AU2031102A - Selective MMP inhibitors having reduced side-effects - Google Patents

Description

a. 1

AUSTRALIA

Patents Act 1990 Darwin Discovery Limited COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Selective MMP inhibitors having reduced side-effects oooo ooQ: o The following statement is a full description of this invention including the best method of performing it known to us:-

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SELECTIVE MMP INHIBTORS HAVING REDUCED SIDE-EFFECTS Field of the Invention This invention relates to selective MMP inhibitors having reduced side-effects.

Background of the Invention Compounds having the ability to inhibit matrix metalloproteinases (MMviPs) and optionally also TNFa release are described in WO-A-9513289, WO-A-9611209, WO-A- 9635711, WO-A-9635712, WO-A-9635714, WO-A-9635687, WO-A-9712902, WO-A- 9719075, WO-A-9738007, WO-A-9805635 and WO-A-9806696. All these specifications are incorporated herein by reference. By way of example, WO-A-96 11209 (Examples 52 and 72) and WO-A-9712902 (Example 6) disclose phthalimido]pentanoyl-L-leucyl-(S)-tert-leucine N-methylamide, 2S-[4-(2,5dioxopyrrolidin-1 -yl)-2-mercaptobutyrylamino]-4-methylpentanoic acid (2,2-dimethyl- IS-methylcarbamoylpropyl)amide, and 2-[2-mercapto-4-(3,4,4-trimethyl-2,5dioxoimidazolidin-1 -yl)butyrylamino]-4-methylpentanoic acid (2,2-dimethyl-1methylcarbamoylpropyl)amide. as racemates. Other compounds of this general type are also known.

MMPs are a group of structurally related endopeptidases that degrade the proteinaceous elements of the extracellular matrix. A number of important features are shared by members of the MMP family and include a zinc atom at the catalytic active site, catalytic activity at neutral pH, initial existence as inactive proenzymes, activation involving removal of an N-terminal domain, structural stabilisation by calcium, and inhibition of the catalytically active forms by a family of specific protein inhibitors called Tissue Inhibitor of Metalloproteinases (TIMPs). The MMP family currently consists of twenty members including MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP- 10, MMP- 11, MMP 12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP- 19 and MMP-20 ("classical MMPs").

The MMPs are a sub-family of a much larger group of zinc-containing proteinases which include the Reprolysins and Serralysins, and the Astacin family. Of particular interest are the Adamalysins, members of the Reprolysin family, which include the metalloproteinase involved in the release of TNFcx and which is presumably inhibited by compounds such as those described in the patent publications/applications identified above.

It has been demonstrated that compounds which inhibit MMPs also have the capability to inhibit a number of other events that are mediated by metalloproteinases and that include the release ofTNF, CSF-1, TGFa, L-selectin, CD30 and Fas Ligand and the shedding of the IL-6, TNF-RI and TNF-RII receptors. See Hooper et al (1997), Biochem.

321:265-279.

MMP inhibitors have been proposed for use in patients with arthritis conditions especially where degradation of cartilage occurs, a process known to involve MMPs. In addition, the use of these compounds in the treatment of various cancers has been advocated. Inhibition of the MMPs, which have been associated with certain disease 10 modalities, offers potential therapeutic benefit. However, the inhibition of other non- MMP metalloproteinases may offer no therapeutic benefit and indeed could be deleterious.

For example, it has been suggested that MMP inhibitors which also inhibit the release of TNFa may have a role in exacerbating liver injury; see Solorzano et al (1997), J.

Immunol., 158:414-419.

Similarly, early clinical evidence from the use ofMMP inhibitors suggests that their use is associated, in many patients, with joint pain. See Wojtowicz-Praga et al, Lombardi Cancer Center, Georgetown University Hospital, Washington DC BBL Anapolis MD, Am. Soc. Clin. Oncol. (May 1996) "The Pharmacokinetics (PK) ofMarimastat (BB- S. 25 16), A Novel Matrix Metalloproteinase Inhibitor (MMPI) administered orally to patients with metastatic lung cancer". The problem may require treatment "holidays", for up to of the course of treatment, or the administration of non-steroidal anti-inflammatory agents (NSAIDs).

Summary of the Invention This invention is based on the appreciation that compounds having particularly valuable properties are of the type which have activity against the classical MMPs, but lack or have little activity against non-MMP metalloproteinase (MP)-related events. This particular profile of activity may confer a therapeutic benefit, particularly regarding the joint pain and tendonitis seen with less selective compounds. Treatment "holidays" may be avoided, e.g. allowing continued treatment. The use ofNSAIDs can also be avoided.

