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    • 专利摘要:
    Optically active compounds of the formulas are described here: II; and III and pharmaceutical compositions thereof. Also described here are the processes for manufacturing these compounds and resolving the racemic mixture or enriching it with one of its enantiomers to produce (R) - and (S) -1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl -4-nitrophenyl) -1-ethyl-N, N'-bis (ethylene) phosphoramidate, and methods of treating cancer comprising administering such compounds.
    公开号:BR112017025778B1
    申请号:R112017025778-5
    申请日:2016-11-15
    公开日:2021-03-16
    发明作者:Jian-Xin Duan；Yeyu Cao；Xiaohong Cai；Hailong Jiao；Jing Yuan Ma；Mark Matteucci
    申请人:Obi Pharma, Inc；
    IPC主号:
    专利说明:

    INVENTION FIELD
    [001] The present invention relates to optically active forms of the compound 1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl-N, N'-bis (ethylene) phosphoramidate suitable as therapeutic agents, pharmaceutical compositions of such compounds and methods of treating cancer, as well as a process for their resolution from, or enrichment in one of its enantiomers, racemic mixture of the compound (R, S) -1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxy-4-nitrophenyl) -1-ethyl- N, N'-bis (ethylene) phosphoramidate, or stereoselective synthesis of (R) and (S) -1- (3- (3-N, N- dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl-N, N'-bis (ethylene) optically pure phosphoramidate. BACKGROUND OF THE INVENTION
    [002] Cancer is a major cause of human morbidity and mortality. Cancer treatment is challenging because it is difficult to kill cancer cells without damaging or killing normal cells. The damage or death of normal cells during cancer treatment is a cause of adverse side effects in patients and can limit the amount of anti-cancer drug administered to a patient. cancer patient.
    [003] The C3 member of the aldo-keto reductase 1 (AKR1C3) family is an enzyme that, in humans, is encoded by the AKR1C3 gene. This gene encodes a member of the aldo / keto reductase superfamily, which consists of more than 40 known enzymes and proteins. These enzymes catalyze the conversion of aldehydes and ketones to their corresponding alcohols using NADH and / or NADPH as cofactors.
    [004] Many cancer cells overexpress AKR1C3 reductase over normal cells (See, for example, Cancer Res. 2010; 70: 1573-1584; Cancer Res. 2010; 66: 28152825). PR 104 has been demonstrated
    as a weak substrate for AKR1C3 and tested in clinical experiments. This compound is not a selective AKR1C3 activated prodrug, as it can also be activated under hypoxic conditions. PR 104 was ineffective in clinical experiments.
    [005] As such, there remains a need for compounds suitable for treating cancer patients, including selective AKR1C3 reductase activated prodrugs to treat cancer patients. The present invention addresses this need. SUMMARY OF THE INVENTION
    [006] In one aspect, compounds of formulas Ia and Ib are described here:
    or an isotopic variant, solvate or hydrate thereof.
    [007] The compounds described here include individual enantiomers, as well as enriched mixtures of enantiomers.
    [008] In another aspect, a pharmaceutical composition comprising a compound described herein and at least one pharmaceutically acceptable excipient is described herein. In another aspect, a unit dose of the pharmaceutical composition described herein is described here.
    [009] In another aspect, a method for treating cancer in a patient is described herein, comprising administering to the patient a therapeutically effective amount of a compound or a pharmaceutically acceptable composition, as described herein. In one embodiment, cancer is one in which the levels of AKR1C3 reductase are elevated or higher than those common in such cancer. In one embodiment, cancer is liver cancer and, more specifically, hepatocellular carcinoma (HCC). In one embodiment, cancer is non-small cell lung cancer or melanoma. In one embodiment, cancer is depressed cancer. In one embodiment, cancer is breast cancer. In one embodiment, cancer is leukemia. In one embodiment, cancer is cancer of the esophagus. In one embodiment, the cancer is renal, gastric, colon, brain, bladder, cervical, ovarian, head and neck, endometrial, pancreatic, sarcoma, or rectal cancer. In another aspect, the method comprises determining the level of cancer AKR1C3 reductase by methods using an AKR1C3 antibody, and administering a therapeutically effective amount of a compound or a pharmaceutically acceptable composition described herein to said patient, if said level is equal to or greater than a predetermined value. In one aspect, the method comprises, prior to administration, determining a level of AKR1C3 intra-tumor reductase in a sample isolated from the patient and selecting the patient for therapy, if the level is equal to or greater than a predetermined level. In some embodiments, a therapeutically effective amount of a cancer treatment other than a treatment comprising administration of a compound or a pharmaceutically acceptable composition described herein is administered if the level does not exceed or is less than said predetermined value. Methods of determining the therapeutically effective amount, appropriate modes of administration of the compounds and compositions described in the document will be apparent to one skilled in the art when reading the description and based on other known methods. AKR1C3 levels are measured after routine methods well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS
    [0010] Figure 1 shows LC chromatogram for the resolution of two (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl-N, N'-bis (ethylene) phosphoramidate by high pressure liquid chromatography in a chiral column CHIRALPAK OZ-H6x250 mm, 5um (Daicel) eluting with 65/35 CO2 / methanol.
    [0011] Figure 2 illustrates the activation of TH 2870 by aldocet reductase, AKR1C3 compared to progesterone.
    [0012] Figure3 illustrates the expression of AKR1C3 in liver cancer cell lines.
    [0013] Figure4 illustrates expression of AKR1C3 in prostate cancer cell lines.
    [0014] Figure 5 illustrates average body weight in each group.
    [0015] Figure 6 illustrates fluorescence images typical of tumor burden in each group.
    [0016] Figure 7 illustrates tumor growth curves in each group.
    [0017] Figure 8 illustrates tumor burden fluorescence images in each group.
    [0018] Figure 9 illustrates average tumor weight in different groups.
    [0019] Figure 10 illustrates changes in body weight in different groups.
    [0020] Figure 11 illustrates the tumor load growth curve in peripheral blood.
    [0021] Figure 12 illustrates the positive percentage of human CD45 antibody in blood, spleen and bone marrow at the end point in different groups. DETAILED DESCRIPTION Definitions
    [0022] The following definitions are presented to assist the reader. Unless otherwise stated, all terms of the technique, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those skilled in the techniques of chemistry and medicine. In some cases, terms with commonly understood meanings are defined here for clarity and / or for immediate reference, and the inclusion of such definitions should not be interpreted as representing a substantial difference from the definition of the term, as generally understood in the art.
    [0023] All numerical designations, for example, pH, temperature, time, concentration, and weight, including ranges for each of them, are approximations that can typically be varied (+) or (-) in increments of 0.1 , 1.0, or 10.0, as appropriate. All numerical designations can be understood as preceded by the term “cercade”. Exemplary reagents described herein and equivalent thereto are known in the art.
    [0024] "One", "one" and "o", "a" include references in the plural, unless the context clearly dictates otherwise. Thus, for example, reference to a compound refers to one or more compounds, or at least one compound. As such, the terms "one" (or "one"), "one or more", and "at least one" are used interchangeably here.
    [0025] The term "about" or "approximately" means an acceptable error for a particular value, as determined by one skilled in the art, which depends, in part, on how the value is measured or determined. In some embodiments, the term "about" or "approximately" means 1,2,3, or 4 standard deviations. In some embodiments, the term "about" or "approximately" means 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, or 0.05% of a given value or range.
    [0026] As used here, the term "comprising" is intended to mean that the compositions and methods include the elements recited, but not excluding others. “Consisting essentially of”, when used to define compositions and methods, must mean excluding other elements of any essential meaning for the composition or method. "Consisting of" should mean excluding more than trace elements from other ingredients for substantial claimed compositions and method steps. Modalities defined by each of these transition terms are within the scope of this invention. Thus, it is intended that the methods and compositions may include additional steps and components (comprising) or, alternatively, including unimportant steps and compositions (consisting essentially of) or, alternatively, targeting only the indicated method steps or compositions (consisting of) .
    [0027] "Departure group" refers to a portion that can be displaced under conditions of nucleophilic displacement well known to those skilled in the art. The leaving groups include, without limitation, halo and -OSO2-R20, where R20 is optionally substituted alkyl, aryl, cycloalkyl, heterocyclyl, or heteroaryl.
    [0028] “Administ rar” or “administration of” a drug to a patient (and grammatical equivalents of the phrase) refers to direct administration, which may include administration to a patient by a healthcare professional or may be self-administration , and / or indirect administration, which may be the act of prescribing a drug. For example, a doctor who teaches a patient to self-administer a drug and / or provides the patient with a prescription for a drug, is administering the drug to the patient.
    [0029] "Cancer" refers to leukemias, lymphomas, carcinomas, and other malignant tumors, including solid tumors, of potentially unrestricted growth, which can expand locally by invasion and systemically by metastasis. Examples of cancers include, but are not limited to, cancer of the adrenal gland, bone, brain, breast, bronchi, colon and / or rectum, gallbladder, head and neck, kidneys, larynx, liver, lung, neural tissue, pancreas, prostate, parathyroid, skin, stomach and thyroid. Some other examples of cancers include, acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, squamous cell carcinomas, giant cell tumor, multiform glioblastoma, hairy cell tumor , intestinal ganglioneuroma, hyperplastic corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemias, lymphomas, malignant carcinoid, malignant melanomas, malignant hypercalcemia, marfanoid habitus tumor, medullary carcinoma, metastatic skin carcinoma, myosomal mucous membrane , mycosis fungoides, neuroblastoma, osteo-sarcoma, osteogen and other sarcoma, ovarian tumor, pheochromocytoma, polythermia vera, primary brain tumor, small cell lung tumor, both ulcerating and papillary squamous cell carcinoma, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomiosarcoma, renal cell tumor, topical skin lesion , ventricular cell sarcoma and Wilm's tumor.
