Treatment or prevention of proliferative conditions

Abstract

Providing novel compounds for use in the treatment or prevention of cancer and other proliferative conditions SOLUTION: The present invention relates to novel compounds for use, for example, in the treatment or prevention of cancers and other proliferation conditions characterized by cells expressing cytochrome P4501B1 (CYP1B1) and allelic variants thereof. The invention also provides pharmaceutical compositions comprising one or more such compounds for use in medical therapy, for example in the treatment or prophylaxis of cancer or other proliferative conditions, as well as methods of treating cancer or other conditions in a human or non-human animal patient. The present invention also provides methods of identifying novel compounds for use in the treatment or prevention of cancers and other prohferative conditions characterized by cells that express, for example, CYP1B1 and allelic variants thereof. The invention also provides a method for determining the effectiveness of a compound of the invention in treating cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to novel compounds for use in the treatment or prevention of, for example, cancer and other proliferative conditions characterized by cells expressing cytochrome P450 1B1 (CYP1B1) and its allelic variants. The present invention also provides pharmaceutical compositions comprising one or more such compounds for use in medical therapy, e.g., the prevention or treatment of cancer or other proliferative conditions, as well as methods for treating cancer or other conditions in human or non-human animal patients. The present invention also provides methods for identifying novel compounds for use in the treatment, e.g., the prevention of cancer and other proliferative conditions characterized by cells expressing CYP1B1 and its allelic variants. The present invention also provides methods for determining the effectiveness of compounds of the present invention in treating cancer. [Background technology]

[0002] As described by Sutter et al. (Non-Patent Document 1), CYP1B1 is a member of the dioxin-inducible CYP1 gene family, which also includes CYP1A1 and CYP1A2. CYP1B1 is a heme thiolate monooxygenase enzyme that can metabolize and activate a variety of substrates, including steroids, xenobiotics, drugs, and / or prodrugs. CYP1B1 protein is frequently expressed in a wide range of human primary and metastatic cancers of different histogenesis types, but is absent or expressed at negligible levels in normal tissues (see, for example, Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4).

[0003] More specifically, CYP1B1 has been shown to be expressed in cancers of the bladder, brain, breast, colon, head and neck, kidney, lung, liver, ovary, prostate, and skin, but not in corresponding normal tissues. For example, Non-Patent Document 5 reported that CYP1B1 was overexpressed in glial tumors, including glioblastoma, anaplastic astrocytoma, oligodendroglioma, and anaplastic oligodendroglioma, but not in unaffected brain tissue; Non-Patent Document 6 reported that CYP1B1 was overexpressed in prostate adenocarcinoma, but not in corresponding normal prostate tissue; Carnell et al. (2004, Non-Patent Document 6) also showed that CYP1B1 was expressed in bladder cancer (n=22, 100%); Non-Patent Documents 7 and 8 reported that CYP1B1 expression was increased in primary and metastatic ovarian cancer, but not in normal ovarian tissue; and Non-Patent Documents 9 and 10 reported that CYP1B1 was overexpressed in colon adenocarcinoma compared to corresponding normal tissue.

[0004] Several studies have shown that CYP1B1 is overexpressed in breast cancer compared to corresponding normal tissues (see, for example, Non-Patent Document 11; Non-Patent Document 12; Non-Patent Document 13).

[0005] In Non-Patent Document 14, it was reported that CYP1B1 was overexpressed in malignant melanoma and disseminated disease, but not in normal skin. In Non-Patent Document 15, Chang et al. reported that CYP1B1 protein was not present in normal liver, but Everett et al. (2007, Non-Patent Document 14) confirmed overexpression of CYP1B1 in stage IV metastasis of melanoma to the liver, but not in adjacent normal liver.

[0006] Greer et al. reported in Non-Patent Document 16 that CYP1B1 was overexpressed during the malignant progression of head and neck squamous cell carcinoma, but not in normal epithelium. do.

[0007] McFadyen et al., in Non-Patent Document 17, detected CYP1B1 in renal cancer but not in corresponding normal tissues.

[0008] Murray et al. (2004, Non-Patent Document 2) used immunohistochemistry to demonstrate overexpression of CYP1B1 in lung cancer cells compared with normal lung tissue. Su et al. (2004, Non-Patent Document 18) used immunohistochemistry to demonstrate overexpression of CYP1B1 in advanced stage IV non-small cell lung cancer compared with earlier stages of the disease.

[0009] It is clear from the numerous disclosures cited above that CYP1B1 expression is characteristic of a range of different cancers and other proliferative conditions, and that CYP1B1 expression can be used to define the extent of such cancers and other conditions. Because normal (non-cancerous) cells do not express significant levels of CYP1B1, it is reasonable to expect that compounds that are cytotoxic in cells that express CYP1B1 but substantially non-toxic to normal cells would be useful as targeted anti-cancer agents in cancers characterized by CYP1B1 expression. "Targeted" means that such compounds can be delivered systemically and are activated only in the presence of cancerous cells that express CYP1B1, remaining substantially non-toxic to the rest of the body.

[0010] Furthermore, some cytochrome P450 enzymes have been shown to metabolize and detoxify various anticancer drugs. McFadyen et al. (Non-Patent Document 19) demonstrated that cells expressing CYP1B1 showed significantly reduced sensitivity to docetaxel compared with cells not expressing CYP1B1. This finding suggests that the presence of CYP1B1 in cells may reduce their sensitivity to certain cytotoxic drugs. Therefore, prodrugs activated by CYP1B1 may be useful for the treatment of cancers in which drug resistance is mediated by CYP1B1.

[0011] Furthermore, the CYP1B1 gene is highly polymorphic in cancer, and several single nucleotide polymorphisms contained within the CYP1B1 gene have been identified that alter the expression and / or activity of the encoded protein. * The 3(4326C>G, L432V) allele was characterized by increased expression and enzyme kinetics of CYP1B1 for several substrates, as described by Sissung et al., Non-Patent Document 20 and the references cited therein. This finding suggests that not only CYP1B1 but also allelic variants of this enzyme may contribute to prodrug activation and cancer targeting.

[0012] Prodrugs have been explored as a means of reducing undesirable toxicity or other negative attributes of drugs without compromising efficacy. Prodrugs are drugs that have been chemically modified to render them inactive, but that are metabolized or otherwise converted to their active form in the body after administration. As reviewed by [2], the overexpression of CYP1B1 in primary tumors and metastatic disease compared with normal tissues presents an excellent opportunity for the development of CYP1B1-activated prodrugs for targeted cancer therapy. Indeed, as reviewed by

[21] , the discovery and development of CYP1B1-activated prodrugs for targeted cancer therapy is likely to offer pharmacological advantages over existing non-targeted cytochrome P450-activated prodrugs in clinical use, such as the alkylating agents cyclophosphamide, ifosfamide, dacarbazine, and procarbazine, which are activated by cytochrome P450s expressed in normal tissues.

[0013] The human cytochrome P450 family contains 57 active isozymes, which function in normal metabolism, affect drug pharmacokinetics, and cause negative outcomes for patients through drug-drug interactions. As described in Non-Patent Document 22, cytochrome P450 isozymes metabolize approximately two-thirds of known drugs in humans, with 80% of this metabolization occurring through five isozymes: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4.

[0014] As reviewed in Non-Patent Document 23, CYP2R1, CYP2W1, CYP2S1, CYP2S1, and CYP2U1 were among the first genes discovered in the Human Genome Project, but the functions, polymorphisms, and regulation of these genes remain to be fully elucidated. In addition to CYP1B1, several of these cytochrome P450 oxidoreductases are extrahepatic and overexpressed in cancer. As described in Non-Patent Document 7, several cytochrome P450s, including CYP1B1, CYP2A / 2B, CYP2F1, CYP2R1, CYP2U1, CYP3A5, CYP3A7, CYP4Z1, CYP26A1, and CYP51, are present at significantly higher levels than in normal ovaries, as determined by immunohistochemistry and light microscopy. Furthermore, as described in Non-Patent Document 2, using a similar detection method in primary colorectal cancer compared with normal colon, several cytochrome P450s, including CYP1B1, CYP2S1, CYP2U1, CYP3A5, and CYP51, were frequently overexpressed. In the same study, several cytochrome P450s, including CYP1B1, CYP2A / 2B, CYP2F1, CYP4V2, and CYP39, were correlated with their presence in primary tumors. According to Non-Patent Document 24, CYP2W1 has also been shown to be overexpressed in colorectal cancer. CYP4Z1 is a gene that is overexpressed in breast cancer and is also associated with the promotion and progression of non-small cell lung cancer, as described in Non-Patent Document 25 and Non-Patent Document 26, respectively.

[0015] A major challenge in this field is elucidating the functions of so-called "orphan" human cytochrome P450s, particularly those with unknown substrates, as reviewed in Non-Patent Document 27. Several substrates of CYP1B1 are known, only a few of which are specifically metabolized by this enzyme. For example, as described in Non-Patent Document 28, 7-ethoxyresorufin undergoes oxidative deethylation when activated by any member of the CYP1 family, including CYP1A1, CYP1A2, and CYP1B1. While several fluorogenic and luminescent probe substrates are available for sensitively assessing cytochrome P450 activity, these substrates exhibit broad specificity and are therefore metabolized by a range of cytochrome P450 enzymes from the CYP1, CYP2, and CYP3 families. For example, Non-Patent Document 29 describes the use of luminescent substrates coupled to firefly luciferase luminescence in a technology called P450-Glo. Another example is 7-ethoxycoumarin, which undergoes cytochrome P450-catalyzed O-deethylation to liberate a highly fluorescent anion, as described by Waxman DJ and Change TKH in "The use of 7-ethoxycoumarin to minister multiple enzymes in the human CYP1, CYP2, CYP3 families" in Non-Patent Document 30.

[0016] (2003) describes an indolequinone model prodrug that is reductively cleaved by cytochrome P450 reductase (not to be confused with cytochrome P450) under anoxic conditions to liberate the 7-hydroxy-4-methylcoumarin anion. This model prodrug is not luminescent at a preselected emission wavelength, and kinetic fluorescence spectroscopy is used to monitor the production of the coumarin anion (λ ex =380nm / λ em = 450 nm), reductive cleavage could be accurately measured.

[0017] Although the interactions of a limited number of compounds (usually less than 100) with cytochrome P450 isozymes have been described, the results of such studies are difficult to compare due to differences in techniques, assay conditions, and data analysis methods, as described in Non-Patent Document 32. Many computational strategies have been advanced to generate predictive activity models of cytochrome P450 isozyme substrates, but they are limited by the lack of a large, single, diverse dataset of cytochrome P450 isozyme activity, as described in Non-Patent Document 33. The authors described the use of quantitative high-throughput screening (HTS) with bioluminescent enzyme substrate inhibition assays to construct a cytochrome P450 bioactivity database and screen 17,143 chemical compounds against five cytochrome P450 isozymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4) expressed in normal tissues, primarily the liver, that are responsible for the so-called phase 1 metabolism of drugs. It is concluded that this database should be useful for building and testing new predictive models of cytochrome P450 activity to support early-stage drug discovery efforts.

[0018] (2009) describes a computational method for predicting CYP2D6 and CYP3A4 inhibition based on a novel Gaussian Kernel-weighted k-nearest neighbor (k-NN) algorithm, which is based on Tanimoto's similarity search using extended connectivity fingerprints. The dataset incorporates modeling of 1153 and 1182 drug candidates tested for CYP2D6 and CYP3A4 inhibition in human liver microsomes. For CYP2D6, 82% of the classified test compounds were predicted into the correct class, and for CYP3A4, 88% of the classified test compounds were correctly classified.

[0019] In theory, it may be possible to use cytochrome P450 HTS to build a large database of the biological activities of cytochrome P450 in tumor and normal tissues, and then develop substrate prediction models as a basis for the design and synthesis of selective CYP1B1-activated prodrugs while examining the pharmacological issues associated with phase I metabolism by normal tissue cytochrome P450. However, implementation simplifications are not clear from the prior art and need to be rationalized in light of the structure of the prodrug and the mechanism of conversion to the active drug upon activation by tumors expressing cytochrome P450.

[0020] In prodrug design, the so-called "trigger-linker-effector" chemistry requires activation of the trigger, initiating linker cleavage and liberating the effector (usually the active drug), whose biological activity is masked in the prodrug form. The modular design of selective prodrugs targeted to tumors expressing cytochrome P450s, such as CYP1B1, requires (1) identification of a selective trigger molecule, (2) use of a biostable linker that is efficiently cleaved (usually by aromatic hydroxylation) following trigger activation, and (3) a suitable effector or drug that does not interfere with the efficiency of the trigger-driven process.

[0021] CYP1B1 mRNA is constitutively expressed in all normal extrahepatic human tissues, but the protein is usually undetectable. In contrast, CYP1B1 protein is expressed at high levels in tumors. It is understood that a wide range of established or immortalized tumor cell lines of human origin that have undergone extensive in vitro passage (such as MCF-7 breast cancer cells) do not constitutively express active CYP1B1 protein. Although CYP1B1 is not constitutively expressed in MCF-7 breast tumor cells, it is possible to induce CYP1 enzyme expression at both the mRNA and protein levels by treating them with aryl hydrocarbon agonists, such as dioxin TCDD.

[0022] US Patent No. 5,999,499 describes prodrugs comprising a drug moiety attached to a carrier backbone, which are activated by CYP1B1 as if hydroxylated to liberate the drug moiety. [Prior art documents] [Patent documents]

[0023] [Patent Document 1] International Publication No. 99 / 40944 [Non-patent literature]

[0024] [Non-Patent Document 1] Sutter et al., J. Biol. Chem., May 6, 1994, Vol. 269, No. 18, pp. 13092-13099 [Non-patent document 2] McFadyen MC, Melvin WT, and Murray Gl, "Cytochrome P450 Enzymes: Novel Options for Cancer Therapeutics," Mol Cancer Ther., 2004, Vol. 3, No. 3, pp. 363-371 [Non-patent document 3] McFadyen MC and Murray GL, "Cytochrome P450 1B1: a Novel Anticancer Therapeutic Target," Future Oncol., 2005, Vol. 1, No. 2, pp. 259-263 [Non-patent document 4] Sissung TM, Price DK, Sparreboom A, and Figg WD, "Pharmacogenetics and Regulation of Human Cytochrome P450 1B1: Implications in Hormone-Mediated Tumor Metabolism and a Novel Target for Therapeutic Intervention," Mol. Cancer Res., 2006, Vol. 4, No. 3, pp. 135-150 [Non-Patent Document 5] Barnett et al., Clin. Cancer Res., 2007, Vol. 13, No. 12, pp. 3559-3567 [Non-patent document 6] Carnell et al., Int.J.Radial Oncol.Biol.Phys., 2004, Volume 58, Issue 2, p.500-509 [Non-Patent Document 7] Downie et al., Clin. Cancer Res., 2005, Vol. 11, No. 20, pp. 7369-7375 [Non-patent document 8] McFadyen et al., Br. J. Cancer, 2001, Vol. 85, No. 2, pp. 242-246 [Non-Patent Document 9] Gibson et al., Mol. Cancer Ther., 2003, Vol. 2, No. 6, pp. 527-534 [Non-Patent Document 10] Kumarakulasingham et al., Clin. Cancer Res., 2005, Vol. 11, No. 10, pp. 3758-3765 [Non-Patent Document 11] Murray GI, Taylor MC, McFadyen MC, McKay JA, Greenlee WF, Burke MD, and Melvin WT, "Tumor-Specific Expression of Cytochrome P450 CYP1B1," Cancer Res., 1997, Vol. 57, No. 14, pp. 3026-3031 [Non-Patent Document 12] Haas S, Pierl C, Harth V, Pesch B, Rabstein S, Bruning T, Ko Y, Hamann U, Justenhoven C, Brauch H, Fischer HP, "Expression of Xenobiotic and Steroid Hormone Metabolizing Enzymes in Human Breast Carcinomas", Int.J.Cancer, 2006, Volume 119, No. 8, p.1785-1791 [Non-licensed Document 13] McKay JA, Murray GI, Ah-See AK, Greenlee WF, Marcus CB, Burke MD, Melvin WT, "Differential Expression of CYP1A1 and CYP1B1 in Human Breast Cancer", Biochem.Soc.Trans., 1996, Volume 24, No. 2, 327S [Non-licensed Document 14] Everett, J.Clin.Oncology, 2007, 25 volumes, 18S [Non-licensed Document 15] Changら, Toxicol.Sci., 2003, Volume 71, No. 1, p.11-19 [Non-licensed Document 16] Greer, Proc.Am.Assoc.Cancer Res., 2004, Volume 45, p.3701 [Non-licensed Document 17] McFadyen, Br.J.Cancer, 2004, Volume 91, No. 5, p.966-971 [Non-licensed Document 18] Suら, Anti-Cancer Res., 2009, Volume 2, 509-515 [Non-licensed Document 19] McFadyen, Biochem Pharmacol. July 15, 2001, Volume 62, No. 2, p.207-212 [Non-licensed Document 20] Sissung et al., Mol Cancer Ther., 2008, Vol. 7, No. 1, pp. 19-26 [Non-Patent Document 21] Patterson LH and Murray GI, Curr Pharm Des., 2002, Vol. 8, No. 15, pp. 1335-1347 [Non-Patent Document 22] Ortiz de Montellano, PR (ed.), Cytochrome P450: structure, mechanism, and biochemistry, New York, Kluwer Academic / Plenum Publishers, 2005. [Non-Patent Document 23] Ingelman-Sundberg, M., Toxicol.Appl.Pharmacol., 2005, vol. 207, p.52-56 [Non-Patent Document 24] Elder et al., Eur. J. Cancer, Vol. 45, No. 4, pp. 705-712 [Non-Patent Document 25] Reiger et al., Cancer Res., 2004, Vol. 64, No. 7, pp. 2357-2364 [Non-Patent Document 26] Bankovic et al., Lung Cancer, 2010, Vol. 67, No. 2, pp. 151-159 [Non-Patent Document 27] Strak K and Guengerich FP, Drug Metab.Rev., 2007, Vol. 39, No. 2-3, pp. 627-637 [Non-patent document 28] Chang TK and Waxman DJ, Methods Mol. Biol., 2006, 320, 85-90 [Non-Patent Document 29] Cali et al., Expert Opinion. Drug Toxicol., 2006, Vol. 2, No. 4, pp. 62-45 [Non-Patent Document 30] Phillips IR and Shephard EA (eds.), Methods in Molecular Biology, 2006, vol. 320, Cytochrome P450 Protocols, 2nd ed. [Non-Patent Document 31] Everett et al., Biochem.Pharmacol., 2002, Vol. 63, pp. 1629-1639 [Non-Patent Document 32] Rendic, S., "Summary of information on human CYP enzymes: human P450 metabolism data" in Drug Metab.Rev., 2002, Vol. 34, pp. 83-448 [Non-Patent Document 33] Veith et al., Nature Biotechnology, 2009, Vol. 27, pp. 1050-1055 [Non-Patent Document 34] Jensen et al., J. Med. Chem., 2007, Vol. 50, pp. 501-511 Summary of the Invention [Problem to be solved by the invention]

[0025] The inventors have surprisingly found that the compounds described herein, which are distinct from those described in WO99 / 40944, are degraded in certain cells, particularly cells expressing cytochrome P450 1B1 (hereinafter CYP1B1), but not in normal cells, as a result of the compounds being degraded by hydroxylation (e.g., by CYP1B1-expressing cells, particularly cancerous cells). [Means for solving the problem]

[0026] Thus, according to a first aspect, the present invention provides a compound of formula (I)

[0027] [ka] [In the formula, X 1 -X 1 -X 2 But -OX 2 , -SX 2 , -SO2-OX 2 , -SO2NZ 10 -X 2 , conjugated alkene methyloxy, conjugated alkene methylthio, conjugated alkene methylSO2-O, conjugated alkene methyl-SO2NZ 10 , or the following formula

[0028] [ka] It is something that consists of -X 2 does not exist or is X 1 -X 2 -Effector,

[0029] [ka] It is like one of each n and m is independently 0 or 1; p is 0, 1 or 2; X 3 is oxygen or sulfur, and when m=0, SO2-O, SO2NZ 10 , conjugated alkene methyloxy, conjugated alkene methylthio, conjugated alkene methyl-SO2-O, or conjugated alkene methyl-SO2NZ 10 But often, Y 1 , Y 2 and Y 3 are each independently carbon or nitrogen, and Y 1 If is nitrogen, Z 1 does not exist, and Y 2 If is nitrogen, Z 3 does not exist, and Y 3 If is nitrogen, Z 5 does not exist, Y 4is an oxygen, carbon, or nitrogen atom, sulfoxide, or sulfone, -Y 5 - is (i) a single bond, (ii) =CH- (=CH- double bond = is Y 4 or (iii) either -CH2- or -CH2CH2-, or a hydrogen atom of (ii) or one or more hydrogen atoms of (iii) is / are bonded to a substituent Z 11 It is one of (ii) to (iii) replaced by Z 11 are independently selected from alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl, nitro, and cyano; Z 1 ~Z 4 each, if present, is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl, nitro, and cyano; Z 5 when present, are independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, carboxy, formyl, nitro, and cyano; or Z 2 and Z 3 , Z 3 and Z 4 , and Z 4 and Z 5 One of the pairs is together with the atom to which it is attached to form an aromatic ring fused with the rest of the compound, provided that Z 1, Z 2 and Z 4 at least one of is hydrogen, Z 6 is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, and aralkyl; Y 6 None of the Y atoms may be nitrogen atoms, or 6 one or two of the groups may be nitrogen atoms and the rest may be carbon atoms; each Z 7 are independently hydrogen, alkyl, or aryl; each Z 8 are independently selected from hydrogen, an electron-withdrawing group, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, and substituted C1-C6 alkoxy, wherein the substituted alkyl or the substituted alkoxy is substituted with one or more groups selected from ether, amino, mono- or di-substituted amino, cyclic C1-C5 alkylamino, imidazolyl, C1-C6 alkylpiperazinyl, morpholino, thiol, thioether, tetrazole, carboxylic acid, ester, amide, mono- or di-substituted amide, N-linked amide, N-linked sulfonamide, sulfoxy, sulfonate, sulfonyl, sulfoxy, sulfinate, sulfinyl, phosphonooxy, phosphate, and sulfonamido; each Z 9 are independently oxygen or sulfur; Z 10 is hydrogen or alkyl, e.g., C 1~4 is alkyl, Effectors are molecules with pharmacological, diagnostic, or screening functions. or a pharmaceutically acceptable salt, ester, amide, or solvate thereof.

[0030] Viewed from a second aspect, the present invention provides a composition comprising a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, and a pharmaceutically acceptable carrier.

[0031] Viewed from a third aspect, the present invention provides a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, for use as a medicament.

[0032] Viewed from a fourth aspect, the present invention provides a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, for use in a method of treating or preventing a proliferative condition.

[0033] Viewed from a fifth aspect, the present invention provides a method of treating or preventing a proliferative condition comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a compound according to the first aspect of the invention or a pharmaceutically acceptable salt, ester, amide, or solvate thereof.

[0034] Viewed from a sixth aspect, the present invention provides the use of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, for the preparation of a medicament for use in a method of treating or preventing a proliferative condition.

[0035] Viewed from a seventh aspect, the present invention provides a method for identifying a compound that is specifically activated by a cytochrome P450 enzyme, comprising the steps of: (a) contacting a set of compounds according to the first aspect of the invention, wherein the effector is a fluorophore, with the cytochrome P450 enzyme, and determining whether the fluorophore is released from one or more compounds of the set as a result of said contacting; (b) contacting the set of compounds with a control tissue, tissue or cell extract, or enzyme, and determining whether the fluorophore is released from one or more compounds in the set as a result of said contacting; (c) identifying the compound that is specifically activated by the cytochrome P450 as any compound in the set of compounds that liberates the fluorophore in step (a) but does not, or does so to a much lesser extent, in step (b); The present invention provides a method comprising:

[0036] Viewed from an eighth aspect, the present invention provides a method for determining whether a compound of the present invention, wherein the effector is a molecule with pharmacological function, is effective in treating cancer, said method comprising administering said compound to an animal having cancer, wherein the cancer has arisen as a result of implantation of either recombinant cells engineered to constitutively express a cytochrome P450 enzyme, tissue taken directly from the tumor or cancer, or cells from an early passage cell line derived from tissue taken directly from the tumor or cancer which express the cytochrome P450 enzyme at a level similar to that of the tumor or cancer from which it originated.

