JP2007277241A - POLYMORPHIC FORM OF (2R,Z)-2-AMINO-2-CYCLOHEXYL-N-(5-(1-METHYL-1H-PYRAZOL-4-YL)-1-OXO-2,6-DIHYDRO-1H-[1,2]DIAZEPINO[4,5,6-cd]INDOL-8-YL)ACETAMIDE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a crystalline polymorphic form of a compound having good physical and pharmacological characteristics and a pharmaceutical composition containing the crystalline polymorphic form. <P>SOLUTION: The present invention provides a new polymorphic forms and amorphous form of (2R,Z)-2-amino-2-cyclohexyl-N-(5-(1-methyl-1H-pyrazol-4-yl)-1-oxo-2,6-dihydro-1H-[1,2]diazepino[4,5,6-cd]indol-8-yl)acetamide and a process for preparing those. The present invention provides, further, use of such polymorphic forms and amorphous form as a component of a pharmaceutical composition for treating a cancer or a mammalian disease condition mediated by protein kinase activity. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [ The invention relates to novel polymorphs of [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide and methods for their preparation. The present invention is also directed to pharmaceutical compositions containing at least one polymorph and therapeutic uses of such polymorphs and compositions.

  Compound (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2 Diazepino [4,5,6-cd] indol-8-yl) acetamide (also referred to as “compound 1”),

ならびに薬学的に許容できるその塩については、２００５年１１月２２日に発行された米国特許第６，９６７，１９８号に記載されており、その開示は、参照により本明細書に組み込まれるものとする。

As well as pharmaceutically acceptable salts thereof are described in US Pat. No. 6,967,198 issued Nov. 22, 2005, the disclosure of which is hereby incorporated by reference. To do.

Many anticancer drugs, as well as radiation therapy, cause DNA damage to cells, particularly cancer cells. CHK1 inhibition enhances the anti-cancer effects of these anti-cancer agents or radiation therapy by inhibiting S and G ₂ arrest of those DNA-damaged cells, resulting in abnormal cell division and cell death of these cells. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] Diazepino [4,5,6-cd] indol-8-yl) acetamide is a potent CHK1 protein kinase inhibitor. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro- in combination with anticancer agents or radiation therapy 1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide, a pharmaceutically acceptable salt or solvate thereof, or a mixture thereof is used in anticancer agents or radiation therapy. It will greatly enhance the anticancer effect.

  The solid compound may exist in an amorphous or crystalline form. Each of the different crystalline forms of the same compound is considered a polymorph of the compound. Crystalline polymorphs are different crystal forms of the same compound. Different polymorphs of the same active pharmaceutical ingredient (API) may have very different physical properties such as thermodynamic stability, solubility, hygroscopicity as well as pharmacological properties such as oral bioavailability. These properties greatly affect drug properties in areas such as shelf life, production costs, consistent dosage, and even drug efficacy. Accordingly, it is desirable to have a polymorph of a compound that has good physical and pharmacological properties.

  In one embodiment, the present invention provides (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1- represented by formula 1 Crystals of oxo-2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide are provided.

In another embodiment, the present invention provides (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6 -Crystals of dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide are provided. The crystal is preferably a substantially pure Form I polymorph. In one aspect of the embodiments, the crystal has a powder X-ray diffraction pattern that includes peaks at diffraction angles (2θ) of 23.6 ± 0.1 and 8.5 ± 0.1. In another aspect of the embodiment, the crystal comprises a powder X-ray diffraction pattern comprising peaks at diffraction angles (2θ) of 23.6 ± 0.1, 8.5 ± 0.1, and 20.7 ± 0.1. Have In another aspect of the embodiments, the crystals are at diffraction angles (2θ) of 23.6 ± 0.1, 8.5 ± 0.1, 20.7 ± 0.1, and 16.4 ± 0.1. It has a powder X-ray diffraction pattern including a peak. In another aspect of the embodiments, the crystals are 23.6 ± 0.1, 8.5 ± 0.1, 20.7 ± 0.1, 16.4 ± 0.1 and 17.0 ± 0. It has a powder X-ray diffraction pattern including a peak at a diffraction angle (2θ) of 1. In another aspect of the embodiment, the crystal has a powder X-ray diffraction pattern that includes a peak at essentially the same diffraction angle (2θ) as shown in FIG. In another aspect of this embodiment, the crystalline form has chemical shifts 175.0 ± 0.1, 137.6 ± 0.1, 134.9 ± 0.1, 110.3 ± 0.1, 106.3. It has a ¹³ C solid state NMR peak pattern including peaks at ± 0.1, 41.1 ± 0.1 and 32.6 ± 0.1 ppm. In another aspect of this embodiment, the crystal has the following chemical shifts 175.0 ± 0.1, 137.6 ± 0.1, 134.9 ± 0.1, 110.3 ± 0.1, 106 It has a ¹³ C solid state NMR peak pattern comprising at least 3 of the 7 peaks at 3 ± 0.1, 41.1 ± 0.1 and 32.6 ± 0.1 ppm. In another aspect of this embodiment, the crystal has the following chemical shifts 175.0 ± 0.1, 137.6 ± 0.1, 134.9 ± 0.1, 110.3 ± 0.1, 106 It has a ¹³ C solid state NMR peak pattern comprising at least 4 of 7 peaks at 3 ± 0.1, 41.1 ± 0.1 and 32.6 ± 0.1 ppm. In another aspect of this embodiment, the crystal has the following chemical shifts 175.0 ± 0.1, 137.6 ± 0.1, 134.9 ± 0.1, 110.3 ± 0.1, 106 It has a ¹³ C solid state NMR peak pattern comprising at least 5 of the 7 peaks at 3 ± 0.1, 41.1 ± 0.1 and 32.6 ± 0.1 ppm. In another aspect of this embodiment, the crystal has the following chemical shifts 175.0 ± 0.1, 137.6 ± 0.1, 134.9 ± 0.1, 110.3 ± 0.1, 106 It has a ¹³ C solid state NMR peak pattern comprising at least 6 of the 7 peaks at 3 ± 0.1, 41.1 ± 0.1 and 32.6 ± 0.1 ppm. In another aspect of this embodiment, the crystal has a chemical shift of 175.0 ± 0.2, 137.6 ± 0.2, 134.9 ± 0.2, 110.3 ± 0.2, 106.3. It has a ¹³ C solid state NMR peak pattern including peaks at ± 0.2, 41.1 ± 0.2 and 32.6 ± 0.2 ppm. In another aspect of this embodiment, the crystal has a chemical shift of 175.0 ± 0.2, 137.6 ± 0.2, 134.9 ± 0.2, 110.3 ± 0.2, 106.3. It has a ¹³ C solid state NMR peak pattern comprising at least three of the seven peaks at ± 0.2, 41.1 ± 0.2 and 32.6 ± 0.2 ppm. In another aspect of this embodiment, the crystal has a chemical shift of 175.0 ± 0.2, 137.6 ± 0.2, 134.9 ± 0.2, 110.3 ± 0.2, 106.3. It has a ¹³ C solid state NMR peak pattern comprising at least 4 out of 7 peaks at ± 0.2, 41.1 ± 0.2 and 32.6 ± 0.2 ppm. In another aspect of this embodiment, the crystal has a chemical shift of 175.0 ± 0.2, 137.6 ± 0.2, 134.9 ± 0.2, 110.3 ± 0.2, 106.3. It has a ¹³ C solid state NMR peak pattern comprising at least 5 of the 7 peaks at ± 0.2, 41.1 ± 0.2 and 32.6 ± 0.2 ppm. In another aspect of this embodiment, the crystal has a chemical shift of 175.0 ± 0.2, 137.6 ± 0.2, 134.9 ± 0.2, 110.3 ± 0.2, 106.3. It has a ¹³ C solid state NMR peak pattern comprising at least 6 of the 7 peaks at ± 0.2, 41.1 ± 0.2 and 32.6 ± 0.2 ppm. In another aspect of this embodiment, the crystal has a ¹³ C solid state NMR peak pattern that includes peaks at essentially the same chemical shift positions as shown in FIG. 4b.

