TW200806671A - Polymorphic forms 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

The present invention relates to novel 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 to processes for their preparation. Such polymorphic forms and amorphous form may be a component of a pharmaceutical composition and may be used to treat a cancer or a mammalian disease condition mediated by protein kinase activity.

Description

200806671 IX. Description of invention:

I: TECHNICAL FIELD OF THE INVENTION FIELD OF THE INVENTION The present invention relates to (2R,Z)_2-amino-2-cyclohexyl_n_(5-(1-methyl-5-1 H-mouth 〇-4-yl) -1 - flavonyl-2,6-diaza-1 H-[ 1,2] bis-oxo-[4,5,6-cd]11-bamboo-8-yl) The new polymorphic form and its preparation method. The invention is also directed to therapeutic uses of pharmaceutical compositions comprising at least one polymorphic form and such polymorphic forms and compositions. 10 BACKGROUND OF THE INVENTION (2R,Z)-2-Amino-2_cyclohexyl_ν·(5·(1·methyl_1H-port ratio. sit-4·* base) small sideoxy-2,6_ Dihydro-1H_[1,2]diindole[4,5,6-cd]dec-8-yl]acetamidamine (also known as "compound"), Η

1 15 and its pharmaceutically acceptable * and its salts are described in U.S. Patent 6,967,198, issued on Jan. 1, 2005. The disclosure of which is incorporated herein by reference. . y kind of m and round-shot treatment on the cells, especially the cancer cells. The inhibition of CHK1 by releasing the S and G2 of the damaged cells of the dnA

20 Suspension, which leads to the mitotic macrophage and cell death of the 箸HA,,, and the cell, so CHK1 inhibition can promote the anticancer activity of these anticancer agents or radiotherapy 5 200806671. (2R,Z)-2.Amino-2-cyclohexyl-indole-(5-(1·indolyl-1H-pyrazole+yl)-small-oxy-2,6-dihydro-1Η-[1 , 2] Dioxin [4,5,6-cd]吲哚-8_yl]Ethylamine is a potent CHK1 protein kinase inhibitor. (2R,Z)-2-Amino-2_cyclohexyl- N-(5-(l-methyl-1H-pyrazol-4-yl)-1-yloxy-2,6-dihydro-5-indole 1,2]diindole[4,5,6- Cd]吲哚-8-yl]acetamide, and a pharmaceutically acceptable salt or solvate or mixture thereof in combination with an anticancer agent or radiotherapy will greatly enhance the anticancer agent or anticancer treatment of radiotherapy The solid compound may exist in an amorphous form or in a crystalline form. Different crystalline forms of the same compound are each considered to be a polymorphic form of the compound. ίο The crystalline polymorph is a different crystalline form of the same compound. Different polymorphic forms of the constituents (API) can have very different physical J* raw shell handles such as thermodynamic stability, solubility, hygroscopicity, and different drug 2 properties such as oral bioavailability. These properties greatly affect the drug. Sexuality = storage life, manufacturing cost, consistent agent The amount of the substance, and the efficacy of the drug, etc., are known as the polymorphic form of the compound. Thus, it is desirable to have a polymorphic form of a compound having good physical properties and pharmacological properties. [Declaration] Summary of the Invention 20 In the practical example, the present invention provides 1 represents the crystalline form of (2R,Z)_2-amino]/houto[4,5,6-cd]吲哚·8-yl]acetamide 6 200806671 Η