[0037] Further aspects and embodiments of the present invention will become apparent from the discussion that follows. [Brief explanation of the drawings]

[0038] [Figure 1] Figure 1 shows Western blots demonstrating detection of CYP1B1 expression in transfected CHO / CYP1B1 / CPR cell lines (Panel A) and CYP1A1 expression in transfected CHO / CYP1A1 / CPR cell lines (Panel B), as detailed in the Experimental Section below. [Figure 2a] FIG. 2a illustrates the mechanism of CYP1B1-induced 3-hydroxylation of a compound of the invention (referred to herein as SU025-04) followed by spontaneous release of the cytotoxic effector molecule (N,N′-bis(2-chloroethyl)phosphorodiamidate (also known as IPM chloride) via 1,4 elimination. [Figure 2b]FIG. 2b illustrates the mechanism of CYP1B1-induced 4-hydroxylation of a compound of the invention (referred to herein as SU025-04) followed by spontaneous release of the cytotoxic effector molecule (N,N′-bis(2-chloroethyl)phosphorodiamidate (also known as IPM chloride) by 1,6-elimination. [Figure 2c] FIG. 2c illustrates the mechanism of CYP1B1-induced 6-hydroxylation of a compound of the invention (referred to herein as SU025-04) followed by spontaneous release of the cytotoxic effector molecule (N,N′-bis(2-chloroethyl)phosphorodiamidate (also known as IPM chloride) via 1,8 elimination. [Figure 3] FIG. 3 illustrates the mechanism of CYP1B1-induced 6-hydroxylation of a compound of the invention (referred to herein as SU024-1-03) followed by spontaneous release of the effector molecule by 1,8 elimination. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention arises from the provision of prodrugs by which so-called effector molecules, which may be cytostatic, cytotoxic, diagnostic, or screening molecules as described in more detail below, can be chemically modified by reacting them to produce compounds of formula (I). The inventors have found that when compounds of formula (I) undergo hydroxylation, particularly CYP1B1-induced hydroxylation, the effector molecule is liberated upon spontaneous breakdown of the compound of formula (I) via direct hydroxylation or hydroxylation via epoxide formation.

[0040] In general terms, the structure of compounds of formula (I) can be considered to comprise three parts: a trigger region, a linker, and an effector molecule. The trigger, which normally serves as a substrate for CYP1B1-induced hydroxylation, comprises the bicyclic moiety and its substituents shown on the left-hand side of formula (I), i.e., Y 1 ~Y5 , Z 1 ~Z 6 , and These moieties can generally be understood to include the compound moiety containing the remaining carbon atoms to which some of the moieties are attached. The trigger region of the compound can be understood to include the C(Z 7 )-X 1 -X 2 The effector molecule is linked to the effector molecule, designated as such, via a linker region comprising the agonist.

[0041] The composition and variability of these three regions of compounds of formula (I), namely the trigger, linker, and effector regions, are now described.

[0042] In the discussion which follows, reference will be made to a number of terms which should be understood to have the meanings set forth below, unless the context dictates otherwise.

[0043] By alkyl herein is meant a saturated hydrocarbyl radical that may be straight-chain, cyclic, or branched (usually straight-chain unless the context dictates otherwise). When an alkyl group has one or more sites of unsaturation, those sites may be constituted by carbon-carbon double bonds or carbon-carbon triple bonds. When an alkyl group contains a carbon-carbon double bond, it is an alkenyl group; when a carbon-carbon triple bond is present, it is an alkynyl group. Typically, alkyl, alkenyl, and alkynyl groups contain 1 to 25 carbon atoms, more usually 1 to 10 carbon atoms, and even more usually 1 to 6 carbon atoms, it being understood, of course, that the lower limit for alkenyl and alkynyl groups is 2 carbon atoms and for cycloalkyl groups is 3 carbon atoms.

[0044] An alkyl, alkenyl, or alkynyl group may be, for example, substituted once, twice, or three times, e.g., once, i.e., one or more hydrogen atoms of the alkyl group are formally replaced. Examples of such substituents are halo (e.g., fluoro, chloro, bromo, and iodo), aryl hydroxy, nitro, amino, alkoxy, alkylthio, carboxy, cyano, thio, formyl, ester, acyl, thioacyl, amido, sulfonamide, carbamate, and the like.

[0045] Carboxy, as used herein, refers to the deprotonated form (CO - ) may also refer to the functional group CO2H.

[0046] Halo is fluoro, bromo, chloro, or iodo.

[0047] Acyl and thioacyl refer to functional groups of the formula -C(O)-alkyl or -C(S)-alkyl, respectively, where alkyl is as defined above.

[0048] By ester is meant a functional group containing an -OC(=O)- moiety.

[0049] Amido refers to a functional group containing an -N(H)C(=O)- moiety, carbamate refers to a functional group containing an -N(H)C(=O)O- moiety, and sulfonamide refers to a functional group containing an -SON(H)- moiety, where each hydrogen atom shown may be replaced (independently in sulfonamides) with alkyl or aryl.

[0050] Alkyloxy (synonymous with alkoxy) and alkylthio moieties are of the formula -O-alkyl and -S-alkyl, respectively, where alkyl is as defined above.

[0051] Similarly, alkenyloxy, alkynyloxy, alkenylthio, and alkynylthio are of the formula -Oalkenyl, -Oalkynyl, -Salkenyl, and -Salkynyl, where alkenyl and alkynyl are as defined above.

[0052] By amino group, we mean herein a group of formula -N(R)2, where each R is independently hydrogen, alkyl, or aryl, e.g., an unsaturated unsubstituted C alkyl group such as methyl or ethyl. 1~6 The two R's attached to the nitrogen atom N are alkyl, or linked together. An example of this is where -RR- forms an alkylene diradical, typically derived formally from an alkane with two hydrogen atoms missing from the terminal carbon atom, thereby forming a ring with the nitrogen atom of the amine. As it is known that the diradical in a cyclic amine need not necessarily be an alkylene, morpholine (-RR- is -(CH2)2O(CH2)2-) is an example from which cyclic amino substituents can be prepared.

[0053] References to amino are understood herein to encompass quaternized or protonated amine derivatives resulting from compounds containing such an amino group, examples of the latter also being understood to be salts such as hydrochlorides.

[0054] Aryl, as used herein, refers to a radical formally formed by the removal of a hydrogen atom from an aromatic compound.

[0055] Arylene diradicals are formally derived from an aromatic moiety by the removal of two hydrogen atoms and may, and usually are, monocyclic, e.g., phenylene, unless the context dictates otherwise. As known to those skilled in the art, heteroaromatic moieties are a subset of aromatic moieties that contain one or more heteroatoms, usually O, N, or S, in place of one or more carbon atoms and their associated hydrogen atoms. Exemplary heteroaromatic moieties include, for example, pyridine, furan, pyrrole, and pyrimidine. Other examples of heteroaromatic rings include pyridyl, pyridazine (two nitrogen atoms in close proximity in a six-membered aromatic ring), pyrazine (two nitrogen atoms in a 1,4-configuration in a six-membered aromatic ring), pyrimidine (two nitrogen atoms in a 1,3-configuration in a six-membered aromatic ring), or 1,3,5-triazine (three nitrogen atoms in a 1,3,5-configuration in a six-membered aromatic ring).

[0056] The aryl or arylene radical may be substituted with an electron-withdrawing group (e.g., halo, cyano (-CN), haloalkyl, amido, nitro, keto (-COR), alkenyl, alkynyl, quaternary amino (-N + R3), ester, amide (-CONR2), N-linked amide (-NR-C(=O)-R), N-linked sulfonamide (-NR-S(=O)2R), sulfoxy (-S(=O)2OH), sulfonate (S(=O)2OR), sulfonyl (S(=O)2R), and sulfonamido (-S(=O)2-NR2), where (each) R is a C1-C6 alkyl group, a C3-C6 alkyl group, a C6-C6 alkyl group, a C7-C6 alkyl group, a C8-C6 alkyl group, a C9-C6 alkyl group, a C10-C6 alkyl group, a C11-C6 alkyl group, a C12-C6 alkyl group, a C13-C6 alkyl group, a C14-C6 alkyl group, a C15-C6 alkyl group, a C16-C6 alkyl group, a C17-C6 alkyl group, a C18-C6 alkyl group, a C19 ... 20 Heterocyclic group, or C3-C 20aryl groups, which are typically C1-C6 alkyl groups), unsubstituted C1-C6 alkoxy, and substituted C1-C6 alkoxy, where the substituted alkyl or alkoxy is selected from ether, amino, mono- or di-substituted amino, cyclic C1-C5 alkylamino, imidazolyl, C1-C6 alkylpiperazinyl, morpholino, thiol, thioether, tetrazole, carboxylic acid, ester, amide, mono- or di-substituted amide, N-linked amide (-NR-C(=O)-R), and substituted with one or more groups selected from N-linked sulfonamido (-NR-S(=O)2-R), sulfoxy (-S(=O)2OH), sulfonate (S(=O)2OR), sulfonyl (S(=O)2R), sulfoxy (S(=O)OH), sulfinate (S(=O)OR), sulfinyl (S(=O)R), phosphonooxy (-OP(=O)(OH)2), phosphate (OP(=O)(OR)2), and sulfonamido (-S(=O)2-NR2), wherein (each) R is a C1-C6 alkyl group, a C3-C6 alkyl group, a C6-C6 alkyl group, a C8-C6 alkyl group, a C9-C6 alkyl group, a C10-C6 alkyl group, a C11-C6 alkyl group, a C12-C6 alkyl group, a C13-C6 alkyl group, a C14-C6 alkyl group, a C15-C6 alkyl group, a C16-C6 alkyl group, a C17-C6 alkyl group, a C18-C6 alkyl group, a C19-C6 alkyl group, a C20-C6 alkyl group, a C21-C6 alkyl group, a C22-C6 alkyl group, a C23-C6 alkyl group, a C24-C6 alkyl group, a C25-C6 alkyl group, a C26-C6 alkyl group, a C25-C6 alkyl group, a C26-C6 alkyl group, a C27-C6 alkyl group, a C28-C6 alkyl group, a C29 ... 20 Heterocyclic group, or C3-C 20 aryl groups.

[0057] The trigger region of the compounds of formula (I) is generally fused to a second ring (Y as shown). 2 and Y 3 and a bicyclic moiety containing an aromatic ring (containing a Y 1 , Y 4 and Y 5 moieties, which may be aromatic or non-aromatic.

[0058] Without being bound by theory, the activity of the compounds of formula (I) as substrates for hydroxylation, for example by CYP1B1, is due to the presence of Z 2 Or Z 4 is hydrogen, or Y 1 -Z 1 This is achieved in part by the structure of the trigger moiety, which is susceptible to hydroxylation when Z is CH; 2 and Z 4 and the carbon atom to which Y is bonded1 (Y 1 Hydroxylation is believed to occur at one of the three carbon atoms of the SU025-04 (where 1 is the carbon atom). As shown in Figure 2, when any of these positions in a representative compound of the present invention, designated SU025-04, undergoes hydroxylation, the compound spontaneously collapses by either a 1,4-, 1,6-, or 1,8-elimination process, depending on which of these positions the hydroxylation occurs.

[0059] From the structure of the compound of formula (I), Z 2 and Z 4 The carbon atom to which is bonded is Y 1 In order to conjugate to the linker moiety via 6 -Y 4 -Y 5 It is noted that this can occur regardless of the nature of the region. Thus, as discussed below, a broad nature of this region of the compounds of formula (I) can be tolerated. Also, the extension of the region of conjugation can occur, particularly in the case of the conjugate X described herein. 1 This is achieved using parts.

[0060] In the compound of formula (I), Y 1 , Y 2 and Y 3 Each atom represented by may independently be a carbon atom or a nitrogen atom. When the atom is a nitrogen atom, each of the substituents (respectively Z 1 , Z 3 or Z 5 In certain embodiments of the present invention, Y 2 or Y 3 is a carbon atom. In certain embodiments of the present invention, Y 2 and Y 3 are both carbon atoms. 2 Or Y 3 is a carbon atom or Y 2 and Y 3 is a carbon atom or Y 2 MoY 3According to an embodiment where no carbon atom is present, Y 1 may be a carbon atom.

[0061] substituent Z 1 , Z 2 and Z 4 may generally be as set forth in claim 1. However, at least one of these moieties is a hydrogen atom to allow for a site for hydroxylation of the compound. In some embodiments of the invention, Z 2 or Z 4 In other embodiments, any one of Z 2 and Z 4 is hydrogen. 2 Or Z 4 is a hydrogen atom or Z 2 and Z 4 are both hydrogen atoms, or Z 2 MoZ 4 In an embodiment where neither Z 1 may be hydrogen. In certain embodiments of the present invention, Z 1 , Z 2 and Z 4 are hydrogen atoms.

[0062] Adjacent substituents on the aromatic ring (i.e., Z 2 Or Z 4 , or Z 3 Or Z 5 ) and Z 3 or Z 4 Any of these substituents, together with the atoms of the aromatic ring to which they are attached, may form an aromatic ring fused with the rest of the compound. 2 and Z 3 is Z 2 The carbon atom to which Y is bonded 2 and may be taken together to form an aromatic ring. 4 , Z 5 , Z 4 The carbon atom to which Y is bonded 3 may be joined together to form an aromatic ring.

[0063] In certain embodiments of the present invention, the substituent Z 2 and Z 3 , Z 3 and Z 4 , and Z 4 and Z 5 Of the pairs of Y, none or only two pairs form a fused aromatic ring together. 2 and Y 3 There is no aromatic ring fused to an aromatic ring containing

[0064] In particular, the substituent Z 3 and Z 5 is typically an aromatic ring fused to the rest of the compound of formula (I). When this is the case, i.e., when these moieties are individual substituents, Z 3 can be alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl, nitro, and cyano; Z 5 may be alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, carboxy, formyl, nitro, and cyano. 3 can be alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl, nitro, and cyano.

[0065] In certain embodiments of the present invention, Z 3 and Z 5 is an individual substituent other than a hydrogen atom. Z 3 and Z 5 are the same substituents or not, Z 3 and Z 5 is, according to certain embodiments of the present invention, an electron donating group such as alkoxy, alkylthioxy, aryloxy, arylthioxy, etc. In certain embodiments of the present invention, Z 3 or Z 5 and both are amino or alkoxy, e.g., C1-C6 alkoxy. Examples of such alkoxy groups include methoxy, ethoxy, isopropoxy, n-propoxy, etc. In certain embodiments of the invention, Z 3 Or Z 5 , or Z 3 and Z 5 is methoxy. In certain embodiments of the invention, Z 3 and Z 5 are the same and are any of the substituents or groups of substituents described immediately above. As noted above, compounds of formula (I) can be prepared by 6 -Y 4 -Y 5 The structure can vary significantly at positions including Y 4 is Z 6 The substituent may be oxygen, sulfur, sulfoxide, or sulfone if it is absent (p=0), nitrogen (where p=0 or 1), or carbon atom if p=1 or 2. In certain embodiments of the invention, p=0 and Y 4 is oxygen, sulfur, sulfone, or sulfoxide. In certain embodiments of the invention, p=0 and Y 4 is oxygen or sulfur. In certain embodiments of the invention, p=0 and Y 4 is oxygen.

[0066] -Y 5 - is (i) a single bond (in this case, Y 5 Since there is no trigger part, Y 2 - and Y3 - is condensed with a 5-membered ring, or (ii) the double bond = is Y 4 Thus, in these embodiments of the invention, the trigger moiety is comprised of two fused aromatic rings, and one of skill in the art would recognize -Y as a ═CH—. 5 - is =CH-, then Y 4 It will be seen that -Y is either a nitrogen atom and p=0, or a carbon atom and p=1. 5 - may be (iii) -CH2- or -CH2CH2-, in which case the trigger moiety is Y 2 and Y 3 In certain embodiments of the present invention, the bicyclic ring system includes a 6- or 7-membered ring fused to an aromatic 6-membered ring substituted with -Y 5 - The only or one or more hydrogens or hydrogen atoms specified in options (ii) and (iii) are Z 11 In certain embodiments of the invention, Z 11 In certain embodiments of the present invention, -Y 5 - is a single bond, for example, p=0 and Y 4 is oxygen, sulfur, sulfone, or sulfoxide, p=0 and Y 4 is oxygen or sulfur, especially p=0 and Y 4 is oxygen.

[0067] Here, the linker moiety CH(Z 7 )-X 1 -X 2 This article describes:

[0068] Z 7 is hydrogen or an alkyl or aryl group, In certain embodiments of the invention, the aryl group is unsubstituted. 7 or each Z 7 is an alkyl group, for example, an unsubstituted alkyl group such as an unsubstituted C1-C6 alkyl group. Z7 Exemplary moieties include methyl and ethyl. In certain embodiments of the invention, Z 7 = hydrogen, -CH(Z 7 )- is methylene. In another embodiment, one or each Z 7 The moiety is a substituted alkyl group, e.g., a substituted methyl or ethyl group. Examples of such embodiments include amino-substituted alkyl groups, e.g., morpholino or piperidinyl alkyl groups, or other groups that enhance aqueous solubility. Alternatively, only one, each, or at least one Z 7 may be an optionally substituted heteroaryl moiety such as pyridyl.

[0069] X 1 can be a variety of linker atoms or divalent linker moieties, for example, X 1 may be oxygen, sulfur, sulfonamide, or sulfonate ester. 1 may be ethane-1,2-diylbis(methylcarbamate) or a conjugated alkene methyloxy moiety.

[0070] A conjugated alkene methyloxy moiety is a group having the formula (=CH-CH) q ═CH—CH—O—, where q is an integer from 0 to 6, e.g., 0 to 3, e.g., 0 or 1. Those skilled in the art will recognize that the oxygen atom shown in the alkene methyloxy moiety may be substituted with a sulfur atom (SO—O or SO—NZ). 10 moiety), thereby resulting in the conjugated alkene methylsulfonate or conjugated alkene methylsulfonamide moieties enumerated above, and the oxygen or sulfur atom, or the sulfonate (SO2-O and SO2-NZ) of the sulfonamide moiety. 10 ) but X 2 , or, if absent, bound to an effector.

[0071] According to a particular embodiment of the present invention, X 1 is oxygen or sulfur. In many embodiments of the present invention, X 1is oxygen.

[0072] X 2 does not exist or is X 1 and the effector moiety.

[0073] X 2 may be composed of the various moieties described herein or may be absent. In certain embodiments of the invention, X 2 does not exist or X 1 -X 2 -Effector,

[0074] [ka] It becomes one of the two.

[0075] For example, X 2 is arylene-CH(Z 7 )X 3 Part (hereinafter -ArCH(Z 7 )X 3 -moiety) and / or amide moiety. When present, -Ar-CH(Z 7 )X 3 The - moiety may contain one or two amide or thioamide groups (C(Z 9 When flanked by one amide or thioamide group, the group is -Ar-CH(Z 7 )X 3 Partial X 1 It may be located directly between the moiety and the aromatic ring (where n=1), or 3 and the effector moiety (where m=1). Alternatively, amide or thioamide groups may be present at both or neither of these positions. In certain embodiments of the invention, n=0 and m=1. X 2 -Ar-CH(Z 7 )X 3-moiety, whether or not it is flanked by one or two amide or thioamide moieties, is attached to the aromatic ring directly or indirectly via an amide or thioamide moiety. 1 The moiety can be located in two positions on the aromatic ring: -Ar-CH(Z 7 )X 3 CH(Z 7 )X 3 The Ar-CH(Z) moiety may be attached at either the ortho or para position. 7 )X 3 -X containing part 2 Strategic placement of these attachment points on the aromatic rings of the X moiety allows for 1,4-, 1,6-, or 1,8-elimination of the effector molecule. 2 In certain embodiments of the present invention, the arylene groups present may be heteroaromatic, that is, one or two or more atoms Y 6 It is understood that Y may be a nitrogen atom and the remaining atoms may be carbon atoms. An example of such a heteroarylene moiety is a heteroaryl group having one Y 6 is a pyridylene in which each Y 6 is a carbon atom, if present.

[0076] X 2 When an arylene group is present in the moiety, the arylene group may have a substituent Z at any of four positions (not connecting the arylene group to the effector and trigger ends of the compound of formula (I)) that may be independently selected as described in claim 1. 8 may be substituted as indicated by

[0077] X 2 is one or more amide or thioamide moieties -CH(Z 9 ) NH, usually (each) Z, if present 9 is oxygen, thereby forming one or more amino acids It becomes the do part, but two or more Z 9 If there is, then for each Z 9 may be independently selected.

[0078] Finally, the effector moiety of a compound of Formula (I) is a moiety that has a desired effect of interest on a cell, typically a cell in which CYP1B1 is expressed. The effector moiety can be any molecule that has a pharmacological, diagnostic, or screening function when released from a compound of Formula (I). By pharmacological or diagnostic function, we mean that the effector moiety, when released, has an appreciable pharmacological or diagnostic effect on the cell in which it is released.

[0079] Those skilled in the art will appreciate that the effector moiety (effector) in the compound of formula (I) when released will form X 1 part of, for example, an oxygen or sulfur atom, or X 2 Part of, for example, X 3 It is understood, however, that the distinction between the trigger, linker, and effector moieties of the compounds of formula (I) is made merely to aid in the description of the compounds of the present invention, and that those skilled in the art will recognize that the effector moiety in the compounds of the present invention will constitute the majority of the effector molecule that is liberated upon hydroxylation-induced decomposition, but that one or some of the atoms in the liberated effector molecule may be present at the X 1 , X 1 or X 2 It will be appreciated that the effector molecule may be provided by any atom described herein as being part of, or indeed elsewhere (e.g., a hydrogen atom stripped from a water molecule). Alternatively, the effector molecule may be attached to the remainder of the compound of formula (I), for example, via a keto or formyl group.

[0080] When an effector molecule has a pharmacological effect, it may be any chemical substance that has, for example, a cytostatic or cytotoxic effect on the cells (e.g., CYP1B1-expressing cells) that it acts to liberate. As is known, a cytotoxic molecule is a molecule that is toxic to cells, whereas a cytostatic is a molecule that inhibits cell growth and / or replication.

[0081] In certain embodiments of the invention, the effector molecule is a cytotoxic agent. Examples of cytotoxic agents that can be used include, but are not limited to, alkylating agents, antimitotic agents, antifolates, antimetabolites, DNA damaging agents, and enzyme inhibitors (e.g., tyrosine kinase inhibitors). Specific examples of possible cytotoxic drug moieties include, but are not limited to, bis(haloethyl)phosphoramidates, cyclophosphamide, gemcitabine, cytarabine, 5-fluorouracil, 6-mercaptopurine, camptothecin, topotecan, doxorubicin, and daunorubicin. duocarmycin, etoposide, duetoposide, combretastatin A-4, vinblastine, vincristine, AQ4N, hydroxyurea, maytansine, enediyene, epothilone, taxane, bleomycin, calicheamicin, colchicine, dacarbazine, dactinomycin, epirubicin, epirubicin derivatives, fludarabine, hydroxyureapentatostatin, methotraxate, mitomycin, mitoxantrone, carboplatin, cisplatin, taxel, 6-thioguanine, vinca alkaloids, platinum coordination complexes, anthracenediones, substituted ureas, methylhydrazine derivatives, and nitrogen mustards.

[0082] In certain embodiments of the invention, the effector molecule is a phosphoramide mustard, i.e., a phosphate derivative in which one or two, usually two, of the hydroxyl groups of the phosphate are replaced with a nitrogen mustard or its analogue containing oxygen or sulfur, and optionally P(=O) is replaced with P(=S). Nitrogen mustards, as defined herein, are mustards in which the sulfur atom is replaced with a nitrogen atom and, optionally, one chloroethyl group is replaced with a nitrogen atom. They are defined as nonspecific alkylated amines structurally related to mustard gas (1,5-dichloro-3-thiapentane) in which the (Chlorethyl) side chain has been replaced with a hydrogen atom or an alkyl group, or one or both terminal chloro substituents have been replaced with a leaving group such as bromo, iodo, or mesylate (-OSO2CH3). Examples of phosphoramide mustards include the compounds known as phosphoramide mustard (PM) and isophosphoramide mustard (IPM).