In another embodiment, the present invention provides (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6 -Crystals of dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide are provided. It is preferred that the crystal is a substantially pure Form II polymorph. In one aspect of this embodiment, the crystal has a powder X-ray diffraction pattern that includes peaks at diffraction angles (2θ) of 25.3 ± 0.1 and 16.0 ± 0.1. In another aspect of the embodiments, the crystals are at diffraction angles (2θ) of 25.3 ± 0.1, 16.0 ± 0.1, 13.9 ± 0.1 and 29.2 ± 0.1. It has a powder X-ray diffraction pattern including a peak. In another aspect of the embodiments, the crystals have 25.3 ± 0.1, 16.0 ± 0.1, 13.9 ± 0.1, 29.2 ± 0.1, and 12.2 ± 0. It has a powder X-ray diffraction pattern including a peak at a diffraction angle (2θ) of 1. In another aspect of the embodiments, the crystals are 25.3 ± 0.1, 16.0 ± 0.1, 13.9 ± 0.1, 29.2 ± 0.1, 12.2 ± 0. It has a powder X-ray diffraction pattern including peaks at 1 and 16.8 ± 0.1 diffraction angles (2θ). In another aspect of the embodiments, the crystals are 25.3 ± 0.1, 16.0 ± 0.1, 13.9 ± 0.1, 29.2 ± 0.1, 12.2 ± 0. It has a powder X-ray diffraction pattern including peaks at diffraction angles (2θ) of 1, 16.8 ± 0.1, 6.9 ± 0.1 and 13.6 ± 0.1. In another aspect of the embodiment, the crystal has a powder X-ray diffraction pattern that includes a peak at essentially the same diffraction angle (2θ) as shown in FIG. In another aspect of the embodiments, the crystalline form has chemical shifts 177.7 ± 0.1, 133.2 ± 0.1, 127.8 ± 0.1, 103.8 ± 0.1, and 22.7. It has a ¹³ C solid state NMR peak pattern including a peak at ± 0.1 ppm. In another aspect of the embodiments, the crystalline form has chemical shifts 177.7 ± 0.1, 133.2 ± 0.1, 127.8 ± 0.1, 103.8 ± 0.1, and 22.7. It has a ¹³ C solid state NMR peak pattern including at least 3 out of 5 peaks at ± 0.1 ppm. In another aspect of the embodiments, the crystalline form has chemical shifts 177.7 ± 0.1, 133.2 ± 0.1, 127.8 ± 0.1, 103.8 ± 0.1, and 22.7. It has a ¹³ C solid state NMR peak pattern including at least 4 out of 5 peaks at ± 0.1 ppm. In another aspect of the embodiments, the crystal has a chemical shift of 177.7 ± 0.2, 133.2 ± 0.2, 127.8 ± 0.2, 103.8 ± 0.2, and 22.7. It has a ¹³ C solid state NMR peak pattern including a peak at ± 0.2 ppm. In another aspect of the embodiments, the crystal has a chemical shift of 177.7 ± 0.2, 133.2 ± 0.2, 127.8 ± 0.2, 103.8 ± 0.2, and 22.7. It has a ¹³ C solid state NMR peak pattern including at least 3 of 5 peaks at ± 0.2 ppm. In another aspect of the embodiments, the crystal has a chemical shift of 177.7 ± 0.2, 133.2 ± 0.2, 127.8 ± 0.2, 103.8 ± 0.2, and 22.7. It has a ¹³ C solid state NMR peak pattern including at least 4 of 5 peaks at ± 0.2 ppm. In another aspect of the embodiment, the crystal has a ¹³ C solid state NMR peak pattern that includes peaks at essentially the same chemical shift positions as shown in FIG. 5b.

In another embodiment, the present invention provides (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6 An amorphous form of -dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide is provided. The amorphous material is preferably substantially pure. In one aspect of the embodiments, the amorphous form has a chemical shift of 163.6 ± 0.2, 138.9 ± 0.2, 131.4 ± 0.2, 129.9 ± 0.2, and 30.8 ±. It has a ¹³ C solid state NMR peak pattern including a peak at 0.2 ppm. In another aspect of the embodiments, the amorphous form has chemical shifts 163.6 ± 0.2, 138.9 ± 0.2, 131.4 ± 0.2, 129.9 ± 0.2, and 30.8. It has a ¹³ C solid state NMR peak pattern including at least 3 of 5 peaks at ± 0.2 ppm. In another aspect of the embodiments, the amorphous form has chemical shifts 163.6 ± 0.2, 138.9 ± 0.2, 131.4 ± 0.2, 129.9 ± 0.2, and 30.8. It has a ¹³ C solid state NMR peak pattern comprising at least 4 of the following peaks at ± 0.2 ppm. In another aspect of the embodiment, the amorphous material has a ¹³ C solid state NMR peak pattern that includes peaks at essentially the same chemical shift positions as shown in FIG.

  In another embodiment, the present invention provides (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6 -Dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide (Compound 1) solid form, wherein the solid form is polymorphic Form I of Compound 1 Including at least two forms selected from polymorphic Form II and amorphous. In one aspect of this embodiment, the solid comprises at least 10% polymorph Form I. More preferably, the solid comprises at least 20% polymorph Form I. More preferably, the solid comprises at least 30% polymorph Form I. Further, it is more preferred that the solid comprises at least 30%, at least 40%, or at least 50% polymorph Form I. Further, it is more preferred that the solid comprises at least 60%, at least 70% or at least 80% polymorph Form I. More preferably, the solid comprises at least 90% polymorph Form I. Further, it is more preferred that the solid comprises at least 95% polymorph Form I.

  In another embodiment, the present invention provides (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo- of the present invention. Polymorphic form I, polymorphic form II, amorphous or solid form of 2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide A pharmaceutical composition is provided.

  In another embodiment, the present invention provides (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo- of the present invention. Provided is a pharmaceutical composition comprising a solid of 2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide.

  In another embodiment, the present invention provides a method of treating cancer in a mammal comprising administering to the mammal in need thereof a pharmaceutical composition of the present invention.

  In another embodiment, the invention provides a method of treating cancer in a mammal, wherein the mammal in need thereof has a therapeutically effective amount in combination with a therapeutically effective amount of an anticancer treatment selected from anticancer agents and radiation therapy. Of administering a pharmaceutical composition of the present invention. In one aspect of this embodiment, the anticancer treatment is an anticancer agent. Anticancer agents include Ara-c, VP-16, cisplatin, adriamycin, 2-chloro-2-deoxyadenosine, 9-β-D-arabinosyl-2-fluoroadenine, carboplatin, gemcitabine, camptothecin, paclitaxel, BCNU, 5-fluorouracil , Irinotecan, and doxorubicin. In another aspect of this embodiment, the anticancer treatment is radiation therapy.

  In another embodiment, the present invention is a method of treating a mammalian disease state mediated by CHK1 protein kinase activity, wherein the mammal in need thereof is selected from anticancer agents, radiation therapy and combinations thereof (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6- of the present invention in combination with anti-cancer therapy Administering polymorphic Form I, polymorphic Form II, amorphous or solid form of dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide Provide a method. In one aspect of this embodiment, the anticancer treatment is an anticancer agent. Anticancer agents include Ara-c, VP-16, cisplatin, adriamycin, 2-chloro-2-deoxyadenosine, 9-β-D-arabinosyl-2-fluoroadenine, carboplatin, gemcitabine, camptothecin, paclitaxel, BCNU, 5-fluorouracil , Irinotecan, and doxorubicin. In another aspect of this embodiment, the anticancer treatment is radiation therapy.

Those skilled in the art will recognize that the chemical shifts of the polymorphic Form I, II or amorphous ¹³ C solid state NMR of Compound 1 may have some differences depending on the external reference used. In the claims of the present invention, ¹³ C solid state NMR chemical shift refers to the chemical shift obtained when the high magnetic field signal of adamantane at 29.5 ppm is used as an external reference.

  The term “in combination with” refers to (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo to a mammal in need thereof. -2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide, pharmaceutically acceptable salts or solvates thereof, or mixtures thereof Refers to the relative timing of administration of the first therapeutic treatment relative to the administration of the second therapeutic treatment, such as an anti-cancer agent or radiation therapy, the relative timing being relative to that normally used in the pharmaceutical field for combination therapy. Timing. In particular, the relative timing may be sequential or simultaneous.

  The term “hyperproliferative disorder” refers to abnormal cell growth (eg, loss of contact inhibition) independent of normal regulatory mechanisms and includes normal cell abnormal growth and abnormal cell growth. This includes, but is not limited to, abnormal growth of both benign and malignant tumor cells (tumors). Examples of such benign proliferative diseases are psoriasis, benign prostatic hypertrophy, human papilloma virus (HPV), and restenosis.

  The term “cancer” includes lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer. Breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer , Parathyroid cancer, adrenal gland cancer, soft tissue cancer, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma , Cancer of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, or a combination of one or more of the foregoing cancers However, it is not limited to these. In another embodiment of the method, the abnormal cell growth is a benign proliferative disease, including but not limited to psoriasis, benign prostatic hypertrophy or restenosis.

  The term “mediated by CHK1 protein kinase activity” refers to a biological or molecular process that is regulated, regulated, or inhibited by CHK1 protein kinase activity.

  The term “polymorph” refers to different crystals of the same compound. “Polymorphs” include hydrates of the same compound (eg, bound water present in the crystal structure) and other solid molecular forms including solvates (eg, bound solvents other than water). However, it is not limited to these.

  The term “pharmaceutically acceptable carrier, diluent, or vehicle” refers to a material (or one or more materials) that may be included with a particular agent to form a pharmaceutical composition, solid or It may be a liquid. Examples of solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Examples of liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, carriers or diluents include delayed release or controlled release materials known in the art such as glyceryl monostearate or glyceryl distearate, ethylcellulose, hydroxypropylmethylcellulose, methyl methacrylate, alone or with a wax. Can be included.

  The term “pharmaceutical composition” refers to a physiological / pharmaceutical of one or more of the compounds or polymorphs described herein, or a physiologically / pharmaceutically acceptable salt or solvate thereof. Refers to a mixture with a pharmaceutically acceptable carrier and excipient. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

  The term “radiotherapy” refers to the medical use of radiation to control malignant cells.

  (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] The term “substantially pure” for a particular polymorph or amorphous form of diazepino [4,5,6-cd] indol-8-yl) acetamide means that the polymorph or amorphous form is (2R, Z) — 2-Amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5, 6-cd] indole-8-yl) means containing less than 10% by weight of impurities, including other polymorphs of acetamide, preferably less than 5% by weight, preferably less than 3% by weight, preferably less than 1% by weight. . Such purity can be determined, for example, by X-ray powder diffraction.