In another embodiment, the invention provides (2R,Z)-2-amino-2-cyclohexyl_N_(5-(1-methyl-111-pyrazole-4-yl)_1_ pendantoxy- 2,6-Dihydro_1Η·[1,2] 5 Dioxo[4,5,6-cd]吲哚-8-yl] acetylamine crystal form. Preferably, the crystal form is substantially pure Form I polymorph. In one aspect of this embodiment, the crystalline form has a powder X-ray diffraction pattern comprising peaks at diffraction angles (2Θ) = 23·6 ± 0·1 and 8.5 soil 0.1. In another aspect of the embodiment, the crystalline form has a powder X-ray diffraction pattern comprising peaks at a diffraction angle (2 Θ) = 10 23 · 6 ± 0 · 1, 8.5 ± 0.1 and 20.7 ± 0.1. In another aspect, the crystalline form has a powder X-ray diffraction pattern included in the diffraction angle (2Θ) = 23·6±0·1, 8·5±0·1, 20·7±0·1, and 16 a peak of 4±0·1. In another aspect of this embodiment, the crystalline form has a powder X-ray diffraction pattern included in the diffraction angle (2Θ) = 23·6±0·1, 8·5± A spike of 0·1, 20·7±0·1, 16.4±0.1, and 15 17·0±0.1. In another aspect of this embodiment, the crystalline form has powder X-rays. The shot pattern comprises a sharp peak substantially equal to the diffraction angle (2Θ) shown in Fig. 1. In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern included in the chemical shift 175.0 ± 0.1 , 137·6±0·1, 134·9±0·1, 110·3±0.1, 106·3±0·1, 41·1±0·1 and 20 32·6±0·1 peaks In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern comprising the following 7 in chemical shifts 7 200806671 175·0±0·1, 137.6 soil 0·1, 134.9 soil 0.1, 110· At least 3 peaks of 3±0·1, 106.3 soil 0.1, 41·1±0·1 and 32·6±0·1 ppm. In another aspect of the embodiment, the crystalline form has The 13 C solid-state NMR peak pattern contains the following seven chemical shifts of 175.0 ± (U, 137.6 0, 134·9 ± 0·1, 5 ll 〇. 3 ± (U, 1 〇 6 · 3 0.1, 41.1 士) At least 4 of the 0. 1 and 32.6 soil peaks of 0. 1 ppm. In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern comprising the following 7 chemical shifts of 175.0 soil 0.1, 137.6 ± 0.1, 134.9 soil 0.1, At least 5 spikes in the peaks of 110.3 ± 0.1, 106.3 ± 0.1, 41·1 ± 0·1, and 32·6 ± 0·1 ppm. In another aspect of the present embodiment 10, the crystalline form has a 13C solid state NMR peak pattern comprising the following seven chemical shifts of 175.0 ± (U, 137.6 0, 134.9 ± (U, 110.3 ± 0·1, 1) At least 6 of the peaks of 〇6·3±0·1, 41·1±0·1 and 32·6±0·1 ppm. In another aspect of the embodiment, the crystalline form has 13C The solid-state NMR peak pattern is included in the chemical shift 175·0±0·2, 15 137·6±0·2, 134·9±0·2, 110·3±0·2, 106·3±0·2, 41 • a peak of 1±0·2 and 32.6±0.2 ppm. In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern comprising the following 7 chemical shifts of 175·0±0·2 , at least 137·6±0·2, 134.9±0·2, 110.3±0·2, 106·3±0·2, 41·1±0·2, and 32·6±0·2 ppm 3 spikes. In another aspect of the 20th embodiment of the present embodiment, the crystalline form has a 13C solid state NMR peak pattern comprising the following 7 at a chemical shift of 175.0 soil 0.2, 137·6 ± 0·2, 134·9 ± 0· 2. At least 4 peaks of the peaks of 0.20.3, 106·3±0.2, 41·1±0·2 and 32.6±0.2 ppm. In another aspect of the embodiment, the crystalline form has a 13C solid state NMR peak pattern comprising the following 7 chemical shifts 8 200806671 175·0±0·2, 137·6±0·2, 134·9±0·2 At least 5 peaks of the peaks of 11 〇 3 soils 0.2, 106.3 soils 0.2, 41·1±0·2 and 32·6±0·2 ppm. In another aspect of the embodiment, the crystallization The form has a 13C solid state NMR peak pattern comprising the following 7 chemical shifts 175.0 ± 0.2, 137.6 ± 0.2, 134.9 soil 0.2, 5 11 〇 · 3 ± 0 · 2, 106 · 3 ± 0 · 2, 41.1 soil 0 · 2 and 32.6 At least 6 of the 0.2 ppm spikes. In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern comprising the same chemical shift position as substantially shown in Figure 4b. Spike. In another embodiment, the invention provides (2R,Z)-2-amino-2-cyclohexyl-10-N-(5-(l-methyl-1H-pyrazol-4-yl) a crystalline form of the small pendant oxy-2,6-dihydro-1H-[1,2]diindole[4,5,6-cd]dec-8-yl]acetamide. Preferably the crystallization Form II is a substantially pure type II polymorph. In one aspect of this embodiment, the 'crystalline form has a powder The X-ray diffraction pattern is included in the diffraction angle (2Θ) = 25·3±0·1 and 16·0±0·1. In another aspect of this embodiment, the 15 crystalline form has a powder X-ray diffraction pattern comprising peaks at diffraction angles (2 Θ) = 25-3 ± 0.1, 16.0 ± 0.1, 13.9 Å 0.1, and 29.2 Å 0.1. In another aspect of this embodiment, the crystalline form has a powder X-ray diffraction pattern included in the diffraction angle (2Θ) = 25·3±0·1, 16.0 soil 0.1, 13·9±0·1, 29 • Peaks of 2±0·1 and 12·2±0·1. In another aspect of this embodiment, the crystalline form has a powder X-ray diffraction pattern included in the diffraction angle (2Θ) = 25·3±0·1, 16.0±0·1, and 13.9±0·1. , 29.2 soil 0.1, 12.2 soil 0.1 and 16·8±0·1 peak. In another aspect of this embodiment, the crystalline form has a powder X-ray diffraction pattern included in the diffraction angle (2Θ) = 25·3±0.1, 16·0 soil 0·1, 13·9±0·1 , 29·2±0·1, 12·2±0·1, 16·8±0·1, 6.9 soil 0.1 and 13.6±0.1 peak. 9 200806671 In another aspect of this embodiment, the crystalline form has a powder X-ray diffraction pattern comprising peaks at a diffraction angle (2 Θ) substantially equal to that shown in Figure 2. In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern included in the chemical shifts 177·7±0·1, 133·2±0·1, 127·8±0·1, 5 1 〇3·8±0·1 and 22·7±0·1 peaks. In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern comprising the following five chemical shifts: 177·7±0·1, 133·2±0·1, 127.8±0.1, 103.8±0.1 And at least 3 of the peaks of 22·7±0·1 ppm. In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern comprising the following 105 chemical shifts 177·7±0·1, 133.2±0”, 127.8±0. Bu 103·8± At least 4 of the spikes of 0·1 and 22_7±0·1 ppm. In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern included in the chemical shifts 177·7±0·2, 133·2±0·2, 127·8±0.2, 103·8± 0.2 ppm spike of 0. 2 and 22.7 soil. In another aspect of this embodiment, the crystalline 15 form has a 13C solid state NMR peak pattern comprising the following five chemical shifts 177·7±0·2, 133·2±0·2, 127·8±0· 2. At least 3 of the spikes of 103·8±0·2 and 22.7±0.2 ppm. In another aspect of this embodiment, the crystalline form has a 13C solid state NMR peak pattern comprising the following five chemical shifts 177·7±0·2, 133·2±0·2, 127·8±0·2 At least 4 of the spikes of 103. 8 ± 0 · 2 and 22.7 ± 0.2 20 ppm. In another aspect of this embodiment, the crystalline form has an nC solid state NMR peak pattern comprising spikes at substantially the same chemical shift position as shown in Figure 5b. In another embodiment, the invention provides (2R,Z)-2-amino-2-cyclohexyl-N-(5-(l-methyl-1H-pyrazol-4-yl)-1- side Oxy-2,6-dihydro_1H-[1,2] 10 200806671 Amorphous form of dioxo[4,5,6-cd]dec-8-yl]acetamide. The crystalline form is substantially pure. In one aspect of this embodiment, the amorphous form has a 13C solid state NMR peak pattern included in the chemical shift of 16·3±0·2, 138·9±0·2, 131· A sharp peak of 4±0·2, 129·9±0·2, 129·9±0·2, and 30.8±0.2 ppm 5. In one aspect of this embodiment, the amorphous form has a 13C solid state NMR peak pattern comprising At least 3 peaks of 5 peaks of chemical shift 16·3±0·2, 138.9 soil 0.2, 131.4 soil 0.2, 129.9±0.2, 129.9±0.2 and 30.8±0.2 ppm. One state in this embodiment In this example, the amorphous form has an nC solid state NMR peak pattern comprising chemical shifts of 16.3 ± 0.2, 10 138.9 ± 0.2, 131 · 4 ± 0 · 2, 129_9 ± 0.2, 129 · 9 ± 0 · 2 and 30.8 ± 0.2 ppm. At least 4 of the 5 peaks. In another aspect of this embodiment, the crystalline form has a 13C solid-state NMR tip. The peak pattern comprises a sharp peak at the same chemical