[0083] [ka] It should be noted that the compound PM may therefore be considered a derivative of phosphoric acid in which one of the hydroxyl groups has been replaced with a nitrogen mustard (the other hydroxyl group has been replaced with an amino group (NH)), and is therefore an example, as well as a name for a class of compounds known as phosphoramide mustards.

[0084] In embodiments of the invention where the effector molecule is a phosphoramide mustard in which one or two, usually two, of the hydroxyl groups of the phosphate derivative have been replaced with an oxygen- or sulfur-containing nitrogen mustard analog, phosphoramide mustard refers to an analog of phosphoramide mustard in which the nitrogen mustard has been replaced with an analog in which one chloroethyl arm is absent and the nitrogen atom has been replaced with a sulfur or oxygen atom.

[0085] In certain embodiments of the invention, the effector molecule is linked to the remainder of the compound via an oxygen or sulfur atom, and the effector is of formula (II):

[0086] [ka] (In the formula, Z 12 is oxygen or sulfur, each X 4 are independently oxygen, sulfur, or NZ13 and each -Z 13 are independently -(CH2)2-Z 14 , -alkyl, or -hydrogen; each Z 14 are independently chloro, bromo, iodo, or mesylate).

[0087] In certain embodiments of the present invention, Z 12 is oxygen. In these and other detailed embodiments, each X 4 In these and other detailed embodiments, each X 4 is NZ 13 In these and other detailed embodiments, each Z 13 is hydrogen. In these and other detailed embodiments of the present invention, each Z 14 are the same and / or are bromo or chloro. In certain embodiments of the invention, each occurrence of Z 14 (2, 3, or 4 Z 14 The aryl group (which can be a moiety) is bromo.

[0088] Alternatively, the effector molecule may serve a diagnostic function, for example, allowing for the identification or better understanding of the nature of tumors in which CYP1B1 is expressed. One example of a class of effector molecules that are diagnostic molecules are fluorophore molecules, which may be useful in the diagnosis of cancerous cells. Examples of fluorophore compounds include coumarin, resorufin, fluorescein, and rhodamine, and indeed the feasibility of the present invention has been demonstrated through several experiments carried out with compounds of the present invention that contain coumarin as an effector molecule (see the Examples section below).

[0089] It will be appreciated that compounds of formula (I) in which the effector performs a diagnostic function may thus be useful in diagnostic methods, and such methods constitute another aspect of the present invention. Accordingly, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, for use in a method for diagnosing a proliferative condition, for example a proliferative condition selected from pre-malignant or malignant cell proliferation, cancer, leukemia, psoriasis, bone disease, fibroproliferative disorders, or atherosclerosis, for example, cancer of the bladder, brain, breast, colon, head and neck, kidney, lung, liver, ovary, prostate, and skin, said method comprising administering to a subject suffering from or suspected of suffering from such a proliferative condition an amount of a compound of formula (I), or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, and monitoring the distribution of free effector molecules in the subject, thereby allowing a diagnosis to be made.

[0090] Alternatively, the effector may serve as a screen, e.g., as part of a model prodrug library collection, to identify trigger-linker combinations that are cleaved upon activation by CYP1B1 and its allelic variants. One example of a class of effector molecules is a fluorophore molecule. Examples of fluorophore compounds include the well-known coumarin, resorufin, fluorescein, and rhodamine. Indeed, the feasibility of the present invention has been demonstrated through several experiments performed with compounds of the present invention containing coumarin as the effector molecule (see Example 1 in the following section). That is, it will be appreciated that compounds of formula (I) in which the effector serves as a screen can be useful in identifying trigger-linker combinations for the design and synthesis of prodrugs activated by CYP1B1, and such methods constitute another aspect of the present invention.

[0091] Thus, compounds of formula (I) in which the effector serves a screening function as part of a model prodrug library collection can be used in conjunction with cytochrome P450 substrate prediction models to predict the activity of, for example, CYP1B1, and CYP1B1 * It is convincing that this can lead to the design and synthesis of prodrugs that are selective for its allelic variants, such as CYP1B1. For clarity, combining a model prodrug library with a substrate prediction model ties substrate specificity to the fundamental design principle of prodrug activation and cleavage by CYP1B1. It is further convincing, therefore, that compounds of formula (I), in which the effector performs a screening function, can be used in combination with a cytochrome P450 substrate prediction model to guide the design and synthesis of prodrugs that are not activated by normal tissue cytochrome P450s, e.g., CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. An example of a substrate prediction model is the Gaussian Kernel-weighted k-NN algorithm, which is based on Tanimoto's similarity search with descriptors such as, but not limited to, extended connectivity fingerprints. Cytochrome P450 substrate prediction models for prodrug design are based on the design of structurally diverse compound cores. The bioactivity database obtained from the cytochrome P450 HTS of the CYP1B1 substrate prediction model can be constructed in the bioactivity database obtained from the CYP1B1 substrate prediction model of ...

[0092] Alternatively, the effector may serve as a screening function as part of a model prodrug library collection to identify trigger-linker combinations that are cleaved upon activation by CYP1B1 and / or other cytochrome P450s and their allelic variants that are overexpressed in cancer and other proliferative conditions. One class of effector molecules is a fluorophore molecule. Examples of fluorophore compounds include coumarin, resorufin, fluorescein, and rhodamine. Examples of cytochrome P450s other than CYP1B1 that are overexpressed in cancer include CYP2A / 2B, CYP2F1, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3A5, CYP3A7, CYP4Z1, CYP26A1, and CYP51.

[0093] Therefore, it is convincing that the compounds of formula (I), in which the effector serves as a screening function as part of a model prodrug library collection, can be used in combination with a cytochrome P450 substrate prediction model to guide the design and synthesis of prodrugs that are selective for CYP1B1 and / or other cytochrome P450s and their allelic variants that are overexpressed in cancer and other proliferative conditions.One example of a class of effector molecules is a fluorophore molecule.Examples of fluorophore compounds include coumarin, resorufin, fluorescein, and rhodamine.Examples of cytochrome P450s other than CYP1B1 that are overexpressed in cancer include CYP2A / 2B, CYP2F1, CYP2R1, CYP2S1, CYP2U1, CYP2W1, CYP3A5, CYP3A7, CYP4Z1, CYP26A1, and CYP51. An example of a substrate prediction model is the Gaussian Kernel weighted k-NN algorithm based on Tanimoto's similarity search with descriptors such as, but not limited to, extended connectivity fingerprints. Cytochrome P450 substrate prediction models for prodrug design can be built on bioactivity databases obtained from cytochrome P450 HTS of structurally diverse compound collections.

[0094] According to aspects and embodiments of the invention in which the effector performs a screening function, e.g., according to the seventh aspect of the invention, the set of compounds will typically comprise a plurality of compounds, e.g., at least 10, e.g., at least 20 compounds. In certain embodiments, the set may comprise 100, 1000, 10,000, or even up to 100,000 compounds. Such classes of sets of compounds, i.e., classes of compounds according to the first aspect of the invention in which the effector is a fluorophore, as well as other classes of compounds in which the effector is not so limited and / or in which the compound may be a pharmaceutically acceptable salt, ester, amide, or solvate, constitute further aspects of the invention.

[0095] According to an embodiment of the seventh aspect of the invention, in the case where the compound liberates a fluorophore in step (a) but not, or to a much lesser extent, in step (b), this means that the P450 enzyme will typically liberate at least 10 times, such as at least 20 times, more of said fluorophore in step (a) than in step (b).

[0096] For example, screening according to an embodiment of the seventh aspect of the invention may identify hits, e.g., and typically, step (a) liberates a fluorophore, but step (b) liberates a fluorophore. If a compound is obtained that is not liberated, or is liberated to a much lesser extent, the method of the seventh aspect of the invention may further comprise the steps of: (d) generating a model of a compound that is structurally identical to the compound identified in step (c), except that the fluorophore is replaced with a molecule that has pharmacological functionality that binds to the active site of the cytochrome P450 enzyme; (e) synthesizing the compound modeled in step (d) that is predicted to be a substrate for said cytochrome P450 enzyme; In some cases,

[0097] Alternatively, these steps ((d) and (e)) may be carried out separately from the essential steps of the seventh aspect of the present invention (i.e., (a) to (c)), thereby constituting yet another embodiment of the present invention.

[0098] Typically, the cytochrome P450 enzyme is selected from the group consisting of CYP1B1, CYP2S1, CYP2W1, CYP4Z1, and allelic variants thereof, such as CYP1B1 and allelic variants thereof, such as CYP1B1.

[0099] One aspect of the present invention is the use of early in vitro passage (<20 passages) human primary tumor cell lines derived from resected cancer specimens. The primary head and neck squamous cell carcinoma cell line UT-SCC, described in Examples 4 and 5 below, constitutively expresses CYP1B1 at the mRNA and protein levels. It can be subcutaneously implanted into immunodeficient mice (e.g., nude mice or severe combined immunodeficiency SCID mice) with high engraftment rates to generate human primary tumor xenografts whose constitutive expression of cytochrome P450 protein matches that of the original tumor in the patient. Therefore, by maintaining cytochrome P450 mRNA / protein expression similar to that of the original tumor in the patient, these human primary tumor xenograft models can be used to evaluate the efficacy of compounds of the present invention, whose effector moieties are pharmacologically active agents, in cancer therapy. Furthermore, in clinical settings, these human primary tumor xenograft models can be used to assess whether the response of compounds of the present invention, whose effector moieties are pharmacologically active agents, correlates with clinical response and outcome, demonstrating their usefulness for personalized chemotherapy. Human primary tumor models can also be used to compare the efficacy of compounds of claim 1, where the effector moiety is a pharmacologically active agent, with standard chemotherapy treatment regimens, and thus identify the most effective treatment regimen for compounds of claim 1, alone or in combination with other chemotherapy drugs.

[0100] Furthermore, as part of the present invention, human primary tumor xenografts can be obtained by directly implanting tumor tissues excised directly from patients into, for example, nude mice, SCID mice, and non-obese diabetic / severe combined immunodeficiency (NOD / SCID) mice subcutaneously. Primary human primary tumor xenografts of a range of different cancers can be generated that retain the histological and genetic characteristics of the original tumor and thus constitutively express CYP1B1 mRNA / protein at levels similar to those of the original tumor. Therefore, these human primary tumor xenograft models can be used to evaluate the efficacy of compounds of the present invention, whose effector moiety is a pharmacologically active agent, in cancer therapy by maintaining CYP1B1 mRNA / protein expression similar to that of the original patient's tumor. Furthermore, in clinical settings, these human primary tumor xenograft models can be used to check whether the response of the compound of claim 1, whose effector moiety is a pharmacologically active agent, correlates with clinical response and outcome, demonstrating its usefulness for personalized chemotherapy. Human primary tumor models can also be used to compare the efficacy of compounds of the invention, where the effector moiety is a pharmacologically active agent, with standard chemotherapy treatment regimens, and thus identify the most effective treatment regimens for compounds of the invention alone or in combination with other chemotherapy agents.

[0101] In accordance with an eighth aspect of the invention, where a cancer results from the implantation of cells from an early passage cell line derived from tissue taken directly from a tumor or cancer which expresses the cytochrome P450 enzyme at a level similar to that of the tumor or cancer from which it originated, a level may be considered to be similar if it is within 10%, for example within 5%, of that from the tumor or cancer from which it originated.

[0102] For use according to the invention, the compounds described herein or physiologically acceptable salts, solvates, esters, or amides thereof may be provided as pharmaceutical formulations comprising the compounds or physiologically acceptable salts, esters, amides, or other physiologically functional derivatives thereof together with one or more pharmaceutically acceptable carriers therefor, and optionally other therapeutic and / or prophylactic ingredients. Any carrier(s) is acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0103] Examples of physiologically acceptable salts of the compounds according to the invention include acid addition salts formed with organic carboxylic acids such as acetic acid, lactic acid, tartaric acid, maleic acid, citric acid, pyruvic acid, oxalic acid, fumaric acid, oxaloacetic acid, isethionic acid, lactobionic acid, succinic acid, etc.; organic sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.; and inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, etc.

[0104] The determination of physiologically acceptable esters or amides, particularly esters, is well within the skill of one in the art.

[0105] It may also be convenient or desirable to prepare, purify, and / or handle corresponding solvates of the compounds described herein that can be used in any one of the uses / methods described. The term solvate is used herein to refer to a complex of solute and solvent, such as a compound or a salt of a compound. If the solvent is water, the solvate can be referred to as a hydrate, e.g., a monohydrate, a dihydrate, a trihydrate, etc., depending on the number of water molecules present per substrate molecule.

[0106] It will be understood that the compounds of the present invention may exist in various stereoisomeric forms, and the compounds of the present invention as defined above encompass all stereoisomeric forms and mixtures thereof, including enantiomers and racemic mixtures. The present invention includes within its scope the use of any such stereoisomeric form or mixture of stereoisomers, including the individual enantiomers of the compounds of formula (I) or (II) as well as fully or partially racemic mixtures of such enantiomers.

[0107] Those skilled in the art will also understand that anti-cancer prodrugs, such as those described herein, can be targeted to specific tumors by attaching a tumor-targeting moiety (e.g., a tumor-targeting peptide), such as a tumor-targeting peptide, e.g., a small peptide identified through the development of a peptide library by phage display. Such peptides or other moieties can help target conjugates containing them to specific cancers, particularly solid tumors. Thus, the provision of such conjugates, i.e., compounds of the invention linked to tumor-targeting moieties, constitutes another aspect of the present invention, as do the compositions, uses, and methods described herein that include or encompass the use of such conjugates.

[0108] The compounds of the present invention can be prepared using reagents and techniques readily available in the art and / or the exemplary methods described below. The compounds of the present invention are cytotoxic in cells that express the CYP1B1 enzyme, but not in normal cells that do not express CYP1B1. The compounds of the present invention have been found to be substantially non-toxic in cells that express the CYP1A1 enzyme. The compounds of the present invention may also be cytotoxic in cells that express the CYP1A1 enzyme. Thus, in effect, the compounds of the present invention are non-toxic prodrugs that are converted (usually by CYP1B1) to cytotoxic drugs.

[0109] The compounds of the present invention are cytotoxic IC 50 Suitably the value is as defined below or less than 10 μM, advantageously less than 5 μM, for example less than 1.0 μM or less than 0.5 μM.

[0110] In some embodiments, the cytotoxicity of compounds of the present invention can be measured by incubating various serial dilutions of the compound with cells engineered to express CYP1B1. In a suitable example, the cells can be Chinese hamster ovary (CHO) cells, which may contain recombinant CYP1B1 and cytochrome P-450 reductase (CPR). When coexpressed with human P-450 reductase, high levels of functional enzyme can be achieved using dihydrofolate reductase (DHFR) gene amplification. Typically, engineered cells are incubated with the compound and, after a suitable time (e.g., 96 hours), further incubated (e.g., 1.5 hours) with an appropriate assay reagent to indicate the number of viable cells in the culture. A suitable assay reagent is MTS (see below), which is bioreduced by cells to a formazan product that is soluble in tissue culture medium. The absorbance of the formazan product can be measured directly at 510 nm, and the quantitative formazan production, as measured by the amount of light absorbance at 490 nm or 510 nm, is directly proportional to the number of viable cells in culture. The IC of the compounds according to the present invention 50 Detailed methods for determining values ​​are described in Example 3 below.

[0111] In comparison, the IC of the compounds of the present invention 50 Values ​​can also be measured in cells that do not contain CYP1B1 (e.g., Chinese hamster ovary cells), e.g., wild-type CHO cells. Compounds of the invention may, in appropriate instances, have a selectivity fold over CYP1B1-expressing cells of at least 200, where "selectivity fold" refers to the ratio of the IC of a given compound in CYP1-non-expressing cells to the IC of a given compound in CYP1-non-expressing cells. 50 values ​​and IC of the same compounds in CYP1B1-expressing cells 50 It is defined as the ratio of the values.

[0112] In some embodiments, the cytotoxicity of compounds of the invention can also be measured by incubating various serial dilutions of the compounds with primary head and neck tumor cells derived from patients with head and neck squamous cell carcinoma, as described in Example 4.

[0113] In some embodiments, the in vivo efficacy of compounds of the invention can also be determined by implanting primary head and neck squamous cell carcinoma tumor cells constitutively expressing CYP1B1 subcutaneously into the flanks of nude mice to generate a human primary tumor xenograft model and measuring the effect of prodrug treatment on tumor growth, as described in Example 5.

[0114] As such, the present invention also encompasses the use of one or more of the compounds of the present invention, including the pharmaceutically acceptable esters, amides, salts, solvates, and prodrugs described above, for use in therapeutic treatment of the human or animal body, particularly in the treatment or prevention of proliferative conditions, such as proliferative disorders and diseases, in humans and non-human animals, including, in certain embodiments of the present invention, proliferative conditions characterized by cells expressing CYP1B1. More particularly, the present invention encompasses the use of one or more of the compounds of the present invention for treating cancers characterized by CYP1B1 expression, in certain embodiments of the present invention.

[0115] By "proliferative condition" herein is meant a disease or disorder characterized by unwanted or uncontrolled cell proliferation of unwanted, excessive or abnormal cells, such as neoplastic or hyperplastic growth, whether in vitro or in vivo. Examples of conditions are premalignant and malignant cell proliferation, including malignant neoplasms and tumors, cancer, leukemia, psoriasis, bone diseases, fibroproliferative disorders (eg, of connective tissue), and atherosclerosis.

[0116] The proliferative condition may be characterized in certain embodiments of the invention by cells expressing CYP1B1.

[0117] The proliferative condition may be selected from cancer of the bladder, brain, breast, colon, head and neck, kidney, lung, liver, ovary, prostate, and skin, hi some embodiments, the proliferative condition may comprise a solid tumor.

[0118] "Treatment," as used herein, means treatment by therapy that achieves some desired therapeutic effect on a proliferative condition, whether the subject is a human or (e.g., in veterinary applications) a non-human animal, such as slowing the rate of progression, preventing the progression of the disorder, including halting the rate of progression, ameliorating the disorder, or curing the condition. Treatment as a preventative measure is also included. As used herein, reference to prevention or prophylaxis does not imply or require complete prevention of the condition; instead, the onset of the condition may be alleviated or delayed by prevention or prophylaxis in accordance with the invention. "Therapeutically effective amount," as used herein, means that amount of one or more compounds of the invention, or a pharmaceutical formulation comprising said compound(s), that is effective to produce such a therapeutic effect, commensurate with a reasonable benefit / risk ratio.

[0119] Therefore, the compounds of the present invention may be used as anti-cancer agents. The term "anti-cancer agent" as used herein refers to a compound that treats cancer (i.e., a compound that is useful in cancer treatment). The anti-cancer effect of the compounds of the present invention may be caused by one or more mechanisms, including regulating cell proliferation, inhibiting angiogenesis, preventing metastasis, preventing invasion, or promoting apoptosis.

[0120] It is understood that the appropriate dosage of the compounds of the present invention may vary from patient to patient. Determining the optimal dosage generally requires balancing the level of therapeutic benefit against any risk or adverse side effects of the treatment of the present invention. The selected dosage level will depend on various factors, including the activity of the particular compound, the route of administration, the timing of administration, the excretion rate of the compound, the duration of treatment, other drugs, compounds, or materials used in combination, and the patient's age, sex, weight, condition, general health, and medical history. The amount and route of administration of the compound(s) are ultimately left to the discretion of the physician, but the dosage will generally achieve a local concentration at the site of action that will produce the desired effect.

[0121] In vivo administration can be carried out in one dose, continuously or intermittently throughout the course of treatment.The method of determining the most effective means and dosage of administration is well known to those skilled in the art, and it varies depending on the formulation used in treatment, the purpose of treatment, the target cell to be treated and the subject to be treated.Single administration or multiple administration can be carried out with the dosage level and pattern determined by the treating physician.

[0122] Pharmaceutical formulations include those suitable for oral, topical (including transdermal, buccal, and sublingual), rectal, or parenteral (including subcutaneous, intradermal, intramuscular, and intravenous), nasal, and pulmonary administration, for example, by inhalation. The formulations may, where appropriate, be conveniently provided in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. Methods usually include combining the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0123] Pharmaceutical formulations suitable for oral administration, in which the carrier is a solid, are most preferably presented as unit dose formulations such as boluses, capsules, or tablets, each containing a predetermined amount of the active compound. Tablets can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing the active compound in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surfactant, or dispersing agent, in a suitable machine. Molded tablets can be prepared by molding the active compound with an inert liquid diluent. Tablets may be optionally coated or, if uncoated, may be optionally scored. Capsules can be prepared by filling the active compound, alone or in admixture with one or more accessory ingredients, into capsule shells, which are then sealed in the usual manner. Cachets are similar to capsules, in that the active compound, along with any accessory ingredient(s), is sealed in a rice paper envelope. The active compound may be formulated as dispersible granules, which can be suspended in water or sprinkled on food before administration. The granules can be packaged, for example, in sachets. Formulations suitable for oral administration, in which the carrier is a liquid, can be provided as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.

[0124] Formulations for oral administration include sustained-release dosage forms, such as tablets in which the active compound is formulated in a suitable sustained-release matrix or coated with a suitable sustained-release film. Such formulations may be particularly advantageous for prophylactic use.

[0125] The pharmaceutical preparation suitable for rectal administration, in which the carrier is solid, is most preferably provided as unit dose suppositories.Suitable carriers include cocoa butter and other materials commonly used in the art.Suppositories can be conveniently produced by mixing the active compound with softened or melted carrier(s), then cooling and shaping in molds.

[0126] Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of the active compounds dissolved or suspended in an aqueous or oily vehicle.

[0127] Injectable preparations can be adapted for bolus injection or continuous infusion.These preparations are conveniently provided in unit-dose or multi-dose containers that are sealed after the preparation is introduced until needed for use.Alternatively, active compound can be in powder form, which is reconstituted with suitable medium such as sterile water without pyrogens before use.

[0128] The active compound may be formulated as a long-acting depot preparation, which can be administered by intramuscular injection or by, for example, subcutaneous or intramuscular implantation. The depot preparation may, for example, comprise a suitable polymeric or hydrophobic material, or an ion exchange resin. Such long-acting formulations are particularly advantageous for prophylactic use.

[0129] Formulations suitable for pulmonary administration via the buccal cavity are prepared such that particles containing the active compound, preferably having a diameter in the range of 0.5 to 7 microns, are delivered into the bronchial tree of the recipient.

[0130] One possibility is that such a formulation is in the form of a finely divided powder, which may conveniently be provided in a suitable pierceable capsule, e.g., made of gelatin, for use in an inhaler device, or alternatively, it may be provided as a self-propelling formulation comprising the active compound, a suitable liquid or gaseous propellant, and optionally other ingredients such as surfactants and / or solid diluents. Suitable liquid propellants include propane and chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Self-propelling formulations, in which the active compound is delivered in the form of droplets of a solution or suspension, are also suitable. Self-propelling formulations may also be used.

[0131] Such self-propelling formulations are similar to those known in the art and can be prepared by established procedures. Suitably, such formulations are provided in a container provided with a manually or automatically operated valve having the desired spray characteristics, and the valve is advantageously of the metered type, delivering a fixed volume, e.g., 25-100 microliters, with each actuation of the valve.

[0132] Alternatively, the active compound may be formulated into a solution or suspension for use in an atomizer or nebulizer, which uses accelerated air or ultrasonic agitation to produce a fine mist of droplets for inhalation.

[0133] Formulations suitable for nasal administration include those generally similar to those described above for pulmonary administration. When delivered, such formulations should desirably have a particle size in the range of 10-200 microns to allow retention in the nasal cavity; this can be achieved by using powders of appropriate particle size or by selecting an appropriate valve, as appropriate. Other suitable formulations include coarse powders in the range of 20-500 microns in particle size, administered by rapid inhalation through the nasal passages from a container held close to the nose, and nasal sprays containing 0.2-5% w / v of the active compound in an aqueous or oily solution or suspension.

[0134] It will be understood that the above-described pharmaceutical formulations may contain, in addition to the aforementioned carrier components, one or more suitable additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), and the like, as well as substances included to render the formulation isotonic with the blood of the intended recipient.