  In general, the term “therapeutically effective amount” refers to the compound being administered, a pharmaceutically acceptable salt or solvate thereof, or to some extent alleviate one or more of the symptoms of the disorder being treated, or Refers to the amount of the mixture. In particular, when the term is used to describe a combination therapy, a “therapeutically effective amount” means 1) enhance the therapeutic effect of another treatment such as an anti-cancer agent or radiation therapy, or 2) other therapies In combination with refers to the amount of a particular treatment that should alleviate one or more of the symptoms of the disorder being treated to some extent. With regard to the treatment of cancer, the symptoms of the disease being treated include: a) reducing the size of the tumor, b) inhibiting tumor metastasis (ie slowing, preferably stopping to some extent), c) tumor growth To some extent (ie, slow to some extent, preferably stop) and d) to some extent reduce (or preferably eliminate) one or more symptoms associated with cancer.

  The term “2-theta value” or “2θ” refers to the peak position based on the experimental setup of the X-ray diffraction experiment described in the present invention, including but not limited to the radiation source used and the wavelength of the radiation source. , A common horizontal axis unit in the diffraction pattern. The experimental setup requires that the reflected beam be recorded with an angle 2 theta (2θ) if the reflected light is diffracted and the incident beam forms an angle theta (θ) with a particular grating plane.

  The terms “treat”, “treating”, and “treatment” refer to a method of reducing or suppressing cancer and / or its attendant symptoms. In particular, with respect to cancer, these terms simply mean that the life expectancy of an individual suffering from cancer is increased or that one or more of the symptoms of the disease are reduced.

The term “essentially the same” as used herein with respect to X-ray diffraction peak positions means to take into account typical peak positions and intensity variations. For example, those skilled in the art will recognize that the peak position (2θ) exhibits some inter-device variation, typically on the order of 0.1 °. In addition, as those skilled in the art will appreciate, relative peak intensities show instrument-to-apparatus variations as well as variations due to crystallinity, preferred orientation, prepared sample surface, and other factors known to those skilled in the art. It should be received only as a qualitative measure. Similarly, “essentially the same” as used herein with respect to solid state NMR spectra and Raman spectra is intended to encompass variations associated with these analytical techniques known to those skilled in the art. For example, the ¹³ C chemical shift measured in solid state NMR typically has a variation of 0.1 ppm, while the Raman shift typically has a variation of 1 cm ⁻¹ .

  In this section, “BOC”, “Boc” or “boc” refers to N-tert-butoxycarbonyl, “CBZ” refers to carbobenzyloxy, “DCE” refers to dichloroethane, and “DCM” refers to , Dichloromethane, “DCC” refers to 1,3-dicyclohexylcarbodiimide, “DIC” refers to diisopropylcarbodiimide, “DIPEA” or “DIEA” refers to diisopropylethylamine, “DMA” refers to N, N-dimethylacetamide, “DMAP” refers to 4-dimethylaminopyridine, “DME” refers to 1,2-dimethoxyethane, “DMF” refers to dimethylformamide, “DMSO” refers to dimethylformamide It refers to sulfoxide, and “DPPP” is 1,3-bis (diphenylphosphate). Fino) propane, “EDC” refers to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and “HATU” refers to hexafluorophosphate O- (7-azabenzotriazole-1- Yl) -N, N, N ′, N′-tetramethyluronium, “HBTU” is O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl hexafluorophosphate Refers to uronium, “HOAc” refers to acetic acid, “HOBt” refers to 1-hydroxybenzotriazole hydrate, “IPA” refers to isopropyl alcohol, “LAH” refers to lithium aluminum hydride “LiHMDS” refers to lithium bis (trimethylsilyl) amide, “MSA” refers to methanesulfonic acid, “MTBE” , Methyl t-butyl ether, “NMP” refers to 1-methyl 2-pyrrolidinone, “TEA” refers to triethylamine, “TFA” refers to trifluoroacetic acid, “TIPS” refers to triisopropylsilyl -, TMSCl refers to trimethylsilyl chloride and "Trt" refers to triphenylmethyl-.

  Compound 1 has been found to exist in two or more polymorphic crystals as well as amorphous. Described herein are methods for producing these polymorphs with high purity and for characterizing these different polymorphs. Also provided is a pharmaceutical formulation comprising Compound 1.

I. Synthesis of Compound 1:
Compound 12, ie, (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [ US Pat. No. 6,967, issued Nov. 22, 2005, for a synthetic route for preparing the HCl salt of 1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide. , 198. In the present invention, the synthesis route is modified. In particular, the reaction conditions were improved and adapted to the scale of the reaction. As shown in Scheme 1, Compound 1 can be prepared directly from Compound 11, the N-Boc precursor of Compound 12. Compound 1 can also be prepared from compound 12 by neutralizing the HCl salt. With either approach, different polymorphs of Compound 1 can be obtained depending on processing and purification conditions.

  As illustrated in Scheme 1, 2-methyl-3,5-dinitrobenzoic acid reacts with N, N-dimethylformamide dimethyl acetal under heating to give compound 2. Suitable solvents for this reaction are DMF, DMA, MTBE, toluene and THF. It is preferred to use THF. The reaction is preferably carried out at about 50 ° C. The reaction mixture is concentrated under reduced pressure followed by the addition of methanol. In that case, compound 2 precipitates as a product.

Compound 2 is then treated with acid in methanol under heating to give compound 3. Typical acids that can be used here are TMSCl, HCl, MSA, H ₂ SO ₄ and TFA. It is preferred to use TMSCl. During the course of the reaction, compound 3 crystallizes out of the reaction solution, thus greatly simplifying the isolation process.

In US Pat. No. 6,967,198, compound 3 was converted to compound 4 by a conventional hydrogenation reaction under Pd / C and H ₂ . However, this conventional hydrogenation reaction condition is difficult to control on a large scale due to the fact that the reduction of two nitro groups is extremely exothermic. In the present invention, compound 3 is converted to compound 4 by a transfer hydrogenation reaction followed by an acid-promoted cyclization reaction. The transfer hydrogenation reaction of compound 3 provides a dose control procedure, thus reducing the thermal hazard. Pd / C and HCO ₂ NH ₄ , Pd (OH) ₂ and BH ₃ . Various transfer hydrogenation reagents such as NMe ₃ , FeCl ₃ / C and hydrazine and Pd / C and ammonium formate can be used. Pd / C and ammonium formate are preferably used. Compound 3 can be reduced using Pd / C and ammonium formate in THF. The crude product is then cyclized under strong acid to give compound 4. A typical condition for this acid-catalyzed cyclization is to use concentrated HCl in MeOH.

  The primary amino group of compound 4 is then BOC protected to give compound 5. In US Pat. No. 6,967,198, this reaction was performed using triethylamine as the base and the reaction was performed in acetonitrile. In the present invention, this reaction is performed using milder conditions such as an inorganic base such as NaOH and a solvent such as THF.

  Compound 5 is converted to compound 6 by a Vilsmeier formylation reaction. In US Pat. No. 6,967,198, this reaction was performed in methylene chloride using 3 equivalents of Vilsmeier reagent. This conversion in methylene chloride was a solid-solid reaction and was quite problematic on a large scale because neither compound 5 nor the reaction intermediate was very soluble in methylene chloride. Further, when compound 5 was treated with 3 equivalents of Vilsmeier reagent, diformylation occurred and an additional hydrolysis step was required to convert diformylated indole to compound 6. In the present invention, the reaction is carried out in a solvent in which the compound 5 has good solubility. In this case, a typical solvent is THF. Since compound 5 is easily soluble in THF, a solution-solid reaction is realized. Mono-C formylation occurred in preference to N-formylation when the amount of Vilsmeier reagent was reduced to 1.1 equivalents, and directly produced compound 6, thus greatly simplifying the reaction procedure.

  Compound 6 is converted to compound 7 by treating compound 6 with hydrazine under acidic conditions. For this reaction, hydrazine monohydrate and 30% aqueous hydrazine can be used. More preferably, the reaction is carried out in an excess, preferably more than 5 equivalents of hydrazine and dilute MeOH solution.

  Compound 7 is then brominated to give compound 8. In US Pat. No. 6,967,198, NBS was used as a bromination reagent. In the present invention, pyridinium tribromide is used as a cheaper alternative.

Compound 8 is then converted to compound 9 by Suzuki coupling. In US Pat. No. 6,967,198, Pd (dppf) Cl ₂ was used. In the present invention, a cheaper alternative Pd (PPh ₃ ) ₄ is used. Compound 8 is treated with ₃ mol% Pd (PPh ₃ ) ₄ and K ₃ PO ₄ in DMA. Usually, the residual palladium content in the crude product is very high (6000-8000 ppm). It has been found that due to the poor solubility of compound 9 in common organic solvents, Florisol treatment for Pd removal is not effective and requires large amounts of solvent. In the present invention, a simple and effective water precipitation Pd removal procedure has been developed: 1) Dissolving the crude product in a water-immiscible solvent such as DMF, DMA, THF, DMSO DMA, preferably DMA, and then obtained N-acetylcysteine is added to the solution and stirred for 1 hour. 2) Water is added to precipitate the product while leaving the N-acetylcysteine-palladium complex in the aqueous solution. After N-acetylcysteine treatment, the residual Pd level drops below 400 ppm, resulting in API compound 1 with a residual Pd of less than 20 ppm.

  Compound 9 is then treated with 4M HCl in dioxane and methylene chloride to give compound 10 as the hydrogen chloride salt. Ion chromatographic analysis revealed that the hydrogen chloride content varies from batch to batch with a ratio of hydrogen chloride to the corresponding free base of 2.1 to 2.8. Other acids such as sulfuric acid, sulfonic acid and TFA can also be used for this reaction.