    [0030] The term "contact" or "contact" means to refer to bringing together a therapeutic agent and cell or tissue, so that a physiological and / or chemical effect occurs as a result of such contact. Contact can occur in vitro, ex vivo, or in vivo. In one embodiment, a therapeutic agent is contacted with a cell in cell culture (in vitro) to determine the effect of the therapeutic agent on the cell. In another embodiment, contact of a therapeutic agent with a cell or tissue includes administering a therapeutic agent to an individual having the cell or tissue to be contacted.
    [0031] The terms "optically active" and "enantiomerically active" refer to a collection of molecules, which have an enantiomeric excess of not less than about 10%, not less than about 20%, not less than about 30%, not less than about 40%, not less than about 50%, not less than about 60%, not less than about 70%, not less than about 80%, not less than about 90% , not less than about 91%, not less than about 92%, not less than about 93%, not less than about 94%, not less than about 95%, not less than about 96%, no less than about 97%, not less than about 98%, not less than about 99%, not less than about 99.5%, not less than about 99.8%, or not less than about 99 , 9%. In some embodiments, the enantiomeric excess for an optically or enantiomerically active compound is not less than about 90%, not less than about 95%, not less than about 98%, or not less than about 99%.
    [0032] When writing an optically active compound, the prefixes R and S are used to denote the absolute configuration of the molecule around its chiral center (s). The (+) and (-) are used to denote the optical rotation of the compound, that is, the direction in which a plane of polarized light is rotated by the optically active compound. The prefix (-) indicates that the compound is levorotatory, that is, the compound rotates the plane of polarized light to the left or counterclockwise.The prefix (+) indicates that the compound is dextrorotatory, that is, the compound rotates plane of polarized light to the right or clockwise. However, the optical rotation signal, (+) and (-), is not related to the absolute configuration of the molecule, R and S.
    [0033] The terms "optically pure" and "enantiomerically pure" refer to a collection of molecules, which have an enantiomeric excess (ee) of not less than about 80%, not less than about 90%, not less than about of 91%, not less than about 92%, not less than about 93%, not less than about 94%, not less than about 95%, not less than about 96%, not less than about 97 %, not less than about 98%, not less than about 99%, not less than about 99.5%, not less than about 99.8%, or not less than about 99.9%. In some embodiments, the enantiomeric excess for an optically or enantiomerically pure compound is not less than about 90%, not less than about 95%, not less than about 98%, or not less than about 99%. An enantiomeric excess of a compound can be determined by any of the standard methods used by one skilled in the art, including, but not limited to, chiro-optical chromatography (gas chromatography, high performance liquid chromatography, and thin layer chromatography) using an optically active stationary phase, isotopic dilution, electrophoresis, calorimetry, polarimetry, NMR resolution methods with chiral derivatization, and NMR methods with a chiral solvent or chiral displacement reagent.
    [0034] The terms "substantially pure" and "substantially homogeneous" mean sufficiently homogeneous to appear free of readily detectable impurities, as determined by standard analytical methods, used by one skilled in the art, including, but not limited to, thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC), gas chromatography (GC), nuclear magnetic resonance (NMR), and mass spectrometry (MS); or sufficiently pure so that another purification would not detectably alter the physical, chemical, biological and / or pharmacological properties, such as enzymatic and biological activities, of the substance. In some embodiments, "substantially pure" or "substantially homogeneous" refers to a collection of molecules, in which at least about 50%, at least about 70%, at least about 80%, at least about 90 %, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% by weight of the molecules are a single stereoisomer of a compound, as determined by standard analytical methods .
    [0035] The term "isotopic variant" refers to a compound that contains an unnatural proportion of an isotope in one or more of the atoms that constitute such compounds. In some embodiments, an "isotopic variant" of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen (1H), deuterium (2H), tritium (3H), carbon-11 (11C) carbon-12 (12C), carbon-13 (13C), carbon-14 (14C), nitrogen-13 (13N), nitrogen-14 (14N), nitrogen-15 (15N), oxygen-14 (14O), oxygen-15 ( 15O), oxygen-16 (16O), oxygen-17 (17O), oxygen-18 (18O), fluorine-17 (17F), fluorine-18 (18F), phosphorus-31 (31P), phosphorus-32 (32P ), phosphorus-33 (33P), sulfur-32 (32S), sulfur-33 (33S), sulfur-34 (34S), sulfur-35 (35S), sulfur-36 (36S), chlorine-35 (35Cl) , chlorine-36 (36Cl), chlorine-37 (37Cl), bromine-79 (79Br), bromine-81 (81Br), iodine-123 (123I), iodine-125 (125I), iodine-127 (127I), iodine-129 (129I), and iodine-131 (131I). In some embodiments, an "isotopic variant" of a compound is in a stable, that is, non-radioactive form. In some embodiments, an "isotopic variant" of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen (1H), deuterium (2H), carbon-12 (12C), carbon-13 ( 13C), nitrogen-14 (14N), nitrogen-15 (15N), oxygen-16 (16O), oxygen-17 (17O), oxygen-18 (18O), fluorine-17 (17F), phosphorus-31 (31P ), sulfur-32 (32S), sulfur-33 (33S), sulfur-34 (34S), sulfur-36 (36S), chlorine-35 (35Cl), chlorine-37 (37Cl), bromine-79 (79Br) , bromo-81 (81Br), and iodine-127 (127I). In some embodiments, an "isotopic variant" of a compound is in an unstable, that is, radioactive form. In some embodiments, an "isotopic variant" of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, tritium (3H), carbon-11 (11C), carbon-14 (14C), nitrogen- 13 (13N), oxygen-14 (14O), oxygen-15 (15O), fluorine-18 (18F), phosphorus-32 (32P), phosphorus-33 (33P), sulfur-35 (35S), chlorine-36 (36Cl), iodine-123 (123I), iodine-125 (125I), iodine-129 (129I), and iodine-131 (131I). It will be understood that, in a compound as described herein, any hydrogen can be 2H, for example, or any carbon can be 13C, for example, or any nitrogen can be 15N, for example, and any oxygen can be 18O, where feasible for according to the judgment of a specialist. In some embodiments, an "isotopic variant" of a compound contains unnatural proportions of deuterium.
    [0036] The phrase "an isotopic variant of the same; or pharmaceutically acceptable salt, solvate, hydrate, or prodrug of the same" has the same meaning as the phrase "an isotopic variant of the compound mentioned in the document; or a pharmaceutically acceptable salt, solvate , or prodrug of the compound mentioned in the document or an isotopic variant of the compound mentioned in the document. "
    [0037] "Patient" and "individual" are used interchangeably to mention a mammal in need of treatment for cancer. Generally, the patient is a human. Generally, the patient is a human diagnosed with cancer. In some modalities, a "patient" or "individual" may refer to a non-human mammal used in screening, characterizing, and evaluating drugs and therapies, such as, a non-human primate, a dog, cat, rabbit, swine, mouse or a mouse.
    [0038] The term "pharmaceutically acceptable carrier," "pharmaceutically acceptable carrier," "physiologically acceptable carrier," or "physiologically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid charge. , diluent, solvent, or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, proportional to a reasonable benefit / risk ratio. See, Remington: The Science and Practice of Pharmacy, 21a. ed .; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 6a. ed .; Rowe et al., Eds .; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3a. ed .; Ash and Ash Eds .; Gower Publishing Company: 2007; and Pharmaceutical Preformulation and Formulation, 2a. ed .; Gibson Ed .; CRC Press LLC: Boca Raton, Fla., 2009.
    [0039] "Prodrug" refers to a compound that, after administration, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A drug, relative to the drug, is chemically modified in a way that makes it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the drug is administered. A prodrug may have, relative to the active drug, altered metabolic or transport stability characteristics, lesser side effects or less toxicity, or improved taste (for example, see Nogrady reference, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug can be synthesized using reagents other than the corresponding drug.
    [0040] "Solid tumor" refers to solid tumors including, but not limited to, metastatic tumors in bone, brain, liver, lungs, lymph node, pancreas, prostate, skin and soft tissue (sarcoma).
    [0041] The term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute, for example, a compound provided herein, and one or more molecules of a solvent, which is present in stoichiometric quantity or not stoichiometric. Suitable solvents include, but are not limited to, water, methanol, ethanol, n-propanol, isopropanol, and acetic acid. In certain embodiments, the solvent is pharmaceutically acceptable. In one embodiment, the complex or aggregate is in a crystalline form. In another embodiment, the complex or aggregate is in a non-crystalline form. Where the solvent is water, the solvate is a hydrate. Examples of hydrates include, but are not limited to, a hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate.
    [0042] "Therapeutically effective amount" of a drug refers to an amount of a drug that, when administered to a cancer patient, will have the desired therapeutic effect, for example, relief, improvement, palliation or elimination of one or more manifestations of cancer in the patient. A therapeutic effect does not necessarily occur by administering a dose, and can occur only after administering a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations.
    [0043] "Treating", "treating" or "therapy of" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviating or ameliorating one or more symptoms of cancer; decrease in the extent of the disease; delay or slowdown in the progression of the disease; improvement, palliation or stabilization of the sick state; or other beneficial results. Cancer treatment can, in some cases, result in partial response or stable disease.
    "Tumor cells" refers to tumor cells of any suitable species, for example, mammalian, such as murine, canine, feline, equine or human. Descriptive modalities
    [0045] The following are composed of formulas Ia and Ib:

    [0046] In another aspect, a process is described here comprising contacting a compound for the preparation of a compound of formula I:
    comprising contacting a compound of formula II:
    with POCl3 and H2NCH2CH2L2., or a salt thereof, to give a compound of formula III,
    wherein L1 and L2 independently are a leaving group, and the compound of formula III is then taken to provide a compound of formula I.
    [0047] Some methods for synthesizing compounds are presented here. Other methods for synthesizing these and other compounds described herein will be evident to the person skilled in the art based on the adaptation of, and the replacement of reagents and reactives in, well-known synthetic methods. See, for example, Hay et al., J. Med. Chem. 2003, 46, 2456-2466 and Hu et al., Bioorganic & Medicinal Chemistry Letters 21 (2011) 3986-3991. The starting materials, usable for preparing the compounds provided herein, are commercially available or can be prepared following routine methods. The reactions are commonly carried out in an inert solvent and heated, if necessary. The person skilled in the art will readily notice that some reactions may require the use of a protection group. Protection groups are well known to the person skilled in the art and described, for example, in Greene's Protective Groups in Organic Synthesis. Peter G. M. Wuts and Theodora W. Greene, 4th. ed. or a later edition, John Wiley & Sons, Inc., 2007. Reaction products can be separated using routine methods, such as crystallization, precipitation, distillation and / or chromatography. The purity of a compound or an intermediate can be determined using well-known methods, such as 1H-NMR, HPLC, TLC and the like.
    [0048] In another embodiment, the present invention relates to a process for optical resolution of a compound of formula I. In view of the pharmaceutical importance of the compounds of formula Ia and Ib of the present invention, it has become imperative to resolve the compound of formula I using an effective industrial process and, especially, with a good yield and excellent chemical and enantiomeric purity.
    [0049] The Applicant has developed a process for the optical resolution of the compound of formula I, which makes it possible to obtain the compound of formulas Ia and Ib with good performance and chemical and enantiomeric purity characteristics. The process of the invention makes it possible to obtain both enantiomers of the compound of formula I in an excellent enantiomeric excess, with high productivity and in an excellent yield while saving the solvents used. More specifically, the present invention relates to a process for the optical resolution of a compound of formula I:
    to give their absolute configuration (R) and (S) enantiomers, respectively of formulas (Ia) and (Ib):
    wherein a racemic or enantiomerically enriched mixture of the compound of formula I is separated into its two enantiomers, (R) -1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl-N , N'-bis (ethylene) phosphoramidate of formula (Ia) and (S) -1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl-N, N'-bis (ethylene) phosphoramidate of formula (Ib), by chiral chromatography.
    [0050] The optical solution is understood to mean the separation of the two enantiomers from a racemic mixture or any mixture of these two enantiomers.
    [0051] The racemic mixture is understood to mean a mixture of two enantiomers in a ratio of 55:45 to 45:55, preferably in a ratio of 50:50.
    [0052] An enantiomerically enriched mixture is understood to mean a mixture of the two enantiomers significantly containing more than one of the enantiomers in a ratio ranging from 55:45 to 90:10.
    [0053] Chiral chromatography is understood to mean an arrangement making it possible to separate the enantiomers of a mixture by means of a chiral stationary phase and a mobile phase composed of a solvent or a mixture of solvents and gases.
    [0054] According to one of the embodiments of the invention, the stationary phase used for chiral chromatography comprises a silica gel impregnated with a functionalized polysaccharide.
    [0055] The mobile phase used for chiral chromatography in one embodiment, comprises a mixture of an alcohol and an organic gas. Among the alcohols that can be used for chiral chromatography, isopropanol, ethanol and methanol can be mentioned, without implying any limitation. In one embodiment, the alcohol used for chiral chromatography is methanol.
    [0056] Among the organic gases, which can be used for chiral chromatography, can be mentioned, without implying any limitation, the organic gases that can be used in high pressure. An organic gas preferably used is CO2. In one embodiment, the mobile phase used for chiral chromatography comprises a mixture of methanol and CO2. In one embodiment of the invention, the mobile phase used for chiral chromatography comprises a mixture of methanol and CO2 in a ratio ranging from 50:50 to 2:98.
    [0057] In one embodiment of the invention, the mobile phase used for chiral chromatography is recycled. In one embodiment of the invention, chiral chromatography is carried out at a temperature of 15 oC to 40 oC inclusive. In one embodiment of the invention, optical resolution is performed in a racemic mixture of 1: 1 of formula (I). In one embodiment of the invention, the (R) enantiomer of 1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl-N, N'-bis (ethylene) phosphoramidate is used. According to an embodiment of the invention, the (S) enantiomer of 1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl-N, N'-bis (ethylene) is used phosphoramidate.
    [0058] In accordance with an embodiment of the invention, a continuous multiple column separation process is used.
    [0059] In accordance with another embodiment of the invention, a simulated moving bed chromatography process is used. Simulated moving bed chromatography is understood to mean a continuous chromatography process that makes it possible to simulate movement of the stationary phase in the opposite direction to the movement of the mobile phase. Such a process makes it possible to separate compounds that are difficult or impossible to separate by conventional chromatography techniques. When a chiral stationary phase is used, such a process is especially usable for the separation of enantiomers. The use of simulated moving bed chromatography makes it possible to perform a continuous resolution of a mixture of enantiomers with high productivity, while reducing the quantities of stationary and mobile phases used compared to batch chromatography processes. List of abbreviations used DMF: Dimethylformamide TEA: Triethylamine RT: Ambient temperature IPM.:Isofosforamide mustard THF: tetrahydrofuran DIAD: diisopropyl azodicarboxylate The Examples below illustrate the invention. EXAMPLES Example 1. Preparation of compound TH 2870.
    [0060] Compounds 2-6 were synthesized as described below. a. Synthesis of compound 3:
    [0061] Compound 1 (3 g, 16.2 mmol) was refluxed in SOCl2 (10 ml) with DMF (3 drops) for 3 h and then SOCl2 was removed in vacuo. The residue was diluted with toluene (5mL) and was used in the next step without further purification.
    [0062] A mixture of MgCl2 (930 mg, 9.8 mmol), TEA (4.7 mL, 33.4 mmol) and dimethyl malonate (1.9 mL, 16.6 mmol) was stirred at RT for 1 hour, 5h followed by addition of the toluene solution above mentioned compound 2. The resulting mixture was stirred at RT for another 1.5 h then conc. (4 ml) was added and stirred for 5 minutes. The mixture was extracted with EtOAc (30 ml x 3), dried (Na2SO4), filtered and concentrated under reduced pressure. To the residue was added 6N HCl (30 ml and the mixture was refluxed overnight. The mixture was extracted with EtOAc (30 ml x 3), dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified via FCC ( silica gel, EtOAc / Hexane) to give Compound 3 as a light yellow solid (1.9 g, 63% yield) .1H NMR (CDCl3,400 MHz) δ: 8.16 (d, J = 8.0 Hz , 1H), 7.86 (t, d = 9.2 Hz, 2H), 2.68 (s, 3H) ppm. b. Synthesis of compound 4
    [0063] To a mixture of compound 3 (1.9 g, 10.4 mmol) in MeOH (20 mL) at -10 ° C was added NaBH4 (418 mg, 11 mmol) in portions. The mixture was stirred at -10 ° C to 0 ° C for 20 minutes, diluted with EtOAc (300 ml), washed with sat. Aqueous NH4Cl solution, brine, dried (Na2SO4). Filtered and concentrated under reduced pressure. The residue was purified via FCC (silica gel, EtOAc / Hexane) to give Compound 4 as a light yellow oil (1.44g, 75% yield). 1H NMR (CDCl3,400 MHz) δ: 8.06 (t, J = 8.4 Hz, 1H), 7.35 (d, J = 11.6 Hz, 1H), 7.30 (d, J = 11.6 Hz, 1H), 5.01-4.99 (m, 1H), 1.52 (d, J = 6.4 Hz, 3H) ppm. c.Synthesis of compound 5
    [0064] To a mixture of compound 4 (1.44g, 7.78 mmol), Br-IPM (2.88g, 9.34 mmol), PPh3 (3.06g, 11.67 mmol) in THF (60 mL ) at 0 ° C DIAD (2.34 g, 11.67 mmol) was added. The mixture was stirred at 0 ° C for 1.5 h, concentrated under reduced pressure and purified via FCC (silica gel, EtOAc / Hexane) to give Compound 5 as a light yellow oil (1.0 g, 27% yield). 1H NMR (CDCl3,400 MHz) δ: 8.09 (t, J = 8.0 Hz, 1H), 8.31 (dd, J = 2.4.13.6 Hz, 2H), 5.52- 5.60 (m, 1H), 3.54-3.19 (m, 8H), 1.63 (d, J = 6.4 Hz, 3H) ppm. d.Synthesis of compound 6
    [0065] A mixture of compound 5 (1g, 2.1 mmol) and Ag2O (3 g) in THF (50 ml) was stirred at 65 ° C for 3h. Filtered and concentrated under reduced pressure. The residue was purified via FCC (silica gel, Acetone / Hexane) to give Compound 6 as a yellow solid (0.6g, 90% yield). 1HRMN (CDCl3,400 MHz) δ: 8.08 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 11.6 Hz, 1H), 7.31 (d, J = 8 , 4 Hz, 1H), 5.70-5.67 (m, 1H), 2.25-2.08 (m, 8H), 1.64 (d, J = 6.4 Hz, 3H) ppm. e.Preparation of compost7
    Preparation of compound 7-2
    [0066] Ac2O (562 ml, 1.5 eq) was added dropwise to a solution of compound 7-1 (150 g, 1.08 mol) in Pyridine (700 ml) at 0 ° C, stirred at room temperature for 6 h. Evaporated, poured into ice water, filtered, the filter cake was dried to give compound 7-2 as a white solid (150 g, 74% yield). 1H NMR (400 MHz, CDCl3): δ ppm 8.00 ~ 7.98 (d, J = 7.6 Hz, 1H), 8.03 (s, 1H), 7.83 (s, 1H), 7 , 51 ~ 7.47 (t, J = 8.0 Hz, 1H), 7.36 ~ 7.34 (dd, J = 8.0 Hz 1.2 Hz, 1H), 2.34 (s, 3H ). Preparation of compound 7-3
    [0067] To a solution of compound 7-2 (150 g, 833 mmol) in DCM (1500 mL), DMF (15 mL) was added, cooled to 0oC followed by the addition of oxalyl chloride (225 mL, 2.50 mol), stirred at room temperature for 4h. Evaporated, the residue was dissolved in DCM (1000 mL) cooled to 0oC followed by the addition of 2M solution of dimethylamine in THF (900 mL, 1.8 mol), stirred at room temperature for 20h. Quenched with H2O (1500 mL), extracted with DCM (2000 mL x 3), evaporated to give crude compound 7-3 as a pale yellow liquid (137 g, 80% yield), 1H NMR (400 MHz, CDCl3): δ ppm 7.43 ~ 7.39 (t, J = 8.0 Hz, 1H), 7.29 ~ 7.28 (d, J = 7.6 Hz, 1H), 7.17 ~ 7.13 ( m, 2H), 3.00 (s, 6H), 2.32 (s, 3H). Preparation of compost 7
    [0068] To a solution of compound 7-3 (137 g, 661 mmol) in MeOH (1000 mL), K2CO3 (276 g, 2 mol) was added, stirred at room temperature for 5 h. Filtered, the filtrate was evaporated. The residue was dissolved in H2O (1000 ml), acidified by 4N HCl to PH6.0, filtered, the filter cake was dried to give compound 7 as a white solid (60 g, 55% yield). 1H NMR (400 MHz, CDCl3): δ ppm 8.25 (s, 1H), 7.19 ~ 7.15 (d, J = 8.0 Hz, 1H), 6.96 ~ 6.95 (t, J = 2.0 Hz, 1H), 6.84 ~ 6.81 (s, 2H), 3.11 (s, 3H), 2.96 (s, 3H). f.Synthesis of TH 2870
    [0069] To a mixture of compound 7 in DMF (60 ml) at oC was added NaH (60%, 0.508 g, 12.7 mmol) in portions. The mixture was stirred at 0 ° C for 0.5 h before Compound 6 (2 g, 6.35 mmol) was added and then stirred at 0 ° C for 2.5 h. The mixture was diluted with EtOAc (500 ml), washed with brine (50 ml x 3), dried over Na2SO4, filtered, concentrated under reduced pressure and purified via FCC (silica gel, Acetone / Hexane) to give TH 2870 as an oil yellow. Final purification of TH 2870:
    [0070] TH 2870, as mentioned above, was purified via semi-prep HPLC. (column C18, acetonitrile / water). The combined collections were concentrated under reduced pressure to give a light yellow oil as the final product. Acetonitrile was added to the evaporations as an azeotropic agent to remove water. 1H NMR (400 MHz, CDCl3): δ ppm 7.98 ~ 7.96 (d, J = 8.4 Hz, 1H), 7.43 ~ 7.39 (m, 1H), 7.27 ~ 7, 21 (m, 2H), 7.10 ~ 7.06 (m, 3H), 5.62 ~ 5.55 (m, 1H), 3.09 (s, 3H), 2.97 (s, 3H) , 2.19 ~ 2.00 (m, 8H), 1.58 ~ 1.57 (d, J = 6.4 Hz, 3H). MS: m / z 460.8 [M + 1] +. PLC: 254 nm: 94.8%. Example 2. Alternative preparation of compound TH2870.
    a.Preparing of compound 3
    [0071] Compound 1 (200 g, 1.08 mol) was refluxed in SOCl2 (700 mL) with DMF (10ml) for 3 h and then SOCl2 was used in the next step without further purification.
    [0072] A mixture of MgCl2 (103g, 1.08 mol), TEA (500ml, 3.60mol) and dimethyl malonate (145g, 1.1mol) was stirred at RT for 1.5 h before the toluene solution above mentioned compound 2 will be added in drops. The resulting mixture was stirred at RT for an additional 1.5 h, washed with H2O (2L), extracted with EtOAc (2L x 5), evaporated, 4N HCl was added to PH6.0 and stirred for 5 minutes. The mixture was extracted with EtOAc (2L x 5), evaporated.
    [0073] To the residue, 6N HCl (1500 mL) was added and the mixture was refluxed overnight.
    [0074] The mixture was extracted with EtOAc (2L x 5), concentrated, purified by column on silica gel (petroleum ether: EtOAc = 20: 1) to give compound 3 as a yellow solid (80g, 41% yield) . b. Preparation of compost 4
    [0075] To a mixture of compound 3 (150 g, 824 mol) in MeOH (2 L) at -10 oC was added NaBH4 (31.2 g, 824 mmol) in portions. The mixture was stirred at -10 ° C to 0 ° C for 20 minutes, diluted with EtOAc (5L), washed with sat. Aqueous NH4Cl solution, brine, dried over Na2SO4, concentrated. The residue was purified by column on silica gel (petroleum ether: EtOAc = 5: 1) to give compound 4 as a yellow oil (90 g, 60% yield). c.Preparing of compound 5
    [0076] To a solution of POCl3 (2ml, 21.6mmol) in DCM (20ml) was added compound 4 (2g, 10.8mmol), then TEA (3.6ml, 27mmol) in DCM (10ml) was added at -40 ° C under N2, stirred at -40 ° C for 5 h. then 2-bromoethylamine hydrobromide (17.6 g, 86.8 mmol) was added, TEA (12 ml, 86.8 mmol) in DCM (40 ml) was added slowly to the above solution at -40oC, stirred for 0.5 h. K2CO3 (10%, 10.4 g, 100 ml) was added, stirred at room temperature for 5 min, extracted with DCM (300ml x 3), evaporated, purified by column on silica gel (EtOAc) to give compound 5 as a yellow oil (2.3 g, 43% yield). d.Preparing of compound 6
    [0077] A mixture of compound 5 (4g, 8.42mmol) and Ag2O (5.85g, 25.26mmol) in THF (40ml) was stirred at 65 ° C for 3 h, filtered and concentrated. The residue was purified by column on silica gel (EtOAc) to give compound 6 as a yellow oil (2.3 g, 87% yield). e.Preparation of compound TH2870
    [0078] To a solution of compound 7 (1.81g, 10.95 mmol) in DMF (10ml), NaH (60%, 438mg, 1095 mmol) was added at 0oC, stirred for 10 min, then compound 6 (2 , 3.7.3 mmol) in DMF (10ml) was added, stirred at 0 ° C for 30 min.
    [0079] It was quenched with H2O, extracted with EtOAc (100ml x 5), washed with H2O (150ml), brine, evaporated, purified by column on silica gel (DCM: MeOH = 40: 1) to give TH2870 compound as a yellow oil (2.3g, 69% yield). Example. 3. Separation of TH2870 enantiomers by chiral preparative chromatography
    [0080] Dissolve 983 mg of compound of formula (I) in 36 ml of methanol, inject 1 ml over a CHIRALPAK OZ-H 4.6x250mm, 5μm (Daicel) in a SFC-80 method site (Thar, Waters), at a flow rate of 3.0 ml / min and a back pressure of 120 bars at a column temperature of 35-40 ° C and elute in this flow rate in a mixture and CO2 / Methanol (65-60 / 35 40). The enantiomer of formula (Ia) (configuration (R)) is obtained in a yield of 86.5% and with an enantiomeric purity of 100%. The enantiomer of formula (Ib) (configuration (S)) is obtained in a yield of 83.8% and with 100% enantiomeric purity. Figure 1 shows a purity check of enantiomer 1 TH2870 (TH 3423) and enantiomer 2 TH 2870 (TH 3424 ouAST 106) after collateral separation by LCMS. Chiral synthesis of TH 3423 and 3424