shift position as shown generally in Figure 6. In another embodiment, the invention provides (2R,Z)-2-amino-2-cyclohexyl-15-N -(5-(l-methyl-1Η-^ϋ-4-yl)-1· sideoxy·2,6-dihydro-1H-[1,2] dioxime[4,5, a solid form of 6-cd]吲哚-8_yl]acetamide (compound oxime), wherein the solid form comprises at least one selected from the group consisting of polymorphic form I, polymorphic form II, and amorphous form of compound 1. In one embodiment of the present embodiment, the solid form comprises at least 10% polymorph Form I. More preferably, the solid form 20 comprises at least 20% polymorph Form I. More preferably, the solid form comprises at least 30% polycrystalline Form I. Still more preferably, the solid form comprises at least 30%, at least 40% or at least 50% polycrystalline Form I. More preferably, the solid form comprises at least 60%, at least 70% or at least 80% More preferably, the solid form comprises at least 90% polymorph Form I. More preferably, the solid form comprises at least 95 Å/〇 polymorph Form I. 11 200806671 In another embodiment, - Pharmaceutical composition comprising (2R,Z)-2-amino-2-cyclol of the invention Hexyl group|(5 servile methyl. mouth ratio κ group) small side oxy group 2,6·dihydro·ιη-[ι52] 叮 叮 [4,5,6_丨嗓丨嗓|基]acetic acid amine Polycrystalline Form I, polymorphic, amorphous or solid form. 5 In another embodiment, the pharmaceutical composition comprises (2R,Z)-2-amino-2-cyclohexyl-indole (5 servylmethyl) small pendant oxy-2,6 of the present invention. - Dihydro-1 Η-[1,2] icosate [4,5,6-valent 嗓8-yl]acetic acid amine solid form. In still another embodiment, the invention provides a method of treating cancer in a mammal, comprising administering to the mammal in need thereof a pharmaceutical composition of the invention. In another embodiment, the present invention provides a method of treating cancer in a mammal comprising administering to a mammal in need thereof a therapeutically effective pharmaceutical composition of the present invention, wherein the therapeutically effective amount is selected from the group consisting of Cancer 15 and anti-cancer treatment for radiotherapy. In one aspect of this embodiment, the anticancer treatment is an anticancer agent. Preferably, the anticancer agent is selected from the group consisting of Ara_c, VP-16, Cis_piatin, yalimycin (tetra) riamydn), 2-chloro-2-deoxyadenosine, 9-(3-D- Arabinoxy)-2-fluoroadenine, carboplatin, gemcitabine, camptotheicin, paclitaxel, BCNU, 5-fluorourine%°疋, a group of irinotecan and doxorubicin. In another aspect of this embodiment, the anti-cancer treatment is a radiotherapy. In another embodiment, the invention provides a method of treating a condition of a mammalian animal mediated by CHK1 protein 12 200806671 plastid kinase activity, comprising administering to the mammal in need thereof (2R,z)-2 _Aminocyclohexyl _n_(5_(bumethyl-1H)pyrazol-4-yl) small side oxy group dihydrogen core called (3): 吖 并 [4,5,6eCd] 吲嗓 基 ] 醯 ] ] a polymorphic Form I, a polymorphic Form II, an amorphous Form 5 or a solid form in combination with an anticancer treatment selected from the group consisting of an anticancer agent, a radiation therapy, and a combination thereof. In one aspect of the embodiment, the The anticancer treatment is an anticancer agent. Preferably, the anticancer agent is selected from the group consisting of Ara-c, VP-16, Heberomycin, 2-chloro-2-deoxyadenosine, 9-(3) -D-arabinosyl)_2_ fluoroadenine, carbobertin, 贞hitabin, camptothecin, paclitaxel, 10 BCNU, 5-fluorouracil, irinotecan and doso rupin In another aspect of the present embodiment, the anti-cancer treatment is radiotherapy. The skilled artisan understands that the polymorphic type I, type II or non-compound of the compound I depends on the external reference used. The chemical shift of the type 13 (solid state 1 PCT 11 may have several k-izations. In the scope of the patent application of the present invention, the uc-solid state NMR 15-displacement refers to the use of adamantane at a field signal of 29.5 ppm as a field signal The chemical shift obtained by external reference. The term "combined with" refers to the administration of a first therapeutic treatment to a mammal in need thereof such as (2R,Z)_2-amino-2_cyclohexyl_N_(5_(1) _Mercapto-1H-pyrazol-4-yl)small oxy-2,6-dihydro-dioxacene 20 [4,5,6-cdH-dudolyl]acetamide, its pharmaceutically An acceptable salt or solvate or fermentate relative to the relative time of administration of a second therapeutic treatment, such as an anticancer agent or radiotherapy, which is the relative time commonly used in the field of combination therapeutic agents. It can be administered sequentially or simultaneously. 0 13 200806671 The term "hyperproliferative disorder" refers to abnormal cell growth (such as loss of contact inhibition) unrelated to normal regulators, including abnormal growth of normal cells and abnormal cells. Growth. Such hyperproliferative disorders include, but are not limited to, benign and malignant tumors. Abnormal growth of cells (tumors). Examples of such benign 5-proliferative diseases are cognac, benign prostatic hypertrophy, human papillomavirus (HPV), and vascular restenosis. "Cancer" - words include, but are not limited to, lung cancer, Bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma or intraocular melanoma, uterine cancer, nest cancer, rectal cancer, cancer in the anal area, stomach cancer, colon cancer, breast cancer, uterine cancer, 10 lose Infected tube cancer, endometrial cancer, cervical cancer, vaginal cancer, female vaginal cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra Cancer, penile cancer, prostate cancer, chronic leukemia or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal cancer, middle sacral nerve system 15 (CNS) tumor, primary A CNS lymphoma, a spinal tumor, a brain-glital cell tumor, a pituitary adenoma or a combination of the aforementioned cancers or cancers. In another embodiment of the method, the abnormal cell growth is a benign proliferative disorder, including, but not limited to, dryness, benign prostatic hypertrophy, or narrowing of blood vessels. 2〇 The term "mediator of CHK1 protein kinase activity" refers to a biological or molecular program that is regulated, regulated or inhibited by CHK1 protein kinase activity. "Polymorph" - the word refers to a different crystalline form of the same compound. The "crystalline form" includes (d) other solid molecular forms limited to the same compound, including hydrates (e.g., bound water present in the crystalline structure) and solvate 14 200806671 (e.g., a combination solvent other than water). The term "pharmaceutically acceptable lay, diluent or vehicle" means a material which can be combined with a particular agent to form a pharmaceutical composition, and which may be a body or a liquid. Examples of solids include lactose, Yan sugar, talc, gelatin, fat, pectin, acacia, magnesium stearate, stearic acid and the like. Examples of liquid carriers are syrup, peanut oil, olive oil, water, and the like. Similarly, the carrier or diluent may comprise a time delay material or a time release material known in the art, such as glyceryl monostearate or succinate (IV) alone, or with earth, ethyl cellulose, hydroxy propyl Group 10 of methyl cellulose, methyl methacrylate and the like. The term "pharmaceutical composition" means one or more of the compounds or polymorphs described herein, or a physiologically/pharmaceutically acceptable salt or solvate thereof, and other chemical components such as physiologically/pharmaceutically acceptable. The purpose of the carrier and excipient t &amp; drug cart composition is to aid in the administration of the compound to the individual. 