  Compound 10 is then coupled with a boc protected hexyl amino acid to give compound 11. EDC is selected as the coupling agent. Many coupling conditions such as EDC, HATU and DCC can be used. In the present invention, EDC is used as a coupling agent together with DMAP as a catalyst. The required amount of DMAP is preferably the same molar amount as the hydrogen chloride present in the starting amine salt. A large excess of DMAP in the reaction mixture can cause significant racemization. Compound 11 is isolated by adding water to the reaction mixture. Chiral HPLC analysis indicated that less than 1% of the racemized byproduct was removed by crystallization in the final step.

Compound 11 is then treated with a strong acid to remove the boc group. All conditions such as HCl / MeOH, HCl / EtOH, TFA / CH ₂ Cl ₂ or MSA / THF can be used. When compound 11 is treated with HCl / MeOH or HCl / EtOH, the reaction mixture is evaporated under reduced pressure to give compound 12, ie (2R, Z) -2-amino-2-cyclohexyl-N- (5- HCl of (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide A salt can be obtained.

Compound 1 can be obtained directly from compound 11 by basic water treatment of de-boc reaction. For example, compound 11 is mixed with MSA and refluxed in THF to remove the Boc group. Once the reaction is complete and the reaction mixture is cooled, an aqueous solution of Na ₂ CO ₃ , K ₂ CO ₃ or NaHCO ₃ can be added to the reaction mixture. Subsequent standard processing gives compound 1 in good purity and yield.

Compound 1 can also be obtained from Compound 12 by mixing Compound 12 with a basic aqueous solution, such as aqueous NaHCO ₃ , stirring the mixture vigorously, and then extracting the mixture with an organic solvent.

II. Preparation of Polymorphs I, II and Amorphous of Compound 1 Polymorph Form I Polymorph Form I of Compound 1 can be prepared directly from Compound 11 by removal of the boc group under strongly acidic conditions followed by basic water treatment. Typical strong acid conditions used here are HCl / MeOH, HCl / EtOH, MSA / THF or TFA. Compound 11 is preferably treated with methanesulfonic acid in THF to remove the boc group. After the reaction is complete, the mixture is basified with an aqueous inorganic base solution, such as an aqueous solution of NaOH, Na ₂ CO ₃ , NaHCO ₃ , K ₂ CO ₃ or KHCO ₃ . Preference is given to using 2M aqueous sodium hydroxide or saturated NaHCO ₃ solution. The organic phase is separated from the aqueous phase and dried over magnesium sulfate. The organic solution is then reduced to a smaller volume and ethanol is added. The resulting solution is reduced to a smaller volume and more ethanol is added. This volume reduction and ethanol addition process is repeated until Compound 1 precipitates as polymorphic Form I.

Polymorphic Form I of Compound 1 can also be prepared from Compound 12. Compound 12 is dissolved in small portions in an aqueous inorganic base solution with vigorous stirring. A typical aqueous inorganic base solution used here is an aqueous solution of NaOH, Na ₂ CO ₃ , NaHCO ₃ , K ₂ CO ₃ or KHCO ₃ . Here, it is preferable to use an aqueous NaHCO ₃ solution. The mixture is extracted with a large amount of an organic solvent such as EtOAc. The resulting organic solution is washed with brine, dried over Na ₂ SO ₄ and filtered. The filtrate is concentrated in vacuo to give a yellow solid that is an amorphous form of Compound 1. The yellow solid is then dissolved in EtOH under heating, preferably at about 75-80 ° C. with vigorous stirring. The solution is preferably cooled to room temperature of about 22 ° C. and left at 22 ° C. for 12 hours. Polymorphic Form I precipitates out.

B. Polymorph Form II Polymorph Form II Compound 1 is described in the previous paragraph, except that Polymorph Form II is formed in methanol at about 4 ° C. during the final processing procedure. Can be obtained from compound 12 or 11 according to similar procedures.

C. Amorphous The amorphous form of Compound 1 can be prepared directly from Compound 12 following a similar procedure for preparing polymorphic Form I. After compound 12 is dissolved in small portions in aqueous base solution with vigorous stirring, the mixture is extracted with copious amounts of EtOAc. The resulting EtOAc solution is washed with brine, dried and filtered. The filtrate is concentrated in vacuo to give a yellow solid that is an amorphous form of Compound 1.

  Amorphous forms of Compound 1 can also be prepared from Compound 11 following a similar procedure for preparing polymorphic Form I. During processing, EtOAc is used as the organic phase. After drying the organic phase, the solvent is evaporated at about 40-70 ° C. to obtain amorphous Compound 1.

  An amorphous form of Compound 1 can also be prepared from Polymorph Form I of Compound 1 by dissolving polymorph Form I in THF until saturation, followed by removal of the solvent at 50 ° C.

III. Characterization and properties of different polymorphs of Compound 1:
Each solid of Compound 1 has an onset of melting point as shown by the following X-ray powder diffraction patterns (ie, X-ray diffraction peaks at various diffraction angles (2θ)), differential scanning calorimetry (DSC) thermogram endotherm (And onset of dehydration for hydrates), Raman spectrum diagram pattern, water solubility, international conference of harmonization (ICH), photostability under high-intensity light conditions, and physical and chemical storage stability Can be characterized by one or more. For example, polymorph I, II and amorphous samples of Compound 1 were each characterized by their peak position and relative intensity in their X-ray powder diffraction pattern.

A. Polymorph X-ray powder diffraction pattern The X-ray powder diffraction pattern for the first batch of polymorphic Form I, Form II and amorphous form of Compound 1 (Figure 3a) is 1.5418 wavelength operating at 40 kV and 40 mA. Measured on a Bruker AXS D8-Discover diffractometer with a Cu Kα ^average radiation source. Samples were analyzed from an angle of 4-40 ° (2θ) using a general area diffraction detector. The detector was placed 30 cm from the sample. As those skilled in the art will recognize, the peak position (2θ) typically exhibits some device-to-device variation on the order of 0.1 °. Therefore, when reporting peak position (2θ), such numbers are intended to encompass such inter-device variation. Furthermore, when a crystal of the present invention is described as having a powder X-ray diffraction pattern that is essentially the same as that shown in a figure, the term “essentially the same” means that at the diffraction peak position. It is intended to encompass such inter-device variation. Table 1 shows the 2θ values for polymorphic Form I and Form II. Amorphous materials show a continuous X-ray powder diffraction spectrum and 2θ values cannot be collected.

  The X-ray powder diffraction pattern of the second batch of amorphous Compound 1 is shown in FIG. The preparation of the second batch of amorphous material is described in Example 4b. Here, the powder X-ray diffraction pattern was prepared with a Bruker D5000 diffractometer using CuKα radiation (wavelength = 1.5406Å). The instrument was equipped with a straight focused X-ray tube. The tube voltage and current were set to 38 kV and 38 mA, respectively. The divergence and scattering slits were set to 1 mm, and the light receiving slit was set to 0.6 mm. The diffracted radiation was detected by a Sol-X energy dispersive X-ray detector. A θ2θ continuous scan at 2.4 ° 2θ / min (1 second / 0.04 ° 2θ step) from 3.0 to 40 ° 2θ was used. The experiment was performed at ambient temperature. An alumina standard (NIST standard reference material 1976) was analyzed and checked for instrument alignment. Data was collected and analyzed using BRUKER AXS DIFFRAC PLUS software version 2.0. The PXRD peak was selected using the peak maximum peak height.

  As those skilled in the art will appreciate, relative peak intensities should show instrumental variations as well as variations due to crystallinity, preferred orientation, prepared sample surface, and other factors known to those skilled in the art. Yes, and should be received only as a qualitative measure.

  When the solid includes two or more polymorphs of the present invention, the X-ray diffraction pattern has a characteristic peak for each individual polymorph of the present invention. For example, a solid comprising two polymorphs has a powder X-ray diffraction pattern that is the product of two X-ray diffraction patterns corresponding to a substantially pure polymorph.

B. Polymorph solid state NMR (SSNMR)
Solid state NMR is a powerful tool for analyzing solids. Different polymorphs often show significant chemical shift differences in solid state ¹³ C cross-polarization and magic angle rotation (CP / MAS) NMR. Solid state ¹³ C CP / MAS NMR was performed on polymorphic Forms I and II, as well as amorphous.

For polymorphic Form I, the first ¹³ C SSNMR spectrum was collected on a 600 MHz Bruker spectrometer and the chemical shift externally referenced the methyl resonance of hexamethylbenzene at 17.36 ppm. Chemical shift using a 500 MHz Bruker spectrometer for the second ¹³ C SSNMR spectrum of polymorph I of compound 1, the first and second spectra of polymorph II and the second batch of amorphous form Externally referenced the high magnetic field signal of adamantane at 29.5 ppm. Table 2a shows the first ¹³ chemical shifts of the C SSNMR spectrum for the first ¹³ C SSNMR spectrum and polymorphic Form II polymorphic Form I. Table 2b shows the chemical shifts of the polymorphic Form I second spectrum, the polymorphic Form II second spectrum and the amorphous second batch spectrum of Compound 1. In Table 2b, peak intensity is defined as peak height and may vary depending on the actual setting of CPMAS experimental parameters.