This selectivity may be defined in terms of a specific range of activity in defined models of MMP and MP effects. In particular, compounds to which this invention relates have characteristics x and y wherein: x MMP inhibition, in terms of IC 0 measured as described in Example A below; and y Inhibition ofMP-related effects, in terms ofICso as described in Examples B-F below; x is below 10" M, preferably below 10" M, more preferably 10' M to 10' 9 M or 10-°0 M; and y is greater than 10 7 M, preferably greater than 10 4 M, more preferably 10 M to 10 M.

An important characteristic related to y is that y is greater or equal to 2 x 10' for 10 at least 4 out of 5 MP-related effects defined by Examples B-F; this is distinct from most prior art compounds having characteristic x.

Description of the Invention It has become clear that MMP inhibitors that are very effective in certain areas, e.g.

as anti-cancer agents, may have undesirable side-effects. In particular, it appears that, by attaining the selectivity described above, compounds of this invention may overcome the known problems ofjoint pain experienced by a proportion, albeit significant of 9 00 patients treated with MMP inhibitors. The selectivity may be determined by the use of an appropriate assay, for example as described in Examples A-F.

Specific examples of compounds showing the desired profile are those of Examples 1 and 2 and cpd. 3 (see below). These three compounds have been tested in the marmoset tendonitis study. This procedure for this study has been defined by Wojtowicz- Praga et al, supra. The study was performed with oral dosing at 30 and 100 mg/kg for 3 months. Repeated plasma sampling showed sustained levels in excess of the enzymes' ICo, up to 9 h post-dose. Continuous clinical observation revealed no adverse effects.

Histological observation revealed no adverse effects on joints and tendons. This is not necessarily seen with all MMP inhibitors. See Hooper et al, supra. It indicates that such compounds are suitable for continued administration.

A compound of the invention may be given in a composition, by a route and/or in an amount that is already known for that compound or for analogues thereof, e.g. as described in the patent specifications identified above. It may be given to patients who have already experienced joint pain (under any circumstances) and to those who are susceptible to joint pain, e.g. those having injured joints. It is within the ability of one skilled in the art to determine which patients are at risk from this complication.

The compound may be used in the treatment or prophylaxis of cancer, inflammation or any condition associated with MMP activity. Many examples are given in the patent publications/applications identified above.

The duration of administration may be conducted on a regular basis, without interruption. This may involve periods of at least 1, 2 3, 6 or more months Example A MMP Inhibition Activity-Fluorimetric Assay The potency of inhibitors of collagenase- (MMP- collagenase-2 (MMP-8), 10 collagenase-3 (MMP-13), gelatinase-A (MMP-2), gelatinase-B (MMP-9) and stromelysin- I (MMP-3) is determined using the following procedure: Inhibitors are dissolved in dimethyl sulphoxide containing 0.02% 3mercaptoethanol, and serial dilutions are prepared. Activated enzyme is incubated in assay *9 buffer containing 50mM Tris, pH 7.4, 5mM CaCI 2 0.002% NaN 3 and Brij 35 in the presence and absence of inhibitor. Samples are preincubated at 37 0 C for 15 minutes before the addition of the fluorimetric substrate (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH 2 to a final concentration of 10 The assay is incubated for 20-30 min at 37°C and then read in a Fluoroscan II at (340nm) and X, (405nm).

The enzyme activity is compared to activity in a control devoid of inhibitor.

Results are reported as that inhibitor concentration effecting 50% inhibition of the enzyme (ICo).

Example B Inhibition of TNFc production The potency of inhibitors of the production of TNFa is determined using the following procedure: A 100 pM solution of the inhibitor being tested or dilutions thereof is incubated at 37* C in an atmosphere of 5% CO 2 with peripheral blood mononuclear cells (PBMC).

PBMC are isolated from buffy coats by standard procedures using Ficoll. The PBMC are suspended in RPM1 1640 medium with 2% fetal calf serum at a cell density of 2 x 10 6 /ml and stimulated with LPS. After 18 hours the supernatant is assayed for the levels ofTNFa.

using a commercially available ELISA kit.

The activity in the presence of 100 pM inhibitor or dilutions thereof is compared to activity in a control devoid of inhibitor. Results are reported as that inhibitor concentration effecting 50% inhibition of the production of TNFa.

Example C Inhibition of L-selectin shedding An assay of L-selectin shedding by peripheral blood mononuclear cells (PBMC) is conducted as follows: PBMC are isolated from buffy coats by standard procedures using Ficoll. A 100 M solution of the inhibitor being tested or dilutions thereof is incubated for 20 min at 37"C in an atmosphere of 5% CO 2 with 4 x I 0/ml PBMC stimulated with PMA. The cells S 10 are centrifuged down and the supernatants tested for sL-selectin using a commercially available ELISA kit.

