WO2011045340A1

WO2011045340A1 - Ido inhibitors and therapeutic uses thereof

Info

Publication number: WO2011045340A1
Application number: PCT/EP2010/065336
Authority: WO
Inventors: Ute Roehrig; Awad Loay; Olivier Michielin; Benoit Van Den Eynde; Luc Pilotte; Vincent Stroobant; Pierre Larrieu; Pierre Vogel; Vincent Zoete
Original assignee: Ludwig Institute For Cancer Research Ltd
Priority date: 2009-10-13
Filing date: 2010-10-13
Publication date: 2011-04-21
Also published as: GB0917926D0

Abstract

Use use of the compounds of formula I, and pharmaceutically acceptable salts thereof (Formula I), in which R₁ represents hydrogen or cyclohexyϊ and R₂ represents hydrogen or alkyl C_1-6 provided that R₁ and R₂ do not both simultaneously represent hydrogen; in the treatment of diseases in which inhibition of IDO plays a therapeutic role, eg in the treatment of cancer.

Description

IDO Inhibitors and Therapeutic uses thereof

This invention relates to inhibitors. of indoleamine 2,3-dioxygenase (IDO) and their use in the treatment of cancer or infections, either alone or in combination with additional therapeutic agents,

The heme-containing enzyme indoleamine 2,3-dioxygenase (IDO, EC 1.13.1 1.52) has been implicated in the establishment of pathological immune tolerance by tumors. IDO catalyzes the initial and rate-limiting step in the catabolism of tryptophan (Tip) along the kynurenine pathway. By depleting Trp locally, IDO blocks the proliferation of T lymphocytes, which are extremely sensitive to Trp shortage. The observation that many human tumors constitutively express IDO introduced the hypothesis that its inhibition could enhance the effectiveness of cancer immunotherapy. Results from in vitro and in vivo studies suggest that the efficacy of therapeutic vaccination of cancer patients may indeed be improved by concomitant administration of an IDO inhibitor.

According to the invention, we provide use of the compounds of formula I, and pharmaceutically acceptable salts thereof,

in which

R₁ represents hydrogen or cyclohexyl and

R₂ represents hydrogen or alkyl C_1-6

provided that R₁ and R₂ do not both simultaneously represent hydrogen;

in the treatment of diseases in which inhibition of IDO plays a therapeutic role, particularly those conditions mentioned herein.

The compound of formula I in which R₁ represents H is known and may be prepared by methods previously described in the literature, for example, Bunce et al, Journal of Organic Chemistry, 52, pp 4214-23 (1987), Soroka et al, Journal of Photochemistry and Photobiology A - Chemistry 64, pp 171-182 (1992) and Knolker et al, Synthesis- Stuttgart 1995, pp 397-408 (1995). The compound of formula I in which Rl is cyclohexyl has not previously been described in the literature.

Accordingly, we provide the compound of formula ΪΑ

and pharmaceutically acceptable salts thereof.

We also provide the use of compound IA and pharmaceutically acceptable salts thereof as a pharmaceutical. We also provide the use of compounds IB and pharmaceutical iy acceptable salts thereof as a pharmaceutical

in which R² is as defined above and preferably represents C¾ The compound of formula IA may be made by reductive amination of a compound of formula i in which Rt represents H with cyclohexanone and a hydride reducing agent, for example, NaBH(OAc)j, in a solvent which is inert to the reactive conditions, such as a polar, aprotic solvent, for example, dicioroethane. Salts of compounds of formula I may be formed by reacting the free acid, or a salt thereof, with one or more equivalents of the appropriate base. The reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, e.g. ethanol, tetrahydrofuran or diethyl ether, which may be removed in vacuo, or by freeze drying. The reaction may also be a metathetical process or it may be carried out on an ion exchange resin.

Pharmaceutically acceptable salts of the compounds of formula 1 thereof include salts with strong acids, e.g., HC1, HBr, etc, and salts with weak acids, eg organic acids, for example carboxylic acids, such as acetic acid, benzoic acids, as well as sulphonic acids. Non-toxic physiologically acceptable salts are preferred, although other salts are also useful, e.g. in isolating or purifying the product. The compounds of formula I may be used alone or in combination with at least one additional therapeutic agent.

