Compounds containing a vascular disrupting agent

Abstract

The present invention relates to compounds, and pharmaceutically acceptable salts thereof, comprising a vascular disrupting agent (VDA) associated and a MMP proteolytic cleavage site. The compounds are useful in the treatment of cancer.

Claims

1. A compound or pharmaceutically acceptable salt thereof, comprising a compound X which promotes the formation of stable microtubules in a manner similar to paclitaxel associated with a peptide Y comprising a matrix metalloproteinase cleavage site comprising the amino acid sequence -Arg-Ser-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 4).

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound X is a taxane.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound X is paclitaxel.

4. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound X which promotes the formation of stable microtubules in a manner similar to paclitaxel is linked directly or indirectly to the peptide.

5. The compound or pharmaceutically acceptable salt thereof according to claim 1, further comprising a linker a directly or indirectly associated with X.

6. The compound or pharmaceutically acceptable salt thereof according to claim 5, wherein the linker a is on the C terminus of the amino acid sequence Y.

7. The compound or pharmaceutically acceptable salt thereof according to claim 5 or 6, wherein the linker a may be removed chemically, enzymatically or decompose spontaneously.

8. The compound or pharmaceutically acceptable salt thereof according to claim 1, further comprising a capping group c to prevent non-specific degradation of the peptide.

9. The compound or pharmaceutically acceptable salt thereof according to claim 8, wherein the capping group c is on the N terminus of the peptide.

10. The compound or pharmaceutically acceptable salt thereof according to claim 8, wherein the capping group c is selected from simple sugars, D-amino acids, proline imino acids, fluorescein, and fluorescein derivatives.

11. The compound or pharmaceutically acceptable salt thereof according to claim 1, further comprising a spacer b directly or indirectly associated with the peptide Y.

12. The compound or pharmaceutically acceptable salt thereof according to claim 11, wherein the spacer b is on the N terminus of the peptide Y.

13. The compound or pharmaceutically acceptable salt thereof according to claim 11, wherein the spacer is selected from a single amino acid, an amino acid sequence and a succinyl group.

14. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound has formula (VI):
X-a-Y-b-c(VI) and each of X, a, Y, b and c are defines as follows: X is a taxane, a is a linker directly or indirectly associated with X, Y is a peptide comprising a matrix metalloproteinase cleavage site comprising the amino acid sequence -Arg-Ser-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 4), b is a spacer directly or indirectly associated with the peptide Y, and c is a capping group to prevent non-specific degradation of the peptide.

15. A compound or pharmaceutically acceptable salt thereof, comprising paclitaxel conjugated via a linker to a peptide comprising a MMP proteolytic cleavage site and comprising the amino acid sequence -Arg-Ser-Cit-Gly-Hof-Tyr-Leu- (SEQ ID NO: 4).

16. A pharmaceutical formulation comprising a compound or pharmaceutically acceptable salt thereof according to claim 1 or 15 and at least one pharmaceutically acceptable excipient, diluent or carrier.

17. A pharmaceutical formulation according to claim 16, further comprising another anticancer agent.

18. A method of treating cancer in a subject comprising administering to the subject an effective amount of a compound or pharmaceutically acceptable salt thereof according to claim 15.

19. A method of treating cancer in a subject comprising administering to the subject an effective amount of compound or pharmaceutically acceptable salt thereof according to claim 1.

20. The method according to claim 18 or 19, wherein the cancer is prostate cancer.

21. A method of treating cancer in a subject comprising administering to the subject an effective amount of a pharmaceutical formulation comprising a compound according to claim 1.

22. The method according to claim 21, wherein the cancer is prostate cancer.

23. The compound of claim 10 wherein the fluorescein derivative is fluorescein isothiocycante (FITC).

24. The compound of claim 13 wherein the single amino acid is -alanine.

Description

(1) The invention will now be described by way of example only with reference to the 0 following Figures in which:

(2) FIG. 1 is a graph showing the metabolism of prodrug-1 versus time in tumour and non-tumour tissues ex vivo;

(3) FIG. 2 is a graph showing accumulation and levels of prodrug-1 in tumour-bearing mice following intraperitoneal administration;

(4) FIG. 3 is a graph demonstrating levels of VDA accumulating following intraperitoneal administration of prodrug-1 to tumour bearing mice;