C. Differential Scanning Calorimetry Test of Polymorph Different polymorphs of Compound 1 were also distinguished using differential scanning calorimetry (DSC). DSC is a thermoanalytical technique that measures the difference in the amount of heat required to raise the temperature of a sample and a standard as a function of temperature. The DSC test is often used to test the melting point of a solid.

  In the present invention, DSC was performed using a TA Instruments Q-1000 DSC. The scan rate for Polymorph Form I is 10 ° C./min from 25 ° C. to 300 ° C., and under such conditions, Polymorph Form I has an onset melting point of 272 ° C. The scan rate for polymorphic Form II was 40 ° C./min from 25 ° C. to 300 ° C. Under such conditions, polymorphic Form II began to melt at 242 ° C. Generated rapidly. We believe that the new crystals produced were polymorphic Form I. This was verified by the following test: heating polymorph Form II to 260 ° C. and examining the resulting solid by PXRD. The PXRD results were identical to the polymorphic Form I PXRD results. When the scan rate for polymorphic Form II is set to 10 ° C / min from 25 ° C to 300 ° C, Polymorphic Form II begins to melt at 243 ° C and rapidly produces new crystals. And then melted at 274 ° C. and we thought the new crystal was also polymorphic Form I.

D. Thermogravimetric analysis of polymorphs Thermogravimetric analysis (TGA) is a test procedure that records changes in the weight of a specimen while it is heated in air or in a controlled atmosphere such as nitrogen. The thermogravimetric curve (thermogram or TGA scan graph) provides information on the solvent and water content of the material and the thermal stability.

  In the present invention, TGA was performed on polymorphic Form I and Form II of Compound 1 using TA Instruments TGA Q500. The temperature was increased from 25 ° C. to 310 ° C. and 350 ° C. at 10 ° C./min for polymorphic Form I and Form II, respectively. Polymorphic Form I TGA analysis indicated that crystalline Form I lost about 0.26% of the total weight by the time the temperature reached 265 ° C. Degradation of polymorph I occurred rapidly immediately after melting. Polymorphic Form II TGA analysis showed about 0.85% reduction in total weight by the time the temperature reached 275 ° C. Degradation of polymorph Form II occurred rapidly after 275 ° C. These TGA results are consistent with the conclusion that both polymorphic Form I and polymorphic Form II are anhydrous / unsolvated forms.

E. Solubility of different polymorphs Equilibrium solubility was tested for both polymorphic Form I and polymorphic Form II. Samples were prepared by dissolving each corresponding polymorph or in ethanol. The sample was stirred overnight at room temperature and centrifuged to remove undissolved solids. The solution was then analyzed by HPLC. The results are listed in Table 3.

F. Hygroscopicity of different polymorphs Different polymorphs of a compound may have different hygroscopic properties. Isothermal water adsorption and desorption experiments of both polymorphic Form I and Form II of Compound 1 were performed on Surface Measurements Systems Dynamic Vapor Sorption-1000 (DVS). Each polymorphic Form I and Form II was loaded into a DVS instrument starting at 25 ° C. with 0% RH. Humidity was increased stepwise from 0% RH to 90% RH by stepping in 10% RH increments. After the sample weight stabilized at 90% RH, the humidity was reduced stepwise from 90% RH to 0% RH to complete the entire cycle. The temperature was kept constant at 25 ° C. during the entire procedure. The weight gain during this experiment was used to determine hygroscopicity. Samples with 0-90% RH and less than 2.0% weight gain are considered non-hygroscopic.

  Polymorphic Forms I and II showed about 1.7% and 1.3% increase in water at 0-90% RH, respectively.

F. Stability of different polymorphs The solid stability of Polymorph Form I of Compound 1 was investigated for 6 weeks at 40 ° C. at 75% relative humidity (RH) in both open and sealed vials. Samples were analyzed by X-ray powder diffraction to determine physical stability, and HPLC was performed for chemical stability. The results are shown in Table 4.

Polymorphs are considered enantiotropic when one form is more thermodynamically stable at one temperature and the other form is more stable at another temperature. The temperature at which both polymorphs are equally stable is known as the transition temperature (T _t ). An enantiotrophic study was conducted on polymorphic Form I and Form II of Compound 1. 10-20 mg of a 1: 1 mixture of Form I and Form II was added to 1-2 mL of ethanol to form a slurry. Samples of such slurries were prepared and stirred at 70 ° C, 60 ° C, 50 ° C, 40 ° C, 30 ° C, room temperature (about 23 ° C) and 3.5 ° C, respectively. The sample was then centrifuged. The supernatant was decanted and the remaining material was dried under vacuum at room temperature. The resulting material was analyzed by PXRD. The results are summarized in Table 5.

IV. Methods Using Polymorphs of the Invention Polymorphs of Compound 1 of the invention are useful in all embodiments where Compound 1 is useful. The method using Compound 1 was described in US Pat. No. 6,967,198 as a method using a compound genus including Compound 1. US Pat. No. 6,967,198 described that the inventive compounds therein can be used in combination with therapeutically effective amounts of antineoplastic agents or radiation therapy to treat neoplasms in mammals. A therapeutically effective amount of an antineoplastic agent as described in US Pat. No. 6,967,198 that the polymorphs and pharmaceutical compositions of Compound 1 of the present invention can be used in combination with the inventive compounds therein Or it can be used in combination with radiation therapy is contemplated by the present invention.

Example 1
(2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] Preparation of diazepino [4,5,6-cd] indol-8-yl) acetamide HCl salt (HCl salt of compound 1) 2- (2-dimethylamino-vinyl) -3,5-dinitro-benzoic acid methyl ester Preparation of compound 2: 2-Methyl-3,5-dinitro-benzoic acid (102.62 g, 0.45 mol) was dissolved in anhydrous THF (1200 mL). N, N-dimethylformamide dimethyl acetal (Aldrich, purity 94%, 3.0 equivalents, 172.4 g, 1.35 mol) was added with stirring over 10 minutes at room temperature under a nitrogen atmosphere. The temperature rose from 22 ° C to 29 ° C. The solution was immediately heated to 50 ° C. and stirred at this temperature for 8 hours behind the shield. The reaction solution was then cooled to room temperature and concentrated on a rotary evaporator to remove most of the THF and unreacted N, N-dimethylformamide dimethyl acetal until approximately 250 g of crude material remained in the flask (water bath temperature was Did not exceed 30 ° C.). Methanol (400 ml) was added and the slurry was stirred at room temperature for 1 hour. The solid was collected by filtration, washed with cold methanol (60 ml) and dried in a stream of air to give 109.08 g of a purple solid (84% yield). ¹ H NMR (300 MHz, CDCl ₃ ) δ 3.01 (s, 6H), 3.93 (s, 3H), 5.99 (d, 1H, J = 13.5 Hz), 6.75 (d, 1H, J = 13.2 Hz), 8.49 (d, 1H, J = 2.4 Hz), 8.57 (d, 1H, J = 2.4 Hz).

Preparation of 2- (2,2-dimethoxy-ethyl) -3,5-dinitro-benzoic acid methyl ester compound 3: Purple solid 2 (92.25 g, 0.313 mol) was suspended in anhydrous MeOH (1000 mL). . TMSCl (70.25 g, 0.65 mol, 2.1 eq) was added over 10 minutes. A clear solution was formed. The solution was heated at 60 ° C. (slow reflux) for 20 hours. HPLC analysis showed the disappearance of starting material. When the reaction mixture was cooled to room temperature, a white solid precipitated (in many cases the product crystallized out of the reaction mixture when the reaction was nearly complete). The reaction mixture was stirred at room temperature for 1 hour. The precipitated solid was collected by filtration, washed with cold MeOH (50 mL) and dried in a stream of air to give 82.71 g of a white solid. The mother liquor was concentrated to remove about 800 ml of solvent. Additional product precipitated out. The mixture was cooled to 0 ° C. and stirred for 1 hour. The orange solid was then collected, washed with cold methanol (25 mL), and dried in a stream of air to give an additional 5.58 g of product. The combined yield of compound 3 was 88.29 g (90%). ¹ H NMR (300 MHz, CDCl ₃ ) δ 3.30 (s, 6H), 3.73 (d, 2H, J = 5.1 Hz), 4.00 (s, 3H), 4.51 (t, 1H, J = 5.1 Hz), 8.65 (d, 1H, J = 2.4 Hz), 8.76 (d, 1H, J = 2.4 Hz).

Preparation of 6-amino-1H-indole-4-carboxylic acid methyl ester hydrochloride compound 4: In a 2 L Erlenmeyer flask, 10% Pd / C wet catalyst (8.6 g), methanol (800 ml) and THF ( 160 ml). Then ammonium formate (139.1 g, 2.21 mol) was added with stirring and the mixture was heated to 35 ° C. Water (100 ml) was added. Compound 3 was then added in small portions over 10 minutes. The reaction temperature was controlled at an addition rate of 3 and maintained at 40 ° C to 45 ° C by appropriate cooling. After the addition was complete, the reaction mixture was stirred for 30 minutes. HPLC analysis indicated that the reaction was complete. The reaction mixture was cooled to room temperature. The catalyst was filtered through a celite pad and the celite pad was washed with MeOH (50 ml). The filtrate was concentrated under reduced pressure to remove volatile components. EtOAc (500 ml) and water (75 ml) were then added and the mixture was stirred for 5 minutes. The organic phase was separated and the aqueous phase was extracted with EtOAc (200 ml). The combined organic solution was concentrated to dryness to give a pale yellow oil (79.17 g). To the oily intermediate was added MeOH (100 ml). The resulting solution was then added to a solution of concentrated hydrochloric acid (37 wt%, 82.8 g) in MeOH (600 ml) while maintaining the temperature at 32 ° C. The reaction mixture was stirred at 35 ° C. for 3 hours. During this time, a solid product precipitated. HPLC analysis indicated that the reaction was complete. The reaction mixture was cooled to room temperature. EtOAc (500 ml) was added and the resulting mixture was stirred for 30 minutes. The solid was collected by filtration to give 45.07 g of product. The filtrate was concentrated to give a paste (331 g). EtOAc (400 ml) was added to the paste and the mixture was stirred for 30 minutes. The solid was then collected by filtration to give a second crop of product 4 (16.28 g). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 3.93 (s, 3H), 6.97 (d, 1H, J = 1.8 Hz), 7.65 (m, 1H), 7.72 (d, 1H, J = 1.8 Hz), 7.80 (s, 1H), 11.81 (s, br, 1H).