15 “Radiotherapy” – the term refers to the medical treatment of radiation to control malignant cells. When referring to (2R,Z)-2-amino-4-cyclohexyl_N-form^methyl-1H" than spiva-4-yl) small side oxy-2,6•dihydro_iH_^, 2] two特定 并 [4'5,6" 各 ] ] 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 特定 ' ' ' ' ' ' ' ' 10/〇, preferably less than 5%, preferably less than 3%, preferably less than 1% by weight of miscellaneous 'including (2R,Z)-2-aminocyclohexyl_ν·(5 servant Base _ihh4-based fluorene-side oxy-2,6-dihydro] gram, 2] bis and [4, 5 6 _ look at the mouth of each base] other polymorphic forms of acetamidine. For example, it can be measured by X-ray powder around 15 200806671. The term "therapeutically effective amount" generally means that a compound, a pharmaceutically acceptable salt or a solvate of a mixture of X or a mixture thereof, will provide a treatment for a condition to be treated. Or a certain degree of symptoms. In particular, when the term "therapeutic has 5 effects" is used to describe a combination therapy, the term refers to a specific treatment that will 1) promote another treatment such as an anticancer agent or radiotherapy. Efficacy; or) combined with other treatments, will alleviate the disease Of one or evening symptoms to some extent. Referring to cancer treatment, the symptoms of the disease treated include a) reducing the size of the tumor; b) inhibiting (in other words, slowing down to a certain extent, squatting as V) tumor metastasis; c) inhibiting (in other words, slowing down to some To the extent that the car is stunned, the tumor grows to a certain extent; and d) relieves (or preferably eliminates) the cancer or a variety of symptoms to some extent. The word "2" or "2" means that the X-ray diffraction based on the present invention includes, but is not limited to, the Han source and the source wave I' used in the diffraction pattern. . Experimental setting 4 When the input beam of the field is formed with a certain lattice plane, the reflected light is diffracted, and the reflected beam is recorded at an angle of 2 。. The terms "treatment" and "," and "therapy" refer to methods of alleviating or eliminating cancer and/or its symptoms. When specifically referring to cancer, these terms simply indicate that the expected life expectancy of an individual with cancer will increase, or one or more symptoms of the disease will be alleviated. As used herein, the term "substantially identical" when referring to the X-ray diffraction peak position is also considered to be &gt;, type peak position and intensity variations. For example, familiar skills, Can * people understand that the peak position (2 Θ) will show a number of devices between 16 200806671 moxibustion 'typically up to 01 degrees. In addition, skilled artisans understand that the relative peak intensity will show a change between the devices, as well as the surface of the sample prepared by crystallinity, preferred directionality, and other factors known to those skilled in the art, so the relative peak intensity is Words are only used as qualitative measurements. Similarly, as used herein, when referring to solid state NMR and Raman spectroscopy, "substantially the same" is also intended to encompass variations that are known to those skilled in the art to be associated with such analytical techniques. For example, the 13C chemical shift measured in solid state NMR typically has a variability of 〇·1 ppm, while the Raman shift has a variability of 2 cm 〇1〇. The simple description of Fig. 1 is (2R, Z)- 2-Amino-2-cyclohexyl-N-(5-(l-methyl ratio)&quot;Sitosteryl) small pendant oxy-2,6-dihydro-1H-[1,2] dioxin [4,5,6-cd]吲哚_8_yl]Ethylamine polycrystalline type I X-ray powder diffraction pattern. Brother 2 is (2R,z)-2_amino-2·cyclohexyl -N-(5-(l-methyl to 15 sinoyloxy-2,6.dihydro_1H-[1,2] dioxin [4,5,6&lt;(1]吲哚- 8-based] acetaminophen polymorph type 11 \ light powder diffraction pattern. Fig. 3a is (2R,Z)-2_amino-2-cyclohexyl-N-(5-(l-fluorenyl)- 1Η^Bizozol-4-yl)_1_sideoxy-2,6-dihydro-1H-[1,2]diindole[4,5,6-cd]indole]ethylamine The amorphous (first batch) X-ray powder is wound around the pattern. Figure 3b is (2R,Z)-2-amino-2-cyclohexyl-indole-(5·(1-methyl-1Η) -π-biazole _4_yl)-1-yloxy-2,6-dihydro-1 fluorene-[1,2]diindole[4,5,6-cd]吲哚-8-yl] X-ray powder diffraction pattern of acetamide amorphous (second batch) 17 200806671 4a (2R,Z)-2-Amino-2-cyclohexyl-N-(5-(l-methyl-1H-pyridin-3-yl)-i-sideoxy-2,6-diaza- Solid-state 13C cross-polarization and magic angle of 1H-[1,2]dioxin and [4,5,6-cd]吲哚-8-yl]polyamine I (first spectrum) Spin (CP/MAS) NMR. 5 Figure 4b shows (2R,Z)-2-amino-2-cyclohexyl-oxime (5-(1·methyl-1H-pyrazol-4-yl)- Polymorphic Form I of 1-oxo-2,6-dihydro-1Η-[1,2]diindole[4,5,6-cd]吲哚_8_yl]acetamide Two-spectrum) solid-state 13c cross-polarization and magic angle spin (CP/MAS) NMR. Figure 5a is (2R,Z)-2-amino-2-cyclohexyl-N-(5-(l-- -1-1H·pyridyl 10-oxazol-4-yl)small oxy-2,6-dihydro-1H-[1,2]diindole[4,5,6-cd]吲哚-8-yl ] Solid state 13C CP/MAS NMR of polymorphic Form II (first spectrum) of acetamide. 苐5b is (2R,Z)-2-amino-2-3⁄4 hexyl-N-(5-(l - Methyl-1Η·σ-Bistazole-4·yl)-1-sideoxy-2,6-dihydro-1Η-[1,2]dioxine 15 [4,5,6-cdHI哚冬基Solid state 13C CP/MAS NMR of polymorphic Form II (second spectrum) of acetamide. Figure 6 is (2R,Z)-2-amino-2-cyclohexyl _]^-(5-(1-methyl-ΙΗ-oral than sial-4-yl) small side oxy-2,6 Solid state 20 13C CP/MAS NMR of the amorphous form (second batch) of _dihydro_1H_[12]diindole[4,5,6-cd]吲哚-8-yl)acetamide. L-Like Mode]|Detailed Description of Preferred Embodiments In this section, "B〇c", "Boc" or "b〇c" means N-tert-butoxycarbonyl, "CBZ" means benzyl ester group, "DCE" means dihaloethane, "DCM" 18 200806671 means dichloromethane, "DCC" means 1,3-dicyclohexylmethyleneimine, "DIC" means diisopropylcarbodiimide, "DIPEA" or "DIEA" means diisopropylethylamine, "DMA" means N-dimethylacetamide, "DMAP" means 4-dimethylamine base bite, "DME" means 1 , 2-dimethyl 5-oxoethane, "DMF" means dimethylformamide, "DMSO" means dimethyl sulfoxide, "DPPP" means 1,3-like diphenylphosphino)propane, "EDC" </ RTI> </ RTI> </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> </ RTI> <RTIgt; ,N,,N,-tetramethylurea hexafluorophosphate, "HBTU" means 〇-benzotriazole small group 10 -n,n,n',n'-tetramethyluronium hexafluorophosphate "h〇Ac" means acetic acid, "HOBt" means 1-hydroxybenzotriazole hydrate, rIPA" Isopropyl alcohol, "LAH" means hydrogenated Zhong Ming, "UHMDS" means martial acid (trimethyl sulphate) lithium amide, "MSA" means methanesulfonic acid; rMTBE" means methyl tertiary butyl ether, "ΝΜΡ" It means 1-methyl-2-pyrrolidinone, "TEA" means triethyl-15-amine, "TFA" means trifluoroacetic acid, and rrTIPS" means triisopropyldecyl-. "TMSCI" means trimethyl decyl chloride, and rTrt" means tris-methyl. Compound 1 was found to exist in a variety of polymorphic forms as well as in amorphous form. Methods of making such polymorphic forms in high purity and methods for determining the characteristics of different polymorphic forms are illustrated herein. A pharmaceutical composition comprising Compound 1 is also provided. I. Synthesis of Compound 1 : Compound 12 and (2R,Z)-2-amino-2-cyclohexyl-N-(5-(l-methyl-1H-pyrazol-4-yl)oxycyloxy _2,6_Dihydro- out of seven,]]Dioxo[4,5,6-cd]Sh-wood-8-yl]acetamide hydrochloride synthesis of the production process via the children 19 200806671 In U.S. Patent No. 6,967,198, issued November 22, 2005. In the present invention, the synthetic route is modified. In particular, the reaction conditions are modified to match the scale of the reaction. As shown in Figure 1, Compound 1 can be compound 11 That is, the N-Boc precursor of the compound 12 is directly produced. The compound 1 can also be produced from the compound 12 by neutralizing the hydrochloride. In either case, the compound 1 can be obtained according to the treatment conditions and purification conditions described later. Different polymorphs. Reaction Figure 1 OMe