(5) FIG. 4 is a graph showing differential metabolism of prodrug-2 in tumour homogenates expressing high MT1-MMP levels (HT1080) versus tumour homogenates expressing low MT1-MMP levels (MCF-7);

(6) FIG. 5 is a graph showing accumulation and levels of prodrug-2 in HT1080 tumour bearing mice following intraperitoneal administration;

(7) FIG. 6 is a graph demonstrating levels of VDA accumulating following intraperitoneal administration of prodrug-2 to HT1080 tumour bearing mice;

(8) FIG. 7 is a schematic of MMP-activated prodrug (compound i) targeting all MMPs showing the amino acid sequence of SEQ ID NO: 6. Hof=homophenylalanine; Cit=citrulline;

(9) FIG. 8 is a schematic of MMP-activated prodrug (compound i) targeting only Membrane-Type WIMPs (MT-MMTPs) showing the amino acid sequence of SEQ ID NO: 7.

EXAMPLE

Materials and Methods

Synthesis of Immobilised Colchicine Derivative

(10) ##STR00001## ##STR00002##

Synthesis of 1

(11) Ammonia solution (35%, 15 mL) was added to colchicine (750 mg, 1.88 mmol, 1.00 eq) and the reaction mixture stirred at room temperature overnight. The crude product was washed with KHSO.sub.4 (1 M, aq), dried with MgSO.sub.4, filtered and concentrated under reduced pressure. It was subsequently purified by flash chromatography on silica gel (gradient elution: CH.sub.2Cl.sub.2/methanol 95:5 to 10:1) to give 1 as a yellow solid (427 mg, 1.11 mmol, 59%). .sub.H (600 MHz, CDCI.sub.3), 7.99 (1H, broad s, NH), 7.56 (1H, d, J 2.1, C8-H), 7.32 (1H, d, J 10.7, C11-H), 6.88 (1H, d, J 11.0, C10-H), 6.52 (1H, s, C4-H), 6.03 (2H, broad s, NH.sub.2), 4.68 (1H, ddd, J 12.6, 6.5 and 6.5, C7-H), 3.93 (3H, s, OCH.sub.3), 3.88 (3H, s, OCH.sub.3), 3.60 (3H, s, OCH.sub.3), 2.47 (1H, dd, J 13.4 and 6.2, C5-CH.sub.2), 2.35 (1H, ddd, J 13.4, 12.7 and 6.9, C5-CH.sub.2), 2.29-2.23 (1H, m, C6-CH.sub.2), 1.98 (3H, s, CH.sub.3), 1.90-1.88 (1H, m, C6-CH.sub.2); ES m/z (%) 385 [M.sup.++H](100).

Synthesis of 2

(12) HCTU (642 mg, 1.55 mmol, 1.50 eq) and diisopropylethylamine (DiPEA, 516 L, 404 mg, 3.11 mmol, 3.00 eq) were added to a solution of Fmoc-tyr(tBu)-OH (714 mg, 1.55 mmol, 1.50 eq) in DMF (10 mL). After stirring at room temperature for 5 minutes, 1 (398 mg, 1.04 mmol, 1.00 eq) was added to the solution. The reaction mixture was stirred at 50 C. for 22 h. DMF was removed in vacuo and the resultant oil was dissolved in CH.sub.2CI.sub.2 (20 mL). The organic phase was washed with KHSO.sub.4 (aq, 220 mL), dried with MgSO.sub.4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (gradient elution: CH.sub.2Cl.sub.2/methanol 100:0 to 99:1 to 98:2) to give 2 as a yellow solid (530 mg, 642 mol, 67%).