  Preparation of 6-amino-1H-indole-4-carboxylic acid methyl ester hydrochloride 4 via direct hydrogenation of 2- (2-dimethylamino-vinyl) -3,5-dinitro-benzoic acid methyl ester compound 2: methanol (27 kg) and 2- (2,2-dimethoxy-vinyl) -3,5-dinitro-benzoic acid methyl ester 2 (10 kg, 33.87 mol) were charged into the reactor through a charge port. A 10% palladium on carbon catalyst (0.4 kg) was then charged to the reactor as a slurry in water (2.5 ± 0.5 kg). Ethyl acetate (123 ± 3 kg) was added, followed by hydrogen in small portions. The internal temperature was maintained at about 0 ° C. by cooling and controlling the hydrogenation rate. After the pressure had become constant over 30 minutes, the hydrogen pressure was maintained at 50-60 psi (about 345-414 kPa) and the temperature was maintained at 0 ° C. for 2 hours. The catalyst was filtered off through an in-line filter (Note: The catalyst is pyrophoric. The catalyst must not be dried). The filtrate was concentrated by vacuum distillation at an internal temperature below 35 ° C. to remove about 95% of the solvent volume. Ethyl acetate (130 L) was added with stirring. Then 37% HCl (10 kg) was slowly added below 10 ° C. The resulting mixture was stirred at 10 ° C. for 1 hour. The solid was filtered on a plate filter and washed with methyl t-butyl ether (15 L). The product (wet cake) was transferred to a vacuum dryer and dried at 23 ± 3 ° C. to give 4.6 kg of product 4 (yield 60%).

Preparation of 6-tert-butoxycarbonylamino-1H-indole-4-carboxylic acid methyl ester 5: THF (600 ml) and 4M aqueous NaOH (138 ml, 0.55 mol) were charged into a reaction flask. Indole HCl salt 4 (61.0 g, 0.269 mol) was then added and the mixture was stirred until the solid was completely dissolved. (BOC) ₂ O (70.43 g, 0.32 mol) was added slowly as a THF (50 ml) solution while maintaining the temperature between 25 ° C. and 32 ° C. during the addition. The resulting reaction mixture was stirred at room temperature for 3 hours. HPLC analysis indicated that the reaction was complete. The aqueous phase was separated. The organic phase was washed with a saturated aqueous ammonium chloride solution (15 ml). The organic solution was then concentrated to give a paste (176 g). THF (40 ml) and heptane (800 ml) were added to the paste and the resulting mixture was stirred for 30 minutes. The solid was collected by filtration to give 69.86 g of product 5 (90% yield). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.50 (s, 9H), 3.89 (s, 3H), 6.83 (s, 1H), 7.41 (m, 1H), 7.89 (S, 1H), 7.91 (s, 1H), 9.38 (s, 1H), 11.24 (s, br, 1H).

Preparation of 6-tert-butoxycarbonylamino-3-formyl-1H-indole-4-carboxylic acid methyl ester compound 6: N, N-dimethylformamide (85 ml) was charged to a 250 ml flask and then in an ice-acetone bath. Cooled to -5 ° C. Phosphorus oxychloride (35.15 g, 0.23 mol) was poured into the flask at 4 ° C. over 10 minutes. After the addition was complete, the mixture was stirred at 0 ° C. for an additional 30 minutes. Indole 5 (60.57 g, 0.21 mol) and anhydrous THF (600 ml) were added in a separate 2 L three-necked flask equipped with an overhead stirrer and thermometer to produce a clear solution. The solution was cooled to −7 ° C. with an ice-acetone bath. While maintaining the temperature at −3 ° C., a cold, pre-formed Vilsmeier reagent was cannulated into the stirred THF solution over 10 minutes. A heavy precipitate formed within a few minutes after the addition was complete. The reaction mixture was further stirred at 0 ° C. for 45 minutes. EtOAc (600 ml) was added to the reaction mixture followed by cold aqueous NaOAc (3M, 310 ml, 0.93 mol) with vigorous stirring. The temperature rose to 10 ° C. after quenching. The cooling bath was removed. The reaction mixture was warmed to 21 ° C. over 20 minutes and stirred at this temperature for 2.5 hours. The solid was completely dissolved. HPLC analysis indicated that all intermediates were converted to product. Stirring was stopped and the aqueous layer was separated. The organic layer was concentrated under reduced pressure to remove volatiles. A paste (226 g) was obtained. Water (30 ml) and EtOAc (70 ml) were added to the paste. The resulting mixture was stirred for 30 minutes. The solid was collected by filtration, washed with EtOAc (25 ml) and dried to give 62.98 g of product (95% yield, 97% purity). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.50 (s, 9H), 3.86 (s, 3H), 7.68 (d, 1H, J = 1.8 Hz), 7.97 (s, 1H), 8.23 (s, 1H), 9.56 (s, 1H), 10.10 (s, 1H), 12.25 (s, br, 1H).

Preparation of 1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-ylcarbamate tert-butyl compound 7: MeOH (1200 ml), acetic acid (100 ml ) And aldehyde 6 (62.98 g, 0.20 mol) were charged into a 2 L flask. Aqueous hydrazine (35 wt%, 88.8 g, 0.97 mol) was then added with stirring. The mixture was heated to 60 ° C. and stirred for 3 hours. HPLC analysis indicated that the reaction was complete. Heating was stopped and the reaction mixture was cooled to room temperature. The mixture was stirred slowly at room temperature for 2 hours. The solid was collected by filtration to give 46.0 g of product. The filtrate was concentrated to give a paste (223 g). While stirring, water (250 ml) was added slowly. The mixture was stirred for an additional 50 minutes to granulate the solid. The solid was collected and dried to give a second crop of product (13.0 g). The combined yield of compound 7 was 99%. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.49 (s, 9H), 7.44 (s, 1H), 7.51 (d, 1H, J = 2.1 Hz), 7.62 (s, 1H), 7.76 (s, 1H), 9.45 (s, 1H), 10.19 (s, 1H), 11.63 (s, br, 1H).

Preparation of tert-butyl 8-bromo-1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-ylcarbamate 8: Compound 7 (45. 0 g, 0.15 mol) and N, N-dimethylformamide (0.45 L) were charged into a 5 L flask equipped with an overhead stirrer, thermometer and nitrogen gas inlet. The mixture was stirred to produce a clear solution. The solution was then cooled to −14 ° C. under nitrogen protection. Pyridinium tribromide (57.6 g, 0.18 mol) was added in portions over 5 minutes under nitrogen protection. During the addition, the reaction temperature was controlled below -5 ° C. After the addition was complete, the reaction mixture was stirred at 0 ° C. for 1.5 hours. HPLC analysis indicated that the reaction was complete. Water (0.225 L) was slowly added to the reaction mixture while maintaining the temperature below 20 ° C. during the addition. EtOAc (0.225 L) is added to the reaction mixture at 15 ° C., followed by aqueous potassium carbonate (preparation: 20.73 g of K ₂ CO ₃ dissolved in 60 ml of water and cooled to 10 ° C. for use). It was. The mixture was stirred for 15 minutes to dissolve the solid. Then, an aqueous sodium sulfite solution (preparation: 3.78 g of Na ₂ SO ₃ was dissolved in 18 ml of water) was added and stirred for 10 minutes. Water (0.5 L) was added over 3 minutes through the addition funnel while maintaining the temperature at 25-30 ° C. A solid gradually precipitated. The mixture was stirred for 20 minutes to granulate the solid. Additional water (0.625 L) was added to complete the precipitation. The mixture was stirred for 60 minutes. The solid was collected by filtration, washed with water (0.3 L) and dried in a stream of air for 90 minutes. The solid was dried in a vacuum oven at 60 ° C. with nitrogen air for 12 hours. The crude product was suspended in acetone (0.19 L) and stirred for 1 hour. Heptane (0.19 L) was slowly added into the mixture. The resulting mixture was stirred for 1.5 hours. The solid was collected by filtration, washed with a mixture of acetone (30 ml) and heptane (30 ml) and dried in a stream of air for 2 hours. The product was further dried in a vacuum oven at 50 ° C. for 12 hours. The solid weighed 45.5 g (yield 80%, purity 98.3%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.53 (s, 9H), 7.31 (s, 1H), 7.70 (d, 1H, J = 1.7 Hz), 7.77 (s, 1H), 9.57 (s, 1H), 10.47 (s, 1H), 12.54 (s, br, 1H).