[0135] Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M, preferably 0.05 M, phosphate buffer, or 0.8% saline. Additionally, pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases.

[0136] Formulations suitable for topical administration may, for example, be presented as gels, creams, or ointments.

[0137] It is also possible to provide a liquid or powder formulation that can be sprayed or sprinkled directly onto the area to be treated, such as a wound or ulcer, or the formulation can be sprayed or sprinkled onto a carrier such as a bandage, gauze, mesh, etc., and then applied to the area to be treated.

[0138] Therapeutic formulations for veterinary use can conveniently be in powder or liquid concentrate form. In accordance with standard veterinary formulation practice, convenient water-soluble excipients, such as lactose or sucrose, can be blended into the powder to improve its physical properties. Thus, particularly suitable powders of the invention contain 50-100% w / w, preferably 60-80% w / w, of the active ingredient(s) and 0-50% w / w, preferably 20-40% w / w, of a conventional veterinary excipient. Such powders can be added to animal feed, for example, as an intermediate premix, or diluted in the animal's drinking water.

[0139] The liquid concentrates of the present invention suitably contain the compound or a derivative or salt thereof, and may optionally include a veterinarily acceptable water-miscible solvent, such as polyethylene glycol, propylene glycol, glycerol, glycerol formal, or such solvents mixed with up to 30% v / v ethanol. The liquid concentrates can be administered to the animal's drinking water.

[0140] In general, a suitable dose of one or more compounds of the invention may range from about 1 μg to about 5000 μg / kg of subject body weight per day, for example, 1, 5, 10, 25, 50, 100, 250, 1000, 2500, or 5000 μg / kg per day. Where the compound(s) is a salt, solvate, prodrug, or the like, the amount administered may be calculated based on the parent compound, and therefore the actual weight used may be increased accordingly.

[0141] In some embodiments, one or more compounds of the present invention can be used in combination therapy, i.e., with other therapeutic agents, to treat the above-mentioned types of proliferative conditions.Examples of such other therapeutic agents include, but are not limited to, topoisomerase inhibitors, alkylating agents, antimetabolites, DNA binders, and microtubule inhibitors (tubulin targeting drugs), such as cisplatin, cyclophosphamide, doxorubicin, etoposide, irinotecan, fludarabine, 5FU, taxanes, or mitomycin C.Other therapeutic agents will be apparent to those skilled in the art.When active compound is combined with other therapies, two or more therapies can be administered individually with different administration schedules and by different routes.

[0142] The combination of the above-listed drugs with the compounds of the present invention will be at the discretion of the physician, who will select the dosage using his or her ordinary general knowledge and dosing regimens known to those skilled in the art.

[0143] When a compound of the invention is administered in combination therapy with one, two, three, four, or more, preferably one or two, preferably one, other therapeutic agents, the compounds may be administered simultaneously or sequentially. When administered sequentially, the agents may be administered closely spaced (e.g., 5 to 10 minutes apart) or more widely spaced (e.g., 1, 2, 3, 4, or more hours apart, or even longer if necessary), with the precise dosing regimen being appropriate to the nature of the therapeutic agent(s).

[0144] The compounds of the invention may also be administered in conjunction with non-chemotherapeutic treatments such as radiation therapy, photodynamic therapy, gene therapy, surgery, and controlled diets.

[0145] The invention will now be illustrated with reference to the following non-limiting examples. [Example]

[0146] Preparation of compounds General matters 1 H, 13 C, and 31 P nuclear magnetic resonance (NMR) spectra were recorded on either a Bruker Avance DPX 500 MHz or a Bruker Avance 300 MHz spectrometer in the solvents indicated. Chemical shifts are expressed in ppm. Signal splitting patterns are described as singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q), multiplet (m), or combinations thereof. Low-resolution electrospray (ES) mass spectra were recorded in methanol / water (95:5) or water. The mobile phase was either acetonitrile (1:1) + 0.1% formic acid and the data were recorded on a Bruker Microtof mass spectrometer operated in the positive ion mode. The electrospray measurements were performed on a Bruker Microtof mass spectrometer. LC-MS analysis was performed on an Agilent HPLC 1100 (Phenomenex Gemini Analysis was performed using a Bruker Microtof mass spectrometer with a 5μ C18 110Å 50×3.0 mm column (eluent: 0-20% MeOH / HO) and diode array detection followed by column chromatography. Column chromatography was performed using silica gel (230-400 mesh) or RediSep™ 4, 12, 40, or 80 g silica pre-packed columns. All starting materials were commercially available and used without further purification. All reactions were performed under anhydrous and inert conditions unless otherwise noted. [bracketed dagger

[0147] [ka] The compounds shown below, marked with a symbol, are not examples of the present invention, but are incorporated herein for a better understanding of the examples of the present invention. 1. Phosphoramidate mustard prodrugs

[0148] [ka] Synthesis of phosphoramidate prodrugs SU025-04 and SU046-04 1. Synthesis of the trigger moiety of the prodrug

[0149] [ka] 2-Bromo-3,5-dimethoxybenzaldehyde(1) 3,5-Dimethoxybenzaldehyde (12.6 g, 76 mmol) was dissolved in acetic acid (350 mL). The resulting colorless solution was cooled to 0 °C. A solution of bromine (3.9 mL) in ethanoic acid (50 mL) was added dropwise over 1 h. Upon completion of the addition, the ice bath was removed, and the resulting pale green solution was stirred at room temperature overnight. Cold water was added to the solution. The resulting white solid was collected by vacuum filtration and rinsed with water. The solid was then redissolved in EtOAc and adsorbed onto silica gel. The product was purified by flash chromatography using hexane / EtOAc (4:1) as the eluent to give 1 (12.5 g, 66%) as a white solid.

[0150] [ka] 2-Hydroxy-3,5-dimethoxybenzaldehyde(2) A three-neck round-bottom flask equipped with a stir bar, septum cap, dropping funnel, thermometer, and argon inlet was charged with morpholine (2.05 g, 24 mmol) and THF (40 mL). The flask was cooled to −50 °C in a dry ice-acetone bath, and a hexane solution of n-BuLi (1.6 M, 15 mL, 24 mmol) was added all at once. After 10 min, a THF (30 mL) solution of 1 (4.9 g, 20 mmol) was added dropwise via syringe over 4 min, and the mixture was cooled to approximately −75 °C over 20 min. A hexane solution of n-BuLi (1.6 M, 20 mL, 32 mmol) was then added dropwise over 45 min, maintaining the temperature at −75 °C. After all the n-BuLi was added, the solution was stirred for 35 min. A solution of nitrobenzene (6.90 g, 46 mmol) in 10 mL of THF was added via a dropping funnel while maintaining the temperature at −75°C. The resulting dark mixture was stirred at −75°C for 4 hours and then warmed to room temperature. It was acidified to pH 1 with 6 N HCl and stirred for 15 minutes. After dilution with brine (100 mL), the THF was removed in vacuo. The aqueous solution was extracted with diethyl ether (4 × 40 mL). The combined organic layers were extracted with 2 N NaOH (3 × 40 mL). The combined NaOH extracts were washed with diethyl ether (3 × 20 mL) and then acidified to pH 1 with concentrated HCl. The resulting mixture was extracted with CHCl (3 × 20 mL), and the combined organic extracts were washed with brine, dried (MgSO), and adsorbed onto silica gel. The product was purified by flash chromatography eluting with 1:2 EtOAc / hexanes. Pure 2 (2.0 g, 55%) was obtained as a yellow solid.

[0151] [ka] 2-(2,2-diethoxyethoxy)-3,5-dimethoxybenzaldehyde (3) To a stirred suspension of 2 (1.1 g, 6.0 mmol) and K2CO3 (1.0 g, 7.2 mmol) in DMF (100 mL) was added bromoacetaldehyde diethyl acetal (0.93 mL, 6.0 mmol) dropwise. The mixture was refluxed for 4 h. After cooling, After filtration, the precipitate was filtered off and the solvent was evaporated in vacuo. The crude residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 3 (1.2 g, 67%) as a clear oil.

[0152] [ka] 5,7-Dimethoxybenzofuran-2-carbaldehyde (4) A stirred solution of 3 (1.2 g, 4.0 mmol) in acetic acid (35 mL) was refluxed for 16 h. After cooling, the solution was evaporated to dryness. The crude product was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (2:1) to give 4 (230 mg, 28%) as a white solid.

[0153] [ka] (5,7-Dimethoxybenzofuran-2-yl)methanol (5) Compound 4 (460 mg, 2.23 mmol) was dissolved in THF (5 mL) and EtOH (1 mL). NaBH (102 mg, 2.68 mmol) was added portionwise at 0 °C with vigorous stirring. The suspension was stirred at 0 °C for 15 min and then at room temperature for 1 h. The solvent was removed in vacuo. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO). The residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (1:1) to give 5 (388 mg, 82%) as an oil.

[0154] [ka] 2. Synthesis of the effector moiety of the prodrug

[0155] [ka] N,N-bis(2-chloroethyl)phosphonamidic acid (6) To a suspension of 2-chloroethylamine hydrochloride (7.2 g, 62 mmol) in CHCl (110 mL) was added POCl (2.84 mL, 31 mmol) over 15 min with vigorous stirring at −78 °C, followed by the addition of a solution of TEA (17.5 mL, 124 mmol) in CHCl (30 mL) over 4 h. The reaction mixture was stirred at −78 °C for 1 h, then warmed to room temperature and stirred for 2 h. The resulting solid was filtered and washed with cold EtOAc. The solid was discarded. The filtrate was concentrated in vacuo to approximately 5 mL, and EtOAc (10 mL) was added. The resulting suspension was filtered and washed with EtOAc (2 × 10 mL). The solid was again discarded. The filtrate was concentrated in vacuo to dryness. The residue was then dissolved in THF (7 mL), followed by the addition of aqueous NaBr (5 g of NaBr in 100 mL of water) over 20 min at 0 °C. The mixture was then warmed to room temperature and stirred in a water bath for 15 hours. A white solid precipitated from the reaction mixture. The mixture was then kept in a freezer at -20°C for 2 hours. The crystalline solid was filtered and washed with cold water (2 x 50 mL, 0°C) and cold EtOAc (2 x 50 mL, 0°C). After drying in vacuo at room temperature overnight, product 6 (1.8 g, 26%) was obtained as a white solid.

[0156] [ka] N,N-bis(2-bromoethyl)phosphonamidic acid (7) Compound 7 was synthesized using the same method as above, and was obtained in 18% yield (1.64 g).

[0157] [ka] 3. Coupling reaction to synthesize SU025-04 and SU046-04

[0158] [ka] 5,7-Dimethoxybenzofuran-2-yl)methyl N,N'-bis(2-chloroethyl)phosphorodiamidate (8) SU025-04 To a suspension of 5 (300 mg, 1.44 mmol), 6 (479 mg, 2.16 mmol), and PPh3 (565 mg, 2.16 mmol) in THF (20 mL) was added dropwise DIAD (0.426 mL, 2.16 mmol) at 0 °C. The resulting suspension was warmed to room temperature and stirred for 2 h. The solvent was removed, and the residue was purified by flash chromatography (70% acetone in toluene) to give 8 (250 mg, 42%) as an oil.

[0159] [ka] 5,7-Dimethoxybenzofuran-2-yl)methyl N,N'-bis(2-bromoethyl)phosphorodiamidate (9) SU046-04 Compound 9 (SU046-04) was synthesized using the same method as above, and was obtained in 25% yield (10 mg).

[0160] [ka] 2. Ether- and thioether-linked model prodrugs

[0161] [ka]

[0162] [ka] Synthesis of ether- and thioether-linked prodrugs. 7-(Benzofuran-2-ylmethoxy)-4-methyl-2H-chromen-2-one (10) TLE-M2-SU010A

[0163] [ka] 2-(Bromomethyl)benzofuran (10) Benzofuran-2-ylmethanol (1.0 g, 6.7 mmol) was dissolved in toluene (50 mL) and pyridine (653 μL, 8.1 mmol) was added. The solution was cooled to 0 °C. PBr (760 μL, 8.1 mmol) was added dropwise over 15 min. The reaction mixture was then allowed to reach room temperature and stirred for 1 h. The mixture was washed with K2CO3 solution and extracted with EtOAc (3 × 30 mL). The EtOAc layer was washed with brine and dried (MgSO4). The solvent was removed in vacuo and the product was purified by flash chromatography eluting with hexane:EtOAc (4:1) to give 10 (780 mg, 55%) as an oil.

[0164] [ka] 7-(Benzofuran-2-ylmethoxy)-4-methyl-2H-chromen-2-one (11) TLE-M2-SU010A Sodium ethoxide (77 mg, 1.13 mmol) was added to DMF (10 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (200 mg, 1.13 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture was added 10 (200 mg, 0.94 mmol) in small portions. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine, water, and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO), and the product was purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 11 (35 mg, 12%) as a white solid.

[0165] [ka] 7-((5-fluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (16)VG015-05

[0166] [ka] 2-(2,2-diethoxyethoxy)-5-fluorobenzaldehyde (12) To a stirred suspension of 2-hydroxy-5-fluorobenzaldehyde (500 mg, 3.57 mmol) and K2CO3 (524 mg, 13.79 mmol) in DMF (10 mL) was added bromoacetaldehyde diethyl acetal (0.6 mL, 3.93 mmol) dropwise. The mixture was refluxed for 4 h. After cooling, the precipitate was filtered off and the solvent was evaporated in vacuo. The crude residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 12 (300 mg, 33%) as an oil.

[0167] [ka] 5-Fluorobenzofuran-2-carbaldehyde (13) A stirred solution of 12 (300 mg, 4.0 mmol) in acetic acid (10 mL) was refluxed for 24 h. After cooling, the solution was evaporated to dryness. The crude product was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 13 (180 mg, 94%) as a white solid.

[0168] [ka] (5-Fluorobenzofuran-2-yl)methanol (14) Compound 13 (180 mg, 1.10 mmol) was dissolved in EtOH (12 mL). NaBH (45 mg, 1.21 mmol) was added portionwise at 0 °C with vigorous stirring. The suspension was stirred at 0 °C for 15 min and then at room temperature for 1.5 h. The solvent was evaporated in vacuo. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO). The solvent was evaporated in vacuo. The residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (3:1) to give 14 (150 mg, 91%) as a white solid.

[0169] [ka] 2-(Bromomethyl)-5-fluorobenzofuran (15) Compound 14 (150 mg, 0.90 mmol) was dissolved in toluene (10 mL) and the solution was cooled to 0 °C. PBr (102 μL, 1.08 mmol) was added dropwise over 15 min. The reaction mixture was then allowed to reach room temperature and stirred for 1 h. The solvent was removed in vacuo. The residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 15 (150 mg, 72%) as an oil.

[0170] [ka] 7-((5-fluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (16)VG015-05 Sodium ethoxide (8.9 mg, 0.13 mmol) was added to DMF (3 ml) at 0° C., and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (25.4 mg, 0.14 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture was added 15 (30 mg, 0.13 mmol) in small portions. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give 16 (9.0 mg, 21%) as a white solid. m / z = 325.20 (M+H).

[0171] 7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (19)VG016-05

[0172] [ka] 2-(2,2-diethoxyethoxy)-3,5-difluorobenzaldehyde (17) To a stirred suspension of 2-hydroxy-3,5-fluorobenzaldehyde (1.0 g, 6.32 mmol) and K2CO3 (960 mg, 6.95 mmol) in DMF (10 mL) was added bromoacetaldehyde diethyl acetal (1.07 mL, 6.95 mmol) dropwise. The mixture was refluxed for 4 h. After cooling, the precipitate was filtered off and the solvent was evaporated in vacuo. The crude residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 17 (380 mg, 22%) as an oil.

[0173] [ka] 2-(Bromomethyl)-5,7-difluorobenzofuran (18) A stirred solution of 17 (380 mg, 1.39 mmol) in acetic acid (10 mL) was refluxed for 24 h. After cooling, the solution was evaporated to dryness. The crude product (300 mg) was dissolved in Et The residue was dissolved in 1HCOOH (5 mL). NaBH4 (73 mg, 1.98 mmol) was added portionwise at 0 °C with vigorous stirring. The suspension was stirred at 0 °C for 15 min and then at room temperature for 1.5 h. The solvent was evaporated in vacuo. The crude residue (280 mg) was dissolved in toluene (20 mL) and the solution was cooled to 0 °C. PBr3 (142 μL, 1.52 mmol) was added dropwise over 15 min. The reaction mixture was then allowed to reach room temperature and stirred for 1 h. The solvent was evaporated in vacuo. The residue was adsorbed onto silica gel and purified by flash chromatography using hexane / EtOAc (4:1) as eluent to give 18 (210 mg, 61%).

[0174] [ka] 7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (19)VG016-05 Sodium ethoxide (8.9 mg, 0.13 mmol) was added to DMF (3 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (25.4 mg, 0.14 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture, 2-(bromomethyl)-5-fluorobenzofuran (30 mg, 0.12 mmol) was added in small portions. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give 19 (8.8 mg, 21%) as a white solid. m / z = 343.12 (M+H).

[0175] 7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (22) (VG017-05)

[0176] [ka] 2-(2,2-diethoxyethoxy)-5-fluoro-3-methylbenzaldehyde (20) To a stirred suspension of 5-fluoro-2-hydroxy-3-methylbenzaldehyde (1.0 g, 6.49 mmol) and K2CO3 (980 mg, 7.10 mmol) in DMF (8 mL) was added bromoacetaldehyde diethyl acetal (1.10 mL, 7.15 mmol) dropwise. The mixture was refluxed for 4 h. After cooling, the precipitate was filtered off and the solvent was removed in vacuo. The crude residue was adsorbed onto silica gel and purified by flash chromatography. The product was eluted with hexane / EtOAc (4:1) to give 20 (350 mg, 20%) as an oil.

[0177] [ka] 2-(Bromomethyl)-5-fluoro-7-methylbenzofuran (21) A stirred solution of 20 (350 mg, 1.30 mmol) in acetic acid (10 mL) was refluxed for 24 h. After cooling, the solution was evaporated to dryness. The crude product (300 mg) was dissolved in THF (5 mL). NaBH4 (78 mg, 2.02 mmol) was added portionwise at 0 °C with vigorous stirring. The suspension was stirred at 0 °C for 15 min and then at room temperature for 1.5 h. The solvent was evaporated in vacuo. The crude residue (260 mg) was dissolved in toluene (20 mL), and the solution was cooled to 0 °C. PBr3 (135 μL, 1.44 mmol) was added dropwise over 15 min. The reaction mixture was then allowed to reach room temperature and stirred for 1 h. The solvent was evaporated in vacuo. The residue was adsorbed onto silica gel and purified by flash chromatography using hexane / EtOAc (4:1) as eluent to give 21 (200 mg, 36%).

[0178] [ka] 7-((5,7-difluorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (22) VG017-05 Sodium ethoxide (8.9 mg, 0.13 mmol) was added to DMF (3 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (25.4 mg, 0.14 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture was added 21 (30 mg, 0.12 mmol) in small portions. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give 22 as a white solid (11 mg, 26%). m / z = 33.20 (M+H).

[0179] 7-((5-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (27) VG027-05

[0180] [ka] 2-(2,2-diethoxyethoxy)-5-methoxybenzaldehyde (23) To a stirred suspension of 2-hydroxy-5-methoxybenzaldehyde (2.0 g, 13.16 mmol) and K2CO3 (2.18 g, 15.79 mmol) in DMF (20 mL) was added bromoacetaldehyde diethyl acetal (2.43 mL, 15.79 mmol) dropwise. The mixture was refluxed for 4 h. After cooling, the precipitate was filtered off and the solvent was evaporated in vacuo. The crude residue was adsorbed onto silica gel and purified by flash chromatography. The product was eluted with hexane / EtOAc (4:1) to give the desired compound 23 (1.10 g, 31%) as an oil.

[0181] [ka] 5-Methoxybenzofuran-2-carbaldehyde (24) A stirred solution of 23 (1.0 g, 3.74 mmol) in acetic acid (10 mL) was refluxed for 16 h. After cooling, the solution was evaporated to dryness. The crude product was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 24 (160 mg, 24%) as a white solid.

[0182] [ka] (5-Methoxybenzofuran-2-yl)methanol (25) Compound 24 (3.5 g, 19.9 mmol) was dissolved in EtOH (20 mL). NaBH4 (957 mg, 25.87 mmol) was added portionwise at 0 °C with vigorous stirring. The suspension was stirred at 0 °C for 15 min and then at room temperature for 1.5 h. The solvent was removed in vacuo. The residue was adsorbed onto silica gel and purified by flash chromatography using hexane / EtOAc (2:1) as eluent to give 25 (3.0 g, 85%) as a white solid. And got it.

[0183] [ka] 2-(Bromomethyl)-5-methoxybenzofuran (26) Compound 25 (40 mg, 0.22 mmol) was dissolved in toluene (5 mL) and the solution was cooled to 0 °C. PBr (21 μL, 0.22 mmol) was added dropwise over 10 min. The reaction mixture was then allowed to reach room temperature and stirred for 1 h. The solvent was removed in vacuo. The residue was adsorbed onto silica gel and purified by flash chromatography using hexane / EtOAc (4:1) as eluent to give 26 (40 mg, 74%) as an oil.

[0184] [ka] 7-((5-methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (27) VG027-05 Sodium ethoxide (12 mg, 0.18 mmol) was added to DMF (5 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (32 mg, 0.17 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. 26 (40 mg, 0.17 mmol) was added in small portions to the mixture. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give 27 (17 mg, 30%) as a white solid. m / z = 337.04 (M+H), 673.13 (2M+H).

[0185] 7-((7-Methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (31)VG029-05

[0186] [ka] 2-(2,2-diethoxyethoxy)-3-methoxybenzaldehyde (28) To a stirred suspension of 2-hydroxy-3-methoxybenzaldehyde (4.0 g, 26.3 mmol) and K2CO3 (4.36 g, 31.60 mmol) in DMF (15 mL) was added bromoacetaldehyde diethyl acetal (4.86 mL, 31.60 mmol) dropwise. The mixture was refluxed for 4 h. After cooling, the precipitate was filtered off and the solvent was evaporated in vacuo. The crude residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 28 (2.60 g, 36%).

[0187] [ka] 7-Methoxybenzofuran-2-carbaldehyde (29) A stirred solution of 28 (2.0 g, 7.46 mmol) in acetic acid (10 mL) was refluxed for 24 h. After cooling, the solution was evaporated to dryness. The crude product was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 29 (450 mg, 34%) as a white solid.

[0188] [ka] 2-(Bromomethyl)-7-methoxybenzofuran (30) Compound 29 (450 mg, 2.56 mmol) was dissolved in EtOH (10 mL). NaBH4 (104 mg, 2.81 mmol) was added portionwise at 0 °C with vigorous stirring. The suspension was stirred at 0 °C for 15 min and then at room temperature for 1.5 h. The solvent was evaporated in vacuo. The resulting crude alcohol residue was dissolved in toluene (5 mL) and the solution was cooled to 0 °C. PBr3 (240 μL, 2.56 mmol) was added dropwise over 10 min. The reaction mixture was then allowed to reach room temperature and stirred for 1 h. The solvent was evaporated in vacuo. The residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 30 (150 mg, 24%) as an oil.

[0189] [ka] 7-((7-Methoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (31)VG029-05 Sodium ethoxide (12 mg, 0.18 mmol) was added to DMF (5 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (32 mg, 0.17 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. 30 (40 mg, 0.17 mmol) was added in small portions to the mixture. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give 31 (14 mg, 25%) as a white solid. m / z 337.04 (M+H), 673.13 (2M+H).

[0190] 7-((5-Bromobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (32)VG035-04

[0191] [ka] 7-((5-Bromobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (VG035-04) Sodium ethoxide (76 mg, 1.10 mmol) was added to DMF (10 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (215 mg, 1.22 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture, 5-bromo-2-(chloromethyl)benzofuran (250 mg, 1.02 mmol) was added in small portions. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give 32 (120 mg, 31%) as a white solid.

