JP2010511040A - 2-{[2- (substituted amino) ethyl] sulfonyl} ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate - Google Patents

Abstract

2-{[2- (substituted amino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, its production and intermediates in its production, pharmaceutical composition containing it And treatment method using the same. The compounds are useful for the treatment of cancer alone or in combination with other anticancer therapeutic agents.

Description

  The present invention relates to 2-{[2- (substituted amino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, a pharmaceutical composition containing the same, and its pharmaceutical use , And its production and intermediates in its production.

［式中、
Lは電子吸引脱離基であり；
S^xは-S(=O)-、-S(=O)₂-、-S(=NH)-、-S(=O)(=NH)-、-S⁺(C₁-C₆アルキル)-、-Se(=O)-、-Se(=O)₂-、-Se(=NH)-もしくは-Se(=O)(=NH)-または-O-C(=O)-もしくは-HN-C(=O)-であり；
R¹、R²およびR³はそれぞれ独立してHまたは非干渉置換基であり；
nは0、1または2であり；
Yは以下の基：

（式中、mは1または2である）
からなる群から選択され；
AA_cはペプチド結合により化合物の残りの部分と連結したアミノ酸である］
で示される化合物およびそのアミド、エステルおよび塩を開示する。 PCT publication number WO 95/09865 has the formula:

[Where:
L is an electron withdrawing leaving group;
S ^x is -S (= O)-, -S (= O) _2- , -S (= NH)-, -S (= O) (= NH)-, -S ⁺ (C ₁ -C ₆ alkyl ) -, - Se (= O ) -, - Se (= O) 2 -, - Se (= NH) - or -Se (= O) (= NH ) - and -OC (= O) - or -HN -C (= O)-;
R ¹ , R ² and R ³ are each independently H or a non-interfering substituent;
n is 0, 1 or 2;
Y is the following group:

(Where m is 1 or 2)
Selected from the group consisting of:
AA _c is an amino acid linked to the remainder of the compound by a peptide bond]
And the amides, esters and salts thereof are disclosed.

  The compounds are stated to be compatible drugs that contain compatible glutathione S-transferase (GST) isozymes and at the same time are useful in the selective treatment of target tissues that increase the level of granulocyte macrophage progenitor cells in the bone marrow. Specific embodiments disclosed for L include those that produce drugs that are cytotoxic to unwanted cells, such as phosphoramidates and phosphorodiamidate mustards.

で示される。
その特許の中ではTER 286として称されており、γ-グルタミル-α-アミノ-β-((2-エチル-N,N,N,N-テトラ(2'-クロロ)エチルホスホロアミデート)スルホニル)プロピオニル-(R)-(-)-フェニルグリシンと命名される。この化合物は、TLK286と後述されるが、非反転（uninverted）CAS名L-γ-グルタミル-3-[[2-[[ビス[ビス(2-クロロエチル)アミノ]ホスフィニル]オキシ]エチル]スルホニル]-L-アラニル-2-フェニル-(2R)-グリシンを有する。中性化合物として、その推奨国際一般名はカンフォスファミド（canfosfamide）であり；その塩酸塩酸付加塩として、その米国採用名はカンフォスファミド塩酸塩である。カンフォスファミドおよびその塩は、GST P1-1およびGST A1-1の作用により活性化されて細胞毒性ホスホロジアミデートマスタード部分を放出する抗がん化合物である。 One such compound is of the formula:

Indicated by
In that patent, it is referred to as TER 286, γ-glutamyl-α-amino-β-((2-ethyl-N, N, N, N-tetra (2'-chloro) ethyl phosphoramidate)) Named as sulfonyl) propionyl- (R)-(−)-phenylglycine. This compound, described below as TLK286, is uninverted CAS name L-γ-glutamyl-3-[[2-[[bis [bis (2-chloroethyl) amino] phosphinyl] oxy] ethyl] sulfonyl] Has -L-alanyl-2-phenyl- (2R) -glycine. As a neutral compound, its recommended international generic name is canfosfamide; as its hydrochloric acid addition salt, its US adopted name is camfosfamide hydrochloride. Camphosfamide and its salts are anticancer compounds that are activated by the action of GST P1-1 and GST A1-1 to release cytotoxic phosphorodiamidate mustard moieties.

  In vitro, camphosfamide is more potent in the M6709 human colon cancer cell line selected for resistance to doxorubicin and the MCF-7 human breast cancer cell line selected for resistance to cyclophosphamide Both have overexpressed GST P1-1 relative to its parental cell line; M7609 xenografts treated with high, medium and low levels of GST P1-1 in mice And the efficacy of camphosphamide hydrochloride was positively correlated with the level of GST P1-1 (Morgan et al., “Ter286 tumor efficacy and bone marrow preliminary properties, cells activated by glutathione S-transferase Toxin ", Cancer Res., 58, 2568-2575 (1998)).

  Camposfamide hydrochloride is currently being evaluated in several clinical trials for the treatment of ovarian cancer, breast cancer, non-small cell lung cancer and colorectal cancer. It shows significant single drug anti-tumor activity and improved survival in patients with non-small cell lung cancer and ovarian cancer, and single drug anti-tumor activity in colorectal and breast cancer. Evidence from in vitro cell cultures and tumor biopsies has shown that camphosphamide is platinum, paclitaxel and doxorubicin (Rosario et al., “Cell response to glutathione S-transferase P1-1 activated prodrugs”, Mol. Pharmacol., 58, 167-174 (2000)) as well as gemcitabine is shown to be non-cross-linking resistant. Patients treated with camphosphamide hydrochloride show a very low prevalence of clinically significant hematologic toxicity.

［式中、
Lは電子吸引脱離基であり；
S⁺はSまたはSeであり；
S^*は-S(=O)-、-S(=O)₂-、-S(=NH)-、-S(=O)(=NH)-、-S⁺(C₁-C₆アルキル)-、-Se(=O)-、-Se(=O)₂-、-Se(=NH)-、-Se(=O)(=NH)-、-O-C(=O)-または-HN-C(=O)-であり；
R¹、R²およびR³はそれぞれ独立してHまたは非干渉置換基であり；
nは0、1または2であり；
Yは以下：

（式中、mは1または2である）
で示される基からなる群から選択され；
AA_cはペプチド結合により化合物の残りの部分と連結するアミノ酸である］
で示される化合物およびそのアミド、エステルおよび塩である中間体も開示する。 PCT publication number WO 95/09865 also has the formula:

[Where:
L is an electron withdrawing leaving group;
S ⁺ is S or Se;
S ^* is -S (= O) -, - S (= O) 2 -, - S (= NH) -, - S (= O) (= NH) -, - S + (C 1 -C 6 alkyl ) -, - Se (= O ) -, - Se (= O) 2 -, - Se (= NH) -, - Se (= O) (= NH) -, - OC (= O) - or -HN -C (= O)-;
R ¹ , R ² and R ³ are each independently H or a non-interfering substituent;
n is 0, 1 or 2;
Y is:

(Where m is 1 or 2)
Selected from the group consisting of:
AA _c is an amino acid linked to the rest of the compound by a peptide bond]
And the intermediates which are amides, esters and salts thereof.

［式中、
Xはハロゲン原子であり；
QはO、SまたはNHであり；
Rは水素、適宜置換されていてもよい低級アルキル、適宜置換されていてもよいアリールもしくは適宜置換されていてもよいヘテロアリールであるか、またはR'CO-、R'NHCO-、R'SO₂-もしくはR'NHSO₂-であるか（ここに、R'は水素、適宜置換されていてもよい低級アルキル、適宜置換されていてもよいアリールまたは適宜置換されていてもよいヘテロアリールである）；またはR-Qは一緒になって塩素である］
で示される化合物およびその塩を開示する。 PCT Publication No. WO 01/83496 is a formula as an antitumor agent:

[Where:
X is a halogen atom;
Q is O, S or NH;
R is hydrogen, optionally substituted lower alkyl, optionally substituted aryl or optionally substituted heteroaryl, or R′CO—, R′NHCO—, R′SO ₂ —or R′NHSO ₂ — (where R ′ is hydrogen, optionally substituted lower alkyl, optionally substituted aryl, or optionally substituted heteroaryl. ); Or RQ together is chlorine]
And the salts thereof are disclosed.