35wt% aq NH2NH2 AcOH, MeOH

OMe BocHN

6 5

Pd(PPh3)4, K3PO4 DMA, 90 °C

H3C, ch3 20 8 200806671

As described in the reaction scheme of Figure 1, 2-methyl-3,5-dinitrobenzoic acid was reacted with N,N-dimethylformamide dimethylacetal under heating to obtain Compound 2. Suitable solvents for the reaction are DMF, DMA, MTBE, toluene and THF. Preferably 5 uses THF. The preferred reaction is carried out at about 50 °C. The reaction mixture was concentrated under reduced pressure and then methanol was added. Compound 2 then precipitated as a product. Compound 2 is then treated with acid in methanol under heating to give compound 3. Typical acids used herein are TMSC1, HQ, MSA, H2S04 and TFA. It is preferred to use TMSC1. During the reaction, Compound 3 was crystallized from the reaction solution, which significantly simplified the single-off procedure. In U.S. Patent 6,967,198, compound 3 is converted to compound 4 by conventional hydrogenation under Pd/C and H2. However, since the reduction of the two nitro groups is practically high, such conventional hydrogenation reaction conditions are difficult to control on a large scale. In the present invention 21 200806671, compound 3 is converted to compound 4 by a transfer hydrogenation reaction followed by an acid-promoting cyclization reaction. The transfer hydrogenation of Compound 3 provides a dose control procedure that reduces the risk of high temperatures. Different transfer hydrogenation reagents can be used, such as HC〇2NH4 and Pd/C, BH3.NMe3 and Pd(OH)2, ruthenium and FeCl3/C, and ammonium amide and Pd/C. Ammonium formate and Pd/C are preferably used. Compound 3 can be reduced with decanoic acid and Pd/C in THF. The crude product is then cyclized under strong acid to give compound 4. Typical conditions for such acid catalyzed cyclization are the use of concentrated hydrochloric acid in methanol. The first amine group of compound 4 is then protected with B〇c to obtain compound 5. In US 6,967,198, the reaction is carried out using triethylamine as the base and the reaction 10 is carried out in acetonitrile. In the present invention, the reaction is carried out under conditions which are more advantageous using an inorganic base such as NaOH and a solvent such as THF. Compound 5 was converted to Compound 6 by the vilsmeier formazonization reaction. In US 6,967,198, the reaction was carried out in dioxane using 3 equivalents of Weissmail reagent. Since either the compound 5 or the reaction intermediate is soluble in the di-methane, this conversion of methylene chloride to a solid reacts with a solid, which is often a problem on a large scale. Further, when Compound 5 was subjected to dimethylation by treatment with 3 vismomeric reagent, an additional hydrolysis step was required to convert dimethyl hydrazine to compound 6. In the present invention, the reaction is carried out in a solvent in which the degree of decomposition of the compound 5 is preferably a solvent. The typical solvent here is thf. The compound 5 is soluble in THF, so that the solution can be reacted with a solid. When the use of the Vismer's reagent is reduced to 1.1 equivalents, the monomethym is more preferred than the mirabiol to directly produce the compound 6, which greatly simplifies the reaction procedure. Compound 6 is converted to compound 7 by treatment of compound 6 with hydrazine under acidic conditions. A hydrazine monohydrate and a 30% hydrazine aqueous solution can be used in the present reaction. 22 200806671 More preferably, the reaction is carried out in an excess of methanol and preferably greater than 5 equivalents in a dilute methanol solution. Compound 7 is then brominated to obtain compound 8. In US 6,967 198, NBS is used as a brominating agent. In the present invention, pyridinium tribromide is used to replace the less expensive alternative. Compound 8 is then converted to compound 9 by a Suzuki coupling reaction. Pd(dPPf)Cl2 is used in US 6,967,198. A less expensive alternative, Pd(PPh3)4, is used in the present invention. Compound 8 was treated with DMA using 3 m〇1% Pd(PPh3)4 and K3P〇4. The residue in the crude product is a very high content (6000-8000 ppm). Since the solubility of the compound 9 in a conventional organic solvent is not good, it has been confirmed that the Fl〇risol treatment is ineffective for removing palladium and requires a large amount of solvent. A simple and effective water precipitation I bar removal procedure has been developed in the present invention: 1) the crude product is dissolved in a water immiscible solvent such as DMF, DMA, THF, DMSO DMA, preferably DMA, then 15 N_ is added Ethyl cysteinyl acid was added to the resulting solution and stirred for 1 hour; 2) water was added to precipitate the product, while N_acetylcysteine _|ba complex was left in the aqueous solution. After N_acetinyl cysteine treatment, the residual palladium content fell below 4 〇〇 ppm. The result was an API compound 1 containing less than 2 〇 ppm of residual palladium. Compound 9 was then dichotomized with 4 hydrazine hydrochloride. The compound was treated with a solution of chlorin and dichlorohydrazine to obtain Compound 10 as the hydrochloride salt. By ion chromatography analysis, it was found that the hydrogenation content varies from batch to batch, from 2.1 to 2.8 of the ratio of hydrogen chloride to the corresponding free base. Other acids such as sulfuric acid, sulfonic acid and TFA can also be used in the present reaction. Compound 10 is then coupled with a b〇c protected hexylamino acid to give compound 11. EDC was chosen as the coupling agent. A variety of coupling conditions can be used, 23 200806671 such as EDC, HATU and DCC. In the present invention, EDC is used as a coupling agent together with the use of DMAP as a catalyst. The amount of DMAP required is preferably that the hydrogen chloride present in the starting amine salt is equal to the molar amount. A large excess of DMAP in the reaction mixture may cause significant racemization. Compound 11 was isolated by adding water to the reaction mixture. HPLC analysis of the palm indicated the presence of about 1% of the racemic by-product, which was removed by crystallization in the final step. Compound 11 was then treated with a strong acid to remove the boc group. Conditions such as HCl/MeOH, HCl/EtOH 'TFA/CH2C12 or MSA/THF can be used. When compound 11 is treated with HCl/MeOH or HCl/EtOH, the mixture of the reverse 10 mixture can be removed by evaporation under reduced pressure to give compound 12 (2R,Z)-2-amino-2-cyclohexyl-N- ( 5-(l-methyl-1H-indazol-4-yl)·1-l-oxy-2,6-dihydro-1Η·[1,2]diindole[4,5,6-cd吲哚-8-yl] acetamidine hydrochloride. The compound η 15 can be directly obtained from compound 1 via an aqueous neutralization treatment for the removal of the boc reaction. For example, compound 11 is mixed with MSA and The THF is refluxed to remove the Boc group. When the reaction is complete and the reaction mixture is cooled, an aqueous solution of ΝΜ〇3, KAO3 or NaHC〇3 can be added to the reaction mixture. Subsequent standard subsequent treatment will yield compound i in good purity and yield. The compound 12 is mixed with an aqueous test solution such as an alkaline solution, and the mixture is vigorously stirred, followed by extracting the mixture with an organic solvent, and the compound 1 can be obtained from the compound 12. The IL compound 丄 crystal type II and amorphous form are prepared by A. polymorph I The type can be directly prepared by compound 11 by removing the b〇c group under strong acidic conditions, followed by alkaline treatment after 24 200806671. Polymorphic Form I. Typical strong acidic conditions used herein are HCl/MeOH, HCl/EtOH, MSA/THF or TFA. Preferably, Compound U is treated with methanesulfonic acid in THF to remove the boc group. After the reaction is completed, the mixture is assayed with an aqueous inorganic base solution such as 5 NaOH, Na2CO3, NaHC03, K2CO3 or KHC03 aqueous solution. Preferably, 2 aqueous sodium hydroxide solution or saturated NaHC〇3 solution is used. The organic phase is separated from the aqueous phase and The sulphuric acid is dehydrated. Then the organic solution is reduced to a small amount, and ethanol is added. The obtained solution is reduced to a small amount, and then ethanol is added. This volume is reduced and the process of adding ethanol is repeated until the compound i is polycrystalline _ 10 precipitate The polycrystalline Form I of Compound 1 can be prepared from Compound 12. Compound 12 is converted into aqueous (4) liquid by vigorous mixing. The typical aqueous inorganic base solution used here is NaOH, Na2C〇3, NaHC〇3. Or an aqueous solution of 15 20 KHC〇3. It is preferred to use NaH (: 〇3 aqueous solution here. Mix L with a large amount of organic (tetra) such as EtQAe. The obtained organic solution is washed with brine and dehydrated with sodium sulfate and filtered. Vacuum thick Obtained yellow = solid is an amorphous form belonging to compound 1. The yellow color is preferably dissolved in ethanol with agitation under heating at about 75 gauge. Newcoming material: dissolves at room temperature of about 22t, and is in 22t dimension (four) H. ^ Precipitate. The species B. polymorph II follows the similar procedure described in the previous paragraphs, and the compound W is obtained by the compound η, but during the final subsequent treatment, the polymorph II It is formed by the formation of methanol at about 4 〇c. 25 200806671 C. Amorphous Form The amorphous form of Compound 1 can be prepared directly from Compound 12 following a similar procedure for the preparation of polymorph Form I. After compound 12 was dissolved in an aqueous solution in several portions with vigorous stirring, the mixture was extracted with a large amount of EtOAc. The resulting EtoAc 5 bath was washed with brine, dehydrated and filtered. The filtrate was concentrated in vacuo to give a yellow solid which was obtained as an amorphous material. The amorphous form of Compound 1 was also prepared from Compound U following a similar procedure for the preparation of polymorph Form I. EtOAc was used as the organic