The activity in the presence of 100 pM inhibitor or dilutions thereof is compared to activity in a control devoid of inhibitor. Results are reported as that inhibitor concentration effecting 50% inhibition of the shedding of L-selectin.

Example D Inhibition of sIL-lRII shedding An assay of slL-lRII shedding by PBMC is conducted as follows: PBMC are isolated from buffy coats by standard procedures using Ficoll. A 100 pM solution of the inhibitor being tested or dilutions thereof are incubated for 18h at 370 C in an atmosphere of 5% CO 2 with 2 x 10'/ml PBMC stimulated with IL-13. The cells are centrifuged down and the supernatants tested for sIL-lRI using a commercially available ELISA kit.

The activity in the presence of 100 pM inhibitor or dilutions thereof is compared to activity in a control devoid of inhibitor. Results are reported as that inhibitor concentration effecting 50% inhibition of the shedding of sIL-1RII.

Example E Inhibition of IL-6R shedding An assay of sIL-6R shedding by HL-60 cells is conducted as follows: PBMC are isolated from buffy coats by standard procedures using Ficoll. A 100pM solution of the inhibitor being tested or dilutions thereof is incubated for 24h at 37* C in an atmosphere of 5% CO 2 with 2 x 10 6 /ml HL-60 cells stimulated with PMA.

The cells are centrifuged down and the supernatants tested for sIl-6R using a commercially available ELISA kit.

The activity in the presence of 100IM inhibitor or dilutions thereof is compared to activity in a control devoid of inhibitor. Results are reported as that inhibitor concentration effecting 50% inhibition of the shedding of L-6R.

Example F Inhibition of TNF RII shedding The potency of inhibitors of the shedding of TNF RII is determined using the following procedure: A 100 pM solution of the inhibitor being tested or dilutions thereof is incubated at 37* C in an atmosphere of 5% CO, with THP-I cells (human monocytes) suspended in *RPM 1640 medium and 20pM B-mercaptoethanol at a cell density of 1 x 10'/ml and 10 stimulated with LPS (lipopolysaccharide). After 18 hours the supernatant is assayed for the levels of sTNF RII using a commercially available ELISA kit.

The activity in the presence of 0.1 mM inhibitor or dilutions thereof is compared S. to activity in a control devoid of inhibitor. Results are reported as that inhibitor concentration effecting 50% inhibition of the shedding of TNF RIL The following Intermediates illustrate stages in the-synthesis of the compound of Example 1.

Intermediate 1 (S)-(2,2-Dimethyl-5-oxo[l,3ldioxolan-4-yl)acetic Acid (L)-Malic acid (200 g) was added to a mixture of acetone (800 mL) and 2,2dimethoxypropane (342 mL) with stirring under nitrogen. Montmorillonite K-10 clay g) was added to the solution and stirred for 17 hours, then the solution was filtered and reduced in volume to approximately 450 mL. The solution was cooled to 5*C, and the resultant crystals of the title compound filtered off(108 g, 42%).

MP 108- 109.5"C Intermediate 2 (S)-5-(2-Hydroxyethyl)-2,2-dimethyl[ 1,3]dioxolan-4-one Intermediate 1 (50 g) was dissolved in tetrahydrofuran (400 mL) and cooled to 2"C. To this solution was carefully added borane-methyl sulfide complex (45.5 mL) and the mixture was stirred for 63 hours. The mixture was evaporated to a clear oil, dissolved in ethyl acetate (600 mL), washed with saturated aqueous sodium bicarbonate (100 mL) and water (100 mL) and dried over magnesium sulfate. Filtration and evaporation in vacuo afforded the title compound as a clear oil (36 g, TLC R 0.41 (Ethyl acetate: Hexane, 1 1) Intermediate 3 esyloxyethyl)-2,2-dimethyl 1,3]dioxolan-4-one Intermediate 2 (24 g) was dissolved in dichloromethane (480 mL) and cooled to 2°C. To this solution was added mesyl chloride (14.5 mL) followed by dropwise addition of triethylamine (25 mL). The solution was then warmed to room temperature and stirred for 40 minutes, washed with water (2 x 120 mL), brine (60 mL) and dried over magnesium sulfate. Filtration and evaporation in vacuo afforded the title compound as a clear oil (37.4 g, 100 TLC R, 0.48 (Ethyl acetate: Hexane, 1 1) Intermediate 4 [2-(2,2-Dimethyl-5-oxo 1,3]dioxolan-4-yl)-ethyll- 10 Intermediate 3 (5 g) was dissolved in N,N-dimethylformamide (50 mL) and potassium succinimide (3.7 g) and sodium iodide (0.6 g) added. The mixture was then heated to 70*C for 15 hours after which time the solvent was removed in vacuo. The ~residue was dissolved in ethyl acetate (100 mL) and filtered through a plug of silica. The plug of silica was washed with further ethyl acetate (300 mL) and the combined organic S"solutions evaporated to an oil, which upon trituration with diethyl ether (5 mL) and *o cooling to 2*C provided crystals which were filtered to give the title compound (2.4 g, 47%).