[0192] [ka] 7-((5-chlorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (36)VG028-05

[0193] [ka] 5-chloro-2-(2,2-diethoxyethoxy)benzaldehyde (33) To a stirred suspension of 5-chloro-2-hydroxybenzaldehyde (5.0 g, 32.1 mmol) and K2CO3 (4.87 g, 35.3 mmol) in DMF (20 mL) was added bromoacetaldehyde diethyl acetal (5.43 mL, 35.3 mmol) dropwise. The mixture was refluxed for 4 h. After cooling, the precipitate was filtered off and the solvent was evaporated in vacuo. The crude residue was adsorbed onto silica gel and purified by flash chromatography. The product was eluted with hexane / EtOAc (4:1) to give 33 (4.10 g, 38%) as an oil.

[0194] [ka] 5-Chlorobenzofuran-2-carbaldehyde (34) A stirred solution of 33 (4.10 g, 15.07 mmol) in acetic acid (20 mL) was refluxed for 24 h. After cooling, the solution was evaporated to dryness. The crude product was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 34 (550 mg, 20%) as a white solid.

[0195] [ka] 2-(Bromomethyl)-5-chlorobenzofuran (35) Compound 34 (160 mg, 0.89 mmol) was dissolved in EtOH (5 mL). NaBH4 (36 mg, 0.98 mmol) was added portionwise at 0 °C with vigorous stirring. The suspension was stirred at 0 °C for 15 min and then at room temperature for 1.5 h. The solvent was evaporated in vacuo. The resulting crude alcohol residue was dissolved in toluene (5 mL) and the solution was cooled to 0 °C. PBr3 (92 μL, 0.98 mmol) was added dropwise over 10 min. The mixture was then heated at room temperature. The mixture was warmed to room temperature and stirred for 1 h. The solvent was removed in vacuo. The residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 35 (128 mg, 57%) as an oil.

[0196] [ka] 7-((5-chlorobenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (36)VG028-05 Sodium ethoxide (60 mg, 0.24 mmol) was added to DMF (5 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (47 mg, 0.27 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. 2-(Bromomethyl)-5-methoxybenzofuran (40 mg, 0.17 mmol) was added in small portions to the mixture. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give the target compound as a white solid (2.7 mg, 3%). m / z 341.10 (M+H).

[0197] 7-((5,7-dimethoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (42) VG035-05

[0198] [ka] 2-Bromo-3,5-dimethoxybenzaldehyde (37) 3,5-Dimethoxybenzaldehyde (12.6 g, 76 mmol) was dissolved in acetic acid (350 mL). The resulting colorless solution was cooled to 0° C. A solution of bromine (3.9 mL) in acetic acid (50 mL) was added dropwise over 1 hour. When the addition was complete, the ice bath was removed, and the resulting pale green solution was stirred at room temperature overnight. Cold water was added to the solution. The resulting white solid was collected by vacuum filtration and rinsed with water. The solid was then redissolved in EtOAc and added to silica gel. The product was purified by flash chromatography eluting with hexane / EtOAc (4:1) to give 37 (12.5 g, 66%) as a white solid.

[0199] [ka] 2-Hydroxy-3,5-dimethoxybenzaldehyde (38) A dry, three-necked round-bottom flask equipped with a stir bar, septum cap, dropping funnel, thermometer, and argon inlet was charged with morpholine (2.05 g, 24 mmol) and THF (40 mL). The flask was cooled to −50 °C in a dry ice-acetone bath, and a hexane solution of n-BuLi (1.6 M, 15 mL, 24 mmol) was added all at once. After 10 min, a solution of 2-bromo-3,5-dimethoxybenzaldehyde 37 (4.9 g, 20 mmol) in THF (30 mL) was added dropwise via syringe over 4 min, and the mixture was cooled to approximately −75 °C over 20 min. A hexane solution of n-BuLi (1.6 M, 20 mL, 32 mmol) was then added dropwise over 45 min, maintaining the temperature at −75 °C. After all the n-BuLi was added, the solution was stirred for 35 min. A solution of nitrobenzene (6.90 g, 46 mmol) in 10 mL of THF was added via a dropping funnel while maintaining the temperature at -75 °C. The resulting dark mixture was stirred at -75 °C for 4 hours and then warmed to room temperature. It was acidified to pH 1 with 6 N HCl and stirred for 15 minutes. After dilution with brine (100 mL), the THF was removed in vacuo. The aqueous solution was extracted with diethyl ether (4 × 40 mL). The combined organic layers were extracted with 2 N NaOH (3 × 40 mL). The combined NaOH extracts were washed with diethyl ether (3 × 20 mL) and then acidified to pH 1 with concentrated HCl. The resulting mixture was extracted with CHCl (3 × 20 mL), and the combined organic extracts were washed with brine, dried (MgSO), and adsorbed onto silica gel. The product was purified by flash chromatography eluting with EtOAc / hexane (1:2) to give 38 (2.0 g, 55%) as a yellow solid.

[0200] [ka] 2-(2,2-diethoxyethoxy)-3,5-dimethoxybenzaldehyde (39) To a stirred suspension of 38 (1.1 g, 6.0 mmol) and K2CO3 (1.0 g, 7.2 mmol) in DMF (100 mL) was added bromoacetaldehyde diethyl acetal (0.93 mL, 6.0 mmol) dropwise. The mixture was refluxed for 4 h. After cooling, the precipitate was filtered off and the solvent was evaporated in vacuo. The crude residue was adsorbed onto silica gel and purified by flash chromatography. The product was eluted with hexane / EtOAc (4:1) to give 39 (1.2 g, 67%) as an oil.

[0201] [ka] 5,7-Dimethoxybenzofuran-2-carbaldehyde (40) A stirred solution of 39 (1.2 g, 4.0 mmol) in acetic acid (35 mL) was refluxed for 16 h. After cooling, the solution was evaporated to dryness. The crude product was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (2:1) to give 40 (230 mg, 28%) as a white solid.

[0202] [ka] (5,7-Dimethoxybenzofuran-2-yl)methanol (41) Compound 39 (460 mg, 2.23 mmol) was dissolved in THF (5 mL) and EtOH (1 mL). NaBH (102 mg, 2.68 mmol) was added portionwise at 0 °C with vigorous stirring. The suspension was stirred at 0 °C for 15 min and then at room temperature for 1 h. The solvent was removed in vacuo. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO). The residue was adsorbed onto silica gel and purified by flash chromatography eluting with hexane / EtOAc (1:1) to give 41 (388 mg, 82%) as a white solid.

[0203] [ka] 7-((5,7-dimethoxybenzofuran-2-yl)methoxy)-4-methyl-2H-chromen-2-one (42) VG035-05 Compound 41 (130 mg, 0.63 mmol) was dissolved in toluene (5 mL) and the solution was cooled to 0 °C. PBr3 (64 μL, 0.69 mmol) was added dropwise over 10 min. The reaction mixture was then allowed to warm to room temperature and stirred for 1 h. The solvent was removed in vacuo. The crude residue was used in the next step. Sodium ethoxide (80 mg, 0.24 mmol) was added to DMF (5 mL) at 0 °C and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (47 mg, 0.27 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture, 2-(bromomethyl)-5-methoxybenzofuran (40 mg, 0.17 mmol) was added in small portions. The resulting reaction mixture was stirred at room temperature for 2 h. The DMF was removed in vacuo, and the residue was purified by flash chromatography eluting with hexane: EtOAc (3:1) to give 42 (2.7 mg, 3%) as a white solid, m / z = 367.05 (M+H), 733.15 (2M+H).

[0204] 4-Methyl-7-(naphthalen-1-ylmethoxy)-2H-chromen-2-one (43) TLE-M1-SU001A

[0205] [ka] 1-Naphthalenemethanol (2.0 g, 12.7 mmol) was dissolved in toluene (30 mL) and pyridine (1.02 mL, 12.7 mmol) was added. The solution was cooled to 0 °C. PBr (1.19 mL, 12.7 mmol) was added dropwise over 15 min. The reaction mixture was then allowed to reach room temperature and stirred for 1 h. The mixture was washed with K2CO3 solution and extracted with EtOAc (3 x 30 mL). The EtOAc layer was washed with brine and dried (MgSO4). The solvent was removed in vacuo to give 1-(bromomethyl)naphthalene (1.5 g, 53%) as a colorless oil. This intermediate was used in the next reaction.

[0206] Sodium ethoxide (169 mg, 2.49 mmol) was added to DMF (5 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (438 mg, 2.49 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. 1-(Bromomethyl)naphthalene (500 mg, 2.26 mmol) was added in small portions to the mixture. The resulting reaction mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine (2 × 50 mL), water (2 × 50 mL), and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4), and the product was purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 43 (200 mg, 28%) as a white solid.

[0207] [ka] 4-Methyl-7-(naphthalen-2-ylmethoxy)-2H-chromen-2-one (44) VG040-03

[0208] [ka] Naphthalen-2-ylmethanol (2.0 g, 12.7 mmol) was dissolved in toluene (30 mL) and pyridine (1.02 mL, 12.7 mmol) was added. The solution was cooled to 0 °C. PBr (1.19 mL, 12.7 mmol) was added dropwise over 15 min. The mixture was then allowed to reach room temperature and stirred for 1 h. The mixture was washed with K2CO3 solution and extracted with EtOAc (3 × 30 mL). The EtOAc layer was washed with brine and dried (MgSO4). The solvent was removed in vacuo to give crude 1-(bromomethyl)naphthalene. This intermediate was used in the next reaction.

[0209] Sodium ethoxide (169 mg, 2.49 mmol) was added to DMF (5 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (438 mg, 2.49 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. 1-(Bromomethyl)naphthalene (500 mg, 2.26 mmol) was added in small portions to the mixture. The resulting reaction mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine (2 × 50 mL), water (2 × 50 mL), and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4), and the product was purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 44 (1.44 g, 36%) as a white solid.

[0210] [ka] 7-(Benzhydryloxy)-4-methyl-2H-chromen-2-one (45) TLE-M1-SU004A

[0211] [ka] Sodium ethoxide (165 mg, 2.43 mmol) was added to DMF at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (428 mg, 2.43 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. To this mixture, diphenylmethyl bromide (500 mg, 2.02 mmol) was added in small portions. The resulting reaction mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine (2 × 30 mL), water (2 × 30 mL), and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4), and the product was purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 45 (200 mg, 29%) as a white solid.

[0212] [ka] 4-Methyl-7-(1-(naphthalen-2-yl)ethoxy)-2H-chromen-2-one (46) VG039-03

[0213] [ka] 1-(Naphthalen-2-yl)ethanol (2.0 g, 11.6 mmol) was dissolved in toluene (30 mL). The solution was cooled to 0 °C. PBr (1.09 mL, 11.6 mmol) was added dropwise over 15 min. The reaction mixture was then allowed to reach room temperature and stirred for 1 h. The mixture was washed with K2CO3 solution and extracted with EtOAc (3 × 30 mL). The EtOAc layer was washed with brine and dried (MgSO4). The solvent was removed in vacuo to give crude 2-(1-bromoethyl)naphthalene. This intermediate was used in the next step.

[0214] Sodium ethoxide (63.4 mg, 0.93 mmol) was added to DMF (5 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (147 mg, 0.84 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. 2-(1-bromoethyl)naphthalene (200 mg, 0.85 mmol) was added in small portions to the mixture. The resulting reaction mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine (2 × 50 mL), water (2 × 50 mL), and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4), and the product was purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 46 (60 mg, 21%) as a white solid.

[0215] [ka] 7-(anthracen-9-ylmethoxy)-4-methyl-2H-chromen-2-one (48)SU06-02

[0216] [ka] 9-(Bromomethyl)anthracene (47) To a stirred suspension of 9-anthracenemethanol (2.0 g, 9.6 mmol) in toluene (100 mL) at 0 °C, PBr (1.2 mL, 12.51 mmol) was added, and the suspension was stirred at 0 °C for 1 h. The reaction mixture was then brought to room temperature and stirred for an additional 1 h. The mixture turned into a yellow solution. KCO (10 mL) was added to quench the reaction. The toluene was evaporated in vacuo. The residue was dissolved in EtOAc, washed with saturated aqueous KCO, water, and brine, and dried (MgSO). The solvent was evaporated in vacuo, and the crude residue was purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 47 (1.4 g, 54%) as a yellow solid.

[0217] [ka] 7-(anthracen-9-ylmethoxy)-4-methyl-2H-chromen-2-one (48) SU06-02 Sodium ethoxide (151 mg, 47 (500 mg, 1.85 mmol) was added in portions. The reaction mixture was stirred at room temperature for 16 hours. The DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine, water, and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4), and the product was purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 48 (200 mg, 30%) as a yellow solid.

[0218] [ka] 7-(bis(4-methoxyphenyl)methoxy)-4-methyl-2H-chromen-2-one (49)SU010-02

[0219] [ka] Bis(4-methoxyphenyl)methanol (2.0 g, 8.2 mmol) was dissolved in toluene (60 mL) and pyridine (661 μL, 8.2 mmol) was added. The solution was cooled to 0 °C. PBr (768 μL, 8.2 mmol) was added dropwise over 15 min. The reaction mixture was warmed to room temperature and stirred for 1 h. The mixture was washed with K2CO3 solution and extracted with EtOAc (3 × 30 mL). The EtOAc layer was washed with brine and dried (MgSO4). The solvent was removed in vacuo to give the crude product 4,4'-(bromomethylene)bis(methoxybenzene) (780 mg, 31%) as a colorless oil, which was used in the next reaction step without further purification. Sodium ethoxide (133 mg, 1.96 mmol) was added to DMF (5 mL) at 0 °C and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (345 mg, 1.96 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture, 4,4'-(bromomethylene)bis(methoxybenzene) (500 mg, 1.63 mmol) was added in small portions. The resulting reaction mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine, water, and 1 M NaOH (2 x 30 mL). The organic layer was dried (MgSO4), and the product was purified by flash chromatography using hexane:EtOAc (2:1) as eluent. Purification by HPLC gave 49 (100 mg, 15%) as a white solid.

[0220] [ka] 7-((1H-Benzo[d]imidazol-2-yl)methoxy)-4-methyl-2H-chromen-2-one (50) VG033-03

[0221] [ka] Sodium ethoxide (82 mg, 1.20 mmol) was added to DMF (10 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (253 mg, 1.44 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture, 2-(chloromethyl)-1H-benzo[d]imidazole (200 mg, 1.20 mmol) was added in small portions. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was evaporated, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give 50 (200 mg, 54%) as a white solid.

[0222] [ka] 7-(benzo[d]thiazol-2-ylmethoxy)-4-methyl-2H-chromen-2-one (51)VG014-04

[0223] [ka] Sodium ethoxide (30 mg, 0.44 mmol) was added to DMF (10 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (77 mg, 0.44 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. 2-(chloromethyl)-1H-benzo[d]imidazole (100 mg, 0.44 mmol) was added in small portions to the mixture. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give 51 (25 mg, 18%) as a white solid. m / z = 324.06 (M+H), 647.12 (2M+H).

[0224] 4-Methyl-7-(4-(thiophen-2-yl)benzyloxy)-2H-chromen-2-one (52) VG015-04

[0225] [ka] Sodium ethoxide (27 mg, 0.40 mmol) was added to DMF (10 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (70 mg, 0.40 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. 2-(chloromethyl)-1H-benzo[d]imidazole (100 mg, 0.40 mmol) was added in small portions to the mixture. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (3:1) as an eluent to give 52 (30 mg, 18%) as a white solid. m / z = 349.09 (M+H), 697.16 (2M+H).

[0226] 6-(Benzhydrylthio)-9H-purine (53) (VG015-02)

[0227] [ka] 6-Mercaptopurine (151 mg, 0.88 mmol) was dissolved in DMF (5 mL). KCO (122 mg, 1.2 mmol) was added, and to the resulting suspension was added diphenyl methylbromide (200 mg, 0.8 mmol). The resulting reaction mixture was stirred at room temperature for 4 h. The mixture was poured onto ice, and the resulting precipitate was isolated by filtration, washed with ether, and dried in vacuo to give 53 (35 mg, 14%) as a white solid.

[0228] [ka] 3. Carbamate-linked nucleoside analogue prodrugs

[0229] [ka] Naphthalen-1-ylmethyl 1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate (55) SU001-03

[0230] [ka] To COCl2 (13.3 mL, as a 20% COCl2 solution in toluene) in THF (30 mL) was added naphthalen-1-ylmethanol (3.0 g, 19.0 mmol) in one portion. The reaction was stirred at room temperature for 2 h. Excess COCl2 and THF were removed under reduced pressure. The solid residue was dissolved in hot hexane and filtered. The hexane was then slowly evaporated in vacuo to give the chloroformate intermediate 54 as a white solid, which was used immediately in the next step. 54 (330 mg, 1.5 mmol) and KHCO3 (252 mg, 2.52 mmol) were added to a solution of cytarabine HCl (243 mg, 0.87 mmol) in dimethylacetamide (5 mL), and the mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo and the product was purified by flash chromatography eluting with a gradient of 2.5% to 12% MeOH in DCM to give 55 (38 mg, 10%) as a white solid, m / z = 428.15 (M+H).

[0231] Naphthalen-1-ylmethyl 1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate (56) SU0023-02

[0232] [ka] To COCl2 (4.4 mL, as a 20% solution of COCl2 in toluene) in THF (20 mL) was added naphthalen-1-ylmethanol (1.0 g, 6.3 mmol) in one portion. The reaction was stirred at room temperature for 2 hours. Excess COCl2 and THF were removed under reduced pressure. The solid residue was dissolved in hot hexane and filtered. The hexane solvent was then slowly removed under vacuum. The mixture was stirred at 100 °C for 16 h. The solvent was evaporated in vacuo and the product was purified by flash chromatography using 3% MeOH in ethyl acetate as the eluent to give 56 (15 mg, 5%) as an oil. m / z = 448.13 (M+H).

[0233] Benzyl 1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate (57) SU0044-2a / 02

[0234] [ka] Gemcitabine HCl (200 mg, 0.67 mmol) was dissolved in HO (2 mL). To this was added KHCO (67 mg, 0.67 mmol) and benzyl carbonochloridate (95 μL, 0.67 mmol), predissolved in ethyl acetate (5 mL). The mixture was stirred at 80° C. for 16 h. The solvent was removed in vacuo, and the product was purified by flash chromatography using 3% MeOH in ethyl acetate as the eluent to give 57 (40 mg, 15%) as an oil. m / z=398.12 (M+H).

[0235] Benzyl 1-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate (58) SU0044-3a / 02

[0236] [ka] Cytarabine HCl (200 mg, 0.72 mmol) was dissolved in HO (2 mL). To this was added KHCO (72 mg, 0.72 mmol) and benzyl carbonochloridate (107 μL, 0.72 mmol) predissolved in ethyl acetate (5 mL). The mixture was stirred at 80° C. for 16 h. The solvent was removed in vacuo, and the product was purified by flash chromatography using 3% MeOH in ethyl acetate as the eluent to give 58. (40 mg, 15%) was obtained as an oil. m / z=378.13 (M+H).

[0237] Benzofuran-2-ylmethyl 4-nitrophenyl carbonate (60) and (5,7-dimethoxybenzofuran-2-yl)methyl 4-nitrophenyl carbonate (61)

[0238] [ka] Benzofuran-2-ylmethyl 4-nitrophenyl carbonate (60) A solution of benzofuran-2-ylmethanol 59 (300 mg, 2.03 mmol) in THF (5 mL) was cooled to 0 °C. TEA (280 μL, 2.03 mmol) was added dropwise, followed by the addition of p-nitrophenyl chloroformate (282 mg, 3.05 mmol). The resulting solution was stirred at room temperature for 2 h. The solvent was removed in vacuo, and the crude residue was purified by flash chromatography eluting with hexane:EtOAc (3:1) to give 60 (350 mg, 54%) as a white solid.

[0239] [ka] (5,7-Dimethoxybenzofuran-2-yl)methyl 4-nitrophenyl carbonate (61) A solution of (5,7-dimethoxybenzofuran-2-yl)methanol 6 (100 mg, 0.48 mmol) in THF (3 mL) was cooled to 0 °C. TEA (69 μL, 0.48 mmol) was added dropwise, followed by the addition of p-nitrophenyl chloroformate (100 mg, 0.72 mmol). The resulting solution was stirred at room temperature for 2 h. The solvent was removed in vacuo, and the crude residue was purified by flash chromatography eluting with hexane:EtOAc (3:1) to give 61 (120 mg, 67%) as a white solid.

[0240] [ka] Benzofuran-2-ylmethyl 1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate (65) SU050-03 and (5,7-dimethoxybenzofuran-2-yl)methyl 1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)methyl 1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate (66) SU048-04

[0241] [ka] 4-Amino-1-(9,9-difluoro-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)pyrimidin-2(1H)-one (62) Gemcitabine HCl (1.0 g, 3.3 mmol) was stirred in pyridine (10 mL) for 10 min (2 × 5 mL). The pyridine was evaporated. Pyridine (10 mL) was added, and 1,1,3,3-tetraisopropyldisiloxane (1.17 mL, 3.63 mmol) was added dropwise. The resulting mixture was stirred at 100 °C for 16 h. Further 1,1,3,3-tetraisopropyldisiloxane (1 mL) was added, and the mixture was stirred at 120 °C for 1 h. The reaction mixture was cooled to room temperature, and the solvent was evaporated in vacuo. The resulting crude solid was recrystallized from EtOAc / ether (1:1) to give 62 (600 mg, 36%) as a white solid. m / z = 506.23 (M+H).

[0242] Benzofuran-2-ylmethyl 1-(9,9-difluoro-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilosin-8-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate (63) To a stirred solution of 62 (300 mg, 0.59 mmol) in THF (5 mL) was added benzofuran-2-ylmethyl 4-nitrophenyl carbonate (223 mg, 0.71 mmol). The resulting solution was stirred at 100 °C for 4 days. The solvent was removed in vacuo and the product was purified by preparative HPLC to give 63 (350 mg, 87%) as an oil. m / z = 680.0 (M+H), 1359.49 (2M+H).

[0243] Benzofuran-2-ylmethyl 1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate (65) SU050-03 Compound 63 (200 mg, 0.29 mmol) was dissolved in THF (1.5 mL). Tetra-n-butylammonium fluoride was added, and the resulting solution was stirred at room temperature for 15 minutes. The solvent was removed in vacuo. The product was purified by flash chromatography using 5% MeOH in EtOAc as the eluent to give 65 (30 mg, 23%) as an oil. m / z = 438.14 (M+H), 874.24 (2M+H).

[0244] (5,7-Dimethoxybenzofuran-2-yl)methyl 1-(3,3-difluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-ylcarbamate (66) SU048-04 To a stirred solution of 62 (108 mg, 0.21 mmol) in THF (5 mL) was added 61 (100 mg, 0.27 mmol). The resulting solution was stirred at 100 °C for 4 days. The solvent was removed in vacuo to give 64 as an oil, which was used in the next step without further purification. Compound 64 (100 mg, 0.14 mmol) was dissolved in THF (1.5 mL). Tetra-n-butylammonium fluoride was added, and the resulting solution was stirred at room temperature for 15 minutes. The solvent was removed in vacuo. The product was purified by flash chromatography using 5% MeOH in EtOAc as the eluent to give 66 (18 mg, 26%) as an oil.

[0245] [ka] 4. Carbamate-linked nitrogen and aniline mustard prodrugs

[0246] [ka] Benzofuran-2-ylmethyl 4-(bis(2-chloroethyl)amino)phenylcarbamate (VG042-04)

[0247] [ka] 2,2'-(4-nitrophenylazanediyl)diethanol (67) To 1-fluoro-4-nitrobenzene (1.0 g, 7.09 mmol) in DMF (30 mL) was added diethanolamine (2.70 mL, 2.5 mmol). The resulting mixture was stirred at 140° C. for 3.5 hours. The solution was cooled to room temperature, and the solvent was removed in vacuo. The residue was dissolved in EtOAc (30 mL), washed with water (3×10 mL) and brine (3×20 mL), and dried (MgSO). The solvent was removed in vacuo, and the product was purified by flash chromatography eluting with EtOAc to give 67 (400 mg, 25%) as a yellow solid.