phase during the subsequent workup. After the organic phase is dehydrated, the solvent is evaporated at about 40-70 ° C to obtain Compound 1 in an amorphous form of 10. The amorphous form of the compound oxime can also be prepared from the polymorph Form I of Compound 1 by dissolving the polymorph Form I in THF to saturation, followed by removal of the solvent at 5 °C. Determination of the characteristics of different polymorphic forms: clear: Each of the solid forms of Compound 1 can be determined by one of the following methods: X-ray powder diffraction pattern (ie, X-rays at different diffraction angles (2 Θ)) Diffraction peaks, such as the beginning of the melting point of the differential scanning calorimetry (DSC) thermal map (and the dehydration starting point of the hydrated form), the Raman spectrogram pattern, the solubility in water, at the International Harmonic Conference (ICH) Light stability under high-intensity light conditions, and physical storage stability, ie chemical storage stability. For example, the polycrystalline 2 〇 type I, type 11 and amorphous forms of the compound 各自 are each determined by the peak position of the X-ray powder diffraction pattern and the relative intensity of the tip ♦. A. Polycrystalline form of X-ray powder diffraction pattern. The X-ray powder diffraction pattern of the first batch (Fig. 3a) of the polycrystalline Form I, Type II and amorphous forms of Compound 1 is based on Bruker AXS D8-Diss 26 200806671. Measurements were performed at 4 〇 kV and 40 mA using a 1.5418 angstrom λ alpha average source. The samples were analyzed by angle (four) degrees (2 Θ) using a general area diffraction detector. The detector was set at a distance of 3 cm from the sample. Skilled people know that the Big Peak position (2Θ) shows variability between several devices, typically up to 度15 degrees. When reporting peak positions (2Θ), this number is intended to cover the variability between several devices. Moreover, when the crystalline form of the invention is described as having the same powder X-ray diffraction pattern as generally indicated by a given pattern, the term "substantially identical" is also intended to encompass such means of diffractive spike patterns. Between variability. Table 1 shows the 2 Θ values of the polycrystalline type 1 and the polycrystalline type II. The amorphous form shows 10 consecutive x-ray powder diffraction spectra without collecting 2 Θ values. The X-ray powder diffraction pattern of the second batch of the amorphous form of Compound 1 is not shown in Figure 3b. The preparation of the second batch of the amorphous form is illustrated in Example 4b. Here, the powder X-ray diffraction pattern was produced using a Bruker D5000 diffractometer using Cu Κα light shot (wavelength = 154 〇 6 Å). The instrument is equipped with a straight line of 15 focal lengths. The tube voltage and amperage are set at 38 kV and 38 mA, respectively. The squashing and diverging slitting system is set at 1 mm, and the receiving slit is set at 0.6 m. The diffracted radiation is detected by a Sol-Χ energy dispersive photodetector. Use from 3·0 to 4〇. 20 Θ 2Θ continuous scanning was performed at 2·4° 2 Θ/min (1 sec/〇·〇 4° 2θ steps). The Oxidation Standard (NIST Standard Reference Material 1976) was calibrated by the Analytical Inspector. Collect and analyze data using Bruker AXS DIFFRAC PLUS Software 2·〇. The maximum peak height of the peak is used to select the PXRD peak. 27 200806671 Table 1: X-ray powder diffraction of polycrystalline Form I and Type II multi-capped polycrystalline type II 2Θ Relative strength 2Θ Relative strength 8.5 33.5 6.9 21.2 10.4 10.1 10.8 9.8 14.4 7.2 12.2 26.3 16.4 21.5 13.6 21.2 17.0 12.6 13.9 28.7 20.7 23.1 16.0 40.0 22.4 11.5 16.8 23.8 23.6 100.0 18.2 11.8 25.0 6.8 20.1 12.0 31.8 8.3 20.8 11.9 32.4 10.9 21.9 10.2 25.3 100.0 27.9 13.1 29.2 28.5 Skilled people know that relative peak strength will show variability between devices and other crystallinity Changes in preferred orientation, preparation of the sample surface, and other factors known to those skilled in the art are only taken as qualitative measurements. When the solid form comprises two or more polymorphs of the invention, the X-ray powder diffraction pattern has characteristic peaks for each of the individual polymorphs of the invention. For example, a solid form comprising two polymorphs will have a powder and a light diffraction pattern, which is a volume of two technical diffraction patterns corresponding to a substantially pure polymorph. Solid State NMR (SSNMR) in 多·polymorphic form Solid state NMR is a powerful tool for analyzing solids. Different polymorphs often show significant chemical shift differences in solid-state nC cross-polarization and magic angle spin (CP/MAS) NMR 5. Solid-state 13C CP/MAS NMR was carried out on polycrystalline Forms I and II and amorphous forms on polycrystalline Form I. The first 13C SSNMR spectra were collected on a 600 MHz Bruker spectrometer with chemical shifts to the external reference hexamethylbenzene. Methyl resonance at 17.36 ppm. A 500 MHz Bruker spectrometer is used for the second 13C SSNMR spectrum of polycrystalline Form I and the second spectrum of the polymorph 10 11 type of Compound 1 and the second batch of the amorphous type. The chemical shift is externally referenced to 29.5 ppm. Adamantane signal on the field. Table 2a shows the chemical shift of the first 13C SSNMR spectrum of the polymorphic Form I and the first 13C SSNMR spectrum of the polymorph II. Table 2b shows the chemical shift of the second spectrum of the polymorph Form I of Compound 1, the second spectrum of Polymorph II, and the spectrum of the second batch of the amorphous form. In Table 2b, the peak intensity is defined as the peak height and varies with the actual settings of the CPMAS laboratory instrument. 29 200806671 Table 2a: Solid state 13C CP/MAS NMR chemical shifts of polymorph Form I (first spectrum) and polymorph II (first spectrum) of Compound 1. Peak number chemical shift chemical shift type I (ppm) type II (ppm) 1 177.1 177.7 2 168.0 165.7 3 142.8 140.7 4 139.6 136.6 5 137.0 135.3 6 130.7 133.2 7 128.3 127.8 8 127.3 125.2 9 116.0 114.4 10 112.4 112.5 11 108.5 108.9 12 64.6 103.8 13 43.2 58.7 14 40.9 39.7 15 34.8 37.9 16 28.9 30.3 17 22.7 30 200806671 Table 2b: Solid state 13C CP/MAS NMR of compound 1 polymorph type I (second spectrum) and polymorph type II (second spectrum) Chemical shift and amorphous (second batch). Peak number 13C chemical shift type I [ppm] intensity 13C chemical shift Π type [ppm] intensity 13C chemical shift amorphous [ppm] intensity 1 175.0 4.65 177.7 6.59 175.8 2.53 2 166.0 4.17 165.7 7.78 166.2 1.3 3 140.8 3.46 140.7 10.95 163.6 5.6 4 137.6 3.59 136.6 9.45 138.9 4.45 5 134.9 6.91 135.3 8.67 135.5 7.42 6 128.7 3.83 133.2 8.21 131.4 6.89 7 126.3 4.89 127.8 12 129.9 7.19 8 125.2 7.25 125.2 8.34 125.9 5.58 9 113.7 6 114.4 10.58 112.2 6.9 10 110.3 4.64 112.5 7.72 108.6 6.9 11 106:3 3.53 108.9 10.43 61.3 2.37 12 62.4 3.62 103.8 6.17 40.1 3.42 13 41.1 4.76 58.7 5.68 37.7 5.75 14 38.8 5.4 39.7 5.57 30.8 4.42 15 32.6 4.35 37.9 8.77 27.1 12 16 26.8 12 30.3 5.62 17 22.7 3.66 C. Polymorph Differential Scanning Calorimetry Test 5 Different polymorphic forms of Compound 1 were also distinguished using Differential Scanning Calorimetry (DSC). DSC is a thermal analysis technique in which the difference in heat required to increase the temperature of a sample and reference is measured as a function of temperature. DSC tests are also commonly used to test the melting point of solids. 31 200806671 In the present invention, the DSC is performed using a helium instrument Q_l〇〇〇 DSC. The scanning rate of the polycrystalline I type is 25. (: to 3 〇〇. 〇 is 1 〇. 〇 / min, under such conditions, polycrystalline Form I has a melting point starting point of 272. 〇. Polycrystalline scanning rate from 25C to 30 (TC is 40 ° C / Minutes, under these conditions, the polycrystalline 丨 type begins to melt at 5 242 C, rapidly forming a new crystal form. The inventors believe that the new crystal formed is polycrystalline type I. It is confirmed by the following experiment. Type 11 is heated to 260C 'The solid obtained by PXRD test. The pXRD result is the same as the pxrd result of polycrystalline j. When the scan rate of polytype is set from 25 〇 to 300 C is l〇°c/min, more The crystal form H is at 243. The enthalpy begins to melt, rapidly forms into a new crystal form, and then melts at 274 ° C. It is believed that the new crystal form is also polycrystalline type I. D. polycrystalline form thermogravimetric analysis thermogravimetry Analysis (TGA) is a measurement procedure in which the weight of a sample 15 is recorded as the sample is heated in air or under a controlled atmosphere such as nitrogen. Thermogravimetric curve (thermograph or TG a scan) Obtain information on the solvent content and water content and thermal stability of the material. In the present invention, TA instrument TGAQ 500 and TGA in compound 1 polymorph type I and type II. For polycrystalline j type and n type from 25 to 3 丨 yc and 25 〇 to 350 C, the temperature is i 〇 ° c / min Elevated. Polycrystalline TGA analysis showed that the temperature did not reach 265. During the time of crystallization, the total weight loss of crystalline type I was about 0.26%. Just after dissolution, the polymorph type quickly degraded. At the time of TGA analysis, the TGA analysis form lost about 0.85% of the total weight. After 275., the polycrystalline π type rapidly degraded. These TGA results are consistent with polymorphism and polymorph II are anhydrous. Conclusions of non-solvent forms. 32 200806671 溶解 · Solubility of different polymorphic forms Equilibrium solubility tests were carried out on both polycrystalline Form I and polymorphs. Samples were prepared by dissolving the corresponding polymorphs in ethanol. The sample was stirred overnight at room temperature and centrifuged to remove any undissolved solids. The solution was then analyzed by HpLC. The results are shown in Table 3. Table 3: Solubility of polycrystalline Form I and Polymorph II