［式中、
Rはそれぞれ独立して水素、C_1-6アルキルまたは-CH₂CH₂X（ここに、Xはそれぞれ独立してCl、Br、C_1-6アルカンスルホニルオキシ、ハロ-C_1-6アルカンスルホニルオキシ、またはハロ、C_1-3アルキル、ハロ-C_1-3アルキル、C_1-3アルキルオキシもしくはハロ-C_1-3アルキルオキシから選択される3までの置換基で適宜置換されていてもよいベンゼンスルホニルオキシである）であるが、但し、それぞれのホスホロジアミデート基における少なくとも2のRは-CH₂CH₂Xであり；
R¹は適宜置換されていてもよいアルキル、適宜置換されていてもよいヘテロアルキル、適宜置換されていてもよいアリール、適宜置換されていてもよいアラルキル、適宜置換されていてもよいヘテロアリールまたは適宜置換されていてもよいヘテロアラルキルであり；
R²は適宜置換されていてもよいアルカンジイル、適宜置換されていてもよいヘテロアルカンジイル、適宜置換されていてもよいアレーンジイル、適宜置換されていてもよいアレーンジアルキル、適宜置換されていてもよいヘテロアレーンジイルまたは適宜置換されていてもよいヘテロアレーンジアルキルである］
で示される化合物およびその塩を開示する。
式：

で示される化合物はPCT公開番号WO 2005/118601の第38頁に化合物16Aとして開示されている。 PCT Publication Number WO 2005/118601 is a formula as an anti-tumor agent:

[Where:
R is independently hydrogen, C _1-6 alkyl or —CH ₂ CH ₂ X (where X is each independently Cl, Br, C _1-6 alkanesulfonyloxy, halo-C _1-6 alkanesulfonyl) Optionally substituted with up to 3 substituents selected from oxy, or halo, C _1-3 alkyl, halo-C _1-3 alkyl, C _1-3 alkyloxy or halo-C _1-3 alkyloxy Is a good benzenesulfonyloxy) provided that at least 2 R in each phosphorodiamidate group is —CH ₂ CH ₂ X;
R ¹ is an optionally substituted alkyl, an optionally substituted heteroalkyl, an optionally substituted aryl, an optionally substituted aralkyl, an optionally substituted heteroaryl or Is a heteroaralkyl optionally substituted;
R ² is optionally substituted alkanediyl, optionally substituted heteroalkanediyl, optionally substituted arenediyl, optionally substituted arenedialkyl, optionally substituted. Heteroarene diyl or optionally substituted heteroarene dialkyl]
And the salts thereof are disclosed.
formula:

Is disclosed as compound 16A on page 38 of PCT Publication No. WO 2005/118601.

で示される化合物を第26頁に化合物13AAおよび14AAとして開示する。 US Patent Application No. 60/588436 published by PCT Publication No. WO 2005/118601 has the formula:

Are disclosed as compounds 13AA and 14AA on page 26.

で示されるスルホニルエチルホスホロジアミデート類を開示する。
該化合物はベータ-脱離により自発的にホスホラミドマスタードを放出し、インビトロにて対応するホスホラミドマスタードよりもV-79チャイニーズハムスター肺線維芽細胞に対してより強力であると言われている。いくつかの化合物はCD2F1マウスにてP388/0（野生）およびP388/CPA（シクロホスファミド耐性）白血病細胞株に対して優れたインビボ抗腫瘍活性を示すと言われた。 Jain et al., "Sulfonyl-containing aldophosphamide analogues as novel anticancer prodrugs targeted against cyclophosphamide-resistant tumor cell lines", J. Med. Chem., 47 (15), 3843-3852 (2004) is the formula:

Disclosed are sulfonylethyl phosphorodiamidates represented by
The compound spontaneously releases phosphoramide mustard upon beta-elimination and is said to be more potent against V-79 Chinese hamster lung fibroblasts than the corresponding phosphoramide mustard in vitro. Some compounds were said to exhibit excellent in vivo anti-tumor activity against CD388 / 0 (wild) and P388 / CPA (cyclophosphamide resistant) leukemia cell lines in CD2F1 mice.

  It would be desirable to develop chemical and pharmaceutically simple (easy to synthesize and formulate) anticancer agents that are as effective and safe as camfosfamide.

［式中、Rはそれぞれ独立してメチル、エチル、プロピルまたはイソプロピルであるか、または-NR₂は一緒になって1-ピロリジニルまたは1-ピペリジニルである］
で示される化合物およびその酸付加塩である。 DISCLOSURE OF THE INVENTION In a first aspect, the present invention provides a compound of formula A:

[Wherein each R is independently methyl, ethyl, propyl or isopropyl, or —NR ₂ together is 1-pyrrolidinyl or 1-piperidinyl]
And an acid addition salt thereof.

  In a second aspect, the invention is a pharmaceutical composition comprising the compound of the first aspect of the invention.

  In a third aspect, the invention treats cancer by administration of the compound of the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention; alone or in combination with other anti-cancer treatments It is a method to do.

  In a fourth aspect, the invention is a method of making the compounds and intermediates of the first aspect of the invention.

  Definition

  “Acid addition salts” are described in the section entitled “Compounds of the invention”.

“Therapeutically effective amount” means an amount sufficient to affect cancer treatment when administered to a human to treat cancer. "Treatment" or "treating" cancer in humans includes one or more of the following:
(1) Limiting / inhibiting cancer growth, ie limiting / blocking its onset,
(2) Reduction / prevention of cancer expansion, that is, reduction / prevention of metastasis,
(3) Cancer alleviation, that is, cancer regression,
(4) Reduction / prevention of cancer recurrence, and
(5) Alleviation of cancer symptoms.

  “Combination therapy” means administration of the compound of the first aspect of the invention and another anti-cancer therapeutic agent during a series of cancer treatments. Such combination therapy can include administration of the compound of the first aspect of the invention before, during and / or after administration of another anticancer therapeutic agent. Administration of the compound of the first aspect of the invention can precede or follow the administration of another anticancer therapeutic agent, separated by time, up to several weeks, but more generally of the invention Administration of the compound of the first aspect is at least one aspect of another anti-cancer therapeutic agent (such as administration of a dose of a chemotherapeutic agent, molecular targeted therapeutic agent, biological therapeutic agent or radiation therapy) within 48 hours. By the most, it will typically accompany within less than 24 hours.

  “Another anticancer treatment” is an anticancer treatment that is not a treatment with the compound of the first aspect of the invention. Such “another anti-cancer treatments” include chemotherapy; molecular targeted therapy; biological therapy; and radiation therapy. These treatments are those used in single treatment or combination treatment.

Chemotherapeutic agents include:
Alkylating agents (alkyl sulfonates such as busulfan,
Ethyleneimine derivatives such as thiotepa,
Nitrogen mustard such as chlorambucil, cyclophosphamide, estramustine, ifosfamide, mechloretamine, melphalan and uramustine,
Nitrosourea, such as carmustine, lomustine and streptozocin,
Triazene such as dacarbazine, procarbazine and temozolamide, and platinum compounds such as cisplatin, carboplatin, oxaliplatin, satraplatin and picoplatin),
Antimetabolites (antifolates such as methotrexate, permetrexed, raltitrexed and trimetrexate,
Purine analogs such as cladribine, chlorodeoxyadenosine, clofarabine, fludarabine, mercaptopurine, pentostatin and thioguanine,
Pyrimidine analogues such as azacitidine, capecitabine, cytarabine, edatrexate, floxuridine, fluorouracil, gemcitabine and troxacitabine);
Anti-tumours such as natural products (such as bleomycin, dactinomycin, mitomycin, mitomycin, mitoxantrone, porphyromycin, and daunorubicin (such as liposomal daunorubicin), doxorubicin (such as liposomal doxorubicin), anthracycline such as epirubicin, idarubicin and valrubicin Antibiotic agents,
Enzymes such as L-asparaginase and PEG-L-asparaginase,
Microtubule polymer stabilizers such as taxanes paclitaxel and docetaxel,
Mitotic inhibitors such as vinca alkaloids vinblastine, vincristine, vindesine and vinorelbine,
Camptothecin topoisomerase I inhibitors such as irinotecan and topotecan, and topoisomerase II inhibitors such as amsacrine, etoposide and teniposide);
Hormones and hormone antagonists (androgens such as fluokimesterone and test lactones,
Antiandrogens such as bicalutamide, cyproterone, flutamide and nilutamide,
Aromatase inhibitors such as aminoglutethimide, anastrozole, exemestane, formestane and letrozole,
Corticosteroids such as dexamethasone and prednisolone,
Estrogens such as diethylstilbestrol,
Antiestrogens such as fulvestrant, raloxifene, tamoxifen and toremifene,
LHRH agonists and antagonists such as buserelin, goserelin, leuprolide and triptorelin Progestins such as medroxyprogesterone acetate and megestrol acetate, and thyroid hormones such as levothyroxine and liothyronine; and various drugs (altretamine, arsenic trioxide, gallium nitrate) , Hydroxyurea, levamisole, mitotane, octreotide, procarbazine, suramin, thalidomide, lenalidomide, photodynamic compounds such as metoxalene and porfimer sodium, and proteasome inhibitors such as bortezomib).