[0248] [ka] N,N-bis(2-(tert-butyldimethylsilyloxy)ethyl)-4-nitroaniline (68) To a cooled solution of 67 (400 mg, 1.77 mmol) and imidazole (481 mg, 7.08 mmol) in DMF (10 mL) was added tert-butyldimethylsilyl chloride (2.72 mg, 3.54 mmol) dropwise. The mixture was allowed to reach room temperature and stirred for 48 h. The solvent was removed in vacuo, and the product was purified by flash chromatography eluting with 10% EtOAc in ether to give 68 (200 mg, 25%) as a yellow solid.

[0249] [ka] Benzofuran-2-ylmethyl 4-(bis(2-chloroethyl)amino)phenylcarbamate (69) VG042-04 Compound 68 (200 mg, 0.44 mmol) was treated with hydrogen in the presence of 10% Pd on carbon (20 mg). After stirring for 16 h, the mixture was filtered through Celite and the solvent was removed in vacuo to give the intermediate aminoaniline product. This was then reacted with triphosgene (195 mg, 0.70 mmol) in the presence of triethylamine (260 μL, 0.70 mmol) in THF (15 mL). After stirring at room temperature for 1 h, the white precipitate was filtered off. The solvent was evaporated in vacuo to give the crude residue of the isocyanate aniline, which was used immediately in the next step. The isocyanate intermediate was dissolved in THF (10 mL). The solution was cooled to 0 °C. Benzofuran-2-ylmethanol 59 (100 mg, 1.35 mmol) was added, and the resulting mixture was stirred at room temperature for 16 h. The mixture was cooled on ice, and TBAF (996 μL, 3.38 mmol) was added dropwise over 5 min. The resulting mixture was allowed to warm to room temperature and then stirred for 20 min. The THF was evaporated in vacuo. The intermediate was dissolved in pyridine (5 mL), and methanesulfonyl chloride (12.5 μL, 0.16 mmol) was added. The mixture was stirred at room temperature for 1 h. The pyridine was evaporated in vacuo, and the crude product was purified by flash chromatography eluting with hexane:EtOAc (3:1) to give 69 (5 mg, 2%) as a white solid. m / z=408.07(M+H),837.16(2M+H).

[0250] Benzofuran-2-ylmethylbis(2-chloroethyl)carbamate (70)VG045-04

[0251] [ka] To a solution of bis(2-chloroethylamine) hydrochloride (227 mg, 1.28 mmol) in pyridine (25 mL) was added a solution of 60 (200 mg, 0.64 mmol) in pyridine (3 mL). The mixture was stirred at room temperature for 16 h. DCM (10 mL) was added, and the mixture was washed with 2% citric acid solution (2 × 50 mL), water (50 mL), brine (50 mL), and dried (MgSO). The solvent was removed in vacuo, and the product was purified by flash chromatography eluting with CHCl:hexane (2:1) to give 70 (125 mg, 62%) as an oil.

[0252] [ka] 5. Ether-linked Topoisomerase I Inhibitor Prodrugs

[0253] [ka] 5,7-Dimethoxybenzofuran-2-yl)methyl-camptothecin (71) SU037-04

[0254] [ka] Compound 41 (100 mg, 0.48 mmol) was dissolved in toluene (5 mL) and the solution was cooled to 0 °C. PBr (46 μL, 0.48 mmol) was added dropwise over 10 min. The reaction mixture was then allowed to reach room temperature and stirred for 1 h. The solvent was removed in vacuo. The crude residue was used in the next step.

[0255] Sodium ethoxide (15 mg, 0.22 mmol) was added to DMF (5 mL) at 0 °C, and the suspension was stirred for 10 min. Camptothecin (81 mg, 0.22 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture, the crude residue from the previous step, 2-(bromomethyl)-5,7-dimethoxybenzofuran (50 mg, 0.18 mmol), was added in small portions. The resulting mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using DCM:EtOAc (2:1) as the eluent to give the desired compound as a white solid (10 mg, 10%). m / z = 555.19 (M+H).

[0256] 6. Ether-linked tyrosine kinase inhibitor prodrugs

[0257] [ka] N-(4-(benzofuran-2-ylmethoxy)quinazolin-2-yl)-4,6,7-trimethylquinazolin-2-amine (72)VG048-04

[0258] [ka] Sodium ethoxide (3 mg, 0.05 mmol) was added to DMF (2 mL) at 0 °C, and the suspension was stirred for 5 min. 2-(4,6-dimethylquinazolin-2-ylamino)quinazolin-4-ol (15 mg, 0.05 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture was added 2-(bromomethyl)benzofuran (16 mg, 0.08 mmol). The resulting mixture was stirred at room temperature for 1 h. DMF was removed in vacuo to give a crude white solid. This was purified by washing with cold ether and EtOAc to give 72 (3 mg, 11%) as a white solid. m / z = 462.2 (M+H).

[0259] 7-(Benzofuran-2-ylmethoxy)-5-isopropyl-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (73)SU01-A-04

[0260] [ka] Sodium ethoxide (7.2 mg, 0.10 mmol) was added to DMF (2 mL) at 0 °C, and the suspension was stirred for 5 min. 5-Isopropyl-2-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (20 mg, 0.10 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture was added 2-(bromomethyl)benzofuran (16 mg, 0.08 mmol). The resulting mixture was stirred at room temperature for 1 h. DMF was removed in vacuo to give a crude white solid. This was purified by semi-preparative HPLC to give 73 (6.3 mg, 19%) as a white solid. m / z = 323.13 (M+H), 645.27 (2M+H).

[0261] 7-(Benzofuran-2-ylmethoxy)-2-methyl-5-((4-methylpyrimidin-2-ylthio)methyl)-[1,2,4]triazolo[1,5-a]pyrimidine (74)SU01-B-04

[0262] [ka] Sodium ethoxide (4.7 mg, 0.07 mmol) was added to DMF (2 mL) at 0 °C, and the suspension was stirred for 5 min. 2-Methyl-5-((4-methylpyrimidin-2-ylthio)methyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (20 mg, 0.07 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture was added 10 (16 mg, 0.08 mmol). The resulting mixture was stirred at room temperature for 1 h. DMF was removed in vacuo to give a crude white solid. This was purified by semi-preparative HPLC to give 74 (5.2 mg, 18%) as a white solid. m / z = 419.09 (M+H), 837.23 (2M+H).

[0263] 7-(Benzofuran-2-ylmethoxy)-1-(2-fluorobenzyl)-4-methyl-1H-[1,2,3]triazolo[4,5-d]pyridazine (75)SU01-C-04

[0264] [ka] Sodium ethoxide (5.2 mg, 0.08 mmol) was added to DMF (2 mL) at 0 °C, and the suspension was stirred for 5 min. 1-(2-Fluorobenzyl)-4-methyl-1H-[1,2,3]triazolo[4,5-d]pyridazin-7-ol (20 mg, 0.08 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture was added 2-(bromomethyl)benzofuran (16 mg, 0.08 mmol). The resulting mixture was stirred at room temperature for 1 h. DMF was removed in vacuo to give a crude white solid. This was purified by semi-preparative HPLC to give 75 (3 mg, 10%) as a white solid. m / z = 390.07 (M+H), 801.12 (2M+Na).

[0265] 7. Carbamate-linked model coumarin prodrugs

[0266] [ka]

[0267] [ka] 7-Isocyanato-4-methylcoumarin (76)

[0268] [ka] A 200 mL three-neck flask equipped with a dry ice condenser and a magnetic stir bar was charged with a solution of 20% phosgene in toluene (2.0 mL) and dioxane (80 mL). To this mixture was added 7-amino-4-methyl-2H-chromen-2-one (2.00 g, 11.4 mmol). The mixture was stirred at 100 °C for 12 h. The initial yellow color disappeared, and a white solid precipitated. An additional 20% phosgene in toluene solution (7.0 mL) was added, and the mixture was heated for an additional 5 h, at which point the solution became clear. Nitrogen gas was bubbled through the solution to remove excess phosgene and traces of HCl. The cloudy solution was filtered to remove unreacted 7-amino-4-methyl-2H-chromen-2-one and concentrated to give 76 (0.5 g, 25%) as a white solid.

[0269] [ka] Naphthalen-1-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (77)VG020-02

[0270] [ka] Naphthalen-1-ylmethanol (56 mg, 0.28 mmol) and 76 (200 mg, 1.27 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / hexane / EtOAc (1:1:1) as eluent to give 77 (3 mg, 5%) as a white solid. m / z = 360.14 (M+H), 719.27 (2M+H).

[0271] (2-Chloroquinolin-3-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (78) SU030-7-03

[0272] [ka] (2-Chloroquinolin-3-yl)methanol (100 mg, 0.52 mmol) and 76 (155 mg, 0.77 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 78 (38 mg, 19%) as a white solid. m / z = 395.08 (M+H).

[0273] Benzhydryl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (79) SU0021-02

[0274] [ka] (2-Chloroquinolin-3-yl)methanol (119 mg, 0.65 mmol) and 76 (70 mg, 0.35 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 79 (50 mg, 37%) as a white solid. m / z = 386.16 (M+H).

[0275] Benzhydryl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (80)SU0021-02

[0276] [ka] (4-Methyl-2-phenylpyrimidin-5-yl)methanol (100 mg, 0.50 mmol) and 76 (151 mg, 0.75 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 80 (70 mg, 35%) as a white solid. m / z = 402.15 (M+H).

[0277] (1H-Benzo[d]imidazol-2-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (81)VG032-03

[0278] [ka] (1H-Benzo[d]imidazol-2-yl)methanol (200 mg, 1.35 mmol) and 76 (272 mg, 1.35 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 81 (80 mg, 17%) as a white solid. m / z = 350.12 (M+H).

[0279] (2H-chromen-3-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (82)SU033-03

[0280] [ka] 2H-Chromene-3-carbaldehyde (500 mg, 3.13 mmol) was dissolved in EtOH (10 mL). NaBH (119 mg, 3.13 mmol) was added portionwise at 0 °C with vigorous stirring. The suspension was stirred at 0 °C for 15 min and then at room temperature for 1.5 h. The solvent was removed in vacuo to give the alcohol intermediate as an oil. This was dissolved in THF (5 mL) and 76 (155 mg, 0.77 mmol) was added. The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. The THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 82 (80 mg, 8%) as a white solid. m / z = 364.12 (M+H), 727.23 (2M+H).

[0281] Naphthalen-2-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (83)VG037-03

[0282] [ka] Naphthalen-2-ylmethanol (200 mg, 1.27 mmol) and 76 (279 mg, 1.39 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 83 (26 mg, 6%) as a white solid. m / z = 360.13 (M+H), 719.25 (2M+H).

[0283] Benzofuran-2-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (84)SU018-03

[0284] [ka] Benzofuran-2-ylmethanol (300 mg, 2.03 mmol) and 76 (407 mg, 2.03 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 84 (130 mg, 18%) as a white solid. m / z = 350.09 (M+H), 699.17 (2M+H).

[0285] Benzo[d]thiazol-2-ylmethyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (85) SU024-3-03

[0286] [ka] Benzo[d]thiazol-2-ylmethanol (200 mg, 1.21 mmol) and 76 (365 mg, 1.8 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 85 (90 mg, 20%) as a white solid. m / z = 367.02 (M+H), 733.13 (2M+H).

[0287] 4-(Furan-2-yl)benzyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (86) SU024-3-03

[0288] [ka] (4-(furan-2-yl)phenyl)methanol (100 mg, 0.57 mmol) and 76 (139 mg, 0.69 mmol) were dissolved in THF (10 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give the desired compound (20 mg, 9%) as a white solid. m / z = 376.11 (M+H), 751.22 (2M+H).

[0289] (5-Methylbenzo[b]thiophen-2-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (87) SU030-4-03

[0290] [ka] (5-Methylbenzo[b]thiophen-2-yl)methanol (100 mg, 0.56 mmol) and 76 (136 mg, 0.67 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 50 °C for 3 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 87 (38 mg, 18%) as a white solid. m / z = 380.09 (M+H), 759.17 (2M+H).

[0291] (5-Methoxybenzofuran-2-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (88)VG032-05

[0292] [ka] (5-Methoxybenzofuran-2-yl)methanol 25 (200 mg, 1.12 mmol) and 76 (190 mg, 0.95 mmol) were dissolved in THF (10 mL). The resulting mixture was stirred at room temperature for 15 min and then at room temperature for 16 h. The THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using hexane / EtOAc (1:1) as the eluent to give 88 (20 mg, 5%) as a white solid. m / z = 380.13 (M+H), 759.26 (2M+H).

[0293] (5-Bromobenzofuran-2-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (89)VG036-05

[0294] [ka] (5-Bromobenzofuran-2-yl)methanol (100 mg, 0.44 mmol) and 76 (106 mg, 0.52 mmol) were dissolved in THF (2 mL). The resulting mixture was stirred at room temperature for 15 min and then at 80 °C for 1 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 89 (5.0 mg, 3%) as a white solid. m / z = 429.10 (M+H).

[0295] (5,7-Dimethoxybenzofuran-2-yl)methyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (90)VG041-05

[0296] [ka] (5,7-Dimethoxybenzofuran-2-yl)methanol 5 (50 mg, 0.24 mmol) and 7-isocyanato-4-methylcoumarin (58 mg, 0.29 mmol) were dissolved in THF (10 mL). The resulting mixture was stirred at room temperature for 16 h. THF was removed in vacuo. The residue was adsorbed onto silica and purified by flash chromatography using CHCl / EtOAc (1:1) as the eluent to give 90 (5.0 mg, 3%) as a white solid. m / z = 410.04 (M+H).

[0297] 8. Extension linker: oxybenzyl ether, carbamate benzyl ether, oxybenzyl carbamate

[0298] [ka]

[0299] [ka] 4-Methyl-7-(4-naphthalen-1-ylmethoxy)benzyloxy)-2H-chromen-2-one (91) TLE-M1-SU001B

[0300] [ka] A suspension of sodium ethoxide (924 mg, 13.6 mmol) in DMF was stirred at 0° C. for 10 minutes. Ethyl 4-hydroxybenzoate (2.26 g, 13.6 mmol) was slowly added, and the resulting mixture was stirred at this temperature for 0.5 hours and then allowed to reach room temperature. To this mixture, 1-(bromomethyl)naphthalene (2.0 g, 9.0 mmol) [pre-dissolved in DMF (5 mL)] was added dropwise. The resulting mixture was stirred at room temperature for 16 hours. DMF was removed in vacuo, and the residue was dissolved in EtOAc and added with brine, water, and 1M NaOH. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO). The EtOAc was evaporated in vacuo to give (4-(naphthalen-1-ylmethoxy)phenyl)methanol (1.3 g, 4.9 mmol) as crude product, which was used in the next reaction step without further purification.

[0301] (4-(Naphthalen-1-ylmethoxy)phenyl)methanol (1.0 g, 3.8 mmol) was dissolved in toluene (30 mL) and pyridine (305 μL, 3.8 mmol) was added. The solution was cooled to 0 °C. PBr (359 μL, 3.8 mmol) was added dropwise over 15 min. The mixture was allowed to reach room temperature and stirred for 1 h. It was washed with K2CO3 solution and extracted with EtOAc (3 × 30 mL). The EtOAc layer was washed with brine and dried (MgSO4). The solvent was removed in vacuo to give 1-((4-(bromomethyl)phenoxy)methyl)naphthalene (660 mg, 53%). This intermediate was used in the next reaction.

[0302] Sodium ethoxide (156 mg, 2.29 mmol) was added to DMF at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (403 mg, 2.29 mmol) was added slowly, and the resulting mixture was stirred for 0.5 h and then allowed to reach room temperature. To this mixture, 1-((4-(bromomethyl)phenoxy)methyl)naphthalene (500 mg, 1.53 mmol) was added in small portions. The resulting mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine (2 × 50 mL), water (2 × 50 mL), and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4) and purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 91 (200 mg, 31%) as a white solid.

[0303] [ka] 7-(4-(benzhydryloxy)benzyloxy)-4-methyl-2H-chromen-2-one (92) TLE-M1-SU004B

[0304] [ka] Sodium ethoxide (661 mg, 9.7 mmol) was added to DMF (5 mL) at 0 °C. The resulting suspension was stirred for 15 min. Ethyl 4-hydroxybenzoate (1.61 g, 9.7 mmol) was slowly added, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. To this mixture, diphenylmethyl bromide (2.0 g, 8.1 mmol) was added in small portions. The resulting reaction mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine, water, and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4), and the solvent was removed in vacuo to give ethyl 4-(benzhydryloxy)benzoate (1.0 g, 3.0 mmol). This was then dissolved in THF (5 mL), and LiAlH4 (114 mg, 3.0 mmol) was added in small portions with vigorous stirring. The suspension was stirred at room temperature for 3 hours. The THF was removed in vacuo. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO). The EtOAc was removed in vacuo to give (4-(benzhydryloxy)phenyl)methanol (760 mg, 2.62 mmol) as a crude product, which was used in the next reaction step without further purification.

[0305] (4-(benzhydryloxy)phenyl)methanol (500 mg, 1.72 mmol) was dissolved in toluene (20 mL) and pyridine (139 μL, 1.72 mmol) was added. The solution was cooled to 0°C. PBr3 (163 μL, 1.72 mmol) was added dropwise over 15 minutes. The resulting mixture was stirred at room temperature for 1 hour. It was then washed with saturated K2CO3 solution and extracted with EtOAc (3 × 30 mL). The EtOAc layer was washed with brine and dried (MgSO4). The solvent was evaporated in vacuo to give ((4-(bromomethyl)phenyl)methanol. (Hexoxymethylene)dibenzene was obtained as an oil (450 mg, 74%). This intermediate was used in the next reaction.

[0306] Sodium ethoxide (87 mg, 1.28 mmol) was added to DMF (3 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (225 mg, 1.28 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. To this mixture, ((4-(bromomethyl)-phenoxy)methylene)dibenzene (300 mg, 1.53 mmol) was added in small portions. The resulting mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine (2 × 50 mL), water (2 × 50 mL), and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4) and purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 92 (80 mg, 21%) as a white solid.

[0307] [ka] 7-(4-(benzofuran-2-ylmethoxy)benzyloxy)-4-methyl-2H-chromen-2-one (94)SU010B-02

[0308] [ka] Ethyl 4-(benzofuran-2-ylmethoxy)benzoate (93) Sodium ethoxide (580 mg, 8.5 mmol) was added to DMF (10 mL) at 0 °C. The resulting suspension was stirred for 15 min. Ethyl 4-hydroxybenzoate (1.4 g, 8.5 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. To this mixture, 10 (1.5 g, 7.1 mmol) predissolved in DMF (5 mL) was added dropwise. The resulting reaction mixture was stirred at room temperature for 2 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine, water, and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4), and the solvent was removed in vacuo to give 93 as a white solid (920 mg, 44%).

[0309] [ka] 7-(4-(benzofuran-2-ylmethoxy)benzyloxy)-4-methyl-2H-chromen-2-one (94)U010B-02 Compound 93 (400 mg, 1.29 mmol) was dissolved in THF (15 mL), and LiAlH4 (49 mg, 1.29 mmol) was added portionwise with vigorous stirring. The suspension was stirred at room temperature for 1 h. THF was removed in vacuo. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO4). EtOAc was removed in vacuo to give (4-(benzofuran-2-ylmethoxy)phenyl)methanol (220 mg, 67%) as crude product, which was dissolved in toluene (10 mL). The solution was cooled to 0 °C. PBr3 (98 μL, 1.04 mmol) was added dropwise over 15 min. The resulting mixture was stirred at room temperature for 1 h. It was then washed with saturated K2CO3 solution and extracted with EtOAc (3 × 30 mL). The EtOAc layer was washed with brine and dried (MgSO4). The solvent was removed in vacuo to give 2-((4-(bromomethyl)phenoxy)methyl)benzofuran as a colorless oil (132 mg), which was used in the next reaction.

[0310] Sodium ethoxide (43 mg, 0.63 mmol) was added to DMF at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (110 mg, 0.63 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. 93 (132 mg, 0.42 mmol) was added portionwise to the mixture. The resulting reaction mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine (2 × 50 mL), water (2 × 50 mL), and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4) and purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 94 (80 mg, 47%) as a white solid.

[0311] [ka] Naphthalen-1-ylmethyl 4-((4-methyl-2-oxo-2H-chromen-7yloxy)methyl)phenylcarbamate (95)VG021-03

[0312] [ka] To a stirred solution of naphthalenemethanol (4.0 g, 25.3 mmol) in THF (30 mL) was added TEA (100 μL). To this was added dropwise ethyl cyanobenzoate (4.0 g, 21.0 mmol) predissolved in THF (10 mL). The resulting solution was stirred at room temperature for 16 h. The solvent was evaporated to give the crude intermediate ethyl 4-((naphthalen-1-ylmethoxy)carbonylamino)benzoate (1.3 g). This was then dissolved in THF (15 mL) and LiAlH4 (141 mg, 3.75 mmol) was added portionwise with vigorous stirring. The suspension was stirred at room temperature for 1 h. The THF was evaporated in vacuo. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO4). The EtOAc was removed in vacuo to give crude naphthalen-1-ylmethyl 4-(hydroxymethyl)phenylcarbamate (500 mg, 1.62 mmol), which was dissolved in toluene (10 mL). The solution was cooled to 0 °C. PBr (154 μL, 1.62 mmol) was added dropwise over 15 min. The resulting mixture was stirred at room temperature for 1 h. The solvent was removed in vacuo to give naphthalen-1-ylmethyl 4-(bromomethyl)phenylcarbamate as an oil (300 mg). This intermediate was used in the next reaction without further purification.

[0313] Sodium ethoxide (44 mg, 0.65 mmol) was added to DMF at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (114 mg, 0.70 mmol) was added slowly, and the mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. To this mixture, 2-((4-(bromomethyl)phenoxy)methyl)benzofuran (200 mg, 0.42 mmol) was added in small portions. The resulting mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with brine (2 × 50 mL), water (2 × 50 mL), and 1 M NaOH (2 × 30 mL). The organic layer was dried (MgSO4) and purified by flash chromatography eluting with hexane:EtOAc (2:1) to give 95 (160 mg, 63%) as a white solid.

[0314] [ka] 4-(Benzofuran-2-ylmethoxy)benzyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (96)SU024-1-03)

[0315] [ka] Compound 93 (300 mg, 1.01 mmol) was dissolved in THF (15 mL) and LiAlH (38 mg, 1.01 mmol) was added portionwise with vigorous stirring. The suspension was stirred at room temperature for 1 h. The THF was removed in vacuo. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO). The EtOAc was removed in vacuo to give (4-(benzofuran-2-ylmethoxy)phenyl)methanol (150 mg, 0.59 mmol) as a crude product, which was used in the next step without further purification.

[0316] Sodium ethoxide (40 mg, 0.59 mmol) was added to DMF at 0 °C, and the suspension was stirred for 10 min. (4-(benzofuran-2-ylmethoxy)phenyl)methanol (150 mg, 0.59 mmol) was added, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. To this mixture was added 76 (130 mg, 0.65 mmol) in small portions. The resulting mixture was stirred at room temperature for 2 h. DMF was evaporated in vacuo. The crude residue was purified by flash chromatography eluting with hexane:EtOAc (2:1) to give the desired compound (20 mg, 8%) as a white solid.

[0317] [ka] 7-(4-((5-methoxybenzofuran-2-yl)methoxy)benzyloxy)-4-methyl-2H-chromen-2-one (97)VG040-05

[0318] [ka] Sodium ethoxide (62 mg, 0.90 mmol) was added to DMF (3 mL) at 0 °C. The resulting suspension was stirred for 15 min. Ethyl 4-hydroxybenzoate (148 mg, 0.90 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h and then allowed to reach room temperature. To this mixture was added 26 (180 mg, 0.75 mmol). The resulting reaction mixture was stirred at room temperature for 1 h. DMF was evaporated in vacuo to give the crude intermediate (150 mg). This was then dissolved in THF (5 mL), and LiAlH4 (34 mg, 0.90 mmol) was added in small portions with vigorous stirring. The suspension was stirred at room temperature for 1 h. THF was evaporated in vacuo. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO4). The EtOAc was removed in vacuo to give (4-((5-methoxybenzofuran-2-yl)methoxy)phenyl)methanol (120 mg) as a crude product, which was dissolved in toluene (4 mL). The solution was cooled to 0 °C. PBr3 (84 μL, 1.04 mmol) was added dropwise over 5 min. The resulting mixture was stirred at room temperature for 1 h. The solvent was removed in vacuo to give 2-((4-(bromomethyl)phenoxy)methyl)-5-methoxybenzofuran as a colorless oil (90 mg). This intermediate was used in the next reaction.

