3 7.35 x 10"1, combination treated spent media vs. 1.31 X 10"3 0.0, control, p=0.0007, pmoles PUT/mL spent media). By day 12, a significant increase in cellular SPD levels were observed (9.05 x 10'3 1.09 x 10'3, combination treated cells vs.
3.93 x 10'3 8.4 x 10"1, control, p=0.007, pmoles SPD/mg protein), with a corresponding decrease in levels of spent media PUT levels (1.65 x 10-3 2.27 x 10"
Z, combination treated spent media vs. 2.12 x 10"3 9.34 x 10"1, control media, pmoles PUT/mL spent media, p=0.013). Intracellular PUT levels in combination treated cells were still significantly higher than controls (p=0.005).
Treatment of PC3 cells with compound (2) therefore results in numerous significant alterations in both intracellular and spent media polyamine levels. After 24 hours of treatment, the increase observed in cellular SPD levels as well as putrescine (PUT) cellular and spent media polyamine levels correlates with the effects expected with MTAP inhibition. MTA is accumulating in the cells, beginning to feedback inhibiting SPN synthase, resulting in accumulations of SPD and PUT, with PUT being significantly excreted into the media, and a slight decrease of SPN in the media. By day 6, cellular SPN levels are significantly reduced in combination treated cells, while maintaining the characteristic elevations in levels of PUT and SPD. Treatment of cells for 12 days shows a significant increase in cellular SPD levels and a slight decrease in spent media PUT levels, pointing to the fact that a compensatory pathway has been activated to make up for the block in MTAP. PUT may be being taken up from the media for SPD synthesis. After combination treatment for approximately 2 weeks, PC3 cells display a cytostatic effect, as determined by the clonogenic assay. Initially, it was believed that MTAP inhibition would lead to MTA
accumulation, causing feedback inhibition of polyamine biosynthesis, resulting in decreases in cellular proliferation. A halt in cellular proliferation is observed, and it is now believed that this is not due only to polyamine depletion.
Figure 24 shows that compound (2) reduces tumour growth and metastasis in TRAMP
mice, but does not alter polyamine levels in vivo. Polyamine levels of mice livers are not significantly altered during short-term treatment (Figure 24A). After extended treatment with compound (2) inhibitor solutions, no significant alterations in either TRAMP liver or GUS polyamine levels were detected (Figures 24B and 24C).
Mass (Table 1) and dimensions of excised genitourinary system tumors were recorded for all members of the treatment groups. Sections of small intestine were also removed for toxicity analysis via H&E staining. Histology of TRAMP mice revealed all animals showed extensive prostate intraepithelial neoplasia involving most prostate acini. However, the size and incidence of preinvasive tumors, as well as the incidence of invasive cancer and metastasis were all decreased in treated TRAMP mice (Table 1). No alterations, inflammations, or irregularities were observed in the intestinal crypts, neither were any hair loss or general Gl problems noted, indicating a lack of drug toxicity.
Table 1: Summary of results for TRAMP mice treated with compound (2) # Tumor Weeks Metastatic Experimental Animals Size treated Cancer Condition (n) (g) Control 16 4.0 t 2.8 32 5 44%
100 M compound (2) 12 1.7 0.8 29 7 8%
Although the invention has been described by way of example, it should be appreciated the variations or modifications may be made without departing from the scope of the invention. Furthermore, when known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in the specification.
severely limits the polyamine biosynthesis and the salvage pathway for adenine in the cells.
Although MTAP is abundantly expressed in normal cells and tissues, MTAP
deficiency due to a genetic deletion has been reported with many malignancies.
The loss of MTAP enzyme function in these cells is known to be due to homozygous deletions on chromosome 9 of the closely linked MTAP and p161MTS1 tumour suppressor gene. As absence of p16/MTS1 is probably responsible for the tumour, the lack of MTAP activity is a consequence of the genetic deletion and is not causative for the cancer. However, the absence of MTAP alters the purine metabolism in these cells so that they are mainly dependent on the de novo pathway for their supply of purines.
MTA has been shown to induce apoptosis in dividing cancer cells, but to have the opposite, anti-apoptotic effect on dividing normal cells such as hepatocytes (E.
Ansorena et al., Hepatology, 2002, 35: 274-280).
MTAP inhibitors may therefore be used in the treatment of cancer. Such treatments are described in WO 03/080620 and WO 2004/018496.
The need for new cancer therapies remains ongoing. For some prevalent cancers the treatment options are still limited. Prostate cancer, for example, is the most commonly diagnosed non-skin cancer in the United States. Current treatment options include radical prostatectomy, radiation therapy, hormonal therapy, and watchful waiting.
Although the therapies may offer successful treatment of an individual's condition, the pitfalls are quite unfavorable and lead to a decrease in a man's overall quality of life.
Surgery may inevitably result in impotence, sterility, and urinary incontinence. Side effects associated with radiation therapy include damage to the bladder and rectum as well as slow-onset impotence. Hormonal therapy will not cure the cancer and eventually most cancers develop a resistant to this type of therapy. The major risk associated with watchful waiting is that it may result in tumour growth, cancer progression and metastasis. It is therefore desirable that a better treatment option is made available to patients diagnosed with prostate cancer.
It is an object of the invention to provide a method of treating cancer, particularly prostate or head and neck cancer, or at least to provide a useful choice.
STATEMENTS OF INVENTION
Ina first aspect, the invention provides a method of treating cancer comprising administering to a patient in need thereof a compound of the formula (I):
V
W
X
Y
OH
(I) wherein:
V is selected from CH2 and NH, and W is selected from NR' and NR2; or V
is selected from NR' and NR2, and W is selected from CH2 and NH;
X is selected from CH2 and CHOH in the R or S-configuration;
Y is selected from hydrogen, halogen and hydroxy, except where V is selected from NH, NR' and NR2 then Y is hydrogen;
Z is selected from hydrogen, halogen, hydroxy, SQ, OQ and Q, where Q
is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
R' is a radical of the formula (II) B
H
N N
A' I
D
~-- G
(II) R2 is a radical of the formula (III) B
N N
A I
/ N E D
~-- G
(III) A is selected from N, CH and CR3, where R3 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen; or R3 is hydroxyl, halogen, NH2, NHR4, NR4R5;
or SR6, where R , R5 and R6 are alkyl, aralkyl or aryl groups, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
B is selected from NH2 and NHR7, where R7 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
D is selected from hydroxy, NH2, NHR8, hydrogen, halogen and SCH3, where R8 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
E is selected from N and CH;
G is selected from CH2 and NH, or G is absent, provided that where W is NR' or NR2 and G is NH then V is CH2, and provided that where V is NR' or NR2 and G is NH then W is CHZ;
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.