(13) .sub.H (600 MHz, CDCI.sub.3), 10.42 (1H, broad s, NH), 9.02 (1H, d J 10.7, C11-H), 7.75 (2H, d, J 7.2, C23-H, C24-H), 7.54 (2H, d, J 7.2, C20-H, C27-H), 7.45 (1H, d, J 11.0, C10-H), 7.39 (2H, dd, J 7.2 and 7.2, C22-H, C25-H), 7.29 (2H, dd, J 6.6 and 6.6, C21-H, C26-H), 7.19 (1H, broad s, C8-H), 7.03 (2H, d, J 7.9, C14-H, C17-H), 6.81 (2H, d, J 7.9, C15-H, C16-H), 6.50 (1H, s, C4-H), 5.88 (1H, broad s, NH), 5.25 (1H, broad s, C12-H), 4.73-4.67 (1H, m, C7-H), 4.43 (1H, dd, J 10.0 and 7.6, C18-CH.sub.2), 4.28 (1H, dd, J 10.0 and 7.2, C18-CH.sub.2), 4.16 (1H, dd, J 7.2 and 6.19, C19-H), 3.93 (3H, s, OCH3), 3.88 (3H, s, OCH.sub.3), 3.62 (3H, s, OCH.sub.3), 3.21 (1H, dd, J 13.1 and 4.8, C13-CH.sub.2), 3.11 (1H, dd, J 13.1 and 5.5, C13-CH.sub.2), 2.53 (1H, dd, J 13.4 and 6.2, C5-CH.sub.2), 2.40 (1H, ddd, J 13.4, 12.7 and 6.9, C5-CH.sub.2), 2.22-2.15 (1H, m, C6-CH.sub.2), 1.88 (3H, s, CH.sub.3), 1.80 (1H, ddd, J 11.5, 11.3 and 6.9, C6-CH.sub.2), 1.22 (9H, s, C(CH.sub.3).sub.3); ES m/z (%) 826 [M.sup.+](100).

Synthesis of 3

(14) TFA (2 mL) was added to a solution of 2 (486 mg, 589 mol, 1.00 eq) and the reaction mixture stirred for 20 min. TLC indicated quantitative conversion to the product. The product was concentrated under reduced pressure, with toluene co-evaporation to give 3 in quantitative yield.

(15) .sub.N(600 MHz, CDCI.sub.3), 10.08 (1H, broad s, NH), 8.99 (1H, d J 10.7, C11-H), 7.71 (2H, d, J 6.2, C23-H, C24-H), 7.55 (1H, s, C8-H), 7.49 (2H, dd, J 6.5 and 6.5, C20-H, C27-H), 7.41 (1H, d, J 10.2, C10-H), 7.33 (2H, dd, J 6.2 and 6.2, C22-H, C25-H), 7.26-7.21 (2H, m, C21-H, C26-H), 6.91 (2H, d, J 8.3, C14-H, C17-H), 6.56 (2H, d, J 7.2, C15-H, C16-H), 6.45 (1H, S, C4-H), 5.93 (1H, broad s, NH), 5.28 (1H, s, NH), 4.95-4.90 (1H, m, C12-H), 4.60 (1H, ddd, J 11.7, 5.8 and 6.9, C7-H), 4.39 (1H, dd, J 8.9 and 8.6, C18-CH.sub.2), 4.29-4.24 (1H, m, C18-CH.sub.2), 4.12 (1H, dd, J 6.9 and 6.9, C19-H), 3.90 (3H, s, OCH.sub.3), 3.84 (3H, s, OCH.sub.3), 3.54 (3H, s, OCH.sub.3), 3.08 (2H, d, J 5.2, C13-CH.sub.2), 2.44 (1H1 dd, J 13.4 and 6.2, C5-CH.sub.2), 2.33-2.26 (1H, m, C5-CH.sub.2), 2.15-2.09 (1H, m, C6-CH.sub.2), 1.82 (3H, s, CH.sub.3), 1.75-1.69 (1H, m, C6-CH.sub.2); ES m/z (%) 770 [M.sup.+](100).

(16) Preparation of 4:

(17) 2-Chlorotrityl chloride resin (Novabiochem, 100-200 mesh, substitution 1.4 mmolg.sup.1, 589 mg, 0.765 mmol, 1.00 eq) was suspended in a solution of 3 (589 mg, 0.765 mmol, 1.00 eq), dimethylaminopyridine (10 mg, 76.5 mol, 0.01 eq), DiPEA (247 mg, 1.913 mmol, 333 L, 2.50 eq) and pyridine (241 mg, 3.061 mmol, 248 L, 4.00 eq) in THF (10 mL) and stirred for 6 hours at 50 C. The resin was subsequently filtered and washed thoroughly with THF. The resin was then capped by washing the resin carefully with methanol (CH.sub.2CI.sub.2:MeOH:DiPEA 17:2:1, 100 mL). Resin 4 was dried overnight over P2O5. Dry resin weight: 593 mg (loading 56%).

(18) General Procedure for Synthesis of Endopeptidase-Activated Pro-Drugs

(19) ##STR00003##

(20) As an example, peptide conjugate 5 was synthesised using conventional solid phase peptide synthesis, from immobilised colchicine derivative 4, using an Fmoc-based strategy.