Molecular targeted therapeutic agents include:
Functional therapeutic agents (gene therapy agents,
Anti-sense therapeutic agent,
Tyrosine kinase inhibitors such as erlotinib hydrochloride, gefitinib, imatinib mesylate and cemaxanib, and retinoids and rexinoids such as adapalene, bexarotene, trans-retinoic acid, 9-cis-retinoic acid and N- (4-hydroxyphenyl) retinamide Gene expression modulators);
Phenotypic targeted therapeutics (monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, ibritumomab tiuxetane, rituximab and trastuzumab,
An antitoxin such as gemtuzumab ozogamicin, and
¹³¹ I-tositumomab); and cancer vaccines.

Biological therapeutic agents include:
Interferons such as interferon-α _2a and interferon-α _2b , and interleukins such as aldesleukin, denileukin diftitox and oprelbekin.

In addition to these drugs that act on cancer cells, anticancer treatments include protective drugs or adjuvants (cytoprotective agents such as amifostine, dexrazoxane and mesna,
Use of phosphonates such as pamidronate and zoledronic acid, and stimulating factors such as epoetin, darbepoetin, filgrastim, PEG-filgrastim and sargramostim.

  The combined cancer treatment plan that can be combined with the compound of the first aspect of the present invention includes two or more anticancer therapeutic agents (anticancer agents) described in the above paragraphs “0022” to “0024” and / or Including all treatment regimes that involve the use of radiation therapy (including the protective and adjuncts described in paragraph “0025” above, as appropriate); the compounds of the first aspect of the invention are described in the literature (Chabner and Longo, eds., "Cancer Chemotherapy and Biotherapy: Principles and Practice", 3rd ed. (2001) and Skele, ed., "Handbook of Cancer Chemotherapy", 6th ed. (2003), both from Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, USA) Can be added to existing anti-cancer treatment plans known for the treatment of various cancers, such as treatment regimens; treatment plans for anti-cancer treatments, especially chemotherapy, are available on the website (National Cancer Institute (www. .cancer.gov), the American Society f or maintained by Clinical Oncology (www.asco.org) and National Comprehensive Cancer Network (www.nccn.org).

  Many combination chemotherapy regimens are known in the art and include platinum compounds and taxane combinations such as carboplatin / paclitaxel, capecitabine / docetaxel, “Cooper regimen”, fluorouracil-levamisole, fluorouracil-leucovorin, methotrexate-leucovorin, etc. , And acronym ABDIC, ABVD, AC, ADIC, AI, BACOD, BACOP, BVCPP, CABO, CAD, CAE, CAF, CAP, CD, CEC, CF, CHOP, CHOP + rituximab, CIC, CMF, CMFP, CyADIC , CyVADIC, DAC, DVD, FAC, FAC-S, FAM-S, FOLFOX-4, FOLFOX-6, M-BACOD, MACOB-B, MAID, MOPP, MVAC, PCV, T-5, VAC, VAD, VAPA , VAP-cyclo, VAP-II, VBM, VBMCP, VIP, VP and the like.

  Combinations of chemotherapy and molecular targeted therapy, biological therapy and radiation therapy are also well known in the art; for example, treatments such as trastuzumab plus paclitaxel, alone or in combination with carboplatin for certain breast cancers, and Many other treatment plans for other cancers; and “Dublin treatment plan” and “Michigan treatment plan” for esophageal cancer, and many other treatment plans for other cancers.

  “Contains” or “contains” and grammatical variations thereof are meant to be inclusive and not limiting, but meant to clarify the presence of the described components, groups, steps, etc., but other components Does not exclude the presence or addition of groups, steps, etc. Thus, “comprising” does not mean “consisting of”, “consisting essentially of” or “consisting solely of”; for example, a pharmaceutical composition “comprising” that a compound does not include that compound Other active ingredients and / or excipients can also be included.

  Compound of the present invention

［式中、Rはそれぞれ独立してメチル、エチル、プロピルまたはイソプロピルであるか、あるいは-NR₂は一緒になってピロリジン-1-イルまたはピペリジン-1-イルである］
で示される化合物およびその酸付加塩である。 In a first embodiment, the present invention provides Formula A:

[Wherein each R is independently methyl, ethyl, propyl or isopropyl, or —NR ₂ together is pyrrolidin-1-yl or piperidin-1-yl]
And an acid addition salt thereof.

Exemplary compounds of the present invention are those wherein each R is independently methyl, ethyl or isopropyl; each R is the same; —NR ₂ is dimethylamino (compound 1A), diethylamino (compound 2A), diisopropylamino (compound 3A), pyrrolidin-1-yl (compound 4A) or piperidin-1-yl (compound 5A) and acid addition salts thereof.

  Acid addition salts (eg, pharmaceutically acceptable acid addition salts) of compounds of Formula A are included in the present invention and are useful in the compositions, methods and uses described herein. Suitable salts are inorganic acids (eg hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and chlorosulfonic acid) or organic acids (eg acetic acid, propionic acid, oxalic acid, malic acid, maleic acid, malonic acid, fumaric acid). Acids, citric acid, tartaric acid, lactic acid, succinic acid and aceturic acid, and alkane- or arenesulfonic acids such as methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, chlorobenzenesulfonic acid and toluenesulfonic acid Such as substituted benzene sulfonic acid, naphthalene sulfonic acid and substituted naphthalene sulfonic acid, naphthalene disulfonic acid and substituted naphthalene disulfonic acid and camphor sulfonic acid) react to form an acid addition salt of the amine group of the compound It is. Such salts are preferably formed with pharmaceutically acceptable acids. See, eg, literature on extensive discussion of pharmaceutical salts, their selection, manufacture and use (Stahl and Wermuth, eds., “Handbook of Pharmaceutically Acceptable Salts”, (2002), Verlag Helvetica Chimica Acta, Zurich, Switzerland). That.

  Compound production

で示されるチオエチルホスホロジアミデートを製造した後、スルフィドを対応するスルホンに酸化し（方法1−合成実施例1〜6に説明）；または
(2) 2-[(2-ヒドロキシエチル)スルホニル]エチル N,N,N',N'-テトラキス(2-クロロエチル)ホスホロジアミデートを製造した後、ヒドロキシ基を脱離基に変換し、式R₂NHのアミンと反応させる（方法2−合成実施例2に説明）
ことにより都合よく製造することができる。 The compound of formula A is
(1) Formula B:

Or the sulfide is oxidized to the corresponding sulfone (as described in Method 1—Synthetic Examples 1-6); or
(2) After producing 2-[(2-hydroxyethyl) sulfonyl] ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, the hydroxy group is converted into a leaving group, Reacting with an amine of formula R ₂ NH (described in Method 2—Synthesis Example 2)
Can be manufactured conveniently.

Method 1 is shown below:

In the first step of Method 1, 2- (NR ₂ ) -ethanethiol is converted to 2-X-ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate (where X Is Cl, Br, C _1-6 alkanesulfonyloxy, halo-C _1-6 alkanesulfonyloxy, or halo, C _1-3 alkyl, halo-C _1-3 alkyl, C _1-3 alkyloxy or halo- as appropriate Elimination of benzenesulfonyloxy optionally substituted with up to 3 groups selected from C _1-3 alkyloxy, such as methanesulfonyloxy, benzenesulfonyloxy, 4-bromobenzenesulfoxy or 4-toluenesulfonyloxy The group is converted to 2-{[2- (NR ₂ ) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate. A typical procedure involves 2- (NR ₂ ) -ethanethiol with a polar solvent such as water, alkanol, dimethylformamide or tetrahydrofuran and a base such as hydroxide, alkoxide, fluoride or hydride or a tertiary amine or amide. Treatment with base to form thiolate anion followed by addition of phosphorodiamidate. 2-{[2- (NR ₂ ) ethyl] thio} ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate is obtained by thiolate substitution of the phosphorylamidate leaving group X. obtain.