Preferably the cancer is prostate cancer or head and neck cancer.
In another aspect, the invention provides a method of treating cancer comprising administering to a patient in need thereof a compound of the formula (IV):
W
X
Y
OH
(IV) wherein:
V is selected from CH2 and NH, and W is selected from NR' and NR2; or V
is selected from NR' and NRZ, and W is selected from CH2 and NH;
X is selected from CH2 and CHOH in the R or S-configuration;
Y is selected from hydrogen, halogen and hydroxy, except where V is selected from NH, NR' and NR2 then Y is hydrogen;
Z is selected from hydrogen, halogen, hydroxy, SQ, OQ and Q, where Q
is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
R' is a radical of the formula (V) B
H
p N N
A I
D
~-- G
(V) R2 is a radical of the formula (VI) B
~ \''N
A I
N
~ E D
goG
(\A) A is selected from N, CH and CR3, where R3 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen; or R3 is hydroxyl, halogen, NH2, NHR4, NR4R5;
or SRs, where R4, R5 and R 6 are alkyl, aralkyl or aryl groups, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
B is selected from NH2 and NHR7, where R7 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
D is selected from hydroxy, NH2, NHR8, hydrogen, halogen and SCH3, where RB is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
E is selected from N and CH;
G is selected from CH2 and NH, or G is absent, provided that where W is NR' or NR2 and G is NH then V is CH2, and provided that where V is NR' or NR2 and G is NH then W is CH2;
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof;
provided that the compound (3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine is excluded.
Preferably Z is SQ.
Preferably Q is an alkyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy. It is further preferred that Q is a Cj-Ce alkyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy. Most preferably Q
is a methyl group Alternatively it is preferred that Z is not methylthio.
It is also preferred that Q is an aryl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy. More preferably Q is a phenyl or benzyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy.
Preferably G is CH2. It is also preferred that V is CH2 and W is NR1. It is further preferred that B is NHZ. It is also preferred that D is H, and it is preferred that A is CH.
Preferably any halogen is chlorine or fluorine.
In another aspect, the invention provides a method of treating cancer comprising administering to a patient in need thereof a compound of the formula (VII):
N
J I
~S N
\N
N
HO
(VI l) where J is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy.
Preferably the cancer is prostate cancer or head and neck cancer.
Preferably J is C1-C7 alkyl. More preferably J is methyl, ethyl, n-propyl, i-propyl, n-butyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl or cycloheptyl.
It is also preferred that J is phenyl, optionally substituted with one or more halogen substituents. More preferably J is phenyl, p-chlorophenyl, p-fluorophenyl or m-chlorophenyl.
It is also preferred that J is heteroaryl. More preferably J is 4-pyridyl.
It is also preferred that J is aralkyl. More preferably J is benzylthio.
Preferably J is -CH2CH2(NH2)COOH.
In another aspect, the invention provides a method of treating cancer comprising administering to a patient in need thereof a compound of the formula (VIII):
N
T S N
N N
HO OH
(VIII) where T is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, carboxy or straight- or branched-chain Cl-Cg alkyl.
Preferably the cancer is prostate cancer or head and neck cancer.
Preferably T is C1-C6 alkyl, optionally substituted with one or more substituents selected from halogen and hydroxy. More preferably T is methyl, ethyl, 2-fluoroethyl, or 2-hydroxyethyl. Most preferably T is methyl.
It is also preferred that T is aryl, optionally substituted with one or more substituents selected from halogen or straight-chain Cl-Ce alkyl. More preferably T is phenyl, naphthyl, p-tolyl, m-tolyl, p-chlorophenyl, m-chlorophenyl or p-fluorophenyl.
It is also preferred that T is aralkyl. More preferably T is benzyl.
Preferably the compound of formula (I) is:
(3R,4R)-1-[(8-aza-9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl) methyl]-3-hydroxy-4-(2-phenylethyl)pyrrolidine;
(3R,4S)-1 -[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzyFthiomethyl)pyrrolidine;
(3R,4S)-1-[(8-aza-9-deazaaden in-9-yl)methyl]-3-hydroxy-4-(benzylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-chlorophenylthiomethyl)pyrrolidine;
(3 R,4R)-1-[(9-deazaadenin-9-yl)methyl]-3-acetoxy-4-(acetoxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(n-butylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-fluorophenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yi)methyl]-3-hydroxy-4-(n-propylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaaden in-9-yl)methyl]-3-hydroxy-4-(cyclohexylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(3-chlorophenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(ethylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(phenylthiomethyl)pyrrolidine;
(3 R,4 S)-1-[(9-deazaaden in-9-yl) methy I]-3-hyd roxy-4-(4-pyridylthiomethyl)pyrrolidine;
(3 R,4S)-1-[(9-deazaadenin-9-yl) methyl]-3-hydroxy-4-n-propylpyrrolidine;
(3R,4S)-1-[(9-deazaaden in-9-yl)methyl]-3-hydroxy-4-(homocysteinylmethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzyloxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaaden in-9-yi)methyl]-3-hydroxy-4-(i-propylthiomethyl)pyrrolidine;
(3 R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methoxymethyl)pyrrolidine;
(3 R,4S)-1-[(9-deazaadenin-9-yl) methyl]-3-hydroxy-4-(cyclohexylmethylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cycloheptylthiomethyl)pyrrolid ine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclopentylthiomethyl)pyrrolid ine;
(3R,4S)-1-[(9-deazaaden in-9-yl)methyl]-3-hydroxy-4-(cyclobutylthiomethyl)pyrrolidine.
The compounds defined above may be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally or via an implanted reservoir. Preferably the compounds are administered orally.
BRIEF DESCRIPTION OF THE FIGURES
Figure la shows the survival of mouse prostate cancer cells (RM1) against increasing concentrations of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methyithiomethyl)pyrrolidine], either in the presence or absence of MTA.
Figure lb shows the survival of human prostate cancer cells (PC3) against increasing concentrations of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine], either in the presence or absence of MTA.
Figure 2 is a time dependent proliferation curve, showing the effect of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine]
and MTA on human prostate cancer cells (PC3).
Figure 3 is a time dependent proliferation curve, showing the effect of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine]
and MTA on SCC25 cells.