    [0081] Compound 1 (65 g) was refluxed in SOCl2 (150 ml) with DMF (2.5 ml) for 5 h to obtain a clear solution and then SOCl2 was removed in vacuo. The residue was diluted with toluene (30mL) and the solvents were removed again. The residue was used in the next step without further purification. Compound 3
    [0082] A mixture of MgCl2 (21.0 g, 221 mmol), TEA (100.0 mL, 717 mmol) and dimethyl malonate (41.0 mL, 359 mmol) was stirred with a mechanical stirrer at RT for 2 h before of compound 2 in THF (80 ml) is added. The resulting mixture was stirred at RT for 4 h before conc. (90 mL) be added and stirred for 30 minutes. The mixture was extracted with EtOAc (300 ml x 3), concentrated under reduced pressure. To the residue, 6N HCl (300 ml) was added and the mixture was refluxed overnight. The mixture was extracted with EtOAc (300 ml x 3), organic layer was washed with NaHCO3 (aq.) And dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was recrystallized from AcOEt / Hex = 1/3 (V / V) to give compound 3 as a light yellow solid (46g) .1H NMR (CDCl3,400 MHz) δ: 8.16 (d, 1H) , 7.86 (t, 2H), 2.68 (s, 3H) Compound 5:
    [0083] Under argon, BH3.THF (1M, 11mL) was added to a solution of compound 4 in 1M toluene (3mL, 3 mmol) at 0oC. The solution was stirred for 30 min. then cooled to -40oC. The solution of compound 3 (1.83g, 10mmol) in THF (40mL) was added slowly over 4 h at -40 oC. The system was stirred at -40 oC for 2 h (TLC showed that SM disappeared). MeOH (20ml) was added to about -40 oC solution and the solution was stirred for 30 min. After the solvents were removed at room temperature the residue was purified by column (Hex / AcOEt = 3/1 (V / V)) to obtain compound 5 (1.6g). 1HRMN (CDCl3,400 MHz) δ: 8.05 (t, 1H), 7.34 (d, 1H), 7.27 (d, 1H), 4.99 (m, 1H), 1.51 (d , 3H). Compound 6
    [0084] To a solution of POCl3 (1.6 mL, 17.25 mmol) in DCM (10 mL) at -40oC under argon was added compound 5 (1.6 g, 8.65 mmol), then TEA (2, 9 mL, 22.9 mmol) in 10 mL DCM. The mixture was stirred at -40 oC for 6 h and then 2-bromoethylamine hydrobromide (14.2 g, 69.3 mmol) was added, TEA (9.6 mL) in DCM (10 mL) was added in drops. The reaction mixture was stirred at -40 ° C to room temperature overnight. K2CO3 (8.3g in 80 ml water) was added and the mixture stirred for 5 min. The mixture was extracted with DCM, dried over Na2SO4, filtered, concentrated and then subjected to silica gel flash chromatography, eluting with Acetone / Hexane = 0-100%) to give compound 6 as yellow oil (2.68g , yield: 65%). 1H NMR (CDCl3,400 MHz) δ: 8.08 (t, 1H), 7.32 (d, 1H), 7.29 (d, 1H), 5.56 (m, 1H), 3.34- 3.56 (m, 2H), 3.32-3.42 (m, 4H), 3.08-3.26 (m, 4H), 1.62 (d, 3H). 31PRMN: 14.44.
    [0085] Alternatively TH3424 can be synthesized by the following procedure:
    [0086] Toluene (11ml / g) was added to a glass bottle with four necks under nitrogen. After stirring, POCl3 was added (1.025eq) to vessel 1 under nitrogen. The contents of vessel 1 were cooled to -2 ~ 2 ° C. The solution of compound 1 was added (1.0eq) and TEA (1.435eq) in toluene (11ml / g) in drops at -2 ~ 2 ° C. The contents of vessel 1 were stirred at -2 ~ 2 C for 1 ~ 2 hours. The content was sampled for HPLC analysis for information. 2-bromoethylamine hydrobromide (3eq) was added to vessel 1. TEA (6eq) was added to vessel 1 in -2 ~ 2C drops. The contents of vessel 1 were stirred at 0C ~ RT overnight. The content was sampled for HPLC analysis. Working procedure was as follows: H2O (19ml / g) was added to vessel 1 and stirred for 5 ~ 10 min. The mixture was extracted with EA (19ml / g) three times. The organic phase was dried over Na2SO4 and filtered. The mother liquor was concentrated at 40 ~ 50 ° C. The crude product was purified by silica gel chromatography to obtain the purified product.
    [0087] Alternatively TH3424 can be synthesized by the following procedure:
    [0088] Toluene (11ml / g) was added to a glass bottle with four necks under nitrogen. Stirring was started and compound 1 (1.0eq) and TEA (1.435eq) were added to vessel 1 under nitrogen. The contents of vessel 1 were cooled to -2 ~ 2 C. POCl3 (1.025eq) was added to vessel 1 under nitrogen in drops at -2 ~ 2 C. The contents of vessel 1 were stirred at -2 ~ 2 C for 1 ~ 2 hours. The content was sampled for HPLC analysis for information. 2-Bromoethylamine hydrobromide (3eq) was added to vessel 1. TEA (6eq) was added to vessel 1 in -2 ~ 2C drops. The contents of vessel 1 were stirred at 0C ~ RT overnight. The content was sampled for HPLC analysis. P work procedure was the same as above.
    [0089] Alternatively TH3424 can be synthesized by the following procedure:
    [0090] DCM (2.2ml / g) and POCl3 (1.91eq) were added to a four-neck glass bottle under nitrogen. Stirring was started and the contents of vessel1 were cooled to -35 ~ -40 ° C under nitrogen. Compound 1 (1.0eq) / DCM solution (4.5g / ml) was added to vessel 1 under nitrogen in drops at -35 ~ -40 C. TEA (4.0eq) / DCM solution (4.5g / ml) were added to vessel 1 under nitrogen in drops at -35 ~ -40 C. The contents of vessel 1 were at -35 ~ -40 C for 4 ~ 6 hours. The content was sampled for HPLC analysis for information. 2-Bromoethylamine hydrobromide (8eq) was added to vessel 1 at -30 ~ -40C. TEA (12eq) was added to vessel 1 in drops at -30 ~ -40C. The contents of vessel 1 were stirred at -30 ~ -40 ° C for 1-2 hours. The content was sampled for HPLC analysis. Working procedure: H2O (15ml / g) was added to vessel 1 and stirred for 5 ~ 10 min. The aqueous phase was extracted with DCM (12.5 ml / g) for one time. The organic phase was dried over Na2SO4 and filtered. The filtrate was concentrated at 20 ~ 30 ° C. The crude product was purified by chromatography to obtain the purified product. 7
    [0091] A mixture of compound 6 (2.68g), Ag2O (3.92 g), in THF (30mL) was stirred at 55 ° C overnight. After removing solvent under vacuum, the residue was separated by flash chromatography on silica gel to give 1.0 g of compound 7 light liquid. 1H NMR (CDCl3,400 MHz) δ: 8.05 (t, 1H) , 7.34 (d, 1H), 7.29 (d, 1H), 5.66 (m, 1H), 2.02-2.24 (m, 8H), 1.61 (d, 3H). 31PRMN: 31.55. TH 3424 (TH 2870 2 enantiomer)
    [0092] A mixture of compound 7 (1.0 g), compound 8 (785 mg), K2CO3 (880 mg) in DMF (8 ml) was stirred at room temperature overnight. The mixture was diluted with water, extracted with DCM, dried over Na2SO4, filtered, concentrated and then subjected to flash chromatography to give compound 9 as a yellow oil (1.1g). 1H NMR (CDCl3,400 MHz) δ: 7.97 (d, 1H), 7.41 (t, 1H), 7187.27 (m, 4H), 7.02-7.12 (m, 3H), 5.59 (m, 1H), 3.08 (s, 3H), 2.97 (s, 3H), 2.01-2.21 (m, 8H), 1.66 (d, 3H) .31P NMR: 31.27. Compound 11
    [0093] Same procedure with compound 5. Compound 8 was used instead of compound 3. Yield: 50% .1H NMR (CDCl3, 400 MHz) δ: 8.05 (t, 1H), 7.34 (dd, 1H ), 7.27 (d, 1H), 4.99 (m, 1H), 1.51 (d, 3H). Compound 12
    [0094] Same procedure with compound 6 (yield: 35%). 1HRMN (CDCl3,400 MHz) δ: 8.08 (t, 1H), 7.32 (d, 1H), 7.29 (d, 1H) , 5.56 (m, 1H), 3.34-3.56 (m, 2H), 3.32-3.42 (m, 4H), 3.08-3.26 (m, 4H), 1 , 62 (d, 3H). 31 PRMN: 14.47. 13
    [0095] Same procedure with compound 7 (yield: 36%). 1HRMN (CDCl3,400 MHz) δ: 8.06 (t, 1H), 7.34 (d, 1H), 7.30 (d, 1H) , 5.67 (m, 1H), 2.02-2.25 (m, 8H), 1.62 (d, 3H). 31PRMN: 31.56. TH3423 (TH 2870 enantiomer 1)
    [0096] Same procedure with compound 9 (yield: 68%). 1H NMR (CDCl3,400 MHz) δ: 7.97 (d, 1H), 7.41 (t, 1H), 718-7.27 (m , 4H), 7.02-7.12 (m, 3H), 5.59 (m, 1H), 3.08 (s, 3H), 2.97 (s, 3H), 2.01-2, 21 (m, 8H), 1.66 (d, 3H). 31 P NMR: 31.25. Example 4. In vitro cytotoxicity assay of human tumor cell lineage
    [0097] In vitro proliferation data on the human tumor cell line from non-H460 lung cancer is reported above in the compound table. IC50 values are reported in micromolar and result from exposure of the compound to various concentrations for 2 h, followed by a washing step and addition of new medium followed by growth and staining of cell viability and comparison with a control treated with only medium.
    [0098] Specifically, cells growing exponentially were seeded at a density of 4 x 103 cells per well in a 96 well plate and incubated at 37oC in 5% CO2.95% air and 100% relative humidity for 24 hours before adding compounds of test. Compounds were solubilized in 100% DMSO at 200 times the desired final test concentration. At the time of drug addition, compounds were further diluted to 4 times the final desired concentration with complete medium. Aliquots of 50 μl of compound in specified concentrations were added to microtiter wells already containing 150 μl of medium, resulting in final registered drug concentration. After adding the drug, the plates were incubated for an additional 2 hours at 37oC, 5% CO2.95% at, and 100% relative humidity, then the drug was washed and new media was added and the plates were incubated for more 70h at 37oC, 5% CO2, 95% air and 100% relative humidity. At the end of this incubation, viable cells were quantified using the AlamarBlue test. The drug concentration resulting in 50% growth inhibition (IC50) was calculated using Prism software (Irvine, CA), and the results were listed in the table.
    [0099] Its anti-proliferation efficacy in H460 lung cancer cells is also tabulated below.