Both 2- (dimethylamino) ethanethiol and 2- (diethylamino) ethanethiol are commercially available as hydrochlorides and are readily available. If 2- (NR ₂ ) -ethanethiol is not available, this can be accomplished by reacting 2- (NR ₂ ) -ethyl halide (chloride shown in the reaction scheme) with thiourea to give 2- (NR ₂ ) -ethyl iso It can be produced by methods such as producing thiourea, and can be isolated as an acid addition salt if desired. When 2- (NR ₂ ) -ethylisothiourea is treated with a base, the corresponding 2- (NR ₂ ) -ethanethiolate is formed in the solution and the resulting solution is 2-{[2- (NR ₂ ) Ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate can be used directly. A typical procedure involves treating 2- (NR ₂ ) -ethyl chloride hydrochloride with thiourea at an elevated temperature in a lower alkanol such as ethanol. Upon cooling, isothiourea precipitates as the dihydrochloride salt, which can be isolated by filtration. Isothiourea is suspended in a lower alkanol, treated with a base to form a thiolate anion, and then phosphorodiamidate is added. 2-{[2- (NR ₂ ) ethyl) thio] ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate is obtained by thiolate substitution of the phosphorylamidate leaving group X. obtain.

In the second step of Method 1, 2-{[2- (NR ₂ ) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate is the corresponding 2- { [2- (NR ₂ ) ethyl] sulfonyl} ethyl Oxidized to N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate. This oxidation can be any method known in the art for the oxidation of sulfides to sulfones, such as peracids (peroxycarboxylic acids), persulfates, perborates, peroxides, ozone, iodosyl. It can be carried out by using a reagent, halogen or the like. When using a peracid, a typical procedure is to use 2-{[2- (NR ₂ ) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate in dichloromethane, This involves adding an excess of peracid (eg, peracetic acid) after dissolving in a solvent such as acetic acid or isopropyl acetate at reduced temperature. Oxidation is performed under conditions that minimize oxidation of the amine nitrogen, for example, at a sufficiently low pH, by stabilizing the amine as an ammonium cation.

Method 2 is shown below:

  In the first step of Method 2, 2-mercaptoethanol is converted to 2-X-ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate (where X is (Similar to the first step of method 1) to 2-{[2- (hydroxy) ethyl] thio} ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate Convert. This reaction can be carried out by any method described in the first step of Method 1.

  In the second step of Method 2, 2-{[2- (hydroxy) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate is converted to 2-{[2- (Hydroxy) ethyl] sulfonyl} ethyl oxidizes to N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate. This oxidation can be carried out by any method described in the second step of Method 1 in paragraph “0039”; however, there is no danger of amine oxidation in Method 2.

  In the third step of Method 2, 2-{[2- (hydroxy) ethyl] sulfonyl} ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate hydroxy group is sulfonated Esterification by reaction with a compound which preferably gives a strong leaving group such as trifluoromethanesulfonic anhydride, fluorosulfonic anhydride or pentafluorobenzenesulfonic anhydride. Typically, 2-{[2- (hydroxy) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate is non-nucleophilic, preferably low, such as dichloromethane. Dissolve in the boiling solvent and add the anhydride, for example at 0 ° C. The sulfonate ester can be isolated if desired, but is more conveniently used directly in the next step.

In the fourth step of Method 2, the sulfonate ester is replaced with excess R ₂ NH. Typically, excess amine is added to the solution from the third step and allowed to proceed until the reaction is complete. 2-{[2- (NR ₂ ) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate is conveniently added to the reaction mixture by adding aqueous acid to the organic layer And neutralized with a weak base (such as solid NaHCO ₃ ) and 2-{[2- (NR ₂ ) ethyl] thio} ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiene The amidate is isolated by re-extraction with an organic solvent and removal of the solvent.

  Compounds of formula A can be converted to acid addition salts by reaction with a suitable acid using techniques well known to those skilled in the art for the formation of acid addition salts. The acid used and reaction conditions can be selected to give a pharmaceutically acceptable acid addition salt having a form convenient for isolation and formulation, eg, a solid form (eg, amorphous or crystalline).

  Pharmaceutical composition and administration

  The second aspect of the present invention is a pharmaceutical composition comprising the compound of the first aspect of the present invention and optionally a pharmaceutically acceptable excipient.

  The compounds of the first aspect of the invention can be administered by any route appropriate to the subject being treated and the nature of the condition of the subject. Routes of administration include administration by injection such as intravenous, intraperitoneal, intramuscular and subcutaneous injection, administration by transmucosal or transdermal delivery, topical application, nasal spray, suppository, etc. or orally Can be administered. The pharmaceutical composition may be a liposomal composition, an emulsion, a composition designed to administer the drug through the mucosa, or a transdermal composition as appropriate. Suitable compositions for these modes of administration can be found, for example, in the literature (Gennaro, ed., “Remington: The Science and Practice of Pharmacy”, 20th ed. (2000), Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, USA). it can. A typical composition will be a solution for oral or intravenous infusion. Typical dosage forms include tablets (such as coated tablets and “caplets”) or capsules for oral administration (such as hard gelatin capsules and “soft gels”), intravenous infusion solutions, and intravenous infusion solutions. It will be a solid for reconstitution (especially lyophilized powder).

  Depending on the intended form of administration, the pharmaceutical composition can be in the form of a solid, semi-solid or liquid dosage form, preferably a unit dosage form suitable for single administration of precise dosages. In addition to an effective amount of the active ingredient, the composition may include suitable pharmaceutically acceptable excipients, such as adjuvants, that facilitate the processing of the active compound into a pharmaceutically acceptable formulation. . “Pharmaceutically acceptable excipient” means an excipient or mixture of excipients that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic when administered to a host. To do.

  For solid compositions, conventional excipients include, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, magnesium carbonate and the like. Pharmaceutically administrable liquid compositions are prepared by, for example, dissolving and dispersing the active compounds described herein and any pharmaceutical adjuvants in excipients such as water, saline, aqueous dextrose, glycerol, ethanol and the like. To form a solution or suspension. If desired, the administered pharmaceutical composition may also contain wetting or emulsifying agents, pH buffering agents and the like, for example, small amounts of non-toxic supplements such as sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate. Excipients can also be included.

  For oral administration, the composition generally takes the form of a tablet or capsule, or can be an aqueous or non-aqueous solution, suspension or syrup. Tablets and capsules are the preferred oral dosage forms. Tablets and capsules for oral use will generally contain one or more commonly used excipients, such as lactose and corn starch. Lubricants such as magnesium stearate are also typically added. If a liquid suspension is used, the active agent can be combined with emulsifying and suspending excipients. If desired, further flavoring, coloring and / or sweetening agents may be added. Optional excipients incorporated into other oral compositions include preservatives, suspensions, thickeners and the like.

  Injectable pharmaceutical compositions can be prepared in conventional forms, liquid solutions or suspensions, solid forms suitable for solubilization or suspension in liquid prior to injection, or emulsion or liposome compositions. . The sterile injectable composition may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, fatty esters or polyols are commonly used as a solvent or suspending medium.

  The pharmaceutical composition of the present invention can also be formulated as a lyophilized powder for parenteral administration. The powder can be reconstituted by the addition of water or other primarily aqueous media and then diluted with a suitable diluent before use. The liquid composition is generally a buffered, isotonic aqueous solution. Examples of suitable diluents are saline, aqueous 5% dextrose solution and buffered sodium or ammonium acetate solutions. Pharmaceutically acceptable solid or liquid excipients can be added to enhance or stabilize the composition, or to facilitate manufacture of the composition.

  Typically, the pharmaceutical composition of the invention is packaged in a container with a label or instructions or both indicating the use of the pharmaceutical composition in the treatment of cancer.