Figure 4 is a time dependent proliferation curve, showing the effect of compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy4-(methylthiomethyl)pyrrolidine]
and MTA on FaDu cells.
Figure 5 shows phase contrast photographs of FaDu cells after 5 days of treatment with compound (2) [(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine] and MTA.
Figure 6 shows a cell cycle and apoptosis analysis of FaDu cells after 6 days of treatment with compound (2) ((3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-(methylthiomethyl)pyrrolidine] and MTA; (1) untreated resufts: G1 83.66%, S
8.08%, G2 8.26%, Apoptosis 6.06%; (2) treated with MTA results: G1 79.67%, S 10.42%, 9.91%, Apoptosis 6.66%; (3) treated with compound (3) results GI 72.06%, S
17.98%, G29.96%, Apoptosis 7.89%; (4) treated with MTA + compound (3) results GI
8.26%, S 31.25%, G2 60.49%, Apoptosis 29.41 %.
Figures 7 to 19 show oral and IP availability of selected compounds that may be used in the methods of the invention.
Figure 20 shows the effects of compound 1[(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzylthiomethyl)pyrrolidine] on FaDu xenografts in NOD-SCID
mice.
Figure 21 shows representative tumours from each of the treatment cohorts for the above NOD-SCID mouse study.
Figure 22 shows MRI images of TRAMP mice (Panels A and B: Control TRAMP
(transgenic adenocarcinoma of mouse prostate) mice, Panels E and F: TRAMP mice treated with 1 mM Compound 1[(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-(benzylthiomethyl)pyrrolidine]) Figure 23 shows that compound (2) and MTA alter polyamine levels and induce cytostasis in PC3 cells (PUT=putrescine, SPD=spermidine, SPN=spermine). PC3 cells were cultured and treated in triplicate as follows: untreated control, substrate (MTA) alone, 1 M compound (2) alone, or a combination of both substrate and inhibitor. Both cells and spent media were harvested at 1, 6, and 12 days for polyamine analysis by HPLC fluorescence.
Figure 24 shows that compound (2) reduces tumour growth and metastasis in TRAMP mice, but does not alter polyamine levels in vivo. C56B1/6 mice were treated with 100 M compound (2) via their drinking water and sacrificed at 24, 48 hours, and 7 days. Livers were immediately removed for polyamine analysis. TRAMP mice were treated approximately 6-8 months with 100 [M compound (2) via their drinking water and control sacrificed. Livers were removed for polyamine analysis.
DETAILED DESCRIPTION
Derinitions The term "alkyl" is intended to include straight- and branched-chain alkyl groups, as well as cycloalkyl groups. The same terminology applies to the non-aromatic moiety of an aralkyl radical. Examples of alkyl groups include: methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, iso-butyl group, sec-butyl group, t-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-ethylpropyl group, n-hexyl group and 1-methyl-2-ethylpropyl group.
The term "aryl" means an aromatic radical having 6 to 18 carbon atoms and includes heteroaromatic radicals. Examples include monocyclic groups, as well as fused groups such as bicyclic groups and tricyclic groups. Some examples include phenyl group, indenyl group, 1-naphthyl group, 2-naphthyl group, azulenyl group, heptalenyl group, biphenyl group, indacenyl group, acenaphthyl group, fluorenyl group, phenalenyl group, phenanthrenyl group, anthracenyl group, cyclopentacyclooctenyl group, and benzocyclooctenyl group, pyridyl group, pyrrolyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, triazolyl group, tetrazolyl group, benzotriazolyl group, pyrazolyl group, imidazolyl group, benzimidazolyl group, indolyl group, isoindolyl group, indolizinyl group, purinyl group, indazolyl group, furyl group, pyranyl group, benzofuryl group, isobenzofuryl group, thienyl group, thiazolyl group, isothiazolyl group, benzothiazolyl group, oxazolyl group, and isoxazolyl group.
The term "halogen" includes fluorine, chlorine, bromine and iodine.
The compounds are useful for the treatment of certain diseases and disorders in humans and other animals. Thus, the term "patient" as used herein includes both human and other animal patients.
The term "prodrug" as used herein means a pharmacologically acceptable derivative of the compound of formula (I), (IV), (VII) or (VIII), such that an in vivo biotransformation of the derivative gives the compound as defined in formula (I), (IV), (VII) or (VIII). Prodrugs of compounds of formulae (I), (IV), (VII) or (VIII) may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to give the parent compound.
Prodrugs include compounds of formulae (I), (IV), (VII) and (VIII), tautomers thereof and/or pharmaceutically acceptable salts thereof, which include an ester functionality, or an ether functionality. It will be clear to the skilled person that the compounds of formulae (I), (IV), (VII) and (VIII) may be converted to corresponding ester or ether prodrugs using known chemical transformations.
Suitable prodrugs include those where the hydroxyl groups of the compounds of formula (I), (IV), (VII) or (VIII) are esterified to give, for example, a primary hydroxyl group ester of propanoic or butyric acid. Other suitable prodrugs are alkycarbonyoxymethyl ether derivatives on the hydroxyl groups of the compounds of formula (I), (IV), (VII) or (VIII) to give, for example, a primary hydroxyl group ether with a pivaloyloxymethyl or a propanoyloxymethyl group.
The term "pharmaceutically acceptable salts" is intended to apply to non-toxic salts derived from inorganic or organic acids, including, for example, the following acid salts: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, p-toluenesulfonate, salicylate, succinate, sulfate, tartrate, thiocyanate, and undecanoate.
It will be appreciated that the representation of a compound of formula (I) or (IV) where B and/or D is a hydroxy group, is of the enol-type tautomeric form of a corresponding amide, and this will largely exist in the amide form. The use of the enol-type tautomeric representation is simply to allow fewer structural formulae to represent the compounds of the invention.
Similarly, it will be appreciated that the representation of a compound of formula (1) or (IV), where B and/or D is a thiol group, is of the thioenol-type tautomeric form of a corresponding thioamide, and this will largely exist in the thioamide form.
The use of the thioenol-type tautomeric representation is simply to allow fewer structural formulae to represent the compounds of the invention.
Detailed Description of the Invention The present invention relates to methods of treating cancer by administering to a patient in need thereof one or more inhibitors of 5'-methylthioadenosine phosphorylase (MTAP). In particular, the invention relates to methods of treating certain cancers, such as prostate cancer or head and neck cancer.
Suitable MTAP inhibitors which may be employed in the method of the present invention and the methods for preparing these inhibitors are described in WO
03/080620 and WO 2004/018496.