[0319] Sodium ethoxide (22 mg, 0.31 mmol) was added to DMF (2 mL) at 0 °C, and the suspension was stirred for 10 min. 7-Hydroxy-4-methylcoumarin (54 mg, 0.31 mmol) was added slowly, and the resulting mixture was stirred at this temperature for 0.5 h. To this mixture, 2-((4-(bromomethyl)phenoxy)methyl)-5-methoxybenzofuran (90 mg, 0.23 mmol) was added in small portions. The resulting reaction mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was purified by flash chromatography using hexane:EtOAc (1:1) as an eluent to give 94 (22 mg, 7%) as a white solid. m / z = 443 (M+H).

[0320] 4-(Benzhydryloxy)benzyl 4-methyl-2-oxo-2H-chromen-7-ylcarbamate (98)SU032-02

[0321] [ka] Sodium ethoxide (300 mg, 4.05 mmol) was added to DMF (10 mL) at 0°C. The resulting suspension was stirred for 10 min. Ethyl 4-hydroxybenzoate (739 mg, 4.05 mmol) was slowly added, and the resulting mixture was stirred at this temperature for 20 min. Diphenylmethyl bromide (1.0 g, 4.05 mmol) was added in small portions to the mixture. The resulting reaction mixture was stirred at room temperature for 16 h. DMF was removed in vacuo, and the residue was dissolved in EtOAc and washed with water and brine. The organic layer was dried (MgSO4), and the solvent was removed in vacuo to give ethyl 4-(benzhydryloxy)benzoate (830 mg). This was then dissolved in THF (5 mL), and LiAlH4 (114 mg, 3.0 mmol) was added in small portions with vigorous stirring. The suspension was stirred at room temperature for 3 h. THF was removed in vacuo. The crude residue was dissolved in EtOAc, washed with water, brine, and dried (MgSO). The EtOAc was removed in vacuo to give (4-(benzhydryloxy)phenyl)methanol (760 mg, 2.62 mmol) as crude product, which was used in the next reaction step without further purification.

[0322] A solution of (4-(benzhydryloxy)phenyl)methanol (100 mg, 0.34 mmol) and 76 in toluene (10 mL) was refluxed for 2 h. The reaction was cooled to room temperature, and the resulting precipitate was filtered and washed with cold ether and EtOAc to give 98 (100 mg, 60%) as a white solid. m / z = 491.55 (M+H).

[0323] biological activity Example 1 CYP1B1 metabolism of prodrugs Effect of Substituents on the Cleavage of Benzofuran Ether- and Carbamate-Linked Coumarins by CYP1 Isoenzymes and Human Liver Microsomes (HLM) Commercially available Supersomal™ CYP1A1, CYP1A2, CYP1B1, and pooled human liver microsomes (supplied by BD Gentest, Oxford, UK) comprised an enzyme screen that, relative to cytochrome P450 enzymes expressed in normal tissues, including the liver, defined structure-activity relationships (SARs) underlying the structural features controlling the efficiency and selectivity of prodrug cleavage by CYP1B1 expressed in cancer. HLMs were derived from human patient livers and, according to the supplier, do not contain CYP1A1 or CYP1B1 but do contain a panel of cytochrome P450s, including CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP4A.

[0324] Exemplary Supersomal™ CYP1A1, CYP1A2, CYP1B1 enzymes In metabolic studies of the element, 10 mmol dm -3 10 pmol enzyme, 100 μmol dm -3 The Supersomal™ enzyme metabolism was performed by adding a stock solution of the prodrug in DMSO to a final concentration of 10 μmol dm -3 The HLM screen was initiated by adding 10 mmol of prodrug and 0.5% DMSO at 37°C, pH 7.4. dm -3 60 microliters of microsomes, 100 μmol dm -3 of NADPH was used in a total reaction volume of 1.5 ml.

[0325] The compounds of the present invention comprise a series of heteroaromatic triggers attached to the hydroxyl group of 7-hydroxy-4-methylcoumarin and to ether and carbamate linkers to 7-amino-4-methylcoumarin, respectively. Another example of the present invention is where the heteroaromatic trigger is attached to the hydroxyl group of 7-hydroxy-4-methylcoumarin via a so-called extended oxybenzyl ether linker (-Ar-CH(Z)). 7 )X 3

[0023] Another example of the invention includes compounds in which a heteroaromatic trigger is attached to the amino group of a 7-amino-4-methylcoumarin via a so-called extended oxybenzyl carbamate linker. Another example of the invention includes compounds in which a heteroaromatic trigger is attached to the hydroxyl group of a 7-hydroxy-4-methylcoumarin via a carbamate benzyl ether linker.

[0326] Both 7-hydroxy-4-methylcoumarin and 7-amino-4-methylcoumarin are partially deprotonated at physiological pH 7.4, and both coumarin anions are highly fluorescent with fluorescence emission maxima of 450 nm and 445 nm, respectively. When the coumarin is attached to a heteroaromatic trigger via a linker described in this invention, the fluorescence of the coumarin anion is quenched. Therefore, the enzymatic hydroxylation of the heteroaromatic trigger and the resulting cleavage of the linker can be monitored in real time by the release of the coumarin anion via kinetic fluorimetry. This prodrug design strategy has been successfully used to monitor the cleavage of so-called bioreductive hypoxia-activated prodrugs by P450 reductases, not to be confused with the monooxygenase enzyme CYP1B1 (see, e.g., Everett SA et al., "Modifying the Fluorescence of Heteroaromatic Triggers," vol. 1, no. 2, pp. 111-114, 2002). rates of reductive elimination of leaving groups from indolequinone prodrugs: a (See, "A key factor in controlling hypoxia-selective drug release," Biochem Pharmacol., 63:1629-39, 2002).

[0327] The release of coumarin anion, an indicator of linker cleavage, was monitored in a Cary Eclipse kinetic fluorometer using a 1 cm pathlength fluorescence cell with excitation and emission slits set at 5 nm. The release of coumarin anion from the compounds of the invention was monitored at an excitation wavelength λ ex = 350 nm and emission wavelength λ em The change in fluorescence intensity was detected at 450 nm using the same instrument settings as for enzyme metabolism, with a concentration of 10 mmol dm -3 The coumarin concentration in potassium phosphate buffer (pH 7.4) was 0 to 3.5 μmol dm -3 ) and quantified by fitting to a linear calibration plot of fluorescence intensity against .

[0328] The specific cleavage activities (pmol coumarin / min / pmol cytochrome P450) for the cleavage and release of coumarin from benzofuran ether-linked and carbamate-linked coumarins activated by CYP1 isoenzymes and HLM are shown in Table 3. The presence of electron-donating (Me, MeO) or electron-withdrawing (Cl, Br, F) substituents at either or both of the 5- and 7-positions of the benzofuran has a significant effect on both cleavage specificity and efficiency. The 4- and 6-positions of the benzofuran are likely to undergo the enzymatic hydroxylation required to induce ether or carbamate linker cleavage according to the proposed mechanism. Since it is a suitable position, it remains unsubstituted (R 4 and R 6 =H). The effects of substituents at the 5- and 7-positions of the benzofuran and the structure-activity relationships (SAR) that influence cleavage efficiency and selectivity by CYP1 isoenzymes are unpredictable for ether or carbamate linker cleavage.

[0329] Z, which has ether-linked coumarin 3 =H, Z 5 SU10A, where =H (see Table 3 below), is cleaved by CYP1A1, CYP1A2, and CYP1B1, as well as HLM. HLM cleaves at 10 μmol dm -3 Contamination with α-naphthoflavone (a CYP1-selective enzyme inhibitor) inhibited SU10A cleavage, suggesting that CYP1A2 is solely responsible for the HLM-mediated coumarin liberation. Thus, benzofurans are common trigger moieties that can promote the cleavage of ether-linked prodrugs by CYP1 isoenzymes.

[0330] Z 3 =F, Z 5 In VG016-04, where Z =F (see also Table 3 below), the electron-withdrawing substituent on the benzofuran inhibits cleavage of the ether linker by CYP1 isoenzymes and HLM. 3 =MeO, Z 5 In VG035-05 (also see Table 3 below), where =MeO, the electron-donating substituent results in no linker cleavage for CYP1A or HLM, and the ether bond is specifically cleaved by CYP1B1. The specific cleavage activity of VG035-05 by CYP1B1 was 13.65 ± 1.00 pmol coumarin / min / pmol cytochrome P450, the highest efficiency among the various benzofuran ether-linked coumarins investigated (see Table 3 below). Thus, the use of a 5,7-dimethoxybenzofuran moiety can specifically induce cleavage of ether-linked prodrugs by the CYP1B1 enzyme, which is overexpressed in cancer.

[0331] In stark contrast to VG035-04 (containing an ether-linked coumarin), Z (containing the corresponding carbamate-linked coumarin) 3 =MeO, Z 5VG041-05, where =MeO, is selectively cleaved by CYP1A1 (but not by CYP1A2 or CYP1B1), with a specific cleavage activity of 5.51±0.06 pmol coumarin / min / pmol cytochrome P450. According to Table 3 below, all compound examples of structure B containing a carbamate linker are cleaved by CYP1A1 but not by CYP1B1. The only exception is X 3 =MeO, Z 5 = MeO, and exhibited a CYP1B1-specific cleavage activity of 1.53 ± 0.09 pmol coumarin / min / pmol cytochrome P450, which is approximately one-sixth of that of VG027-05, which contains an ether linker.

[0332] Example 2 Combining a model prodrug library with a CYP1B1 substrate prediction model links substrate specificity to prodrug activation and cleavage Conducting a high-throughput screen (HTS) to build a bioactivity dataset for CYP1B1 The target enzyme, CYP1B1, was screened against two commercially available libraries, including the ChemDiv Diversity collection of 50,000 test compounds and the ChemDiv Kinase Targeted collection of 10,000 test compounds, with the goal of identifying underlying structures that drive activity differences and a large bioactivity data set from which to build substrate specificity models. HTS was performed in a miniaturized 384-well format using a liquid handler (Beckman FXp), a bulk dispenser (Matrix Wellmate), and a luminescence plate reader (Molecular Devices Analyst AD plate Reader). P450-Glo™ Assays measure cytochrome P450 activity. This paper provides a luminescent method for measuring CYP1B1 activity. The conventional reaction is performed by incubating the recombinant enzyme, human Supersomal CYP1B1 plus reductase (BD Gentest™, UK), with a luminescent cytochrome P450 substrate, i.e., luciferin 6'-chloroethyl ether (luciferin-CEE), which is a substrate for CYP1B1 but not luciferase. Luciferin-CEE is converted to a luciferin product, which is detected in a second reaction with Luciferin Detection Reagent (CYP1B1 Luminescent Assay Kit, P450-Glo™, Promega, Madison, USA). This reagent simultaneously quenches the cytochrome P450 reaction, generating a stable luminescent signal with a half-life of more than 2 hours. The amount of light produced in the second reaction is proportional to the activity of CYP1B1. The biochemical endpoint is determined by measuring the apparent Km of luciferin-CEE (20 μmol dm -3 The assay was characterized by substrate inhibition of the enzyme (0.5 pmol / well) acting at 100 kJ / min. When performed in a 384-well format, this assay is characterized by excellent Z' factors, typically greater than 0.6 (Z' = 1.0 indicates a perfectly robust and reproducible assay). The negative control was the level of activity defined for the unaltered state of the enzyme target, while the positive control was the level of activity defined for the hit. The negative control contained the CYP1B1 / KPO4 / NADPH / substrate reaction mixture plus 1% DMSO, an equivalent concentration used to solubilize the test compounds. The positive control for the assay was the CYP1B1 / KPO4 / NADPH substrate reaction mixture plus 1% DMSO, a final concentration of 5 μmol dm -3 The test compound was placed in the remaining 320 wells. The definition of a hit was 0.5 μmol dm -3 The test compound is a substrate inhibitor that inhibits CYP1B1 activity by 80 to 100% at a concentration of 100 mg / kg.

[0333] To simplify and integrate the large amount of data to identify SARs from the CYP1B1 HTS, we used Pipeline Pilot (Scitegic, San Diego, USA) with the assistance of computational scientists at UCSF SMDC. This software was used to (1) identify preliminary SAR results for hits versus non-hits, (2) determine the physicochemical properties of the hit population, such as molecular weight, calculated logP, and H-atom donor / acceptor interactions, (3) determine the occurrence of cyclic fragments and functional groups, and (4) define an in silico model for predicting CYP1B1 substrate inhibition as a basis for future prodrug design. Importantly, a significant number of hits (approximately 10%) had molecular weights between 400 and 500, the maximum molecular weight of test compounds available in both compound collections. This information defined the maximum molecular weight for prodrugs that would be acceptable while maintaining CYP1B1 substrate specificity. Structural analysis of the hit scaffolds in SARvision v2 in CHEMAPPS™ (La Jolla, CA, USA) confirmed that the test compounds did not bear the correct functional groups (e.g., hydroxymethyl substituents on the trigger) for direct integration into the coupling chemistry outlined in Scheme 1. However, by identifying cyclic fragments with high occurrence in the hits versus non-hits, it was possible to identify templates for the trigger moiety that could be subsequently functionalized appropriately for coupling reactions.

[0334] In silico models to predict cytochrome P450 substrate inhibition to aid prodrug design A major challenge in prodrug design is defining a strategy that integrates trigger, linker, and effector chemistries while maintaining substrate specificity for the target enzyme. While the CYP1B1 HTS was extremely valuable in identifying potential trigger moieties, the subsequent "hit-to-lead" chemistry incorporating linker and effector drugs sometimes meant that the final prodrug structure might not be optimal for activation by the target enzyme. Two HTs (totaling 60,000 test compounds) Optimal use of the vast amount of structural data from the S screen was achieved by developing an in silico predictive model of cytochrome P450 1B1 substrate inhibition using a Gaussian kernel-weighted k-nearest neighbor (k-NN) algorithm based on Tanimoto's similarity search with extended connectivity fingerprints. Optimal parameters for the CYP1B1 kernel-weighted k-NN model were selected using leave-one-out cross-validation on training sets selected from 45,000 and 9,000 test compounds from the ChemDiv Diverse and Kinase libraries. The remaining test compounds, totaling 6,000, were used as an internal test set to confirm the accuracy of the model in predicting substrate inhibition. Any test compound showing greater than 20% but less than 80% inhibition was designated as unclassified. The model accurately predicted 89% of classified non-substrate inhibitors and 95% of classified substrate inhibitors. The CYP1B1 substrate prediction model protocol was uploaded to Scitegic Web Port to facilitate the coupling of putative prodrug structures with standard chemical drawing packages such as ChemDraw / IsisDraw via an interface.

[0335] Validation of a CYP1B1 substrate prediction model using an external test set of compounds A 384-well stock plate constituting the external test set for the CYP1B1 substrate prediction model was constructed. This stock plate contained (1) known CYP1B1 substrate inhibitors (e.g., tetramethoxystilbene, β-estradiol, α-naphthoflavone, ethoxyresorufin, resveratrol, etc.), (2) compounds that are not CYP1B1 substrate inhibitors (e.g., quinidine (a potent and specific inhibitor of CYP2D6) and sulfaphenazole (a potent and specific inhibitor of CYP2C9)), (3) model prodrugs VG016-05 and VG035-05, and (4) phosphoramidate mustard prodrugs SU025-04 and SU046-04. The external test set storage concentration was 10 mmol dm in DMSO. -3and 0.5 mmol dm using the same method described for the primary CYP1B1 HTS. -3 The percent CYP1B1 substrate inhibition at final concentrations of 0.01% was determined. Experiments were performed in triplicate to obtain the mean % substrate inhibition of CYP1B1 activity ± standard deviation. All external test set structures were submitted as queries to the CYP1B1 substrate prediction model via the Scitegic Web Port to generate predicted % substrate inhibition of CYP1B1 and compared to the actual biochemically measured % substrate inhibition.

[0336] The comparative CYP1B1 observed and predicted % substrate inhibition values ​​were as follows:

[0337] [ka] The CYP1B1 substrate prediction model was accurately predictive of % CYP1B1 substrate inhibition across several classes of compounds, with model validation using an external test set of compounds to confirm broad activity. Importantly, in terms of inventive step, the model was able to accurately predict the activity of two model prodrugs, VG016-05 and VG035-05, with respect to CYP1B1 substrate inhibition, which can be directly correlated to the efficiency of prodrug cleavage and the release of the 7-hydroxy-4-methylcoumarin anion. According to Table 3, the R of the benzofuran trigger 5 and R 7 Electron-donating or electron-withdrawing substituents in R trigger aromatic hydroxylation and cleavage of these model prodrugs. 5 and R 7 When R =F, an electron-withdrawing substituent, the model prodrug is not activated by CYP1B1, as predicted, resulting in no linker cleavage. In sharp contrast, when R =F, as in VG035-05, 5 and R 7When =MeO, an electron-donating substituent, the model prodrugs were activated by CYP1B1 as predicted, and the linker was cleaved with good efficiency. Incorporation of a dimethoxybenzofuran trigger moiety into the phosphoramidate mustard prodrugs SU025-04 and SU046-04 produced compounds that were accurately predicted to be good substrate inhibitors of CYP1B1. In conclusion, combining a model prodrug library with a CYP1B1 substrate prediction model based on a database of CYP1B1 biological activity facilitates the design of specific prodrugs activated by CYP1B1.

[0338] Example 3 Prodrug cytotoxicity in wild-type CHO cells and CHO cells engineered to express CYP1A1 and CYP1B1 isoenzymes. Engineered CHO cells were used to demonstrate selective cell killing mediated by CYP1 expression. In the experiments described below, compounds were exposed to wild-type CHO cells engineered to express either the CYP1A1 (CHO / CYP1A1) or CYP1B1 (CHO / CYP1B1) enzymes.

[0339] CHO cells: Chinese hamster ovary (CHO) DUKXB cells were cultured in α-MEM supplemented with 10% FCS, 1 unit / ml each of hypoxanthine and thymidine, penicillin (100 IU / ml) and streptomycin (100 μg / ml) according to a literature method (Ding S et al., Arch. Biochem. Biophys. 348:403-410, 1997, the contents of which are incorporated herein by reference). 11 cells were grown under standard cell culture conditions. Cells were grown at 37°C in a humidified atmosphere supplemented with 5% CO2.

[0340] CHO / CYP1A1 and CHO / CYP1B1 cells: P450 reductase-containing CHO cells co-expressing recombinant CYP1A1 and recombinant CYP1B1, i.e., (CHO / CYP1A1) and (CHO / CYP1B1), respectively, were cultured in standard CHO cell medium supplemented with 0.4 mg / ml G418 bisulfate and 0.3 μM methotrexate (Sigma / Aidrich Co., Gillingham, Dorset, UK) according to the method described in the literature (ibid.). Cells were grown at 37°C in a humidified atmosphere containing 5% CO2.

[0341] Recombinant CYP1A1 and CYP1B1 expression Amplification of the dihydrofolate reductase (DHFR) gene from human cDNA CYP1A1 or cDNA CYP1B1 in CHO cells was used to obtain high levels of functional enzyme coexpressed with human P450 reductase (ibid.; Ding S et al., Biochem J., 356(2):613-9, 2001). The modified CYP1A1 or CYP1B1 cDNA was digested and ligated into the mammalian expression vector pDHFR to generate the plasmids pDHFR / 1A1 and pDHFR / 1B1, respectively (ibid.). Cell culture and DNA transfection into CHO DUKXB11 were performed according to published methods, and transfected cells were selected for the DHFR+ phenotype by growth in nucleoside-free medium (ibid.). To amplify the transfected CYP1A1 or CYP1B1 cDNA, DHFR+ clones were pooled and grown at gradually increasing MTX concentrations (0.02–0.1 μM). Cell clones surviving 0.1 mM MTX selection were isolated and further selected with 0.3 μM MTX. The resulting cell lines were analyzed for CYP1A1 or CYP1B1 expression by immunoblotting. Cell lines expressing high levels of each enzyme were stably transfected with the plasmid pcDNA / HR containing the full-length human cytochrome P450 reductase (CPR) cDNA and selected with G418 (0.8 mg / ml) and MTX (0.3 μM) according to the method described previously (ibid.). After isolation of resistant clones, the G418 concentration was reduced to 0.4 mg / ml, and cloning was repeated to ensure cell line homogeneity. The CHO cell line transfected with a plasmid carrying cDNA CYP1A1 and subsequently transfected with CPR cDNA was designated CHO / CYP1A1, and the CHO cell line transfected with a plasmid carrying cDNA CYP1B1 and subsequently transfected with CPR cDNA was designated CHO / CYP1B1.

[0342] Immunochemical detection of CYP1A1 and CYP1B1 Cells were harvested and lysed by sonication using standard methods (Ding S et al., 1997, the contents of which are incorporated herein by reference). Proteins (usually 50 μg of lysate) were separated by SDS / PAGE, transferred to nitrocellulose membranes, and probed using standard methods (Paine MJ et al., Arch. Biochem. Biophys., 328:380-388, 1996, the contents of which are incorporated herein by reference). Human CYP1A1 Plus Reductase Supersomes™, Human CYP1A2 Plus Reductase Supersomes™, and CYP1B1 Plus Reductase Supersomes™ (BD Biosciences, Oxford, UK) were used as positive controls (usually 0.03-0.3 pmol) for immunochemical detection of enzyme expression in cell lines. WB-1 B1 primary antibody (1:1500 dilution, BD Biosciences, Oxford, UK) and anti-CYP1A2 antibody (1:2000 dilution, Cancer Research Technology, London, UK), which cross-reacts with CYP1A1, were used to detect CYP1B1 and CYP1A1 expression, respectively. The secondary antibody was goat anti-rabbit IgG, used at a dilution of 1:500. Enhanced Chemiluminescence (ECL) Immunoblots were developed using a Western blot detection kit (GE Healthcare Life Sciences, Amersham, Buckinghamshire, UK).

[0343] Western blot characterization of CYP1A1 and CYP1B1 expression in engineered CHO cells Figure 1a of the accompanying drawings is a typical Western blot showing the detection of CYP1B1 protein expression in lysates from a CHO / CYP1B1 cell line that is not detectable in either untransfected CHO DUKXB11 cells or the CHO / CYP1A1 cell line. The band corresponds to a molecular weight of 56 kDa and is consistent with the band of the human CYP1B1 Supersomal™ enzyme. Figure 1b is a typical Western blot showing the detection of CYP1A1 protein expression in lysates from a CHO / CYP1A1 cell line that is not detectable in either untransfected CHO DUKXB11 cells or the CHO / CYP1B1 cell line. The band corresponds to a molecular weight of 60 kDa and is consistent with the band of the human CYP1A1 Supersomal™ enzyme detected by cross-reactivity of an anti-CYP1A2 antibody.

[0344] Functional CYP1 enzyme activity The ethoxyresorufin O-deethylation (EROD) assay is widely used to confirm functional CYP1 activity (Chang TK and Waxman DJ, "Enzymatic Analysis of cDNA-Expressed Human CYP1A1, CYP1A2, and CYP1B1 with 7-Ethoxyresorufin as Substrate," Methods Mol. Biol. 320:85-90, 2006, the contents of which are incorporated herein by reference). This assay measures the O-dealkylation of 7-ethoxyresorufin by CYP1A1, CYP1A2, and CYP1B1 to yield the enzyme product resorufin, which is continuously monitored by fluorescence emission at 580 nm. Another assay for measuring enzyme activity is the commercially available Promega P450-Glo™ Assay, which utilizes luciferin-CEE as a luminescent substrate for CYP1 enzyme, as described in Cali JJ et al., Expert. Opin. Drug Metabolism Toxicol., 2(4):629-45, 2006, the contents of which are incorporated herein by reference. The EROD assay and the Promega P450-Glo™ Assay were used with selective and non-selective CYP1 inhibitors to confirm that the CHO cell lines referenced above functionally express the predicted CYP1 enzyme.