The at least one additional therapeutic agent may be an antineoplastic chemotherapy agent. Suitable antineoplastic chemotherapeutic agent is selected from the group consisting of cyclophosphamide, methotrexate, fluorouracil, doxorubicin, vincristine, ifosfamide, cisplatin, gemcytabine, busulfan, ara-C, and combinations thereof.

Alternatively, the at least one additional therapeutic agent may be radiation therapy. The radiation therapy may be localized radiation therapy delivered to the tumour or may be total body Irradiation.

The compounds of the invention may be used as an adjuvant to the therapeutic vaccination of various cancers. Cancers that may be mentioned include melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, ieukemia, brain tumours, lymphoma, sarcoma, ovarian cancer, and Kaposi's sarcoma. Other cancers and tumours that may be mentioned include adrenocorticocancer, basal cell carcinoma, bladder cancer, bowel cancer, brain and CK!S tumors, breast cancers, B- cell lymphoma, carcinoid tumours, cervical cancer, childhood cancers, chondrosarcoma, choriocarcinoma, chronic myeloid leukemia, rectal cancers, endocrine cancers, endometrial cancer, esophageal cancer, Ewing's sarcoma, eye cancer, gastric cancer or carcinoma, gastrointestinal cancers, genitourinary cancers, glioma, gynecological cancers, head and neck cancers, hepatocellular cancer, Hodgkins disease, hypopharynx cancer, islet cell cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer (including small-cell lung carcinoma and non-small-cell carcinoma), lymphoma, male breast cancer, melanoma, mesothelioma, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkins lymphoma, non-melanoma skin cancer, osteosarcoma, ovarian cancer, pancreas cancer, pituitary cancer, prostate cancer, renai cell carcinoma, retinoblastoma,, rhabdomyosarcoma, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer or seminoma, thymus cancer, thyroid cancer, transitional cell cancer, trophoblastic cancer, uterine cancer, vaginal cancer, Waldenstrom's macroglobulinemia, and Wilm' s tumor, colorectum, cervix, endometrium, ovary, testis, mesothelial lining, white blood cell (including lymphoma and leukemia) esophagus, muscle, connective tissue, adrenal gland, bone, glioblastoma, and cutaneous basocelluiar carcinoma.

In addition to cancers, IDO plays a role in several diseases, including Clamydia psittaci infection and Streptococcus pyogenes infection, systemic lupus erythematosus, rheumatoid arthritis, Alzheimer's disease, Huntington's disease, Parkinson's disease, lyme neuroborreliosis, late lyme encephalopathy, Tourette's syndrome, systemic sclerosis, multiple sclerosis, coronary heart disease, T-cell mediated immune diseases, chronic infections (viral, bacterial, fungal and microbial), depression, neurological disorders, cancer tumors, and cataracts. Inhibitors of IDO may be used to treat these diseases. Other diseases that IDO inhibitors may be used to treat include, but are not limited to, human immunodeficiency virus (HIV) and AIDS-reiated cancers. The compounds may also be used as adjuvants to bone marrow transplantation or peripheral blood stem cell transplantation and in immunotherapy by adoptive transfer,

Where the compounds are used in the treatment of an infection, the infection may be selected from the group consisting of a viral infection, infection with an intracellular parasite, and infection with an intracellular bacteria.

Particular viral infections include human immunodeficiency virus or cytomegalovirus, Particular intracellular parasite infections may be selected from the group consisting of Leishmania donovani, Leishmania tropica, Leishmania major. Leishmania aethiopica, Leishmania mexicana, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Particular intracellular bacterial infections may be selected from the group consisting of Mycobacterium leprae, Mycobacterium tuberculosis, Listeria monocytogenes, and Toxplasma gondii.