F. Hygroscopicity of different polymorphs 10 15 Different polymorphic forms of the compounds have different hygroscopic properties. The constant temperature water adsorption and desorption experiments of the combined polycrystalline Form I and Type II are based on Surface Measurements Systems Dynamic Vapor Sdrpti〇n-D (DVS) get on. Polycrystalline j and 卩 loads are loaded onto the DVS instrument starting at 25. (:, 0% relative humidity. The humidity is gradually increased from 〇% to 9〇% relative humidity in increments of 1〇% relative humidity., 卯% relative humidity sample weight is stabilized, humidity*9〇%RH Step by step = 0% RH to complete a complete cycle. During the entire procedure, the temperature is maintained = set at 25. The weight gain during the experiment is used to determine the hygroscopicity. Samples with a weight gain of less than 2.0% are considered non- Hygroscopicity Polycrystalline Forms I and II showed an increase in water content from 0-90% RH [7% and 1.3%. Stomach ° 33 20 200806671 G. Different polymorphic forms of stability of compound 1 polymorph I The solid-state stability of the type is 4 〇. The 〇 is studied in open vials and closed vials for 7 weeks at 75% relative humidity (RH). The sample is examined by diffracted powder diffraction analysis to check the physical stability and chemical stability by HpLC test. The results are shown in Table 4. Table 4 · Solid state stability of polycrystalline type I