[0345] In the absence of inhibitors, CHO / CYP1A1 and CHO / CYP1B1 (but not wild-type CHO cells) converted 7-ethoxyresorufin to resorufin or luciferin-CEE to luciferin, thereby confirming functional CYP1 expression in these cells (see Table 1 below).

[0346] As expected, the addition of the broad-spectrum CYP1 inhibitor α-naphthoflavone abolished activity in both CYP1-expressing cell lines (see Table 1 below). The selective inhibitor tetramethoxystilbene is 30-fold more selective for CYP1B1 than for CYP1A1 (Chun YJ, Kim S, Kim D, Lee SK, and Guengerich FP, "A New Selective and Potent Inhibitor of Human Cytochrome P450 1B1 and Its Application to Antimutagenesis," Cancer Res 61(22):8164-70, 2001). At high concentrations, tetramethoxystilbene abolished activity in both CYP1-expressing cell lines, and at lower concentrations, it preferentially reduced activity in CYP1B1-expressing cells (compared to CYP1A1-expressing cells) (see Table 1 below).

[0347] These results independently confirm that the expression levels of CYP1A1 and CYP1B1 are as expected.

[0348] Cytotoxic IC in CHO, CHO / CYP1A1, and CHO / CYP1B1 cell lines 50 Determining Values Single-cell suspensions of CHO, CHO / CYP1A1, or CHO / CYP1B1 cells in 100 μl of the required cell culture medium were seeded into 96-well plates at a cell density of 1500 cells per well and incubated at 37°C for 24 hours. DMSO stock solutions of test compounds were then added at concentrations ranging from 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0 μM. A final DMSO concentration of 0.2% was found to have no effect on the growth characteristics of the various CHO cell lines. Cells were incubated with test compounds for 72 or 96 hours, after which the media was aspirated and replaced with 100 μl of fresh media to compensate for media loss due to evaporation. Cells were then incubated with 20 μl of MTS assay reagent for 1.5 hours, and the absorbance per well at 510 nm was measured using a plate reader. The mean absorbance and standard deviation for each test compound concentration were calculated for (a) cells plus medium, (b) cells plus medium containing 0.2% DMSO, (c) medium only, and (d) cells plus medium containing 0.2% DMSO and 0–100 μmol dm -3 Cytotoxicity was calculated against a series of controls including medium containing test compound at concentrations ranging from 0.01 to 0.01. 50 Values ​​were calculated by plotting the percentage of cell proliferation (100% cell proliferation corresponds to untreated control cells) against test compound concentration.

[0349] Cytotoxic IC 50 The IC value is defined herein as the concentration of a compound that kills 50% of the cells, and fold selectivity is the IC value in non-CYP1-expressing cells. 50 IC in CYP1A1- or CYP1B1-expressing cells 50 It is calculated by dividing by the differential cytotoxicity IC 50 The ratio is the compound IC in normal CHO cells. 50 IC in CHO cells transfected with CYP1A1 or CYP1B1 50 It is calculated by dividing by .

[0350] Promega™ CellTiter 96® Aqueous Non-Radioactive Cell Proliferation (MTS) Assay The commercially available MTS assay is a homogeneous colorimetric method for determining viable cell numbers in proliferation, cytotoxicity, or chemosensitivity assays. The assay consists of a solution of the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt, MTS] and the electron-coupling reagent PMS (phenazine methosulfate). MTS undergoes bioreduction by cells to a formazan product that is soluble in tissue culture medium. The absorbance of the formazan product at 510 nm can be measured directly from 96-well assay plates. The amount of formazan product, as measured by the amount of absorbance at 490 nm or 510 nm, is directly proportional to the number of viable cells in culture.

[0351] Two compounds of the present invention (SU025-04 and SU046-04) are designed to liberate the phosphoramidate mustards N,N'-bis(2-chloro-ethyl)phosphoramide (Cl-IPM) and N,N'-bis(2-bromo-ethyl)phosphoramide (Br-IPM), respectively, upon activation by CYP1B1. The high toxicity of the two phosphoramidate mustards, Cl-IPM and Br-IPM, is significantly reduced when incorporated into the prodrugs SU025-04 and SU046-04, respectively. Both SU025-04 and SU046-04 exhibited cytotoxic IC values ​​of 1.0 and 1.0 in wild-type CHO cells after 72 or 96 hours of exposure. 50 Value is 10 μmol dm -3 and cytotoxic IC50 in wild-type CHO cells after 72 hours of exposure. 50 The value is 0.007 μmol dm -3This is in stark contrast to Cl-IPM and Br-IPM, which are less than 0.01 (see Table 2 below). The mechanism by which the two prodrugs are activated is reflected in their relative cytotoxic IC in CHO-wild-type (lacking CYP1 enzyme expression), CHO / 1A1, and CHO / CYP1B1 cells. 50 For example, SU025 and SU046 exhibit low toxicity in wild-type CHO cells but are highly toxic to CHO / 1B1 cells, with differential cytotoxicity IC values ​​of 0.05 and 0.05 at 72 h exposure. 50 The ratios are 1689 and 5075, respectively. At a longer exposure time of 96 hours, the CYP1B1-selective prodrugs SU025-04 and SU046-04 are 3367- and 5400-fold more toxic to CYP1B1-expressing cells than non-CYP1B1-expressing cells (see Table 2 below). Thus, compounds SU025-04 and SU046-04 are demonstrated to be CYP1B1-activated prodrugs. SU025-04 and SU046-04 also exhibited similarly low cytotoxicity against wild-type CHO and CHO / CYP1A1 cells, with differential cytotoxicity IC at 72 hours of exposure. 50 The ratio is less than 1, suggesting that highly toxic phosphoramidate mustards are not liberated by CYP1A1 activation (see Table 2 below). As predicted by the literature, ifosfamide and cyclophosphamide, two clinically used prodrugs, similarly yield alkylated isophosphoamidate mustards when activated by CYP2B6 and CYP3A4 but are not activated by CYP1 enzymes (e.g., McFadyen et al., 2004). MC, Melvin WT, and Murray GL, "Cytochrome P450 Enzymes: Novel Options for Cancer Therapeutics," Mol Cancer Ther., 3(3):363-71, 2004. The 100 μmol dm -3 At the highest concentration and longest exposure time of 96 hours, both are non-toxic (again, see Table 2 below).

[0352] Example 4 Cytotoxicity of prodrugs in human primary tumor cell lines Cytotoxicity of prodrugs in a human primary head and neck squamous cell carcinoma tumor cell line (UT-SCC-14) constitutively expressing CYP1B1 Greer et al. reported in Proc. Am. Assoc. Cancer Res., Vol. 45:3701, 2004, that CYP1B1 was overexpressed during malignant progression of head and neck squamous cell carcinoma (HNSCC), but not in normal epithelium. The primary UT-SCC-14 tumor cell line was isolated from a patient with HNSCC (see, e.g., Yaromina et al., Radiother Oncol., Vol. 83:304-10, 2007, and Hessel et al., Int J Radiat Biol., Vol. 80:719-27, 2004). The patient was a 25-year-old man with HNSCC characterized by the following clinicopathological parameters: location: tongue squamous cell carcinoma; T3, N1, M0; site: tongue; lesion: primary; stage: G2. The UT-SCC-14 cell line constitutively expresses CYP1B1 at the mRNA and protein levels and was used to demonstrate the cytotoxicity of compounds in cancer cells derived from human cancers characterized by overexpression of CYP1B1 (Greer et al., Proc. Am. Assoc. Cancer Res. 45:3701, 2004).

[0353] UT-SCC-14 tumor cells: Cultured according to the method described in Hessel et al., Int J Radiat Biol., 80:719-27, 2004, the contents of which are incorporated herein by reference, in a 50 ml suspension containing fetal bovine serum (50 ml), non-essential amino acids (100x, 5 ml), sodium pyruvate (100 mmol dm -3 , 5ml), L-glutamine (200mmol dm -3 HNSCC cell lines were grown under standard cell culture conditions in EMEM (500 ml) supplemented with penicillin 100 IU / ml / streptomycin (100 μg / ml, 5 ml) plus 500 ml of EMEM.

[0354] Cytotoxic IC of prodrugs in primary head and neck tumor cell lines 50 Determine the value Fresh medium was added, if necessary, to a 2000-cell UT-SCC-14 tumor cell suspension per well of a 96-well plate to give a total volume of 100 μl per well. The cells were allowed to attach for 4 hours in an incubator. After 4 hours, cells were confirmed by microscopy to be adherent to the bottom of the 96-well plate, and the medium was then removed and replaced with fresh medium containing a stock solution of test compound to give final concentrations of 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μmol dm in a final volume of 100 μl per well. -3 A final concentration of 0.2% ethanol was found to have no effect on the growth characteristics of the UT-SCC-14 cell line. UT-SCC-14 cells were incubated with test compounds for 72 hours, after which the cells were aspirated and replaced with 100 μl of fresh medium to compensate for medium loss due to evaporation. Cells were incubated with 20 μl of MTS assay reagent for 1.5 hours, and the absorbance per well at 510 nm was measured using a plate reader. The mean absorbance and standard deviation for each test compound concentration were calculated for (a) cells plus medium, (b) cells plus medium containing 0.2% ethanol, (c) medium alone, and (d) cells plus medium containing 0.2% ethanol and 0–100 μmol dm -3 Cytotoxicity was calculated against a series of controls including medium containing test compound at concentrations ranging from 0.01 to 0.01. 50 Values ​​were calculated by plotting the percentage of cell proliferation (100% cell proliferation corresponds to untreated control cells) against test compound concentration.

[0355] Cytotoxic IC 50 The value is defined herein as the concentration of compound that kills 50% of UT-SCC-14 tumor cells. The commercially available MTS assay is a homogeneous colorimetric method for determining viable cell number in proliferation, cytotoxicity, or chemosensitivity assays, and was used as previously described in this Example 3 above.

[0356] Two compounds of the present invention (SU025-04 and SU046-04) are designed to release the phosphoramidate mustards N,N'-bis(2-chloro-ethyl)phosphoramide mustard (Cl-IPM) and N,N'-bis(2-bromo-ethyl)phosphoramide mustard (Br-IPM), respectively, when activated by CYP1B1. Cytotoxic IC values ​​for SU025-04 and SU046-04 in UT-SCC-14 tumor cells after 72 hours of exposure were 50 The values ​​are 0.05 ± 0.01 μmol dm -3 and 0.02 ± 0.01 μmol dm -3 The data demonstrate the potent cytotoxicity of SU025-04 and SU046-04 in the UT-SCC14 cell line from a cancer patient suffering from HNSCC, which overexpresses CYP1B1.

[0357] SU025-04 and SU046-04 were evaluated in three additional primary head and neck cell lines, including UT-SCC-8, UT-SCC-9, and UT-SCC-10, cultured under the same conditions as UT-SCC-14. For SU025-04, cytotoxic IC 50 is μmol dm -3 In units, the cytotoxicity IC of SU025-04 was 0.31 ± 0.06 for UT-SCC-8, 0.43 ± 0.07 for UT-SCC-9, and 0.22 ± 0.03 for UT-SCC-10 after 72 hours of exposure. 50 is μmol dm -3 In units, UT-SCC-8 (0.06 ± 0.02), UT-SCC-9 (0.15 ± 0.02), and UT-SCC-10 (0.09 ± 0.03). The data suggest that across a range of primary head and neck cell lines constitutively expressing CYP1B1, SU046-04 is a more potent cytotoxin than SU025-04.

[0358] One compound of the present invention, SU037-04, was designed to release camptothecin when activated by CYP1B1. SU037-04 had a cytotoxic IC of 0.01 for each primary tumor cell line after 72 hours of exposure. 50 is μmol dm -3 In units, UT-SCC-8 (0.56±0.04), UT-SCC-9 (0.22±0.08), U T-SCC-10 (0.21±0.04), UT-SCC-14 (0.12±0.07).

[0359] One compound of the present invention, SU048-04, was designed to release gemcitabine when activated by CYP1B1. Cytotoxicity IC for SU048-04 in the UT-SCC-14 tumor cell line 50 was 0.94 ± 0.02 μmol dm after 72 h of exposure. -3 10 μmol dm -3 When co-incubated with α-naphthoflavone (a potent CYP1B1 inhibitor), the toxicity of SU048-04 increased to 12.2 ± 0.2 μmol dm -3 significantly decreased, thereby providing indirect evidence that the prodrug was activated by CYP1B1, which is constitutively expressed in the cells.

[0360] Example 5 Antitumor activity of SU046-04 in human primary tumor xenograft models constitutively expressing CYP1B1 Primary UTSCC-14 cell line 3 × 10 6 The tumors were implanted subcutaneously into the flank of nude mice. 3Once the mice reached 100 mg / kg, they were randomized to 10 mice per group. SU046-04 was administered intraperitoneally in PBS at 12, 25, or 50 mg / kg, or vehicle alone, for two cycles of 5 days daily / 2 days off. Tumor volume was measured every 4 days using calipers. Significant inhibition of tumor growth was observed in all three treatment groups compared to vehicle alone. Tumor growth delay at 28 days was 31% at 12 mg / kg, 56% at 25 mg / kg, and 90% at 50 mg / kg, with 4 / 10 complete responses. No adverse effects or significant weight loss were observed after the highest exposure of 250 mg / kg.

[0361] [Table 1]

[0362] [Table 2]

[0363] [Table 3]

[0364] According to a preferred embodiment of the present invention, for example, the following is provided: (Item 1) Compounds of formula (I) [ka] [In the formula, X 1 -X 1 -X 2 But -OX 2 , -SX 2 , -SO2-OX 2 , -SO2NZ 10 -X 2 , conjugated alkene methyloxy, conjugated alkene methylthio, conjugated alkene methylSO2-O, conjugated alkene methyl-SO2NZ 10 , or the following formula [ka] It is something that consists of -X 2 does not exist or is X 1 -X 2 -Effector, [ka] It is like one of each n and m is independently 0 or 1; p is 0, 1 or 2; X 3 is oxygen or sulfur, and when m=0, SO2-O, SO2NZ 10 , conjugated alkene methyloxy, conjugated alkene methylthio, conjugated alkene methyl-SO2-O, or conjugated alkene methyl-SO2NZ 10 But often, Y 1 , Y 2 and Y 3 are each independently carbon or nitrogen, and Y 1 If is nitrogen, Z 1 does not exist, and Y 2 If is nitrogen, Z 3 does not exist, and Y 3 If is nitrogen, Z 5 does not exist, Y 4 is an oxygen, carbon, or nitrogen atom, sulfoxide, or sulfone, -Y 5 - is (i) a single bond, (ii) =CH- (=CH- double bond = is Y 4 or (iii) either -CH2- or -CH2CH2-, or a hydrogen atom of (ii) or one or more hydrogen atoms of (iii) is / are bonded to a substituent Z 11 It is one of (ii) to (iii) replaced by Z 11are independently selected from alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl, nitro, and cyano; Z 1 ~Z 4 each, if present, is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, halo, carboxy, formyl, nitro, and cyano; Z 5 when present, are independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, carboxy, formyl, nitro, and cyano; or Z 2 and Z 3 , Z 3 and Z 4 , and Z 4 and Z 5 together with the atoms to which they are attached form an aromatic ring fused with the rest of the compound, with the proviso that Z 1 , Z 2 and Z 4 at least one of is hydrogen, Z 6 is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, and aralkyl; Y 6 None of the Y atoms may be nitrogen atoms, or 6 one or two of the groups may be nitrogen atoms and the rest may be carbon atoms; each Z 7are independently hydrogen, alkyl, or aryl; each Z 8 are independently selected from hydrogen, an electron-withdrawing group, unsubstituted C1-C6 alkyl, substituted C1-C6 alkyl, unsubstituted C1-C6 alkoxy, and substituted C1-C6 alkoxy, wherein the substituted alkyl or the substituted alkoxy is substituted with one or more groups selected from ether, amino, mono- or di-substituted amino, cyclic C1-C5 alkylamino, imidazolyl, C1-C6 alkylpiperazinyl, morpholino, thiol, thioether, tetrazole, carboxylic acid, ester, amide, mono- or di-substituted amide, N-linked amide, N-linked sulfonamide, sulfoxy, sulfonate, sulfonyl, sulfoxy, sulfinate, sulfinyl, phosphonooxy, phosphate, and sulfonamido; each Z 9 are independently oxygen or sulfur; Z 10 is hydrogen or alkyl, e.g., C 1~4 is alkyl, Effectors are molecules with pharmacological or diagnostic functions. or a pharmaceutically acceptable salt, ester, amide, or solvate thereof. (Item 2) The only Z 7 or each Z 7 is hydrogen, or a pharmaceutically acceptable salts, esters, amides, or solvates thereof. (Item 3) X 1 The compound according to item 1 or item 2, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is oxygen. (Item 4) Y 1 4. The compound according to any one of items 1 to 3, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is carbon. (Item 5) Z 15. The compound according to item 4, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is alkoxy or amino. (Item 6) Z 1 5. The compound according to item 4, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is hydrogen. (Item 7) Z 2 and / or Z 4 7. The compound according to any one of items 1 to 6, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is hydrogen. (Item 8) Y 4 8. The compound according to any one of items 1 to 7, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is nitrogen, oxygen, or sulfur. (Item 9) Y 4 9. The compound according to item 8, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is oxygen or sulfur and p=0. (Item 10) Y 4 10. The compound according to item 9, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is oxygen. (Item 11) -Y 5 11. The compound according to any one of items 1 to 10, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein - is a single bond. (Item 12) Y 2 and Y 3 12. The compound according to any one of items 1 to 11, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein: (Item 13) Z 3 and Z 5 13. The compound according to item 12, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein each is alkoxy or amino. (Item 14) Z 3and Z 5 But, respectively, C 1~6 13. The compound according to item 12, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, which is alkoxy. (Item 15) Z 3 and Z 5 and R are each methoxy; or a pharmaceutically acceptable salt, ester, amide, or solvate thereof. (Item 16) Z 3 is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, alkyloxy, alkenyloxy, alkynyloxy, aryloxy, aralkyloxy, alkylthioxy, alkenylthioxy, alkynylthioxy, arylthioxy, aralkylthioxy, amino, hydroxy, thio, carboxy, formyl, nitro, and cyano; or a pharmaceutically acceptable salt, ester, amide, or solvate thereof. (Item 17) X 1 But -X 1 -X 2 Ga-OX 2 , -SX 2 , -SO2-OX 2 , or -SO2NZ 10 -X 2 17. The compound according to any one of items 1 to 16, wherein or a pharmaceutically acceptable salt, ester, amide, or solvate. (Item 18) X 1 But -X 1 -X 2 Ga-OX 2 18. The compound according to any one of items 1 to 17, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein: (Item 19) X 2 19. The compound according to any one of items 1 to 18, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein (Item 20) X 2 does not exist or X 1 -X 2 -Effector is [ka] 19. The compound according to any one of items 1 to 18, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, consisting of: (Item 21) 21. The compound according to item 19 or item 20, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein one of n and m is 0, or both of n and m are 0. (Item 22) The only Z 9 or each Z 9 22. The compound according to any one of items 19 to 21, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is oxygen. (Item 23) Which Y 6 is not nitrogen or Y 6 23. The compound according to any one of items 19 to 22, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein one of is nitrogen. (Item 24) X 2 19. The compound or a pharmaceutically acceptable salt, ester, amide, or solvate according to any one of items 1 to 18, wherein (Item 25) 25. The compound or a pharmaceutically acceptable salt, ester, amide, or solvate according to any one of items 1 to 24, wherein said effector is a cytotoxic or cytostatic agent. (Item 26) The effector is linked to the remainder of the compound via an oxygen or sulfur atom, and the effector has the formula (II) [ka] [In the formula, Z12 is oxygen or sulfur, each X 4 are independently oxygen, sulfur, or NZ 13 and each -Z 13 are independently -(CH2)2-Z 14 , -alkyl, or -hydrogen; each Z 14 are independently chloro, bromo, iodo, or mesylate] 26. The compound according to item 25, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, consisting of: (Item 27) Z 12 27. The compound according to item 26, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is oxygen. (Item 28) each X 4 NZ 13 28. The compound according to item 26 or 27, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein (Item 29) each Z 13 29. The compound according to item 28, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is hydrogen. (Item 30) each Z 14 30. The compound according to any one of items 26 to 29, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is bromo or chloro. (Item 31) each Z 14 31. The compound according to item 30, or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, wherein is bromo. (Item 32) 32. A composition comprising a compound according to any one of items 1 to 31 or a pharmaceutically acceptable salt, ester, amide, or solvate thereof, together with a pharmaceutically acceptable carrier. (Item 33) 32. A compound according to any one of items 1 to 31 or a pharmaceutically acceptable salt, ester, amide, or solvate thereof for use in medicine. (Item 34) 32. A compound or a pharmaceutically acceptable salt, ester, amide, or solvate according to any one of items 1 to 31 for use in the treatment or prevention of or method for a proliferative condition. (Item 35) The proliferative condition may be pre-malignant or malignant cell proliferation, cancer, leukemia, psoriasis, bone disease, fibrosis, or the like. 35. The compound or a pharmaceutically acceptable salt, ester, amide, or solvate for use in the treatment or prevention or method according to item 34, wherein the disorder is a proliferative disorder, or atherosclerosis. (Item 36) 36. The compound or a pharmaceutically acceptable salt, ester, amide, or solvate for use in the treatment or prevention or method according to item 35, wherein the proliferative condition is selected from cancer of the bladder, brain, breast, colon, head and neck, kidney, lung, liver, ovary, prostate, and skin. (Item 37) 32. A method for treating or preventing a proliferative condition, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a compound or a pharmaceutically acceptable salt, ester, amide, or solvate according to any one of items 1 to 31. (Item 38) 32. Use of a compound or a pharmaceutically acceptable salt, ester, amide, or solvate according to any one of items 1 to 31 for the preparation of a medicament for use in the treatment or prevention or method of a proliferative condition. (Item 39) 25. The compound according to any one of items 1 to 24, wherein the effector is a molecule with diagnostic function. (Item 40) 40. The compound according to item 39, wherein the effector is a fluorophore. (Item 41) 41. The compound according to item 40, wherein the fluorophore is selected from the group consisting of coumarin, resorufin, fluorescein, and rhodamine. (Item 42) 42. The compound according to item 41, wherein the fluorophore is a coumarin. (Item 43) 1. A method for identifying a compound that is specifically activated by a cytochrome P450 enzyme, comprising: (a) contacting a set of compounds according to item 40 with the cytochrome P450 enzyme and determining whether the fluorophore is released from one or more compounds of the set as a result of the contact; (b) contacting the set of compounds with a control tissue, tissue or cell extract, or enzyme, and determining whether the fluorophore is released from one or more compounds in the set as a result of the contacting; (c) identifying the compound that is specifically activated by the cytochrome P450 as any compound in the set of compounds that releases the fluorophore in step (a) but not, or to a much lesser extent, in step (b); A method comprising: (Item 44) 44. The method of claim 43, wherein the compound specifically activated by the P450 enzyme liberates at least 10 times more of the fluorophore in step (a) than in step (b). (Item 45) 45. The method of claim 43 or 44, wherein the set of compounds comprises at least 10 different compounds. (Item 46) 46. ​​The method of any one of items 43 to 45, wherein the set of compounds comprises at least 20 different compounds. (Item 47) (d) generating a model of a compound that is structurally identical to the compound identified in step (c), except that the fluorophore is replaced with a molecule that has pharmacological functionality that binds to the active site of the cytochrome P450 enzyme; (e) synthesizing the compound modeled in step (d) that is predicted to be a substrate for the cytochrome P450 enzyme. (Item 48) 48. The method of any one of items 43 to 47, wherein the cytochrome P450 enzyme is selected from the group consisting of CYP1B1, CYP2S1, CYP2W1, CYP4Z1, and allelic variants thereof. (Item 49) 10. A method for determining whether the compound according to item 1, wherein the effector is a molecule with pharmacological function, is effective in treating cancer, the method comprising administering the compound to an animal having cancer, the cancer resulting from implantation of either recombinant cells engineered to constitutively express a cytochrome P450 enzyme, tissue taken directly from the tumor or cancer, or cells from an early passage cell line derived from tissue taken directly from the tumor or cancer that express the cytochrome P450 enzyme at a level similar to that of the tumor or cancer from which it originated.

Claims

[Claim 1] Pharmacological function of the effector.

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