When the compounds are used in combination, the at least one additional therapeutic agent may be a vaccine, for example, an anti-viral vaccine, a vaccine against HIV, a vaccine against tuberculosis, a vaccine against malaria. The vaccine may also be a tumour vaccine or a melanoma vaccine. Preferably, the tumour vaccine comprises genetically modified tumour cells or genetically modified cell lines. In such cases, preferably the genetically modified tumour cells or genetically modified cell line has been transfected to express granulocyte-macrophage stimulating factor (GM-CSF),

Alternatively, the vaccine may comprise one or more immunogenic peptides, preferably immunogenic peptides of cancer-testis antigens (CTAgs). Such CTAgs and immunogenic peptides thereof are well known in the art, see Scanlan et ai, Cancer Immun. 2004; 4:1 and Simpson et al ., Nat Rev Cancer. 2005; 5:615. CTAg proteins include MAGE, BAGE, GAGE, SSX, NY-ESO-1, LAGE, SCP, CTSP, CT7, CT8, CT9, CT10, CT 11, SAGE, OY-TES-1, NY-SAR-35 and NY-BR-1. Several MAGE proteins are known, including MAGE-A1, A3, A4, A5, A6, A8, A9, A10, A12, Bl , B2, B3, B4, C1, C2 and C3 proteins- Several SSX proteins exist, including SSX1, SSX2, SSX3 and SSX5.

Further, the tumour vaccine may comprise dendritic cells.

Further, the additional therapeutic agent may be a cytokine, for example a granulocyte- macrophage colony stimulating factor (GM-CSF) or flt3-Hgand.

According to the invention we further provide a method of treating a subject receiving a bone marrow transplant or peripheral blood stem cell transplant comprising administering a therapeutically effective amount of compound of formula 1 or a pharmaceutically acceptable salt thereof to such a subjeet.

Preferably, the compound of formula I or a pharmaceutically acceptable salt thereof is administered in an amount effective to increase the delayed type hypersensitivity reaction to tumour antigen, delay the time to relapse of post-transplant malignancy, increase relapse free survival time post-transplant, and/or increase long-term post- transplant survival. Preferably, the compound of formula I or a pharmaceutically acceptable salt thereof is administered prior to full hematopoetic reconstitution.

The compounds of formula I or a pharmaceutically acceptable salt thereof for use in the method will generally be administered in the form of a pharmaceutical composition.

Thus, according to a further aspect of the invention there is provided a pharmaceutical composition including preferably less than 80% w/w, more preferably less than 50% w/w, e.g. 0.1 to 20%, of the compound of formula I or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable diluent or carrier. We also provide a process for the production of such a pharmaceutical composition which comprises mixing the ingredients. Examples of pharmaceutical formulations which may be used, and suitable diluents or carriers, are as follows;

for intravenous injection or infusion - purified water or saline solution;

for inhalation compositions - coarse lactose;

for tablets, capsules and dragees - macrocrystalline cellulose, calcium phosphate, diatomaceous earth, a sugar such as lactose, dextrose or mannitoi, talc, stearic acid, starch, sodium bicarbonate and/or gelatin;

for suppositories - natural or hardened oils or waxes.

When the compounds of formula I or a pharmaceutically acceptable salt thereof is to be used in aqueous solution, e.g. for infusion, it may be necessary to incorporate other excipients, In particular there may be mentioned chelating or sequestering agents, antioxidants, tonicity adjusting agents, pH-modifying agents and buffering agents.

Solutions containing the compound of formula I or a pharmaceutically acceptable salt thereof may, if desired, be evaporated, e.g, by freeze drying or spray drying, to give a solid composition, which may be reconstituted prior to use.

When not in solution, the compound of formula 1 or a pharmaceutically acceptable salt thereof preferably is in a form having a mass median diameter of from 0.01 to Ι Ομm. The compositions may also contain suitable preserving, stabilising and wetting agents, solubilisers, e.g. a water-soluble cellulose polymer such as hydroxypropyl methylce!lulose, or a water-soluble glycol such as propylene glycol, sweetening and colouring agents and flavourings. Where appropriate, the compositions may be formulated in sustained release form. The content of the compound of formula I or a pharmaceutically acceptable salt thereof in a pharmaceutical composition is generally about 0.01 -about 99.9wt%, preferably about 0.1 -about 50wt%, relative to the entire preparation.