Certain MTAP inhibitor compounds are surprisingly effective for treating prostate and head and neck cancers. These are compounds of general formula (VII).
N
~N
NS N
HO
(VI l) This sub-class of MTAP inhibitors incorporates an adenine-like base moiety and a pyrrolidine moiety having an alkyl- aryl- or aralkylthiomethyl group at the 4-position.
Other MTAP inhibitor compounds are also surprisingly effective for treating prostate and head and neck cancers. These are compounds of general formula (VIII).
T~s N
N N
HO OH
(VIII) This sub-class of MTAP inhibitors also incorporates the adenine-like base moiety but has an iminoribitol moiety with an alkyl- aryl- or aralkylthiomethyl group at the 5'-position.
Examples of the first sub-class of inhibitors include compounds (1) and (2).
H NH= PN
NS N
HO HO
Compound (1) Compound (2) BT-DADMe-ImmA MT-DADMe-ImmA
The present studies show that compounds (1) and (2) are effective both in vitro and in vivo against a variety of cell lines (PC3, RM1, SCC25 and FaDu). These compounds are therefore particularly useful in the treatment of prostate and head and neck cancers.
The MTAP inhibitor compounds inhibit cell growth in vitro of the prostate cancer cell lines PC3 and RM1 and the head and neck cancer cell lines SCC25 and FaDu. An enhanced cell-killing effect is seen in vitro with combined administration of the MTAP
inhibitor compound plus MTA. Examples of this effect are shown in Figures 1 to 6.
Furthermore, the inhibitor compounds, when co-administered with MTA, exhibit a cytostatic effect on PC3 cells in vitro.
In order to determine whether the inhibition is selective for malignant cells, normal human fibroblast cells (GM02037) were also treated with compound (2) and MTA
for 3 weeks. No cytotoxicity was observed. Compound (2) is therefore cytotoxic for human HNSCC (human head and neck squamous cell carcinoma) cells at doses that exhibit minimal toxicity for normal cells. This selectivity is a further indication that the MTAP inhibitors described above are useful agents for the treatment of head and neck cancer.
The present in vivo studies further demonstrate the surprising efficacy of the compounds. In a NOD-SCID mouse model, compound (2) significantly delays the growth of established FaDu xenografts. The effect is seen either with or without co-administration of the inhibitor compound with MTA.
In addition, prostate cancer progression in the TRAMP mouse model is inhibited in mice treated with compound (1), either alone or in combination with MTA.
An example of the second sub-class of inhibitors is compound (3).
N
N
N
HO Compound (3) MT-ImmA
This compound also inhibits prostate cancer progression in the TRAMP mouse model, when administered either alone or in combination with MTA.
For the above in vivo models, the inhibitor compounds exhibit activity when administered with exogenous MTA and when administered alone. There is not a significant enhancement observed when the inhibitors are administered together with MTA. However, the in vitro results clearly demonstrate a surprising enhancement in activity when the inhibitors are administered in conjunction with MTA. Thus, the combined administration method provides a potential alternative treatment method for patients suffering from cancer, where the administration of an MTAP inhibitor is indicated.
The MTAP inhibitor compounds of formulae (I), (IV), (VII) and (VIII) (in particular the compounds of formulae (VII) and (VIII)) provide an effective alternative treatment option for cancer sufferers, especially for patients diagnosed with prostate and head and neck cancers.
General Aspects The MTAP inhibitor compounds are useful in both free base form and in the form of salts.
Figures 7, 9, 10, 12, 13, 15 and 16-19 show that the MTAP inhibitor compounds used in the methods of the present invention are orally available, and may therefore be formulated for oral administration. The compounds may also be administered by other routes. For example, the MTAP inhibitors may be administered to a patient orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally or via an implanted reservoir. The amount of compound to be administered will vary widely according to the nature of the patient and the nature and extent of the disorder to be treated. Typically the dosage for an adult human will be in the range less than 1 to 1000 milligrams, preferably 0.1 to 100 milligrams. The specific dosage required for any particular patient will depend upon a variety of factors, including the patient's age, body weight, general health, sex, etc.
For oral administration the active compounds can be formulated into solid or liquid preparations, for example tablets, capsules, powders, solutions, suspensions and dispersions. Such preparations are well known in the art as are other oral dosage regimes not listed here. In the tablet form the compounds may be tableted with conventional tablet bases such as lactose, sucrose and corn starch, together with a binder, a disintegration agent and a lubricant. The binder may be, for example, corn starch or gelatin, the disintegrating agent may be potato starch or alginic acid, and the lubricant may be magnesium stearate. For oral administration in the form of capsules, diluents such as lactose and dried comstarch may be employed. Other components such as colourings, sweeteners or flavourings may be added.
When aqueous suspensions are required for oral use, the active ingredient may be combined with carriers such as water and ethanol, and emulsifying agents, suspending agents and/or surfactants may be used. Colourings, sweeteners or flavourings may also be added.
The compounds may also be administered by injection in a physiologically acceptable diluent such as water or saline. The diluent may comprise one or more other ingredients such as ethanol, propylene glycol, an oil or a pharmaceutically acceptable surfactant.
The compounds may also be administered topically. Carriers for topical administration of the compounds include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. The compounds may be present as ingredients in lotions or creams, for topical administration to skin or mucous membranes. Such creams may contain the active compounds suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyi alcohol and water.
The compounds may further be administered by means of sustained release systems.
For example, they may be incorporated into a slowly dissolving tablet or capsule.
Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).
EXAMPLES
Inhibitor Compounds Inhibitors of MTAP were synthesized as described earlier (Singh, V., Shi, W., Evans, G. B., Tyler, P. C., Furneaux, R H, Almo, S C, and Schramm, V L (2004) Biochemistry 43, 9-18; Evans G B, Furneaux R H, Lenz D H, et al., J Med Chem 2005:48, 4679-89). Solutions were standardized by the UV
absorbance of the 9-deazaadenine ring. Sterile solutions of inhibitors were prepared by filtration.
Example 1: Clonogenic Assays (Figure 1) PC3 cells were grown in equal (1:1) portions of Dulbecco's modified Eagle's medium and F12 containing 10% fetal bovine serum, 10 U/mL penicillin-G and 10 g/mL
streptomycin in monolayers to near confluency at 37 C. Cells were lysed in 50 mM
sodium phosphate pH 7.5, 10 mM KCI and 0.5% Triton X-100.