(21) N.sup.-Fmoc strategy synthesis of peptide acids was achieved manually using 2-chlorotrityl-derivatised resin 4. The resin was swelled thoroughly in DMF, followed by removal of the N-Fmoc protecting group by treatment with 20% v/v piperidine in DMF (33 min). All couplings were performed in DMF, employing 2.5-fold molar excesses of N.sup.-Fmoc protected amino acids (with appropriate side-chain protecting groups), and activated using HCTU/HOBt/DiPEA. N.sup.-Fmoc deprotections were performed using 20% piperidine in DMF (33 min). The success of couplings and de-protections was monitored using the ninhydrin-based Kaiser test. Unsuccessful couplings were repeated. After the final N.sup.-Fmoc deprotection, the peptide chain was endcapped with fluorescein isothiocyanate (2.50 eq, in the presence of DiPEA1 1.50 eq). The success of this reaction was also monitored by the Kaiser test.

(22) An additional -alanine residue was incorporated into the sequence to overcome incompatability of the thiourea linkage and the acidic conditions of cleavage (the thiourea can rearrange, and the carbonyl carbon of the preceding amide bond can undergo nucleophilic attack by the sulphydryl-like function so formed. This leads to cleavage of the amide bond, with concomitant formation of a cyclic thiazolinone. The thiazolinone can undergo rearrangement in the presence of aqueous acid to form a thiohydantoin).

(23) On completion of the sequence, the resin was washed (DMF, CH2CI2, CH2CI2/MeOH) and dried in vacuo over KOH to constant weight. Peptides were cleaved from the resin by mild acidolysis using TFA-H20-triisopropylsilane 95:2.5:2.5 for 2 h at RT, with simultaneous side-chain de-protection. Following cleavage, the TFA was removed under reduced pressure. The crude product was extracted into 95% aqueous acetic acid and lyophilised. The crude peptide was subsequently analysed using reversed phase HPLC and purified using preparative HPLC (purity>97%). Pure fractions were combined and lyophilised. Identity was confirmed by mass spectrometry.

(24) Potential Attachment of Parent Colchicine to a Peptide Sequence Through the B-Ring

(25) To enable attachment of a colchicine moiety through the peptide N-terminus, the following strategy will be used. The B-ring amine can be de-masked using published methods. Acylation with aspartic acid will introduce a carboxylic acid to the molecule (from the amino acid side chain) thereby enabling conjugation to the free amine at the peptide N-terminus (see below).

(26) ##STR00004##

(27) Acetylation of the amide bond will also be examined, to assess whether parent colchicine is released following MMP activation and subsequent exopeptidase degradation.

(28) ##STR00005##
Strategies for the Attachment of Doxorubicin to a Peptide Sequence

(29) To enable attachment of doxorubicin through the peptide N-terminus (following peptide synthesis using the immobilised colchicine derivatised resin previously described) it must first be modified to introduce a carboxylic acid. Examples include reaction with succinic anhydride (strategy 1, below). However, by utilising the side chain of aspartic acid (both natural amino acids), as shown below (strategy 2) a natural amino acid (as opposed to a foreign chemical entity) is released on metabolism:

(30) Strategy 1: (Succinyl Spacer)

(31) ##STR00006##
Strategy 2: (Amino Acid Spacer)

(32) ##STR00007##
C-Terminal Doxorubicin Linkage

(33) Protection of the amine group of doxorubicin with Dde (a commonly used protecting group in peptide chemistry and by our group) would allow immobilisation of the agent onto a trityl-based (or otherwise) resin. Subsequent removal of the Dde group would de-mask the amine, allowing a peptide sequence to be constructed from this point (i.e. through the C-terminus). Standard Fmoc-based solid phase synthesis would produce a peptide sequence. An appropriately derivatised VDA could then be conjugated through the N-terminus. Resin cleavage and purification would be as previously described.

(34) ##STR00008##
Incorporation of Glycosylated Amino Acids (to Enhance Physicochemical Properties)

(35) Amino acids with appropriate side chain functionality (e.g. serine, tyrosine, threonine) can be glycosylated (with mono-, di- or trisaccharides) to produce peptides with enhanced aqueous solubility. Such a carbohydrate-derivatised moiety could be used in place of serine, for example (see scheme below).