TLC: R,0.88 (Ethyl acetate Methanol, 8 2, one drop of acetic acid).

Intermediate 5 (S)-4-(2,5-Dioxopyrrolidin-1-yl)-2-hydroxybutyric Acid Intermediate 4 (2.3 g) was suspended with Dowex 50WX4-400 resin (1.0 g) in methanol (23 mL) and water (4.6 mL) then stirred for 19 hours. The resin was removed by filtration and solvent removed in vacuo to give an oil. The oil was seeded with some crystals of the product and cooled to -25C, upon complete crystallisation, the solid was washed on a filter bed with diethyl ether (10 mL) and ethyl acetate (1 mL) affording the title compound (1.5 g, 78%).

TLC Rf 0.20 (Ethyl acetate Mlethanol, 8 2, one drop of acetic acid).

Intermediate 6 (R)-2-Bromo-4-(2,5-dioxopyrrolidin-l-yl)butyric Acid Intermediate 5 (18.4 g) was dissolved in 30% hydrobromic acid acetic acid (111 mL) under nitrogen, and the mixture stirred at ambient temperature for 35 minutes. After this time the reaction was poured into water (300 mL) and extracted with ethyl acetate (4 x 400 mL). The combined extracts were washed with brine (100 mL), dried over S. S

S

magnesium sulfate, filtered and evaporated to an oil. This was then purified on silica (450 g) with an eluent of dichloromethane and ethyl acetate (3 1) with 2% acetic acid. The correct fractions were combined and evaporated in vacuo to afford an oil which was dissolved in heptane. Re-evaporation in vacuo afforded the title compound as a white powder (10.9 g, TLC Rf 0.16 (99% Ethyl acetate, 1% acetic acid).

Intermediate7 (S)-2-Acetylsulfanyl-4-(2,5-dioxopyrrolidin-1-yl)butyric Acid Intermediate 6 (10.5 g) was dissolved in methanol (53 mL) and cooled to 0 C under nitrogen. To this solution was added potassium thioacetate (4.9 g) and the mixture warmed with stirring to ambient temperature, then stirred for 90 minutes. The solvent was then removed in vacuo and the residue resuspended in dichloromethane (200 mL) and water (30 mL). The aqueous layer was extracted with dichloromethane (3 x 200 mL) and the combined organic layers washed with brine (50 mL) and dried over magnesium sulfate.

Filtration and evaporation afforded an oil which was mixed with diethyl ether (10 mL).

After cooling to 2 0 C, crystals of the title compound were isolated by filtration (8.2 g, MP 110- 112 0

C.

Intermediate 8 Thioacetic Acid IS-lIS-(2,2-Dimethyl-IS-methylcarbamoylpropylcarbamoyl)-3-methylbutylcarbamoyl]-3-(2,5-dioxopyrrolidin-l-yl)propyl Ester Intermediate 7 (1.28 g) was dissolved in dichloromethane (32 mL) and cooled to 0°C under nitrogen. To this solution was added 2-amino-4-methylpentanoic acid (2,2-dimethyl-l-methylcarbamoylpropyl)amide (1.33 g) rapidly followed by 1hydroxybenzotriazole hydrate (0.667 g) and 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (1.14 after which the mixture was stirred at 0°C for minutes, then at ambient for 3.5 hours. The reaction was then diluted with dichloromethane (35 mL), washed with saturated aqueous sodium bicarbonate (20 mL), 1 M hydrochloric acid (20 mL) and water (30 mL), and dried over magnesium sulfate.

Filtration and evaporation gave the crude title compound as a glassy solid (2.5 g, 100%).

TLC: Rr 0.23 Ethyl acetate).