Example 2: Effect of Compound 2 and MTA on PC3 cells (Figure 2) PC3 cells were maintained in MEM Eagle's media supplemented with 10% fetal bovine serum, 100 units/mi penicillin, 100 g/mL streptomycin, 0.1 mM non essential amino acids and 1 mM sodium pyruvate.
Cell survival was evaluated using the WST-1 assay (Kicska G A, long Li, Horig H, et al. Proc Natl Acad Sci USA 2001;98:4593-98). Cells were seeded onto 96 well plates at a density of 104 cells per well, with either no additions, 1 M compound 2, MTA or 1 M compound 2 + 20 M MTA. IC50 was determined following the manufacturer's protocol (Roche Applied Science, IN). Cells were grown and measured in triplicate or quadruplicate and the error bars show the mean t SD
of the multiple samples.
Example 3: Effect of Compound 2 and MTA on SCC25 cells (Figure 3) SCC25 cells were maintained in MEM Eagle's media supplemented with 10% fetal bovine serum, 100 units/mI penicillin, 100 g/mL streptomycin, 0.1 mM non essential amino acids and 1 mM sodium pyruvate.
Cell survival was evaluated using the WST-1 assay (Kicska G A, long Li, Horig H, et al. Proc Naf/ Acad Sci USA 2001;98:4593-98). Cells were seeded onto 96 well plates at a density of 10 cells per well, with either no additions, 1 M MT-compound 2, 20 M MTA or 1 M compound 2 + 20 M MTA. IC50 was determined following the manufacturer's protocol (Roche Applied Science, IN). Cells were grown and measured in triplicate or quadruplicate and the error bars show the mean SD
of the multiple samples.
Example 4: Effect of MT-DADMe-ImmA (Compound 1) and MTA on FaDu cells (Figure 4) FaDu cells were maintained in MEM Eagle's media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 g/mL streptomycin, 0.1 mM non essential amino acids and 1 mM sodium pyruvate.
Cell survival was evaluated using the WST-1 assay (Kicska G A, long Li, Horig H, et al. Proc Natl Acad Sci USA 2001;98:4593-98). Cells were seeded onto 96 well plates at a density of 10d cells per well, with either no additions, 1 M compound 2, MTA or 1 M compound 2 + 20 M MTA. IC5o was determined following the manufacturer's protocol (Roche Applied Science, IN). Cells were grown and measured in triplicate or quadruplicate and the error bars show the mean SD
of the multiple samples.
Example 5: Phase Contrast Microscopy of FaDu Cells (Figure 5) FaDu cells were subjected to six days in culture using the same conditions described as for Example 4.
Example 7: Cell Cycle and Apoptosis Analysis of FaDu cells (Figure 6) FaDu cells were subjected to six days in culture using the same conditions described as for Example 4, before staining with propidium bromide and FACS cell sorting analysis.
Example 8: Oral Availability (Method for Compound (2)) Two groups of 3 C57BL6 mice received a single oral dose of compound (2) dissolved in sterile, deionized water, pippeted onto a crumb of food. Treated food was fed to each mouse individually under close observation at time zero. Two different single doses of inhibitor were administered: 50 g and 100 g. Mice were individually fed and closely observed for consumption of food. At specific time points, 4 L
blood samples were collected from the tail vein. The blood was mixed with 4 L 0.6%
Triton X-1 00 in PBS and stored at -80 C until time of analysis.
The amount of adenine produced was measured by the following MTAP activity assay:
Cells were harvested, washed three times with PBS and lysed with RIPA buffer.
The reaction mixture for MTAP activity assays contained the following: - 75 gg protein from cell lysates, 50 mM HEPES pH 7.4, 50 M MTA, and 20,000 dpm [2,8-3H]MTA.
Labeled MTA was synthesized from [2,8-3H]S-adenosylmethionine by a known method. Products of the MTAP reaction were resolved using TLC silica plates with 1 M ammonium acetate, pH 7.55, and 5% isopropanol. Adenine spots were excised and counted for label incorporation.
Example 9: FaDu Xenograft Studies (Figures 20 and 21) FaDu cells were injected into the dorsum of the foot of NOD-SCID mice. Groups were fed with 250 and 500 M of compound 2 p.o. or given i.p. injections of 100 p.l of compound 2, daily. Differences between treatment cohorts were determined using Student's t test. Compound 2 significantly delayed the growth of established FaDu xenografts.
Example 10: MRI Studies (Figure 22) MRI experiments were performed using a 9.4T 21 cm bore horizontal bore magnet (Magnex Scientific) Varian INOVA MRI system (Fremont, CA) equipped with a 28 mm inner diameter quadrature birdcage coil. Mice were anesthetized with isoflurane inhalation anesthesia (1-1.5% in 100% 02 administered via a nose cone) and positioned in the MRI coil. Body temperature was maintained (37-38 C) using a homeothermic warming system. After acquiring scout images, multi-slice spin-echo imaging with an echo time of 18 ms and a repetition time of 400ms ms was performed. A 40 mm field of view with a 256 x 256 matrix size was used. Nine to 15 slices along the transverse, sagittal, and coronal planes were acquired in each multi-slice experiment with a slice thickness of 1 mm and the gap between slices of 0.5 mm. MRI data were processed off-line with MATLAB-based MRI analysis software.
Example 11: Quantitation of Polyamines in Cells, Spent Media and Tissue Samples (Figure 23) Spent media and perchloric acid extracts of both PC3 cells and tissue samples were subjected to purification via cation exchange chromatography and dansyl-derivatized with minor changes. Disposable 10 ml BIO-RAD columns were centrifuged at 4,000 rpm for 3 minutes. Sodium carbonate used for derivatization was adjusted to pH
9.3 and the concentration of dansyl-chloride was adjusted to 100 mM. Dansyl-polyamines were quantitated by a Waters HPLC/ Fluorescence system. A
Phenomenex Luna 5 p, C18 column was used with a mobile phase of 30%
acetonitrile in a 50 mM ammonium acetate buffer at pH 6.8 (eluent A) and 100% acetonitrile (eluent B). Fluorescence detection was monitored by excitation at 338 nm and emission at 500 nm.
Example 12: Treatment of TRAMP Mice (Table 1, Figure 22) Short-Term: Mice were treated with sterile solutions of 100 M compound (2) (pH -6.4). Water bottles were autoclaved prior to filling with sterile inhibitor solutions.
Mice were sacrificed at 1, 2, and 7 days, with three mice in each group, with the control group sacrificed after 7 days. Livers were immediately removed upon sacrifice for polyamine analysis, conducted as described above.