    [00100] The H460 data above demonstrate a substantial antitumor effect with inhibition at decreasing and low nanomolar levels for various compounds during an exposure of just 2 hours. Example 5. Activation of TH 2870 by aldocet reductase, AKR1C3
    [00101] Recombinant human AKR1C3 was diluted to 25 μg / mL in phosphate buffered saline (PBS), pH 7.4 (37oC), containing 2 mM NADPH. TH2870 or progesterone (positive control) in 30% methanol / 70% water was added to a reaction mixture at a final concentration of 5 μM and incubated at 37 oC for 120 minutes. At various times up to 120 min, 50 μL of a reaction mixture was taken and 200 μL of propranolol containing acetonitrile as an internal standard was added, vortexed and centrifuged for 10 min. The resulting supernatant (5 μL) was injected into an LC / MS / MS to quantify the remaining% of TH 2870 and progesterone. The compounds were tested in duplicates.
    [00102] The data in Figure 2 demonstrate the rapid disappearance of TH 2870 in the presence of AKR1C3 while the substrate Progesterone is reduced slowly. Buffer controls containing NADPH but without enzyme showed no reaction with both compounds (data not shown). Example 6. In vitro cytotoxicity assay of human tumor cell lineage
    [00103] TH 2870, TH 3423 and TH 3424 were also tested on different cancer cell lines using the materials and procedures as follows. Cell lysate buffer 10 * (Cell Signaling Technology, Catalog Number 9803); Protease inhibitor cocktail for mammalian tissues (Sigma, Catalog Number P8340); phosphatase inhibitor cocktails for serine / threonine phosphatases and alkaline phosphatase L-isozymes (Sigma, Catalog Number P0044); tyrosine phosphatase inhibitor cocktails protein phosphatases, acid and alkaline phosphatases (Sigma, Catalog Number P5726); BCA kit (Thermo, Catalog Number 23225); primary antibody, mouse monoclonal antibody AKR1C3 (clone NP6.G6.A6; Sigma-Aldrich); primary antibody, α-tubulin (clone B-5-1-2; Sigma-Aldrich); secondary antibody, HRP IgG anti-conjugated goat mouse (A4416; Sigma-Aldrich) were used. The cells passed through two generations in good condition and digested. The appropriate number of cells was inoculated into 6 cm cell culture discs, and incubated at 37 ° C, 5% CO2 overnight. When the cells were grown at 80% density, the disc was removed from the incubator. The medium was aspirated, washed twice with ice-cold PBS, and residual PBS was removed. An appropriate volume of ice-cold 1 * cell lysate was added and incubated on ice for 10 minutes. Cell lysate was transferred to ice-cooled microcentrifuge tubes, 4C, 12,000 rpm and centrifuged for 15 minutes. Supernatant was transferred to another microcentrifuge tube. Cell lysates were diluted by a 10 * cell lysate, and protease inhibitor cocktail for mammalian tissues (Sigma, # P8340), phosphatase inhibitor cocktails for serine / threonine phosphatases and alkaline phosphatase L-isozymes, cocktails phosphatase inhibitor for tyrosine protein phosphatases, and acid and alkaline phosphatases. The BCA protein quantification kit for protein quantification was used with 1 * cell lysate to dilute the cell lysate to the same concentration. Corresponding samples were added in a 5 * SDS loading buffer, heated to 85 ° C for 10 minutes, and briefly centrifuged. The samples were stored at -20 C or used directly for protein electrophoresis. The samples were stored at -20C or used directly for protein electrophoresis. These samples were electrophoresed according to standard practice, transferred to a membrane, the primary antibodies and then secondary antibodies were applied according to the manufacturers' instructions. The Odyssey infrared laser imaging system was used to scan the signals.
    [00104] The results are shown below in Figures 3 and 4 and listed in the following tables: Table: Sensitivity to TH 2870, TH 3423 and TH 3424 in cancer cell lines of, liver, prostate, esophagus and leukemia