5- (1-Methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-ylcarbamic acid Preparation of tert-butyl 9: Compound 8 (308.24 g, 0.81 mol) and N, N-dimethylformamide (2.5 L) were added to a 10 L flask equipped with an overhead stirrer, thermometer and nitrogen gas inlet. . A clear solution was formed. Water (0.625 L) was added to the DMA solution with stirring while controlling the temperature at 30 ° C. Then 1-methyl-4- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -1H-pyrazole (186 g, 0.89 mol) and potassium phosphate (431. 3 g, 2.03 mol) was added to the flask with stirring. The mixture was degassed by vacuum suction of the flask followed by a nitrogen flush. The degassing process was repeated 3 times over 20 minutes. Tetrakis (triphenylphosphine) palladium (28.2 g, 0.024 mol) was added under nitrogen protection. The reaction mixture was degassed 3 times over 20 minutes by vacuum / nitrogen cycle. The reaction was heated at 90 ° C. under nitrogen protection for 5 hours. HPLC analysis indicated that the reaction was complete. The reaction mixture was cooled to room temperature. N-acetylcysteine (30 g, 0.18 mol) was added into the flask and the mixture was stirred for 2 hours. While controlling the temperature at 30 ° C., water (5 L) was added over 30 minutes with stirring. A solid precipitated out. The mixture was stirred for an additional 2 hours to granulate the solid. The solid was collected by filtration and washed with a mixture of DMA (0.1 L) and water (0.3 L), water (0.7 L), and a mixture of acetone (0.3 L) and water (0.3 L). The filter cake was dried in a stream of air overnight and then in a vacuum oven at 60 ° C. for 16 hours. The crude product was dissolved in DMA (1.5 L). N-acetylcysteine (30 g, 0.18 mol) was added into the DMA solution and the solution was stirred for 2 hours. While controlling the temperature at 30 ° C., water (0.8 L) was added over 10 minutes with stirring. A solid precipitated out. The mixture was stirred for 20 minutes to granulate the solid. Additional water (2.2 L) was added over 5 minutes to complete the precipitation. The mixture was stirred for 1 hour. The solid was collected by filtration and washed sequentially with a mixture of DMA (0.2 L) and water (0.4 L), water (0.8 L), and a mixture of acetone (0.4 L) and water (0.4 L). The compound was dried in a vacuum oven at 60 ° C. until the water content was reduced to less than 1.5 wt%. The product weighed 286 g (93% yield, 98% apparent purity). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.49 (s, 9H), 3.92 (s, 3H), 7.57 (s, 1H), 7.65 (d, 1H, J = 1. 5 Hz), 7.68 (d, 1 H, J = 1.5 Hz), 7.90 (s, 1 H), 8.27 (s, 1 H), 9.40 (s, 1 H), 10.14 (s , 1H), 11.75 (s, br, 1H).

Preparation of 8-amino-5- (1-methyl-1H-pyrazol-4-yl) -2H- [1,2] diazepino [4,5,6-cd] indole-1 (6H) -one hydrochloride 10 : 5- (1-Methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-ylcarbamine Tert-butyl acid 9 (236.06 g, 0.62 mol) was charged into a 10 L flask equipped with an overhead stirrer, thermometer and bubbler. Dichloromethane (3.5 L) was added and the mixture was stirred for 10 minutes. The suspension was then cooled to 10 ° C. followed by the addition of 4M HCl in dioxane (2.4 L) over 10 minutes. The temperature did not exceed 25 ° C. The reaction mixture was stirred at room temperature for 16 hours. HPLC analysis indicated that about 98% of the starting material was consumed. The solid was collected by centrifugation under nitrogen protection and washed with dichloromethane (2.2 L). The product was then dried in a vacuum oven at 40 ° C. to give the product (239.77 g). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 3.94 (s, 3 H), 7.47 (d, 1 H, J = 1.8 Hz), 7.58 (d, 1 H, J = 1.8 Hz), 7.66 (s, 1H), 8.00 (s, 1H), 8.40 (s, 1H), 10.42 (s, 1H), 12.51 (s, br, 1H).

(R) -1-cyclohexyl-2- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5, Preparation of 6-cd] indol-8-ylamino) -2-oxoethylcarbamate tert-butyl 11: 8-amino-5- (1-methyl-1H-pyrazol-4-yl) -2H- [1,2 Diazepino [4,5,6-cd] indole-1 (6H) -one hydrochloride 10 (224.35 g, 0.64 mol, containing 25.7% chloride as determined by ion chromatography) anhydrous Suspended in DMF (2.2 L). 4-Dimethylaminopyridine (197.08 g, 1.65 mol) was added and the mixture was stirred for 30 minutes. Boc-D-cyclohexylglycine (179.80 g, 0.7 mol) was then added and the mixture was heated to 35 ° C. 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide (158.5 g, 0.83 mol) was added in one portion and the reaction mixture was heated at 50 ° C. for 1 hour. HPLC analysis indicated that the reaction was complete. The reaction mixture was cooled to room temperature. Water (2.2 L) was added over 10 minutes while maintaining the temperature at 30 ° C. The mixture was stirred for 20 minutes to granulate the solid. Additional water (4.4 L) was added over 5 minutes to complete the precipitation and the mixture was stirred for an additional 30 minutes. The solid was collected by filtration and washed with a mixture of DMF (0.5 L) and water (1.5 L), then water (1.0 L). The product was dried in a vacuum oven at 60 ° C. for at least 48 hours to give 293.91 g of product (HPLC purity 94.2%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ1.1 (m, 5H), 1.36 (s, 9H), 1.60 (m, 6H), 3.93 (s, 3H), 3.96 (M, 1H), 7.59 (s, 1H), 7.60 (s, 1H), 7.92 (s, 1H), 8.07 (s, 1H), 8.30 (s, 1H) 10.05 (s, 1H), 10.20 (s, 1H), 11.86 (s, br, 1H).

(2R) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [ Preparation of 4,5,6-cd] indol-8-yl) acetamide 1: Methanesulfonic acid (44.4 g, 0.46 mol) was added to THF (690 mL) in a 2 L flask. (R) -1-cyclohexyl-2- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5, 6-cd] Indol-8-ylamino) -2-oxoethylcarbamate tert-butyl 11 (30.0 g, 57.74 mol) was added via a powder funnel. THF (60 mL) was used to flush the funnel and flask sides. The flask was purged with nitrogen and the reaction was heated to 65 ° C. and stirred for 18-24 hours. HPLC analysis indicated that the reaction was complete. The reaction was cooled to room temperature using a water bath. 2M NaOH (255 mL) was added over 30 minutes while maintaining the temperature at 20 ± 5 ° C. After stirring for 5 minutes, the mixture was transferred to a 2 L separatory funnel using THF to wash away. The layers were separated. The aqueous phase was extracted with THF (60 mL). The organic fractions were combined and washed twice with saturated aqueous NaCl (2 × 60 mL). MeOH (80 mL) and MgSO ₄ (42 g) were added. The mixture was stirred for 75 minutes and then filtered through celite. The cake was washed with 9: 1 THF / MeOH (3 × 50 mL) and the solution was transferred to a 2 L distillation flask. The solution was concentrated to a volume of 300 mL by distillation at 1 atmosphere. EtOH (450 mL) was added slowly and the solution was cooled to 50 ° C. to crystallize the product. Once the product crystallized, the mixture was reheated and distilled to a volume of 450 mL. The reaction solvent ratio was monitored by ¹ H NMR. The distillation was stopped when the THF content was reduced to 5 mol%. The resulting yellow suspension was cooled to 25 ° C. over 60 minutes and vacuum filtered on paper. The filter cake was washed with EtOH (2 × 75 ml). The solid was transferred to a crystallization dish and dried under vacuum at 55 ° C. for 16 hours. 13.55 g of compound 1 (54%) was obtained as a yellow solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 1.14 (m, 5H), 1.62 (m, 6H), 3.10 (d, 1H, J = 5.7 Hz), 3.93 (s, 3H), 7.58 (s, 1H), 7.59 (s, 1H), 7.91 (s, 1H), 8.11 (s, 1H), 8.29 (s, 1H), 10. 19 (s, 1H), 11.83 (s, br, 1H).

(Example 2)
(2R) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [ Preparation of 4,5,6-cd] indol-8-yl) acetamide polymorph Form I:
From the HCl salt of Compound 1: (2R) -2-amino-2-cyclohexyl-N- (5- (5- (5); 5%; 5 eq; ca. 18 mL) with vigorous stirring in small portions. 1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide hydrochloride ( 1 g) was added. To the mixture was added 500 mL of ethyl acetate and 80 mL of water. The suspension was stirred vigorously for 10-15 minutes and stirring was stopped. The organic phase was carefully decanted into another Erlenmeyer and the process was repeated three more times with 500 mL of ethyl acetate each time. The combined organic phases (about 2000 mL) were washed with brine (100 mL), dried over sodium sulfate and filtered. The volatiles were removed in vacuo and the resulting yellow solid was dried overnight to yield 670 mg of Compound 1 polymorph I (yield approximately 84%).

  From an amorphous form of Compound 1: (2R) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide amorphous (100 mg) was mixed with ethyl alcohol (1.5 mL) to form a suspension. The suspension was heated at 75-80 ° C. for 5 hours with vigorous stirring and left at 22 ° C. for 12 hours. The solid was filtered and washed with 0.5 mL ethyl alcohol. Then, it was dried in a house vacuum for 24 hours, and then further dried at 25 to 30 ° C. for 12 hours with a vacuum pump to obtain 66 mg of a crystalline substance.