Long-Term: Sterile solutions of 100 M compound (2) (pH -6.4). Water botties were autoclaved prior to filling with sterile inhibitor solutions. Water consumption was monitored every other day, with fresh inhibitor solution being administered to prevent bacterial growth. Mice were control-sacrificed and tissues (genitourinary system, liver, lungs) were collected for histology and polyamine analysis. Mass and dimensions of excised genitourinary system tumours were recorded. Sections of small intestine were also removed for toxicity analysis via H&E staining.
Discussion of the Examples Figure 1 a shows the effect of the addition of compound (2) to cultured mouse prostate cancer cells (RM1). Figure lb shows the effect of the addition of compound (2) to cultured human prostate cancer cells (PC3). Compound (2) was added either alone or in the presence of 20 ttM MTA. Figures 2, 3 and 4 show the effects of MTA
alone, compound (2) alone and MTA with compound (2) in time dependent cell proliferation experiments (PC3 cells, SCC25 cells and FaDu cells). The combination of compound (2) and MTA reduces cell proliferation. These data demonstrate that the compounds which are used in the methods of the present invention inhibit cell growth in vitro, when administered in combination with MTA.
Figure 5 further demonstrates, showing phase contrast photographs of FaDu cells after 5 days of treatment with compound (2)/compound (2) + MTA, that the inhibitor compound + MTA is effective in inhibiting cell growth.
Thus, administration of MTA in circumstances where its degradation by MTAP is inhibited by an MTAP inhibitor leads to greater circulatory and tissue levels of MTA
and consequently an enhanced effect in the treatment of cancer.
Figure 6 shows that compound (2) in combination with MTA is also effective for stopping cell cycling (for FaDu cells) such that the cells become apoptotic.
Figures 20 and 21 show the results of in vivo studies. FaDu cells were injected into the dorsum of the foot of NOD-SCID mice. Groups were fed with 250 and 500 M
of compound (2) p.o. or given i.p. injections of 100 i of 4 mM compound (2), daily.
Figure 21 shows representative tumours from each of the treatment cohorts.
Differences between treatment cohorts were determined using Student's t test.
Figure 20 is a summary of the data for all treatment cohorts. The results show that compound (2) significantly delays the growth of established FaDu xenografts.
Longitudinal MRI provides a noninvasive means of monitoring prostate tumour growth in mice (Gupta S, Hastak K, Ahmad N, Lewin J S, Mukhtar H Proc Nat/ Acad Sci USA
2001 Aug 28;98(18):10350-5; Eng M H, Charles L G, Ross B D, Chrisp C E, Pienta K
J, Greenberg N M, Hsu C X, Sanda M G Urology 1999 Dec:54(6):1112-9; Song S K, Qu Z, Garabedian E M, Gordon J I, Milbrandt J, Ackerman J J Cancer Res. 2002 Mar 1:62(5):1555-8.).
MRI is used in the present case to evaluate prostate tumour growth and progression longitudinally in TRAMP mice (either untreated or treated with a compound that may be used according the methods of the invention). Mice were imaged approximately monthly from 12-33 weeks of age. Representative MRI images comparing untreated control TRAMP and treated TRAMP mice at approximately 30 weeks of age are shown in Figure 22.
Panels A and B show results from control mice. Panel A shows a coronal section through of a 30 week old TRAMP mouse with a large tumour (bright tissue) that weighed 8.76 g upon dissection at 34 weeks of age. The inset shows a more posterior coronal section. The bright tumour is smaller in this section but metastasis to the liver is observed (white arrow). Panel B shows a coronal section through the prostate region of a 30 week old TRAMP mouse. The seminal vesicles (SV) are enlarged. A large tumour (weighing 4.89 g upon dissection at 36 weeks of age) that spanned from the kidney to bladder (BL) is visible in the transverse section shown in the inset (white arrow).
Panels E and F show results for mice treated with 1 mM compound (1). Panel E
shows a coronal section through the prostate region of a 30 week old treated TRAMP
mouse. The tumour, weighing 0.41 g upon dissection at 34 weeks of age, was not observed during the imaging session. Panel F shows a similar section through a week old treated TRAMP mouse that exhibited a 0.64 g tumour upon dissection at weeks of age. The tumour is indicated by the white arrow in the MRI image shown in this panel.
Untreated TRAMP mice therefore demonstrate primary prostate tumour growth.
However, prostate cancer progression in the TRAMP mouse is inhibited in mice treated with compound (1), either alone or in combination with MTA.
Figure 23 shows that compound (2) and MTA, administered together, alter polyamine levels and induce cytostasis in PC3 cells. Combination treatment of PC3 cells with compound (2) and MTA for 1 day resulted in a significant 6-fold increase in intracellular PUT levels (3.03 x 10'3 t 2.86 x 10-2, combination treated cells vs. 5.04 X
10'2 f 1.08 x 10-2, control, p= 0.001, pmoles PUT/mg protein), a 2-fold increase in spent media PUT levels [1.19 x 10'3 2.04 x 10-1, combination treated media vs. 5.85 x 10'2 5.09 x 10"0, control media, p= 0.0001, pmoles PUT/mL spent media, as well as roughly a 2.5-fold increase in intracellular SPD levels (7.19 x 10'3 4.38 x 10"2, combination treated cells vs. 3.05 x 10'3 6.3 x 10'2, control, p=0.001 pmoles SPD/mg protein). SPN levels in combination treated spent media also slightly decreased (p=0.02). After 6 days of treatment, cellular SPN levels were decreased roughly 0.5-fold (4.0 x 10"3 7.38 x 10-2, combination treated cells vs. 6.87 x 10-3 9.68 x 10-2, control, p= 0.005, pmoles SPN/mg protein), with both PUT and SPD
elevated (p= 0.02 and p= 0.01, respectively in compa(son to controls). Most significantly, levels of PUT in spent media were almost double that of the control (2.41 x 10'3 7.35 x 10"1, combination treated spent media vs. 1.31 X 10"3 0.0, control, p=0.0007, pmoles PUT/mL spent media). By day 12, a significant increase in cellular SPD levels were observed (9.05 x 10'3 1.09 x 10'3, combination treated cells vs.
3.93 x 10'3 8.4 x 10"1, control, p=0.007, pmoles SPD/mg protein), with a corresponding decrease in levels of spent media PUT levels (1.65 x 10-3 2.27 x 10"
Z, combination treated spent media vs. 2.12 x 10"3 9.34 x 10"1, control media, pmoles PUT/mL spent media, p=0.013). Intracellular PUT levels in combination treated cells were still significantly higher than controls (p=0.005).