  The pharmaceutical composition may contain one or more other pharmacologically active substances in addition to the compound of the present invention. These additional active substances will typically be useful for the treatment of cancer or for enhancing the treatment of cancer with the compounds of the present invention.

  How to use the compound

  The compounds of the first aspect of the invention have activity against human cancer cell lines as described in the in vitro and in vivo examples below, and therefore human cancer for the treatment of human cancer It is considered useful as a chemotherapeutic agent.

  Accordingly, a third aspect of the invention is a method of treating cancer in a human by administering to the human a therapeutically effective amount of a compound of the first aspect of the invention or a pharmaceutical composition of the second aspect of the invention; And the use of a compound of the first aspect of the invention in the manufacture of a medicament for the treatment of cancer in humans. In some cases, the method further includes treatment of the human with another anti-cancer treatment, such as a therapy that is already normal for the cancer being treated.

  Cancers that are particularly treatable by the methods of the present invention are cancers that are sensitive to apoptosis-inducing agents, more specifically those that express, particularly overexpress, one or more glutathione S-transferase isozymes. Cancers that express or overexpress one or more glutathione S-transferase isozymes when treated with other anti-cancer compounds or combination cancer chemotherapy regimens can be specifically treated by the methods of the invention. Such cancers include brain cancer, breast cancer, bladder cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, kidney cancer, lung cancer, liver cancer, ovary Cancer, pancreatic cancer, prostate cancer and gastric cancer; leukemias such as ALL, AML, AMML, CLL, CML, CMML and hairy cell leukemia; Hodgkin and non-Hodgkin lymphoma; mesothelioma, multiple myeloma; and bone and Examples include soft tissue sarcomas. Particularly cancers that can be treated by the methods of the present invention include breast cancer, ovarian cancer, colorectal cancer and non-small cell lung cancer.

  The amount of compound of the first aspect of the invention administered to a human (alone or more usually in the composition of the second aspect of the invention) alone or in combination with another anticancer treatment (of the invention) The therapeutically effective amount used when the compound of the first aspect is administered in combination with another anti-cancer treatment); similarly, the amount of another anti-cancer treatment administered to the mammal ( When the compound of the first aspect of the invention is administered in combination with another anti-cancer therapy) should be a therapeutically effective amount used in combination with the compound of the first aspect of the invention. However, the therapeutically effective amount of the compound of the first aspect of the invention and the amount of another anticancer treatment when administered in combination with cancer chemotherapy are each when delivered alone to a human. It can be less than an amount that would be therapeutically effective. However, it is common in cancer treatment to use the maximum tolerated dose of that or each treatment that is alleviated only by augmenting the general toxicity of the treatment used or the toxicity of one treatment with another treatment . For example, the relative lack of some common chemotherapeutic agents and clinically severe toxicities, especially the lack of camphorsfamide cross-resistance with clinically severe hematologic toxicities Thus, it is anticipated that the compound of the first aspect of the present invention can be administered essentially as its single tolerated dose at its maximum tolerated dose, and no reduction in the amount of another anticancer treatment would be necessary. The

Accordingly, a compound of the first aspect of the invention or a pharmaceutical composition of the second aspect of the invention is a cancer in a human in need of such treatment by administering a therapeutically effective amount of a selected compound or composition. Used to treat. Therapeutically effective amount of the compound of the present invention 10~10,000 mg / m ^2, for example in the range of 30-3000 mg / m ² or 100~1000 mg / m ^2. Dosing every 1 to 35 days; eg once every 2, 3 or 4 weeks, 1 to 5 weeks, especially 1, 2, 3 or 4 weeks or number (eg 5 or 7) once a day More frequently such as about 500-1000 mg / m ² , or continuous infusion for 6-72 hours with repeated doses every ² , 3 or 4 weeks. Appropriate doses and frequency of administration can be readily determined by one skilled in the art and as described herein. When the compounds of the present invention are administered according to the present invention, no unacceptable toxicological effects are expected.

Appropriate administration for other anti-cancer therapies (when combined with the compound of the first aspect of the invention) would be an administration already established for the treatment described in the literature described in paragraph “0026”. . Such administration varies greatly with treatment: for example, capecitabine (2500 mg / m ² oral) twice daily for 2 weeks, not for 1 week, imatinib mesylate (400 or 600 mg oral) daily administration, rituximab weekly administration, paclitaxel (135~175 mg / m ²⁾ and docetaxel administered every (60~100 mg / m ²⁾ every week to 3 weeks, carboplatin (4~6 mg / mL · min) 3 Or once every 4 weeks (but can be divided and administered over several days), a nitrosourea alkylating agent such as carmustine is administered once every 6 weeks. Radiation therapy can be administered weekly (or divided into smaller doses and within daily doses).

  Those skilled in the art of cancer treatment will be able to treat the compounds of the first or second aspect of the invention for a given stage of cancer and disease based on personal knowledge and disclosure of the present application without undue experimentation. An effective amount and a therapeutically effective amount of another anticancer treatment could be ascertained.

  As the combination therapy, the compound of the first aspect of the present invention and a platinum compound such as carboplatin or cisplatin, and further combined with gemcitabine or a taxane such as docetaxel or paclitaxel; combined use with gemcitabine; combined use with taxane; doxorubicin or liposomal doxorubicin A combination with an anthracycline such as: oxaliplatin, optionally further combined with capecitabine or fluorouracil / leucovorin; a combination with gemcitabine or a platinum compound such as carboplatin or cisplatin, and a vinca alkaloid such as vinorelbine.

  Example

  The following examples illustrate the preparation of the compounds of the invention and their activity in predictive in vitro and in vivo anticancer assays.

  Example of synthesis

  The compounds of the present invention are prepared by conventional methods of organic chemistry. See, for example, Larock, “Comprehensive Organic Transformations” (1989), Wiley-VCH, New York, New York, U.S.A. The compounds of the invention can be synthesized according to the synthetic schemes generally described earlier in this application as shown in the following examples, or by modifying the synthesis exemplified by methods known to those skilled in the art. .

  Synthesis Example 1: 2-{[2- (Diisopropylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, compound 3A, its preparation as hydrochloride

2-{[2- (Diisopropylamino) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate. A mixture of 2- (diisopropylamino) ethyl chloride hydrochloride (1.0 g, 5.0 mmol) and thiourea (380 mg, 5.0 mmol, 1.0 eq.) In 4 mL of ethanol was heated to 120 ° C. for 5 minutes and then cooled to room temperature. I let you. The white precipitate of 2- (diisopropylamino) ethylisothiouronium dihydrochloride formed was filtered, washed with ethanol, placed in a round bottom flask containing 50 mL of methanol and sodium hydroxide (5.0 M solution in methanol 5.0 M). mL, 20 mmol, 4.0 eq.). The mixture was stirred at room temperature for 30 min and 2- (4-bromobenzenesulfonyloxy) ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate (7.0 mL of a 0.7 M solution in toluene, 4.9 mmol, 0.98 eq.). All 2- (4-bromobenzenesulfonyloxy) ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate is consumed as measured by HPLC-ELSD (Evaporative Light Scattering Detection) The mixture was stirred until room temperature. The mixture was acidified with 1.0 M aqueous H ₃ PO ₄ to pH 7 and concentrated in vacuo to a high viscosity slurry. The slurry was taken up in 800 mL ethyl acetate and washed with 5% aqueous NaHCO ₃ (2 × 800 mL). The organic layer was separated, dried over anhydrous MgSO ₄ , filtered, concentrated under reduced pressure, 2-{[2- (diisopropylamino) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) ) The phosphorodiamidate was obtained as a clear oil (2.3 g, 86% yield).