The dose of the compound of formula I or a pharmaceutically acceptable salt thereof is determined in consideration of age, body weight, general health condition, diet, administration time, administration method, clearance rate, combination of drugs, the level of disease for which the patient is under treatment then, and other factors.

While the dose varies depending on the target disease, condition, subject of administration, administration method and the like, for oral administration as a therapeutic agent for the treatment of cancer in a patient suffering from such a disease is from 0.01 mg - 10 g, preferably 0.1 - 100 mg, is preferably administered in a single dose or in 2 or 3 portions per day. Where the compound of formula I or a pharmaceutically acceptable salt thereof is used in combination with other therapeutic agents, these may be used at their normal therapeutic doses, e.g., as set out in pharmacopoeias or prescribing guides, such as the Physicians' Desk Reference (PDR). In certain cases, the compound of formula I or a pharmaceutically acceptable salt thereof supplements the activity of the additional therapeutic agent(s) in a synergistic fashion, such that the additional therapeutic agent(s) can be administered at a lower dose than is normally used.

The potential activity of the compound of formula 1 or a pharmaceutically acceptable salt thereof in the treatment of cancer or infections has been demonstrated in the following predictive experiments, which demonstrate that the compound of formula I is an IDO inhibitor.

Experimental Methods

Chemistry Reagents were purchased from Acros, Fluka, Senn, Sigma-Aldrich or Merck and used without further purification. For extraction and chromatography all solvents were distilled prior to use. Anhydrous THF, diethyl ether, and toluene were distilled from sodium and benzophenone, dichloromethane from CaH₂, and methanol from magnesium. Reactions were monitored by thin layer chromatography (TLC) using silica gel plates (Merck 60 F254). Developed TLC plates were visualized with UV light (254 nm) or molybdic reagent (21 g of (ΝΗ₄)₆Μ0₇O₂₄.4Η₂O, 1 g of Ce(S0₄)₂, 31 ml of H₂S0₄ and 470 ml of H₂0). Flash chromatography (FC) was conducted using silica gel 60 A, 230-400 mesh (Merck 9385). Melting points were measured with a Mettler FP52 and are uncorrected. Optical rotations were measured with a JASCO DIP-370 digital polarimeter. UV spectra were recorded on a Kontron Uvikon 810 CW spectrophotometer. IR spectra were recorded on a Perkin Elmer Paragon 1000 FT-IR spectrometer. Mass spectra were recorded on a Nermag R 10- IOC in chemical ionisation mode. Electrospray mass analyses were recorded on a Finnigan MAT SSQ 7 IOC spectrometer in positive ionisation mode. ¹H-NMR spectra were recorded on a Bruker DPX-400 FT, Bruker ARX-400 FT, or Bruker AMX-600 spectrometer. All ¹H signal assignments were confirmed by COSY spectra. ^!3C-NMR spectra were recorded on a Bruker DPX-400 FT (100.61 MHz) or on a Bruker ARX-400 FT (100.61 MHz) spectrometer. All ¹³C signal assignments were confirmed by HMQC spectra. Chemical shifts are given in ppm, relative to an internal standard such as residual solvent signals. Coupling constants are given in Hertz. High resolution mass spectra were recorded by ESI-TOF-HRMS or MALDI-TOF-HRMS.