(36) ##STR00009##
Results
1) MMP-activated prodrug (compound i) pan MMP targeted
Structure:

(37) ##STR00010## a) Prodrug-1 has been screened using normal mouse plasma, normal mouse liver homogenate and experimental human tumour model homogenate (HT1080 xenograft; known to express majority of MMPs) ex vivo. Prodrug cleavage and metabolism were detected using LC/MS/MS. a. Prodrug-1 was stable in plasma and liver, supporting systemic stability of these therapeutics (FIG. 1). b. Prodrug-1 was metabolised in tumour homogenate, supporting activation of these therapeutics in tumours expressing MMPs (FIG. 1). b) Prodrug-1 is cleaved rapidly at the Glycine-Homophenylalanine (Gly-Hof) by recombinant MMP-2, MMP-9, MMP-10 and MMP-14 at least. Demonstrated by LC/MS/MS and mass spectrometry (data not shown) c) Prodrug-1 was administered in vivo via the intraperitoneal route to mice bearing subcutaneous HT1080 tumour model (expression of majority of MMPs). Plasma, tissues and tumours were collected at regular intervals post-dosing. Levels of prodrug and VDA2 were assessed by LC/MS/MS. a. Prodrug-1 accumulated and was detected in all tissues analysed (FIG. 2). b. Highest prodrug-1 levels were observed in the tumour (FIG. 2). c. Prodrug-1 not detectable after 24 hours post-dosing. (FIG. 2) d. VDA2 was detectable at low levels in normal tissues following prodrug-1 administration (FIG. 3) e. VDA2 levels were detected at high levels in tumour tissue following prodrug-1 administration (FIG. 3) f. VDA2 was still detectable at high levels in tumour and was undetectable in normal tissues after 24 hours post-dosing with prodrug-1 (FIG. 3)
2) MMP-activated prodrug (compound i) targeted to Membrane-type WIMPs (MT-MMPs)
Structure:

(38) ##STR00011## d) Compound 1 was modified in order to change the MMP-targeting of the compound from being pan-MMP to MT-MMP selective (Prodrug-2) a. Arginine was incorporated in place of the Glutamic acid at the P4 position b. Proline was removed and replaced with Serine at the P3 position e) Prodrug-2 has been screened using normal mouse plasma, normal mouse liver homogenate and experimental human tumour model homogenates demonstrating high MT1-MMP (MMP-14) expression and activity (HT1080) and low MT1-MMP expression and activity (MCF-7) ex vivo. Prodrug-2 cleavage and metabolism were detected using LC/MS/MS. a. Prodrug-2 remained intact in plasma supporting systemic stability of this therapeutic. b. Prodrug-2 remained stable in murine liver homogenates c. Prodrug-2 was metabolised rapidly in tumour homogenate expressing high MT-MMP levels (HT1080) relative to tumour homogenate expressing low MT-MMP levels (MCF7) (FIG. 4). d. These data support the systemic stability of this prodrug and the selectivity of activation by MT-MMPs. f) Prodrug-2 is cleaved differentially by MMPs as shown by LC/MS/MS and mass spectrometry (data not shown): a. Cleaved rapidly at the Glycine-Homophenylalanine (Gly-Hof) by recombinant MMP-14. b. Cleaved slowly at the Homophenylalanine-Tyrosine (Hof-Tyr) by recombinant MMP-2. Demonstrating different cleavage to that observed with prodrug-1. c. Prodrug-2 is not cleaved by recombinant MMP-9, in contrast to prodrug-1 d. These data support the MMP selective cleavage of prodrug-2 g) Prodrug-2 was administered in vivo via the intraperitoneal route to mice bearing subcutaneous HT1080 tumour model (MT1-MMP positive). Plasma, tissues and tumours were collected at regular intervals post-dosing. Levels of prodrug-2 and VDA2 were assessed by LC/MS/MS. a. Prodrug-2 accumulated and was detected in all tissues analysed (FIG. 5). b. Highest prodrug-2 levels were observed in the tumour (FIG. 5). c. Liver was representative of all normal tissues analysed. (FIG. 5) d. VDA2 was undetectable in plasma following administration of prodrug-2 (FIG. 6) e. High concentrations of VDA2 were detected in tumour following prodrug-2 administration (FIG. 6) f. Levels of VDA2 in tumour were 10 times higher than that detected in normal tissues following administration of prodrug-2 (FIG. 6) g. High levels of prodrug-2 and VDA2 were still detectable in tumour 48 hours post administration.