    Example 7. In vivo human tumor xenograft models and antitumor activity
    [00105] Three human xenograft anti-tumor models using H460 non-small cell lung, A549 non-small cell lung, and A375 melanoma models were used to demonstrate the effectiveness of the compounds described herein.
    [00106] Nude female mice, homozygous, free of specific pathogens (nu / nu, Charles River Laboratories) were used. The mice received feed and water ad libitum and were housed in 'microisolator' cages. Animals from four to six weeks of age were identified by microchips (Locus Technology, Manchester, MD, USA) at the time of the experiments. All animal studies have been approved by the Institutional Committee for the Care and Use of Animals at Threshold Pharmaceuticals, Inc.
    [00107] All cell lines were from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in the suggested medium with 10% fetal bovine serum and kept in a humidified environment at 5% with CO2 at 37 ° C.
    [00108] The cells were mixed with Matrigel (30% in H460) and 0.2 ml per mouse was subcutaneously implanted in the animals' flank area. When the tumor size reached 100-150 mm3, mice were randomized into experimental groups or vehicle with 10 mice / group and treatment was started (Day 1). The tested compounds were formulated in 5% DMSO in D5W. All compounds were given by IP, QDx5 / week (5 days with, 2 days without) as a cycle, for a total of 2 cycles. Tumor growth and body weight were measured twice a week. The tumor volume was calculated as (length x width2) / 2. The efficacy of the drug was evaluated as inhibition of tumor growth (TGI) and delay in tumor growth (TGD). TGI was defined as (1-ΔT / ΔC) x 100, where ΔT / ΔC presented the reason for the change in mean tumor volume (or median, if the variation within the group was relatively large) of the treated group and the control group . TGD was calculated as the additional days for the treated tumor to reach 500mm3, as compared to the control group. Animals were slaughtered when individual tumor size exceeded 2000mm3 or mean tumor volume exceeded 1000mm3 in the group. Data are expressed as mean ± SEM. One-way analysis of variance with Dunnett's post-comparison test (GraphPad Prism 4) or paired Student's t test were used for analysis. A level P <0.05 was considered statistically significant. Example 8. In vivo efficacy results:
    [00109] This study employed a model of xenograft and human tumor of melanoma A375 and the compounds provided for here were compared with thiotepa and the approved anti-melanoma drug, Abraxane. The antitumor effects and security of administration are graphically illustrated below. Mpk refers to mg / kg.