Example 1 2S-I4-(2,5-Dioxopyrrolidin-l-yl)-2S-mercaptobutyrylamino]-4methylpen tanoic Acid (2,2-D imethyl-IS-methylcarbamoylpropyl)amide Intermediate 8 (2.3 g) was dissolved in methanol (115 mL) and degassed. To this solution was added dithiothreitol (0.071 Degassing was performed again. Aqueous ammonia (3.0 mL) was added and the mixture stirred at ambient temperature for **foe:minutes. The methanol was removed in vacuo to give an oil which was purified by column 0 chromatography (ethyl acetate: methanol, 95 The correct fractions were combined and evaporated in vacuo at 40*C to provide the title compound as a glassy solid (1.8 g, 86%).

NW 81 84 0

C

TLC RfO0.

3 7 (Ethyl acetate methanol, 95 :Example 2 2S-12S-M ercapto-4-(3,4',4-trimethyl-2,5-dioxoimidazolidin-1yl)butyrylaminoj-4-methylpeinta-noic Acid (2,2-Dimethyl-ISmethylcarbamoylpropyl)amide Was prepared as described for Example 6 in WO-A-97 12902, by resolution of an intermediate involved in the synthesis or by separation of the distereoisomers of Example 6 in WO-A-9712902 by hplc.

Biolo2ical Data The following Table reports results (in riM) from the assays described above, for the compounds of Examples 1 and 2, and also for the compound (Cpd. 3) of Example 52 of WQ-A-96 11209, i.e. (S)-N-[2-mercapto-5-phthalimidopentanoyl-L-leucyl-(S)-4ertleucine N-methylamide.

0 *0

S

Assay Cpd. 3 Ex. 1 Ex. 2 Example A MMP-1 0.028 0.012 0.025 MMP-8 0.009 0.010 0.010 MMP-13 0.002 0.004 0.004 MMP-2 0.015 0.130 0.041 MMP-9 0.005 0.053 0.025 MMP-3 0.179 0.381 0.157 Example B 10 IA IA 10 Example C IA IA IA Example D IA 38 IA Example E IA IA IA Example F IA IA IA 15 IA means no activity at the highest concentration tested, which was 50 or 100 pM.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step. or group of elements, integers or steps.

Claims (13)

1. Use of a matrix metalloproteinase (MMP) inhibitor for the manufacture of a medicament for the treatment of cancer or inflammation, by regular administration over a period of at least 3 months, wherein the inhibitor exhibits values of x below 10-' and y greater than 10- 7 M, x being the MMP inhibition, in terms of ICs, measured as described in Example A herein, and y being the inhibition of MP-related effects, in terms of IC 5 o measured as described in Examples B-F herein, provided that y is at least 2x10 5 M in 4 or 5 of these Examples. 10 2. Use according to claim 1, wherein x is below 10"6 M.

3. Use according to claim 1 or claim 2, wherein y greater than 10- 1 M.

4. Use according to claim 1, wherein the inhibitor is phthalimido]pentanoyl-L-leucyl-(S)-tert-leucine-N-methylamide. Use according to claim 1, wherein the inhibitor exhibits enzyme inhibition selectively substantially the same as that exhibited by the inhibitor defined in claim 4.

6. Use according to any one of claims 1 to 5, for the treatment of cancer.

7. Use according to any one of claims 1 to 6, for the treatment of Sinflammation. 000

8. Use according to any one of claims 1 to 7, wherein the inhibitor is administered regularly over a period of at least 6 months.

9. Use according to any one of claims 1 to 8, without the concomitant use of non-steroidal anti-inflammatory agent. A method of treating cancer or inflammation, the method including administering to a subject requiring treatment, by regular administration over a period of 3 months, a matrix metalloproteinase (MMP) inhibitor, wherein the inhibitor exhibits values of x below 10-' and y greater than 10- 7 M, x being the MMP inhibition, in terms of IC,, measured as described in Example A herein, and y being the inhibition of MP-related effects, in terms of ICo measured as described in Examples B-F herein, provided that y is at least 2x10' 5 M in 4 or 5 of the Examples.

11. The method according to claim 10 wherein x is below 106.

12. The method according to claims 10 or 11, wherein y is greater than 10 6 M.

13. The method according to claim 10, wherein the inhibitor is 12

14. The method according to claim 13, wherein the inhibitor exhibits enzyme inhibition selectively substantially the same as that exhibited by the inhibitor defined in claim 13. The method according to any one of claims 10 to 14, for the treatment of cancer.

16. The method according to any one of claims 10 to 14, for the treatment of inflammation.

17. The method according to any one of claims 10 to 16, wherein the inhibitor is administered regularly over a period of at least 6 months. 10 18. The method according to any one of claims 10 to 17, without the concomitant use of non-steroidal anti-inflammatory agent. Dated this 4th day of March 2002 DARWIN DISCOVERY LIMITED Patent Attorneys for the Applicant: F .B RICE CO