Protein expression and purification of recombinant human IDO

The coding region for human IDO (Ala2-Gly403) was cloned into a derivative of plasmid pET9 (Novagen). The recombinant plasmid, pETIDO, encodes a histidine tag at the N-terminus of IDO. The bacterial strain BL21 AI (Invitrogen) was used for overexpression of IDO and transformed with the plasmid pETIDO. The transformed cells were grown on a rotary shaker at 37 C and 220 rpm to an OD600 of 1.2, in LB medium supplemented with kanamycin 25 μg/ml, L-tryptophan 50 μg/ml and bovine hemin (Sigma) 10 μΜ. The culture was cooled in a water/ice bath and supplemented again with L-tryptophan 50 μg/ml, bovine hemin 10 μΜ. The expression of his-tagged IDO was induced by the addition of 1% (w/v) arabinose. Induced cells were grown at 20 C, 60 rpm for 20 h. Cells (1 1 culture) were collected by centrifugation, resuspended in 40 ml of Mes 25 mM, KC1 150 mM, imidazole 10 mM, protease inhibitors (complete EDTA free, Roche Applied Science), pH 6.5, and disrupted with a French press. The extract was clarified by centrifugation and filtration on a 0.22 μηι filter. The enzyme was purified by IMAC using Ni^2÷ as ligand and an IMAC HITRAP column (5 ml; GE Healthcare). Briefly, the extract was loaded on the column with buffer Mes 25 mM, KC1 150 mM, imidazole 10 mM, pH 6.5. The column was washed with 50 ml of the same buffer with imidazole adjusted to 100 mM. Finally, the protein was eluted with Mes 25 mM, KC1 150 mM, EDTA 50 mM, pH 6.5. The buffer was then exchanged to Mes 25 mM, KCi 150 mM, pH 6.5 using a HITRAP desalting column (GE Healthcare). The purity of the enzyme was estimated to be > 95% based on SDS PAGE gel and Coomassie blue staining. The ratio of absorbance 404 nm/280 nm of the protein was around 1,9.

4-(CyclohexyIamino)-1-naphthol hydrochloride (Compound of Formula IA).

The compound of formula IΑ was synthesized from 4-amino-1-naphthol hydrochloride and cyclohexanone according to the reductive amination procedure to afford the hydrochloric salt as a white solid in 82% yield. Mp = 276-278 °C. TLC R = 0.42 (40% diethyl ether/pentane). ¹H NMR (400 MHz, DMSO-d6 + D20): δ 8.47 (d, 1H, ³J(H- C(8), H-C(7)) = 8.5, H-C(8)); 8.19 (d, 1H, ³J(H-C(5), H-C(6)) = 8.5, H~C(5)); 7.78- 7.64 (m, 4H, H-C(2), H-C(3), H-C(6), H-C(7)); 3.50 (m, 1H, H-C(l ')); 2.15 (d, 2H, ³ J(H-C(2'), H-C(l ')) - ³J(H-C(6'), H-C(l ')) = 8.5, H-C(2')-ax , H-C(6')-i?x); 1.77 (d, 2H, ³J(H-C(2'), H-QT)) = ³J(H-C(6'), H-C(l ')) = 8.5, H-C(2')-eq, H-C(6')-<w); 1.68- 1.54 (m, 2H, H-C(4')); 1.30-1.06 (m, 4H, H-C(3'), H-C(5')). ¹³C NMR (100.6 MHz, DMSO-d6): δ 151.7 (s, C(l)); 128.2 (d, ^!J(C,H) = 156, C(7)); 127.2 (s, C(4)); 126.9 (d, 'J(C,H) = 162, C(6)); 126.7 (s, C(9)); 126.6 (d, V(C,H) - 129, C(8)); 124.0 (d, ^SJ(C,H) = 128, C(5)); 121.9 (d, 'J(C,H) = 156, C(2)); 121.9 (d, ^!J(C,H) = 156, C(3)); 1 17.0 (s, C(10)); 61.3 (d, 'J(C,H) = 125, C(F)); 29.3 (t, 'J(C,H) = 137, C(2^!), C(6')); 25.0 (t, ¹ J(C,H) = 137, C(3'), C(5')); 24.4 (t, 'J(C,H) - 137, C(4')). ESI-TOF-HRMS m/z calcd. for (M + H) C16H20NO 242.1545, found 242.1544. Enzymatic Assay

The enzymatic inhibition assays were performed as described by Takikawa et al. [Takikawa, O.; Kuroiwa, T.; Yamazaki, F.; Kido, R. J Biol Chem, 1988, 263, 2041- 2048.] with some modifications. Briefly, the reaction mixture (100 μΐ) contained potassium phosphate buffer (100 mM, pH 6.5) ascorbic acid (20 raM), catalase (200 units/ml), methylene blue (10 μΜ), purified recombinant IDO (l ng/μl), and L- Trp (200 μΜ). The inhibitors were serially diluted ranging from 0.1 to 1000 μΜ. The reaction was carried out at 37 °C for 60 min and stopped by the addition of 30% (w/v) trichloroacetic acid (40 μΐ). To convert the product of Trp dioxygenation by IDO, N- formylkynurenine, to spectroscopically detectable kynurenine, the tubes were incubated at 50 °C for 30 min, followed by a centrifugation at 1OOOOg for 20 min. Lastly, 100 μΐ of supernatant from each probe was transfered to another tube for HPLC analysis. The mobile phase for HPLC measurements consisted of 50% sodium citrate buffer (40 mM, pH 2.25) and 50% methanol with 400 μΜ SDS. The flow rate through the S5-ODS1 column was chosen to be 1 ml/min, and kynurenine was detected at a wavelength of 365 nm.