    [00110] Taken together, these studies demonstrate significant anti-tumor efficacy in 3 different tumor cell lines with respect to standard chemotherapy. Example 9.TH3424 in mouse model of human liver cancer
    [00111] Female athymic nude mice (6 weeks old) were used in this study. The animals were purchased from Beijing HFK Bioscience, Co., Ltd and kept in a filtered environment with high efficiency air particulate filter (HEPA) with cages, feed and platform sterilized by irradiation or autoclaving. A total of 32 nude mice were used for the study. HepG2-GFP human hepatocellular carcinoma cells (AntiCancer, Inc., San Diego, CA) were incubated with RPMI-1640 (Gibco-BRL, Life Technologies, Inc.), which contained 10% FBS. Cells were cultured in a CO2 incubator with a water jacket (Forma Scientific) maintaining the air atmosphere at 37 ° C and 5% CO2 / 95%. Cell viability was determined by trypan blue exclusion analysis. Five female athymic nude mice were injected subcutaneously with a single dose of 5 x 10 6 HepG2-GFP cells. Tumors were collected when their size reached 1cm3 and the tumor tissues were then cut into small 1mm3 fragments. Forty female nude mice were orthotopically implanted with a single piece of tumor fragment, which was derived from a subcutaneous tumor mode of human hepatocellular carcinoma HepG2-GFP. The tumor tissue was orthotopically implanted in the right lobe of the liver in each mouse by SOI (surgical orthotopic implant). Briefly, a 1 cm upper abdominal incision was made under anesthesia. The right lobe of the liver was exposed and part of the liver's surface was mechanically wounded by scissors. Then, a piece of the tumor fragment was fixed inside the liver tissue, the liver was returned to the peritoneal cavity and the abdominal wall was finally closed. The mice were kept in laminar flow cabinets under specific pathogen-free conditions.
    [00112] Treatment was started three days after the tumor implantation when the implanted tumors reached an average size around 1 mm2. The 32 mice with tumor were randomly divided into four experimental groups of 8 mice each. Each cage was clearly marked for its group with four mice per cage. Each mouse had an ear tag for identification. The table below shows the study design. Treatment groups and protocol

    Note: Treatment was started on day 3 after tumor implantation.
    [00113] During the study period, all experimental mice were checked daily for mortality or signs of morbidity. The animals were observed until day 38 after tumor implantation. The body weights of the mice were measured twice weekly during the study period. Images of tumor growth and progression were taken twice a week during the study period with the FluorVivo imaging system, Model 300 / Mag (INDEC, CA, USA). All experimental animals were euthanized by injecting a super-dose of sodium pentobarbital on day 38 after tumor implantation. The livers were exposed for imaging, after the tumors were removed and weighed with an electronic scale (Sartorius BS 124 S, Germany). Tumor tissues were kept in formalin for further analysis. Comparisons of body weights and tumor loads in different groups were analyzed using Student's t test with an α = 0.05 (paired). After injecting the test agents intravenously, the mice did not lie down and showed no reduction in autonomous activity. The experimental animals were generally kept in good condition. The changes in body weight in each group are shown in Figure 5 and in the Table below. Table. Comparison of average body weight of mice at the end of the study

    [00114] As shown in Figure 5 and in the table above, on day 35 of the study, the average body weight of the mice in each group was increased by 21% to 41%. There was no statistically significant difference between the TH2870-2 groups and the negative control group. This suggested that there was no obvious acute toxicity to the experimental mice by intraperitoneal administration of low dose or TH2870-2 minutes. In the group treated with sorafenib, however, the average body weight was statistically significantly lower than in the negative control group. This suggested that a degree of toxicity was noted for experimental mice by administering sorafenib at the tested dosage. Tumor progression in each group.
    [00115] Progressions of HepG2-GFP hepatocellular carcinoma in different groups were monitored by images in real time. Images were acquired twice a week. The typical tumor images at the end of the study in each group and tumor growth curves derived from the tumor fluorescence signals, which were analyzed using the Power Station software (INDEC Biosystems, CA, USA), are shown in Figure 6 and Figure 7, respectively. Table. Average tumor size in each group (mm2)

    [00116] As shown in Figure 6, Figure 7 and the table above, the average tumor size in the positive control group was about 39% smaller than in the negative control group, but there was no statistically significant difference between the 2 control groups (P = 0.0577).
    [00117] In TH3424 groups (2.5 mg / kg) and TH3424 groups (5 mg / kg), the areas of medium fluorescence image reading were significantly less significant than in the negative control group, showing strong inhibitory effects and an obvious dose-effect relationship. Among these, the fluorescence image reading area was 0 in the TH3424 group (5mg / kg). In this group, dosing was discontinued after 3 treatment cycles; fluorescence image reading of the tumor was still 0 until the end of the experiment. In the TH3424 group (2.5 mg / kg), however, drug administration was stopped for 1 week after 3 treatment cycles and then restarted in 3 treatment cycles. The average fluorescence image reading area on day 35 was 1.2 ± 1.1, which was around 8% of that in the negative control group and was 2.0 ± 2.2 (~ 8%) of the negative control group on day 49 after treatment. Figure 8 shows all tumors at the end of the study and Figure 9 shows the average tumor weight value in each of the experimental groups. Table. Average tumor weight in each group

    [00118] As shown in Figure 8, Figure 9 and the table above, the average tumor weight of the positive control group was less than that of the negative control group (P = 0.0159), which showed that the drug of positive control (sorafenib) had an inhibitory effect on mouse model of human hepatocellular carcinoma HepG2-GFP orthotopic at the tested dosage.
    [00119] The average tumor weight in the TH3424 (2.5mg / kg) and TH3424 (5mg / kg) group was 0.0081g and 0g, respectively. The whole was statistically less significant than in the negative control group. Among these, the average weight was 0g in the high dose group, and it was only 0.0081 ± 0.0088g in the low dose group, which was 0% and 5.2% of that in the negative control group, respectively. This suggested that TH3424 had a very strong inhibitory effect on the mouse model of human hepatocellular carcinoma HepG2-GFP orthotopic at the tested doses and also showed a clear dose-effect relationship.
    [00120] Tumor IR was calculated based on the final average weight of tumors according to the formula: IR (%) = (1-treatment (t) / control (c)) x 100 IR for each treatment group: IR (%) = (1-PC / NC) x100 = (1-0.0637 / 0.1543) x 100-58.7% IR (%) = (1-TH2870-2LT / NC) x100 = (1- 0.0081 / 0.1543) x100 «94.8% IR (%) = (1-TH2870-2HT / NC) x100 = (1-0.0000 / 0.1543) x100« 100% Note: NC represents the negative control group PC positive control group
    [00121] The tumor inhibition rate was 58.7% in the sorafenib-treated group (P = 0.0159 versus control), showing a strong inhibitory effect in a mouse model of human hepatocellular carcinoma HepG2-GFP at the tested dose. However, in this group, the average body weight of the experimental mice was statistically less significant than that of the negative control group. This suggested that a certain degree of toxicity is noted for experimental mice by administering sorafenib at the tested dosage. The tumor inhibition rate of TH3424 (2.5 mg / kg) and TH3424 (5 mg / kg) was 94.8% and 100%, respectively, showing a very strong tumor inhibitory effect and a clear dose-effect relationship in the mouse model of human hepatocellular carcinoma HepG2-GFP orthotopic. No obvious toxicity was noted for experimental mice at the tested dosages of TH3424 Example 10.TH3424 in animal models of T-cell leukemia
    [00122] Leukemic cells were prepared by thawing ~ 150x106 of AL7473 LN2 cells (about 2-3 vials) and placing in a 37 ° C water bath quickly. All cells were transferred in a 50 ml falcon tube with 40 ml of pre-heated complete medium. The cells were centrifuged at 1200rpm for 5min. The cells were resuspended with 40ml RPMI1640. The cells were then counted. The cells were centrifuged at 1200 rpm for 5 min. and resuspended with ice-cooled PBS to 2 million cells per 100ulPBS. 100ul of saline solution containing 2X106 cells prepared above were used to inject into each mouse by IV. The FACS analysis was made for blood weekly during the experimental period by transferring the samples in FACS tubes, adding human CD45 FITC and isotypes in corresponding tubes for all samples and incubating the cells on ice for 30 minutes in the dark. 2 ml of red blood cell lysing buffer was added to each tube and incubated on ice for another 30 minutes in the dark. All samples were vortexed several times during this process and the samples were centrifuged for 5 min. at 1500 rpm at 4 ° C. The supernatants were discarded and 2 ml of ice-cooled wash buffer was added to each tube. The samples were centrifuged for 5 min at 1500 rpm at 4 ° C and these steps were repeated. The cells were resuspended in 150 μl of wash buffer / PBS to acquire FACS. Samples were analyzed on a BD Calibur using Cell Quest or Flowjo and the results were analyzed using Prism 5.0.
    [00123] Blood samples for FACS were collected on day 26,33,42 after inoculation of cells in the pre-group. After samples from the group, FACS was done weekly until the study was completed (Day 50.57.64 after cell inoculation). Samples of 10 μl of plasma were collected for each mouse and 3 blood smears were obtained from each group (Group1: # 7, # 43, # 52; Group2: # 11, # 15, # 23; Group3: # 5, # 9 , # 48; Group4: # 25, # 28, # 36) on day 57 after cell inoculation. The sample list is summarized below. Table: Collection of blood at the termination point for all animals.