2-{[2- (diisopropylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate hydrochloride. 2-{[2- (diisopropylamino) ethyl] thio} ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate (2.3 g, 4.3 mmol) in 30 mL of dimethylformamide The mixture was treated with trifluoroacetic acid (1.6 mL, 21.5 mmol, 5.0 eq.) At 0 ° C., and further stirred at 0 ° C. for 10 minutes. After adding peracetic acid (726 μL of a 32% / wt solution in acetic acid, 10.8 mmol, 2.5 eq.) Over 5 minutes, the mixture was allowed to warm to room temperature. Analysis of aliquots by LC / MS showed 2-{[2- (diisopropylamino) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate 2-{[2- Stirring was continued until complete conversion to (diisopropylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate (typically about 2 hours) was shown. The reaction mixture was diluted with 800 mL ethyl acetate and washed with a 1: 1 mixture of 0.2 M aqueous Na ₂ S ₂ O ₄ and saturated aqueous NaHCO ₃ (2 × 800 mL). The organic layer was separated, dried over anhydrous MgSO ₄ , filtered and treated with excess hydrochloric acid (4.0 M in dioxane). The resulting turbid solution was concentrated under reduced pressure to give 2-{[2- (diisopropylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate hydrochloride as a clear oil As a product (2.53 g, 100% yield). MS (ES ⁺ ): m / z = 564 [C ₁₈ H ₃₉ Cl ₄ N ₃ O ₄ PS + H]; ¹ H NMR (CD ₃ OD): δ = 1.44 (t, J = 6.3 Hz, 12H), 3.41 3.52 (m, 8H), 3.64 3.87 (m, 16H), 4.53 (q, J = 5.5 Hz, 2H); ³¹ P NMR (CD ₃ OD): δ = 18.01.

  Synthesis Example 2: 2-{[2- (Pyrrolidin-1-yl) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, Compound 4A, its hydrochloride As manufacture

Using the procedure of Synthesis Example 1, 2-{[2- (pyrrolidin-1-yl) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate was converted to 1 Prepared from-(2-chloroethyl) pyrrolidine hydrochloride. MS (ES ⁺ ): m / z = 536 [C ₁₆ H ₃₃ Cl ₄ N ₃ O ₄ PS + H]; ¹ H NMR (CD ₃ OD): δ = 2.02 2.09 (m, 2H), 2.15 2.22 (m , 2H), 3.15 3.22 (m, 2H), 3.42 3.53 (m, 8H), 3.68 3.75 (m, 16H), 4.51 (dd, J = 5.5, 5.9 Hz, 2H); ³¹ P NMR (CD3OD): δ = 18.01.

  Synthesis Example 3: 2-{[2- (Piperidin-1-yl) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, Compound 5A, its hydrochloride salt As manufacture

Using the procedure of Synthesis Example 1, 2-{[2- (piperidin-1-yl) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate Prepared from-(2-chloroethyl) piperidine hydrochloride. MS (ES ⁺ ): m / z = 548 [C ₁₇ H ₃₅ Cl ₄ N ₃ O ₄ PS + H]; ¹ H NMR (CD ₃ OD): δ = 1.50 1.58 (m, 1H), 1.74 1.88 (m , 3H), 1.94 2.04 (m, 2H), 3.04 (t, J = 3.1 Hz, 2H), 3.41 3.52 (m, 8H), 3.61 3.77 (m, 16H), 4.51 (dd, J = 5.5, 6.3 Hz , 2H).

  Synthesis Example 4: 2-{[2- (Diethylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, Compound 2A, Preparation as its Hydrochloride

To a 20 mL methanol solution of 2- (diethylamino) ethanethiol hydrochloride (840 g, 5 mmol), NaOH (3.8 mL of a 4M solution in methanol, 15 mmol, 3 eq.) And 2- (4-bromobenzenesulfonyloxy) Ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate (7.0 M solution in toluene 7.0 mL, 4.9 mmol, 0.98 eq.) Was added. The mixture was stirred at room temperature until 2- (4-bromobenzenesulfonyloxy) ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate was consumed by HPLC-ELSD. The mixture was acidified with 1.0 M aqueous H ₃ PO ₄ to pH 7 and concentrated in vacuo to a viscous slurry. The slurry was taken up in 400 mL ethyl acetate and washed with 5% aqueous NaHCO ₃ (2 × 400 mL) followed by aqueous HCl (2 × 400 mL). The acidic aqueous layer was collected, adjusted to pH> 9 with solid NaHCO ₃ and extracted with ethyl acetate (2 × 800 mL). The ethyl acetate solution was separated, dried over anhydrous MgSO ₄ , filtered, concentrated under reduced pressure, 2-{[2- (diethylamino) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) ) The phosphorodiamidate was obtained as a clear oil (1.05 g, 42% yield). 2-{[2- (diethylamino) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate is converted to 2-{[2- (diethylamino) ethyl] sulfonyl} ethyl N , N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate and isolated as the hydrochloride salt using the procedure of Synthesis Example 1. MS (ES ⁺ ): m / z = 536 [C ₁₆ H ₃₅ Cl ₄ N ₃ O ₄ PS + H]; ¹ H NMR (CD ₃ OD): δ = 1.36 (t, J = 7.4 Hz, 6H), 3.28 3.34 (m, 2H), 3.40 3.50 (m, 10H), 3.65 3.77 (m, 14H), 4.51 (dd, J = 5.1, 6.3 Hz, 2H); ³¹ P NMR (CD ₃ OD): δ = 18.02 .

  Synthesis Example 5: 2-{[2- (Dimethylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, Compound 1A, its preparation as hydrochloride

Using the procedure of Synthesis Example 4, 2-{[2- (dimethylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate was converted to 2- (dimethyl Prepared from amino) ethanethiol hydrochloride. MS (ES ⁺ ): m / z = 508 [C ₁₄ H ₃₁ Cl ₄ N ₃ O ₄ PS + H]; ¹ H NMR (CD ₃ OD): δ = 2.98 (s, 6H), 3.30 3.31 (m, 4H), 3.43 3.50 (m, 8H), 3.66 3.77 (m, 14H), 4.51 (dd, J = 5.1, 6.7 Hz, 2H); ³¹ P NMR (CD ₃ OD): δ = 18.08.

  Synthesis Example 6: 2-{[2- (Diethylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, Compound 2A, Preparation as Citrate

2-{[2- (Diethylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate. To a 5 L three-necked flask equipped with an overhead stirrer, thermometer and 1 L addition funnel was added 2- (diethylamino) ethanethiol hydrochloride (170 g, 447 mmol). A solution of NaOH (42.0 g, 1.05 mol) in methanol 480 mL was added and the resulting solution was cooled to 5 ° C. in an ice bath. 2- (4-Bromobenzenesulfonyloxy) ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate (320 g of a 50% solution in toluene, 263 mmol) is placed in a 1 L flask and the solvent Was distilled off under reduced pressure. The resulting oil was dissolved in 320 mL of methanol and added dropwise to the reaction mixture over 50 minutes maintaining the temperature below 10 ° C., after which a white solid precipitate was formed. The obtained white slurry was stirred at 10 to 20 ° C. for 4 hours, and further stirred at room temperature for 2 hours. Disappearance of 2- (4-bromobenzenesulfonyloxy) ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate and 2-{[2- (diethylamino) ethyl by analysis by HPLC-ELSD Based on the appearance of the thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate product, the reaction was shown to be> 95% complete. The reaction mixture was filtered to remove white precipitate, and the solvent was distilled off from the filtrate under reduced pressure. The resulting viscous white oil was dissolved in 3000 mL of ethyl acetate and washed with water (2 × 1500 mL). This solution is a 5 L three-necked flask equipped with an overhead stirrer, thermometer, and 1 L addition funnel containing 1 L water solution of 2KHSO ₅ .KHSO ₄ .K ₂ SO ₄ (Oxone®, 257.0 g, 418 mmol) Put in. Oxone® was added in approximately 250 mL portions over 3 hours and monitored by HPLC-ELSD analysis of the biphasic reaction mixture and 2-{[2- (diethylamino) ethyl] thio} ethyl N, N, N ′ , N'-Tetrakis (2-chloroethyl) phosphorodiamidate to 2-{[2- (diethylamino) ethyl] sulfonyl} ethyl N, N, N ', N'-tetrakis (2-chloroethyl) phosphorodiamidate Converted. Once the reaction was complete, as determined by the disappearance of sulfide and intermediate sulfoxide and the appearance of the product, Na ₂ S ₂ O ₄ (400 mL of 1M aqueous solution) was added to quench the reaction. The organic layer was removed and washed with water (2 × 1 L) and 1M aqueous NaOH (2 × 1 L), then the solvent was distilled off and dried under reduced pressure to give 2-{[2- (diethylamino) ethyl] sulfonyl } Ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate was obtained as a clear oil (128.5 g, 91% yield, 95% purity by HPLC-ELSD).