Treatment of PC3 cells with compound (2) therefore results in numerous significant alterations in both intracellular and spent media polyamine levels. After 24 hours of treatment, the increase observed in cellular SPD levels as well as putrescine (PUT) cellular and spent media polyamine levels correlates with the effects expected with MTAP inhibition. MTA is accumulating in the cells, beginning to feedback inhibiting SPN synthase, resulting in accumulations of SPD and PUT, with PUT being significantly excreted into the media, and a slight decrease of SPN in the media. By day 6, cellular SPN levels are significantly reduced in combination treated cells, while maintaining the characteristic elevations in levels of PUT and SPD. Treatment of cells for 12 days shows a significant increase in cellular SPD levels and a slight decrease in spent media PUT levels, pointing to the fact that a compensatory pathway has been activated to make up for the block in MTAP. PUT may be being taken up from the media for SPD synthesis. After combination treatment for approximately 2 weeks, PC3 cells display a cytostatic effect, as determined by the clonogenic assay. Initially, it was believed that MTAP inhibition would lead to MTA
accumulation, causing feedback inhibition of polyamine biosynthesis, resulting in decreases in cellular proliferation. A halt in cellular proliferation is observed, and it is now believed that this is not due only to polyamine depletion.
Figure 24 shows that compound (2) reduces tumour growth and metastasis in TRAMP
mice, but does not alter polyamine levels in vivo. Polyamine levels of mice livers are not significantly altered during short-term treatment (Figure 24A). After extended treatment with compound (2) inhibitor solutions, no significant alterations in either TRAMP liver or GUS polyamine levels were detected (Figures 24B and 24C).
Mass (Table 1) and dimensions of excised genitourinary system tumors were recorded for all members of the treatment groups. Sections of small intestine were also removed for toxicity analysis via H&E staining. Histology of TRAMP mice revealed all animals showed extensive prostate intraepithelial neoplasia involving most prostate acini. However, the size and incidence of preinvasive tumors, as well as the incidence of invasive cancer and metastasis were all decreased in treated TRAMP mice (Table 1). No alterations, inflammations, or irregularities were observed in the intestinal crypts, neither were any hair loss or general Gl problems noted, indicating a lack of drug toxicity.
Table 1: Summary of results for TRAMP mice treated with compound (2) # Tumor Weeks Metastatic Experimental Animals Size treated Cancer Condition (n) (g) Control 16 4.0 t 2.8 32 5 44%
100 M compound (2) 12 1.7 0.8 29 7 8%
Although the invention has been described by way of example, it should be appreciated the variations or modifications may be made without departing from the scope of the invention. Furthermore, when known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in the specification.
Claims (41)
1. A method of treating cancer comprising administering to a patient in need thereof a compound of the formula (I):
wherein:
V is selected from CH2 and NH, and W is selected from NR1 and NR2; or V
is selected from NR1 and NR2, and W is selected from CH2 and NH;
X is selected from CH2 and CHOH in the R or S-configuration;
Y is selected from hydrogen, halogen and hydroxy, except where V is selected from NH, NR1 and NR2 then Y is hydrogen;
Z is selected from hydrogen, halogen, hydroxy, SQ, OQ and Q, where Q
is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
R1 is a radical of the formula (II) R2 is a radical of the formula (III) A is selected from N, CH and CR3, where R3 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen; or R3 is hydroxyl, halogen, NH2, NHR4, NR4R5;
or SR6, where R4, R5 and R6 are alkyl, aralkyl or aryl groups, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
B is selected from NH2 and NHR7, where R7 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
D is selected from hydroxy, NH2, NHR8, hydrogen, halogen and SCH3, where R8 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
E is selected from N and CH;
G is selected from CH2 and NH, or G is absent, provided that where W is NR1 or NR2 and G is NH then V is CH2, and provided that where V is NR1 or NR2 and G is NH then W is CH2;
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.
wherein:
V is selected from CH2 and NH, and W is selected from NR1 and NR2; or V
is selected from NR1 and NR2, and W is selected from CH2 and NH;
X is selected from CH2 and CHOH in the R or S-configuration;
Y is selected from hydrogen, halogen and hydroxy, except where V is selected from NH, NR1 and NR2 then Y is hydrogen;
Z is selected from hydrogen, halogen, hydroxy, SQ, OQ and Q, where Q
is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
R1 is a radical of the formula (II) R2 is a radical of the formula (III) A is selected from N, CH and CR3, where R3 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen; or R3 is hydroxyl, halogen, NH2, NHR4, NR4R5;
or SR6, where R4, R5 and R6 are alkyl, aralkyl or aryl groups, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
B is selected from NH2 and NHR7, where R7 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
D is selected from hydroxy, NH2, NHR8, hydrogen, halogen and SCH3, where R8 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
E is selected from N and CH;
G is selected from CH2 and NH, or G is absent, provided that where W is NR1 or NR2 and G is NH then V is CH2, and provided that where V is NR1 or NR2 and G is NH then W is CH2;
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.
2. A method of treating cancer comprising administering to a patient in need thereof a compound of the formula (IV):
wherein:
V is selected from CH2 and NH, and W is selected from NR1 and NR2; or V
is selected from NR1 and NR2, and W is selected from CH2 and NH;
X is selected from CH2 and CHOH in the R or S-configuration;
Y is selected from hydrogen, halogen and hydroxy, except where V is selected from NH, NR1 and NR2 then Y is hydrogen;
Z is selected from hydrogen, halogen, hydroxy, SQ, OQ and Q, where Q
is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
R1 is a radical of the formula (V) R2 is a radical of the formula (VI) A is selected from N, CH and CR3, where R3 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen; or R3 is hydroxyl, halogen, NH2, NHR4, NR4R5;
or SR6, where R4, R5 and R6 are alkyl, aralkyl or aryl groups, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
B is selected from NH2 and NHR7, where R7 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
D is selected from hydroxy, NH2, NHR8, hydrogen, halogen and SCH3, where R8 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
E is selected from N and CH;
G is selected from CH2 and NH, or G is absent, provided that where W is NR1 or NR2 and G is NH then V is CH2, and provided that where V is NR1 or NR2 and G is NH then W is CH2;
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof;
provided that the compound (3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine is excluded.