    [00124] Results of body weight changes in mice are shown in Figure 10. Peripheral blood was collected weekly for human CD45 antibody FACS detected during treatment because treatment started on Day 43 after cell inoculation. The mice were dosed at Dia 43,50,57 and 64 respectively (group 1 and group 2 dosing only) after cell inoculation. The tumor load growth curve after clustering is shown in Figure 11. The percentages of human CD45 + human leukemia cells in peripheral blood in each group increased as the disease progressed, and the percentage curves fell within 7 days after the first dosage, except group2 (P> 0.05). After the second dosage, the treatment group (group 2-4) was significantly smaller than the vehicle group (group 1) (P <0.001). All mice (total of 4 groups, 10 mice in each group) were sacrificed 6 days after the fourth dosage. Blood, spleen and bone marrow from all mice were collected for detected human CD45 FACS. Spleen and bone and 3 mice in each group were collected for FFPE. The details data and sample lists at the termination point are summarized in Appendix 10.3. The cargatumor in peripheral blood, spleen and bone marrow of mice at the study termination point is shown in Figure 12. Respectively compared to blood, spleen and bone marrow in group1, except bone marrow in group2 showed slightly significant differences (P <0, 05), the entire treatment group (groups 2-4) showed significant differences (P <0.01). Example 10. Study of acute toxicity and pharmacokinetics in non-naive monkeys
    [00125] TH 3423 and TH 3424 were tested in non-naive monkeys (1 male and 1 female for each compound at 2 mg / kg) with 30 min intravenous infusion, TK sampling: on Day 1 and Day 15: 0, 25, 0.5, 0.75, 1 and 2 h after starting the infusion. Serum chemistry and hematology: pre-dose (Day 1), day 5, day 8 (pre-dose), day 15 (pre-dose), day 22 and day 28. Clinical observation: daily during the study, total 35 days . Feed consumption: daily during the study, total 35 days. Body weight measurement: twice weekly for five weeks.
    [00126] The TK parameters are listed in the following tables:



    [00127] The serum chemistry is shown in the following table:



    [00128] The hematology data are shown in the following table:




    [00129] The changes in body weight are listed in the following table:

    [00130] It should be understood that, although the present invention has been specifically described by certain aspects, modalities and optional aspects, modification, improvement and variation of such aspects, modalities and optional aspects can be adopted by those skilled in the art and such modifications, improvements and variations are considered to be within the scope of this disclosure.
    [00131] The inventions were described in the document in a broad and generic way. Each of the narrower subgeneric groupings and species, relevant to the generic description, is also part of the invention. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also described here in terms of any individual member or subgroup of members of the Markush group.
    权利要求:
    Claims (15)
    [0001]
    1. COMPOSITION, characterized by the fact that it is (R) - 1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxy-4-nitrophenyl) -1-
    [0002]
    2. COMPOSITION, characterized by the fact that it is (S) - 1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1- ethyl-N, N'-bis (ethylene) phosphoramidate:
    [0003]
    3. Compound according to claim 1 or 2, characterized by the fact that the compound has an enantiomeric excess of not less than 80%.
    [0004]
    4. Compound according to claim 3, characterized by the fact that the compound has an enantiomeric excess of not less than 90%, or not less than 95%.
    [0005]
    5. Compound according to claim 1 or 2, characterized by the fact that the compound is substantially pure.
    [0006]
    6. Compound according to claim 5, characterized by the fact that the compound has a purity of at least 50%, or at least 90%.
    [0007]
    Compound according to claim 1 or 2, characterized in that it is for use in treating or ameliorating one or more symptoms of a proliferative disease in an individual, comprising administering to the individual a compound according to claim 1 or 2 .
    [0008]
    8. Compound according to claim 1 or 2, characterized by the fact that the disease is cancer including liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer, melanoma, prostate cancer, breast cancer , leukemia, esophageal cancer, kidney cancer, gastric cancer, colon cancer, brain cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreatic cancer, a sarcoma cancer, and rectal cancer.
    [0009]
    Compound according to claim 1 or 2, characterized in that it comprises use in a method of inhibiting the growth of a cell comprising contacting the cell with a compound according to claim 1 or 2.
    [0010]
    10. Compound according to claim 1 or 2, characterized by the fact that the use of the method, according to claim 9, comprises the fact that the cell is a cancer cell.
    [0011]
    11. PHARMACEUTICAL COMPOSITION, characterized by the fact that it comprises the compound according to claim 1 or 2, and a pharmaceutically acceptable excipient.
    [0012]
    12. MANUFACTURING PROCESS, of the compound of formula I:
    [0013]
    13. PROCESS FOR RESOLUTION IN ONE OF THE COMPOUND RACEMATE ENANTIOMERS, according to claim 1, or for the enrichment of a mixture with any enantiomeric excess of said compound of formula (I), characterized by the fact that it comprises the following steps: a) subject the compound of formula (I): to an optical resolution process in which the enantiomerically-enriched racemic mixture 1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl-N, N '- bis (ethylene) phosphoramidate is separated into its two enantiomers (S) -1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl-N, N'-bis (ethylene ) phosphoramidate and (R) -1- (3- (3-N, N-dimethylaminocarbonoyl) phenoxyl-4-nitrophenyl) -1-ethyl- N, N'-bis (ethylene) phosphoramidate by chiral chromatography containing a stationary phase and a mobile phase, in which the stationary phase comprises a silica gel impregnated with a functionalized polysaccharide, and in which the mobile phase comprises an alcohol and another solvent.
    [0014]
    14. Process according to claim 13, characterized by the fact that alcohol is methanol.
    [0015]
    15. Process according to claim 13, characterized by the fact that the other solvent is CO2.
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    同族专利:
    公开号 | 公开日
    AU2016357728A1|2017-11-30|
    AU2016357728B2|2020-08-27|
    WO2017087428A1|2017-05-26|
    EP3390415A1|2018-10-24|
    ES2781398T3|2020-09-01|
    JP2018517710A|2018-07-05|
    EP3390415B1|2020-02-26|
    HK1250988A1|2019-01-18|
    EP3390415A4|2019-05-22|
    BR112017025778A2|2018-08-14|
    TW201726695A|2017-08-01|
    KR20170130615A|2017-11-28|
    CN108290911A|2018-07-17|
    IL256569D0|2018-02-28|
    CN108290911B|2020-05-08|
    CA2990696A1|2017-05-26|
    JP6695360B2|2020-05-20|
    KR101985778B1|2019-06-04|
    TWI702222B|2020-08-21|
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    法律状态:
    2020-07-28| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|
    2021-02-23| B09A| Decision: intention to grant|
    2021-03-16| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 15/11/2016, OBSERVADAS AS CONDICOES LEGAIS. |
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    US201562255905P| true| 2015-11-16|2015-11-16|
    US62/255,905|2015-11-16|
    US201662324259P| true| 2016-04-18|2016-04-18|
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    PCT/US2016/062114|WO2017087428A1|2015-11-16|2016-11-15|- and -1-phenoxyl-4-nitrophenyl)-1-ethyl-n,n'-bis phosphoramidate, compositions and methods for their use and preparation|
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