2-{[2- (Diethylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate citrate. 2-{[2- (diethylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate (90.0 g, 167.5 mmol) in 300 mL of ethyl acetate The acid (1M ethanol solution 167.5 mL, 167.5 mmol) solution was added with vigorous stirring. After stirring at room temperature for 6 hours, the reaction mixture became a beige viscous slurry. Citrate was obtained as a white amorphous powder (80 g, 65.5% yield for the first yield, 98% purity by HPLC-ELSD) by filtration, washing the precipitate with ethyl acetate and drying under high vacuum. MS (ES ⁺ ): m / z = 536 [C ₁₆ H ₃₅ Cl ₄ N ₃ O ₄ PS + H].

  Synthesis Example 7: 2-{[2- (Diethylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate, Compound 2A, as its hydrochloride according to Method 2 Manufacturing

2-{[2- (hydroxy) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate. 2-mercaptoethanol (400 μL, 5.7 mmol) was dissolved in 8 mL of methanol and NaOH in methanol (4M 3.56 mL, 14.25 mmol) was added. The solution was cooled to 0 ° C. and 2- (4-bromobenzenesulfonyloxy) ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate (8.35 mL of a 0.82 M solution in toluene, 6.84 mmol) ) And the reaction mixture was allowed to warm to room temperature. After 12 hours, the mixture was filtered, neutralized to pH 7 with 1M aqueous H ₃ PO ₄ and concentrated in vacuo to a viscous syrup. The syrup was diluted with 200 mL isopropyl acetate and washed with water (3 × 200 mL). The isopropyl acetate layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure to give a clear oil. The oil was purified on a 30 mm × 150 mm silica gel column using a gradient of 70:30 ethyl acetate / hexanes to 100% ethyl acetate to give 2-{[2- (hydroxy) ethyl] thio} ethyl N, N , N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate was obtained as a clear oil (1.41 g, 55% yield). ¹ H NMR (CDCl ₃ ): δ1.64 (bs, 1H), 2.76 (t, 2H, J = 5.9 Hz), 2.84 (t, 2H, J = 6.3 Hz), 3.40 3.47 (m, 8H), 3.62 3.69 (m, 8H), 3.76 (t, 2H, J = 5.9 Hz), 4.18 4.23 (m, 2H); ³¹ P NMR (CDCl ₃ ): δ17.58.

2-{[2- (hydroxy) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate. 2-{[2- (hydroxy) ethyl] thio} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate (4.53 g, 10 mmol) is dissolved in 10 mL of isopropyl acetate, Cooled to 0 ° C. and peracetic acid (32% in acetic acid, 8 mL, 30 mmol) was added over 5 minutes. The reaction mixture was warmed to room temperature and maintained for 3 hours. The reaction mixture was diluted with 200 mL of isopropyl acetate and washed with 0.1M aqueous Na ₂ S ₂ O ₃ (2 × 200 mL) and 200 mL of water. The isopropyl acetate layer was dried over MgSO ₄ , filtered, concentrated under reduced pressure, and 2-{[2- (hydroxy) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiami The date was obtained as a clear oil (3.89 g, 81% yield). MS (ES ⁺ ): m / z = 481 [C ₁₂ H ₂₅ Cl ₄ N ₂ O ₅ PS + H]; ¹ H NMR (DMSO d ⁶ ): δ 3.26 3.36 (m, 12H), 3.67 3.82 (m , 10H), 4.28 (dd, J = 5.9, 6.3 Hz, 2H), 5.19 (t, J = 5.1 Hz, 1H); ³¹ P NMR (DMSO d ⁶ ): δ17.22.

2-{[2- (Diethylamino) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate hydrochloride. 2-{[2- (hydroxy) ethyl] sulfonyl} ethyl N, N, N ′, N′-tetrakis (2-chloroethyl) phosphorodiamidate (150 mg, 0.31 mmol) is dissolved in 2 mL of dichloromethane and 0 Cooled to 0 ° C., N, N-diisopropylethylamine (108 μL, 0.62 mmol) and trifluoromethanesulfonic anhydride (55 μL, 0.33 mmol) were added and the reaction mixture was left for 15 minutes. The reaction mixture was added to diethylamine (64 μL, 0.62 mmol) in 2 mL of isopropyl acetate and allowed to stand for 30 minutes. The reaction mixture was diluted with 10 mL of isopropyl acetate and washed with 5% aqueous NaHCO ₃ (2 × 10 mL) and 1M aqueous HCl (2 × 5 mL). The aqueous acidic fraction was collected and washed with 10 mL of dichloromethane, and solid NaHCO ₃ was added to pH> 9. The mixture was extracted with isopropyl acetate (2 × 10 mL). The isopropyl acetate extracts were collected, dried over MgSO ₄ , filtered, diluted with HCl (4.0 M in dioxane, 1 mL), concentrated in vacuo and 2-{[2- (diethylamino) ethyl] sulfonyl} ethyl N, N , N ′, N′-Tetrakis (2-chloroethyl) phosphorodiamidate hydrochloride was obtained as a light brown oil (32 mg, 19% yield). MS (ES ⁺ ): m / z = 536 [C ₁₆ H ₃₄ Cl ₄ N ₃ O ₄ PS + H].

  Other compounds of formula A can be prepared analogously.

  Using a method similar to that used to produce the citrate salt of Compound 2A, treating Compound 1A with citric acid produced the citrate salt of Compound 1A as a solid and the hydrochloride salt (Synthesis Example 5) was prepared as an oil. Using a method similar to that used to prepare the citrate, reacting compound 2A with the corresponding acid yields the fumarate, tartrate and p-toluenesulfonate of compound 2A as a solid. And maleate, methanesulfonate, phosphate, succinate and sulfate were prepared as oils and hydrochloride (Synthesis Example 4) was prepared as an oil. Citrate, fumarate, hydrochloride, maleate, methanesulfonate, phosphate, succinate and sulfate of compound 3A were converted into oils in the same manner as hydrochloride (Synthesis Example 1). Manufactured. Compound 5A citrate and tartrate were prepared as solids, maleate, succinate and p-toluenesulfonate salts were similarly prepared as oils and hydrochloride (Synthesis Example 3) was prepared as a solid. . Other salts of the compound of formula A can be prepared by using a suitable acid, preferably in a solvent that allows the acid addition salt to be isolated as a solid.

  Compound 2A hydrochloride had a water solubility of at least 50 mg / mL, citrate had a solubility of about 19 mg / mL, and tartrate was slightly more soluble than citrate. The hydrochloride salt of Compound 1A was also highly soluble in water, and the citrate salt was somewhat more soluble than the citrate salt of Compound 2A.

  In vitro Example 1: Cytotoxicity / growth inhibition assay

  The following examples illustrate the beneficial effects of the compounds of the present invention on human cancer cell lines in vitro. These results are expected to predict efficacy in human cancer chemotherapy, as other anticancer agents tested in these assays show anticancer activity in humans.

  Human cancer cell lines DLD-1 (colorectal adenocarcinoma), LNCaP (prostate cancer) and MIA PaCa-2 (pancreatic cancer) were obtained from the American Type Culture Collection, Manassas, Virginia, USA. MX-1 (Breast cancer) was obtained from the National Cancer Institute, Bethesda, Maryland, USA. The CellTiter-Glo assay kit was obtained from Promega Corporation, Madison, Wisconsin, U.S.A. All products were used according to manufacturer's policy. All assays were performed in triplicate wells of dimethyl sulfoxide (DMSO) solvent control. The degree of cell proliferation was expressed as a percentage of signal from the solvent control wells.

Log phase cells were trypsinized, collected by centrifugation, resuspended in a small amount of fresh medium, and the density of viable cells was determined by trypan blue staining. Dilute cells in fresh medium (3 × 10 ³ cells / mL for DLD-1, MIA PaCa-2 and MX-1 and 6 × 10 ³ cells / mL for LNCaP), 96-well plate at 150 μL / well In addition, it was incubated for several hours and allowed to adhere in the case of adherent cells. Compound 1A-5A dissolved in DMSO as its hydrochloride salt is diluted 50-fold with fresh medium and the diluted solution is immediately added to the cell suspension at 50 μL / well to a final compound concentration of 0.1 μM-200 μM and 0.5 % Final DMSO concentration. Cells were cultured for approximately 3 doubling times (3 days for MIA PaCa-2 and MX-1 and 4 days for DLD-1 and LNCaP). Cells were then collected by centrifugation and 100 μL of culture supernatant was replaced with CellTiter-Glo reagent. After 10 minutes incubation at room temperature, the plate was recorded with a luminometer. Many compounds of formula A were tested in this assay and found to be active. The compound was found to be as effective as camfosfamide, and compounds 2A and 3A were more effective in all assays.