(Example 3)
(2R) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [ Preparation of 4,5,6-cd] indol-8-yl) acetamide polymorph Form II:
Compound 1, (2R) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2 The polymorphic Form I (50 mg) of diazepino [4,5,6-cd] indol-8-yl) acetamide was slurried in 1 mL of methanol at 4 ° C. for 23 days. The slurry was centrifuged at 14,000 RPM to separate the solid. The solid was dried in a low temperature vacuum oven to obtain Form II.

Example 4a
(2R) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [ Preparation of a first batch of amorphous 4,5,6-cd] indol-8-yl) acetamide amorphous:
1.3 g of compound 11 (2HCl) was dissolved in water (200 mL) at 22 ° C., followed by addition of saturated aqueous sodium bicarbonate (50 mL) and 500 mL of ethyl acetate. An additional 2 mL of sodium bicarbonate and an additional 50 mL of water were added. After vigorous stirring, the mixture was filtered and the phases were separated. The aqueous phase was re-extracted with ethyl acetate (2 x 200 mL). The combined organic phases were washed with brine and dried over sodium sulfate. Filtration followed by evaporation of the volatiles to 10 mL gave a yellow solid that was filtered and dried. The total amount of the amorphous material obtained was 690 mg.

(Example 4b)
(2R) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [ Preparation of second batch of amorphous of 4,5,6-cd] indol-8-yl) acetamide amorphous:
749.2 mg of polymorphic Form I of Compound 1 was dissolved in 850 mL of USP grade ethanol. The solution was vacuum filtered using a Buchner funnel and Whatman type 2 filter paper. The filtered solution was transferred to a 190 mm x 100 mm crystallization dish and evaporated under ambient conditions for 63 hours. 589.1 mg of orange solid was recovered as a second batch of Compound 1 amorphous.

(Example 5)
¹³ C solid state NMR of polymorphic Form I (second spectrum), Form II (second spectrum) and amorphous form (second batch) of Compound 1
The sample was filled in a ZrO ₂ rotor. Form I and amorphous samples were filled into 4 mm rotors, and Form II samples were filled into 7 mm rotors. Carbon spectra were collected at ambient conditions with a Bruker-Biospin 4 and 7 mm CPMAS probe placed in a wide bore Bruker-Biospin Advance DSX 500 MHz NMR spectrometer. The rotor was placed at the magic angle and rotated at 15.0 kHz (4 mm rotor) and 7.0 kHz (7 mm rotor). The fast rotation speed minimized the strength of the spinning sideband. The number of scans was adjusted to obtain a sufficient S / N. One-dimensional ¹³ C spectra of Form I and amorphous samples are collected using ¹ H- ¹³ C crossed bipolar magic angle rotation (CPMAS), followed by complete suppression of spinning sidebands for Form II samples. (TOSS) was performed. TOSS was applied to suppress spinning sidebands. In order to optimize signal sensitivity, the cross-polarization contact time was adjusted to 2.0 ms and the decoupling field was set to about 75 kHz. For type I, 512 scans were acquired with a 30 s recycle delay, for type II 256 scans were acquired with a 30 s recycle delay, and for an amorphous sample, 2048 scans were acquired with a 3 s recycle delay. All spectra were referenced using an external sample of adamantane whose high magnetic field signal was set at 29.5 ppm.

(2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] FIG. 4 is an X-ray powder diffraction pattern of polymorphic Form I of diazepino [4,5,6-cd] indol-8-yl) acetamide. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] FIG. 3 is an X-ray powder diffraction pattern of polymorphic Form II of diazepino [4,5,6-cd] indol-8-yl) acetamide. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] It is a figure which shows the X-ray powder diffraction pattern of the amorphous body (1st batch) of diazepino [4,5,6-cd] indol-8-yl) acetamide. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] It is a figure which shows the X-ray powder diffraction pattern of the amorphous body (2nd batch) of diazepino [4,5,6-cd] indol-8-yl) acetamide. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] FIG. 3 is a solid state ¹³ C cross-polarization and magic angle rotation (CP / MAS) NMR (first spectrum) of polymorphic Form I of diazepino [4,5,6-cd] indol-8-yl) acetamide. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] FIG. 3 is a solid state ¹³ C cross-polarization and magic angle rotation (CP / MAS) NMR (second spectrum) of polymorphic Form I of diazepino [4,5,6-cd] indol-8-yl) acetamide. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] FIG. 3 is a diagram showing solid form ¹³ C CP / MAS NMR (first spectrum) of polymorphic form II of diazepino [4,5,6-cd] indol-8-yl) acetamide. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] FIG. 3 is a diagram showing solid ¹³ C CP / MAS NMR (second spectrum) of polymorphic Form II of diazepino [4,5,6-cd] indol-8-yl) acetamide. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] FIG. 3 is a diagram showing solid ¹³ C CP / MAS NMR of an amorphous form (second batch) of diazepino [4,5,6-cd] indol-8-yl) acetamide.

Claims (25)

  (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] A crystal of diazepino [4,5,6-cd] indol-8-yl) acetamide.   The crystal of claim 1, which is a polymorph of Form I.   The crystal body according to claim 2, which has a powder X-ray diffraction pattern including peaks at diffraction angles (2θ) of 23.6 ± 0.1 and 8.5 ± 0.1.   The crystal of claim 2 having a powder X-ray diffraction pattern comprising a peak at essentially the same diffraction angle (2θ) as shown in FIG. Chemical shifts 175.0 ± 0.2, 137.6 ± 0.2, 134.9 ± 0.2, 110.3 ± 0.2, 106.3 ± 0.2, 41.1 ± 0.2 and The crystal of claim 2 having a ¹³ C solid state NMR peak pattern including a peak at 32.6 ± 0.2 ppm. Chemical shifts 175.0 ± 0.2, 137.6 ± 0.2, 134.9 ± 0.2, 110.3 ± 0.2, 106.3 ± 0.2, 41.1 ± 0.2 and The crystal of claim 2 having a ¹³ C solid state NMR peak pattern comprising at least 3, at least 4, at least 5 or at least 6 of the 7 peaks at 32.6 ± 0.2 ppm. The crystal of claim 2 having a ¹³ C solid state NMR peak pattern comprising peaks at essentially the same chemical shift positions as shown in Figure 4b.   8. A crystal as claimed in any one of claims 2 to 7 which is a substantially pure Form I polymorph.   The crystal of claim 1, which is a polymorph of Form II.   The crystal body according to claim 9, which has a powder X-ray diffraction pattern including peaks at diffraction angles (2θ) of 25.3 ± 0.1 and 16.0 ± 0.1.   The crystal of claim 9 having a powder X-ray diffraction pattern comprising a peak at essentially the same diffraction angle (2θ) as shown in FIG. ¹³ C solid state NMR peaks including peaks at chemical shifts 177.0 ± 0.2, 133.2 ± 0.2, 127.8 ± 0.2, 103.8 ± 0.2 and 22.7 ± 0.2 ppm. The crystal body according to claim 9, having a pattern. Chemical shifts at least of 5 peaks at 177.7 ± 0.2, 133.2 ± 0.2, 127.8 ± 0.2, 103.8 ± 0.2 and 22.7 ± 0.2 ppm The crystal of claim 9 having a ¹³ C solid state NMR peak pattern comprising three or at least four. The crystal of claim 9 having a ¹³ C solid state NMR peak pattern comprising a peak at essentially the same chemical shift position as shown in Figure 5b.   15. A crystal as claimed in any one of claims 9 to 14 which is a substantially pure Form II polymorph.   (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro-1H- [1,2] Amorphous form of diazepino [4,5,6-cd] indol-8-yl) acetamide. ¹³ C solid state NMR including peaks at chemical shifts 163.6 ± 0.2, 138.9 ± 0.2, 131.4 ± 0.2, 129.9 ± 0.2, and 30.8 ± 0.2 ppm The amorphous body according to claim 16, which has a peak pattern. Of the five peaks at chemical shifts 163.6 ± 0.2, 138.9 ± 0.2, 131.4 ± 0.2, 129.9 ± 0.2, and 30.8 ± 0.2 ppm The amorphous body according to claim 16, which has a ¹³ C solid state NMR peak pattern including at least 3 or at least 4. The amorphous body of claim 16 having a ¹³ C solid state NMR peak pattern comprising peaks at essentially the same chemical shift positions as shown in FIG.   20. Amorphous according to claims 16-19, which is substantially pure.   (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazole-) comprising at least two forms selected from polymorph I, polymorph II and amorphous 4-yl) -1-oxo-2,6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide solid.   The solid of claim 21 comprising at least 80% polymorph Form I.   23. (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2, according to any of claims 1 to 22. A pharmaceutical composition comprising the form of 6-dihydro-1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide.   24. A method of treating cancer in a mammal, wherein the mammal in need is treated with a therapeutically effective amount of the pharmaceutical composition of claim 23 in combination with a therapeutically effective amount of an anticancer treatment selected from anticancer agents and radiation therapy. A method comprising administering.   A method of treating a mammalian disease state mediated by CHK1 protein kinase activity, wherein the mammal in need thereof is combined with a therapeutically effective amount of an anticancer treatment selected from an anticancer agent and radiation therapy. To (2R, Z) -2-amino-2-cyclohexyl-N- (5- (1-methyl-1H-pyrazol-4-yl) -1-oxo-2,6-dihydro Administering a form of -1H- [1,2] diazepino [4,5,6-cd] indol-8-yl) acetamide.

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