wherein:
V is selected from CH2 and NH, and W is selected from NR1 and NR2; or V
is selected from NR1 and NR2, and W is selected from CH2 and NH;
X is selected from CH2 and CHOH in the R or S-configuration;
Y is selected from hydrogen, halogen and hydroxy, except where V is selected from NH, NR1 and NR2 then Y is hydrogen;
Z is selected from hydrogen, halogen, hydroxy, SQ, OQ and Q, where Q
is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
R1 is a radical of the formula (V) R2 is a radical of the formula (VI) A is selected from N, CH and CR3, where R3 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen; or R3 is hydroxyl, halogen, NH2, NHR4, NR4R5;
or SR6, where R4, R5 and R6 are alkyl, aralkyl or aryl groups, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
B is selected from NH2 and NHR7, where R7 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
D is selected from hydroxy, NH2, NHR8, hydrogen, halogen and SCH3, where R8 is alkyl, aralkyl or aryl, each of which is optionally substituted with one or more substituents selected from hydroxy and halogen;
E is selected from N and CH;
G is selected from CH2 and NH, or G is absent, provided that where W is NR1 or NR2 and G is NH then V is CH2, and provided that where V is NR1 or NR2 and G is NH then W is CH2;
or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof;
provided that the compound (3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methylthiomethyl)pyrrolidine is excluded.
3. A method as claimed in claim 1 or claim 2 where Z is SQ.
4. A method as claimed in claim 3 where Z is not methylthio.
5. A method as claimed in claim 3 where Q is an alkyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy.
6. A method as claimed in claim 3 where Q is an aryl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy.
7. A method as claimed in claim 3 where Q is a C1-C6 alkyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy.
8. A method as claimed in claim 7 where Q is a methyl group.
9. A method as claimed in claim 6 where Q is a phenyl or benzyl group, optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy.
10. A method as claimed in claim 1 where G is CH2.
11. A method as claimed in claim 10 where V is CH2 and W is NR1.
12. A method as claimed in claim 10 where B is NH2.
13. A method as claimed in claim 10 where D is H.
14. A method as claimed in claim 10 where A is CH.
15. A method as claimed in claim 1 where any halogen is chlorine or fluorine.
16. A method of treating cancer comprising administering to a patient in need thereof a compound of the formula (VII):
where J is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.
where J is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, or carboxy;
or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.
17. A method as claimed in claim 16 where the cancer is prostate cancer or head and neck cancer.
18. A method as claimed in claim 16 or claim 17 where J is C1-C7 alkyl.
19. A method as claimed in claim 16 or claim 17 where J is methyl, ethyl, n-propyl, i-propyl, n-butyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl or cycloheptyl.
20. A method as claimed in claim 16 or claim 17 where J is phenyl, optionally substituted with one or more halogen substituents.
21. A method as claimed in claim 16 or claim 17 where J is phenyl, p-chlorophenyl, p-fluorophenyl or m-chlorophenyl.
22. A method as claimed in claim 16 or claim 17 where J is heteroaryl.
23. A method as claimed in claim 16 or claim 17 where J is 4-pyridyl.
24. A method as claimed in claim 16 or claim 17 where J is aralkyl.
25. A method as claimed in claim 16 or claim 17 where J is benzylthio.
26. A method as claimed in claim 16 where J is -CH2CH2(NH2)COOH.
27. A method of treating cancer comprising administering to a patient in need thereof a compound of the formula (VIII):
where T is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, carboxy or straight- or branched-chain C1-C6 alkyl;
or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.
where T is aryl, aralkyl or alkyl, each of which is optionally substituted with one or more substituents selected from hydroxy, halogen, methoxy, amino, carboxy or straight- or branched-chain C1-C6 alkyl;
or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.
28. A method as claimed in claim 27 where the cancer is prostate cancer or head and neck cancer.
29. A method as claimed in claim 27 or claim 28 where T is C1-C6 alkyl, optionally substituted with one or more substituents selected from halogen and hydroxy.
30. A method as claimed in claim 27 or claim 28 where T is methyl, ethyl, 2-fluoroethyl, or 2-hydroxyethyl.
31. A method as claimed in claim 27 or claim 28 where T is methyl.
32. A method as claimed in claim 27 or claim 28 where T is aryl, optionally substituted with one or more substituents selected from halogen or straight-chain C1-C6 alkyl.
33. A method as claimed in claim 27 or claim 28 where T is phenyl, naphthyl, p-tolyl, m-tolyl, p-chlorophenyl, m-chlorophenyl or p-fluorophenyl.
34. A method as claimed in claim 27 or claim 28 where T is aralkyl.
35. A method as claimed in claim 27 or claim 28 where T is benzyl.
36. A method as claimed in claim 1 where the compound of formula (I) is:
(3R,4R)-1-[(8-aza-9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(2-phenylethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzylthiomethyl)pyrrolidine;
(3R,4S)-1-[(8-aza-9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-chlorophenylthiomethyl)pyrrolidine;
(3R,4R)-1-[(9-deazaadenin-9-yl)methyl]-3-acetoxy-4-(acetoxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(n-butylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-fluorophenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(n-propylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclohexylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(3-chlorophenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(ethylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(phenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-pyridylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-n-propylpyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(homocysteinylmethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzyloxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(i-propylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methoxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclohexylmethylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cycloheptylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclopentylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclobutylthiomethyl)pyrrolidine.
(3R,4R)-1-[(8-aza-9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(2-phenylethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzylthiomethyl)pyrrolidine;
(3R,4S)-1-[(8-aza-9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-chlorophenylthiomethyl)pyrrolidine;
(3R,4R)-1-[(9-deazaadenin-9-yl)methyl]-3-acetoxy-4-(acetoxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(n-butylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-fluorophenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(n-propylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclohexylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(3-chlorophenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(ethylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(phenylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(4-pyridylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-n-propylpyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(homocysteinylmethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(benzyloxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(i-propylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(methoxymethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclohexylmethylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cycloheptylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclopentylthiomethyl)pyrrolidine;
(3R,4S)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxy-4-(cyclobutylthiomethyl)pyrrolidine.
37. A method as claimed in claim 1 where the cancer is prostate cancer or head and neck cancer.
38. A method as claimed in claim 16 where the cancer is prostate cancer.
39. A method as claimed in claim 16 where the cancer is head and neck cancer.
40. A method as claimed in any one of claims 1, 16 or 27 where the compound is administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally or via an implanted reservoir.
41. A method as claimed in claim 40 where the compound is administered orally.
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