Cellular Assay

Cell Lines

A plasmid construct encoding murine IDO was transfected into mouse mastocytoma line P815B. Clone P185B-mIDO clone 6, [Uyttenhove et al, Nat Med, 9(10) ppl269- 1274 ((2003)] which overexpresses IDO, was selected and used for the cellular assay. Mouse IDO shares a 62% sequence identity with human IDO, and the active site residues mentioned in the Introduction are 100% conserved. For the assays evaluating inhibition of human IDO, a plasmid construct encoding human IDO was transfected into human HEK-293 cells, and clone 293-hIDO clone 17 [Uyttenhove et al] was selected and used in the assay.

Assay

The assay was performed in 96-well flat bottom plates seeded with 2 x 10⁵ cells in a final volume of 200 μΐ. To determine whether compounds were significant IDO inhibitors, the cells were incubated overnight at 37 °C in HBSS (Hanks Balanced Salt Solution, Invitrogen) supplemented with 50 μΜ L-Trp and 2, 20 or 200 μΜ of the compound. To determine the IC , the cells were incubated for 8 h at 37 °C in HBSS supplemented with 80 μΜ L-Tryptophan and a titration of the compound ranging from 0.312 to 80 μΜ or from 1.56 to 400 μΜ. The plates were then centrifuged for 10 min at 300 g, and 150 μl of the supernatant were collected. The supernatant was analyzed by HPLC to measure the concentration of tryptophan and kynurenine. For HPLC analysis, 50 μΐ of supernatant were mixed with 500 μl acetonitrile to precipitate the proteins. After centrifugation, the supernatant was collected, concentrated on a speedvac, resuspended in a final volume of 100 μΐ water and injected in the HPLC (CI 8 column). Trp was detected at an absorption wavelength of 280 nm, kynurenine at 360 nm. In the conditions used for IC₅₀ estimation, about 50% of the initial amount of tryptophan was degraded in the absence of inhibitor, and an equimolar amount of kynurenine was produced. The percentages of inhibition of tryptophan degradation and kynurenine production by the compounds were calculated in reference to this maximal activity. The initial wells containing the cells in the remaining volume of 50 μΐ were used to estimate cell viability in a classical MTT assay. To that end, 50 μΐ of culture medium (Iscove medium with 10% FCS and amino acids) were added to the wells together with 50 μΐ of MTT. After 3-4 h of incubation at 37 °C, 100 μΐ of SDS/DMF were added to dissolve the crystals of formazan blue. The absorbance at 570 nm/650 nm was measured after overnight incubation at 37 °C.

Experimental Results

In the enzymatic model of IDO inhibition, the following activities were observed:

Claims

1. Use of the compounds of formula I, and pharmaceutically acceptable salts thereof,

in which

Ri represents hydrogen or cyclohexyl and

R.2 represents hydrogen or alkyl C_1-6

provided that R_\ and R₂ do not both simultaneously represent hydrogen;

2. A compound of formula I according to claim 1 in combination with at least one additional therapeutic agent.

3. Use of a compound of formula I according to claim 1 as an adjuvant to the therapeutic vaccination of a cancer or tumour, including melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain tumours, lymphoma, sarcoma, ovarian cancer, and Kaposi's sarcoma.

4. The compound of formula I A

and pharmaceutically acceptable salts thereof.

5. Use of compounds IB and pharmaceutically acceptable salts thereof as a pharmaceutical

in which R₂ is as defined in claim 1 and preferably represents CH₃