The compound of formula A showed the following activity in this assay:

  In vivo example

  In vivo Example 1: MX-1 xenograft assay, intraperitoneal administration

  Female athymic nu / nu mice (Harlan, Indianapolis, Indiana, USA or similar), 6-8 weeks old (approximately 20 g), from similar nu / nu mice previously implanted with MX-1 tumor The collected 20-30 mg pieces of MX-1 tumor were transplanted into the mammary fat pad on the right front flank. Mice were assigned to treatment groups so that each treatment group had a similar average tumor weight at the start of treatment, approximately 7-10 days after tumor implantation when the tumor weight was approximately 50-200 mg. Groups of mice with compounds 2A and 3A (50 mg / Kg) and compounds 1A, 2A, 3A and 5A (100 mg / Kg), in all cases as hydrochloride dissolved in aqueous 5% dextrose, with vehicle control Treated by 1 / day intraperitoneal injection for 5 consecutive days. Tumor volume estimated from volume was measured twice weekly; tumor growth inhibition was measured when the average tumor volume in the control group first exceeded 2000 mg. In this assay, all compounds were compared to vehicle at 70 mg and 23% for compounds 2A and 3A, respectively, and at 100 mg / Kg for compounds 1A, 2A, 3A, and 5A, respectively. Active at 100%, 100%, 100% and 91% tumor growth inhibition.

  In vivo Example 2: MX-1 xenograft assay, oral administration

  Studies similar to those described in Example 1 in vivo were conducted using oral administration of compounds 2A and 3A. Groups of mice are treated with 100, 150, 200 or 300 mg / Kg of Compound 2A or 150 mg / Kg of Compound 3A in each case as a hydrochloride salt dissolved in water once every 5 days with vehicle control. Treated by daily oral gavage. Both compounds were active in this assay and compound 2A resulted in dose-dependent tumor inhibition. Compound 2A resulted in a dose-dependent inhibition of tumor growth from 92% (100 mg / Kg) to 100% (300 mg / Kg) versus vehicle, and Compound 3A resulted in 98% (150 mg / Kg) inhibition .

  In vivo Example 3: MX-1 xenograft assay, intravenous administration

  Studies similar to those described in Example 1 in vivo were performed using intravenous administration of Compound 2A. Groups of mice were treated as hydrochloride salt dissolved in 40% and 80 mg / Kg of compound in aqueous 5% dextrose with vehicle control by single / day tail vein injection for 5 consecutive days. In this assay, Compound 2A was active, resulting in 53% (40 mg / Kg) and 99% (80 mg / Kg) inhibition of tumor growth compared to vehicle.

  In vivo Example 4: MiaPaCa-2 xenograft assay, intraperitoneal administration

  Male athymic nu / nu mice, 6-8 weeks old (approximately 20 g), 20-30 mg pieces of MIA PaCa-2 collected from similar nu / nu mice previously implanted with MIA PaCa-2 tumor Tumors were implanted subcutaneously on the right front flank. Mice were assigned to treatment groups so that each treatment group had a similar average tumor weight at the start of treatment, approximately 7-10 days after tumor implantation when the tumor weight was approximately 50-200 mg. Groups of mice are treated with 100 mg / Kg of compounds 2A, 3A and 5A, in all cases as hydrochloride dissolved in aqueous 5% dextrose, once daily for 7 consecutive days with vehicle control by intraperitoneal injection did. In this assay, all compounds were active with 89%, 89% and 62% inhibition of tumor growth for compounds 2A, 3A and 5A, respectively, compared to vehicle; all animals had some weight loss It was seen.

  All test compounds were safe and nontoxic at the doses tested.

  Formulation and treatment examples

  Formulation Example 1. Pharmaceutical composition for oral administration

Solid pharmaceutical compositions for oral administration include the following substances:
Compound of the present invention 25.0% w / w
Magnesium stearate 0.5% w / w
Starch 2.0% w / w
Hydroxypropyl methylcellulose 1.0% w / w
Microcrystalline cellulose 71.5% w / w
And the mixture is compressed into tablets, or filled into hard gelatin capsules containing, for example, 100 mg of the compound of the invention. If desired, tablets may be coated by applying a suspension of a film former (eg, hydroxypropylmethylcellulose), a dye (eg, titanium dioxide) and a plasticizer (eg, diethyl phthalate) and drying the film by evaporation of the solvent. can do.

  Formulation Example 2. Pharmaceutical composition for IV administration

  A pharmaceutical composition for IV administration is prepared by dissolving the compound of the present invention, for example, as a pharmaceutically acceptable salt in phosphate buffered saline in a concentration of 1% w / v; the solution is sterilized by, for example, sterile filtration, For example, it was sealed in a sterile container containing 100 mg of the compound of the present invention.

  Alternatively, the lyophilized composition may be prepared by dissolving the compound of the present invention again in a suitable buffer such as a pharmaceutically acceptable salt, for example, a phosphate buffer of the above-mentioned phosphate buffered saline, and sterilizing the solution. Prepare by diffusing in a clean sterile vial, lyophilizing the solution to remove water and sealing the vial. The lyophilized composition can be reconstituted by the addition of sterile water and the reconstituted solution can be further diluted for administration by a solution such as 0.9% sodium chloride intravenous infusion or 5% dextrose intravenous infusion.

  Therapeutic example. Treatment with the compounds of the present invention

The compound of the present invention diluted with 5% dextrose intravenous infusion is intravenously administered to patients suffering from metastatic ovarian cancer at an initial dose of 100 mg / m ² over 30 minutes; this dose is 250 mg Increase to / m ² , 500 mg / m ² , 750 mg / m ² and 1000 mg / m ² . The compound is administered at weekly intervals. The same dose increase is administered to other patients suffering from the same cancer at 2 and 3 week intervals.

Claims (18)

Formula A:

[Wherein each R is independently methyl, ethyl, propyl or isopropyl, or —NR _{2 taken} together is pyrrolidin-1-yl or piperidin-1-yl]
Or an acid addition salt thereof.   The compound of claim 1 in solid form.   The compound according to claim 1 or 2, wherein each R is independently methyl, ethyl or isopropyl.   4. A compound according to claim 3, wherein each R is the same.   5. A compound according to claim 4, wherein each R is methyl.   5. A compound according to claim 4, wherein each R is ethyl.   5. A compound according to claim 4, wherein each R is isopropyl.   8. A compound according to any of claims 1 to 7, which is an acid addition salt of a compound of formula A.   9. A compound according to claim 8 in solid form.   A pharmaceutical composition comprising the compound according to any one of claims 1 to 9 and an excipient.   Use of the compound according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 10 in the manufacture of a medicament for the treatment of cancer. Formula A:

[Wherein each R is independently methyl, ethyl, propyl or isopropyl, or —NR ₂ together is pyrrolidin-1-yl or piperidin-1-yl]
A process for producing a compound represented by the formula:
(a) Formula B:

Comprising the step of oxidizing the corresponding compound of formula A to a compound of formula A, and then optionally one or more of the following steps:
(e) forming an acid addition salt of the compound of formula A;
(f) converting an acid addition salt of the compound of formula A to another acid addition salt of formula A; and
(g) A method comprising the step of converting the acid addition salt of the compound of formula A to the non-salt form of the compound of formula A. Formula B:

[Wherein each R is independently methyl, ethyl, propyl or isopropyl, or —NR ₂ together is pyrrolidin-1-yl or piperidin-1-yl]
Or an acid addition salt thereof.   14. A compound according to claim 13, wherein each R is independently methyl, ethyl or isopropyl.   15. A compound according to claim 14, wherein each R is the same.   16. A compound according to claim 15, wherein each R is methyl.   16. A compound according to claim 15, wherein each R is ethyl.   16. A compound according to claim 15, wherein each R is isopropyl.