The physical stability measured by PXRD and DSC is determined by HPLC. The stability of stability is unchanged. 99.3% is unchanged. 99.3°/❻ is unchanged. The thermodynamic stability of the form is higher at one temperature, while the other 10 is higher. 15 ==:: The solid temperature is relatively safe, and the polymorph is regarded as enantiomeric owing > The temperature of the day-to-day equivalent stability is called the transition temperature == the polycrystal type 1 and the _ row of the compound 1 . (4) ^ Add 1-2 ml of ethanol to the mixture to form a slurry to the fox (,,, spoon 23 〇 and 3.5 c to prepare and transfer. The liquid is decanted, the rest of the material is at room temperature, The results are summarized in Table 5. One (10). The obtained material by PXRD is divided into 34 200806671 Table 5: Conversion temperature between Type I and π type Conversion direction Time conversion completeness 7〇 °C II Type 41 type 1 曰 Complete 6 〇t Type II -> Type 1 曰 Complete 5 (TC II type - Μ type 6 曰 Complete 4 (TC II type 5 曰 Complete 30 ° C Type II -> Type 1 11 Daily Remaining Small Volume II Type 23〇C(RT) Type I-&gt; Type 11 12曰 Incomplete 3.5t: Type I - Type II 7曰 Remaining Small Volume I Form IV. Usage of the Polymorph of the Present Invention The polymorphic form of Compound 1 can be used in all five useful forms of the compound. The use of Compound 1 is described in US 6,967, 198 as a use of a broad class of such compounds containing Compound 1. The invention is illustrated in US 6,967,198. The compounds may be combined with a therapeutically effective amount of an anti-tumor agent or radiation therapy to treat a tumor in a mammal. Within the scope of the invention, the compounds of the invention! The polymorphic form and the pharmaceutical composition can be used in combination with a therapeutically effective amount of an antitumor agent or 10 radiotherapeutic, as described in US 6,967, 198, which can be used in combination with the compounds of the invention. Example 1: (2R,Z)-2-Amino- 2·cyclohexyl-N_(5-(l•methyl-111_pyrazol-4-yl)·1_ pendant oxy-2,6-dihydro-1H-[1,2]diindole[4 ,5,6_cd]吲π朵_8_15 base) Preparation of acetamidine hydrochloride (hydrochloride salt of compound 1) 2·(2-dimethylamino-vinyl)-3,5-dinitrate Methyl-benzoic acid methyl ester, Preparation of Compound 2 · 2_Methyl_3,5-dinitro-benzoic acid (102.62 g, 0 45 35 200806671 mol) was dissolved in anhydrous THF (1200 mL). ν,ν·dimercaptocarbamide dimethyl acetal (Aldrich, 94% purity, 3.0 equivalents, 172.4 g, 1.35 mol) was added over 10 minutes at room temperature with stirring under a nitrogen atmosphere. The temperature is raised from 22 C to 29. (3. The solution is immediately heated to 5 ° C, and the temperature is 5 at the back of the screen for 8 hours. Then the reaction solution is cooled to room temperature and concentrated on a rotary evaporator. Removal of most of THF and unreacted N,N-dimethylformamide Aldehyde until about 250 g of crude product remained in the reaction flask (water bath temperature is not allowed to 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 to give cold methanol (60 ml). Washing, drying in a stream of 10 to give 109.08 g of a purple solid (84% yield). 4 NMR (300 MHz, CDC13) δ 3.01 (s, 6H), 3.93 (s, 3H), 5.99 (d, 1H, 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). 2-(2-Dimethylamino-vinyl)-3,5-dinitro-benzoic acid formazan, Preparation of compound 3: purple solid 2 (92.25 g, 0.313 mol) suspended in anhydrous methanol ( 1000 ml). TMSC1 (70.25 grams, 0.65 moles, 2.1 equivalents) was added over 10 minutes. A clear solution is formed. The solution was heated (slow reflux) at 60 ° C for 20 hours. HPLC analysis indicated the disappearance of the starting material. The reaction mixture was allowed to cool to room temperature and a white solid precipitated (often, when the reaction was near completion, the product was crystallized from the reaction mixture). The reaction mixture was stirred at room temperature for 1 hour. The precipitated solid was collected by suction <RTI ID=0.0> The mother liquor was concentrated to remove about 8 ml of solvent. Extra product Shen Dian. 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 air stream 36 200806671 to obtain an additional 5.58 g product. The combined yield of Compound 3 was 88.29 g (90%). 4 NMR (300 MHz, CDC13) δ 3.30 (s, 6 Η), 3.73 (d, 2 Η, J = 5.1 Hz), 4.00 (s, 3H), 4.51 (t, 1H, 5.1 Hz), 8.65 (d, 1H) , J = 2.4 Hz), 8.76 (d, 1H, J = 2.4 Hz). 5 6-Amino-1H-indole-4-carboxylic acid methyl ester hydrochloride, preparation of compound 4:

A 2 liter Erlenmeyer flask was charged with 10% Pd/C wet catalyst (8.6 g), methanol (800 ml) and THF (160 ml). Ammonium formate (139.1 g, 2.21 mol) was then added with stirring and the mixture was heated to 35. Add water (100 ml). Compound 3 was then added in small portions over a period of 10 minutes. By controlling the rate of addition of compound 3 and appropriate, the reaction temperature is maintained between 40 ° C and 45 ° C when cooled. After the addition was completed, the reaction mixture was stirred for 30 minutes. HPLC analysis indicated completion of the reaction. The reaction mixture was cooled to room temperature. The catalyst was filtered through a pad of Celite and the Celite pad was washed with methanol (50 mL). The filtrate was concentrated under reduced pressure to remove volatile components. Then EtOAc (500 mL) and water (75 mL) were added and the mixture was stirred for 15 min. The organic phase was separated and EtOAc (EtOAc) The combined organic solution was concentrated to dryness to give a pale yellow oil (79.17 g). Methanol (100 mL) was added to the oil intermediate. The resulting solution was then added to a solution of concentrated aqueous hydrochloric acid (37 wt%, 82.8 g) in methanol (600 ml) while maintaining the temperature at 32 °C. The reaction mixture was stirred at 35 ° C for 3 hours. During this time, the solid product precipitate 20 is deposited. HPLC analysis indicated completion of the reaction. The reaction mixture was cooled to room temperature. EtOAc (500 mL) was added and the mixture was stirred 3 min. The solid was collected by filtration to give 45.07 g of product. The filtrate was concentrated to give a syrup (331 g). EtOAc (400 mL) was added to a broth and mixture was stirred for 3 min. The solid was then collected by filtration to give a second crop of product 4 (16.28 g). 4 NMR 37 200806671 (300 MHz, DMSO-d6) δ 3.93 (s, 3H), 6.97 (d, 1H, 1·8 Hz), 7.65 (m, 1H), 7.72 (d, 1H, 1.8 Hz), 7.80 (s, 1H), 11.81 (s, br, 1H). Preparation of 6-Amino-1H_decade-4-carboxylate methyl ester hydrochloride 4 by direct hydrogenation of compound 2··Methanol (27 kg) and 2-(2-dimethylamino-vinyl _3,5_ Dinitro-benzoic acid methyl ester 3 (10 kg, 33.87 mol) was fed into the reactor through the feed port. A 10% palladium on carbon catalyst (〇·4 kg) was then fed to the reactor as a slurry of water (2.5 〇·5 kg). Ethyl acetate (123 soil 3 kg) was added and then hydrogen was added in portions. The internal temperature 10 is maintained at about 〇 ° C by cooling and controlling the rate of hydrogen addition. After the pressure was constant for more than 30 minutes, the hydrogen pressure was maintained at 5 〇 6 psi and the temperature was maintained at 0 ° for 2 hours. The catalyst is filtered through an in-line filter (Note • The catalyst is pyrolyzed. The catalyst is not allowed to dry). The filtrate was below 35. The internal temperature of 〇 is concentrated by vacuum distillation to remove about 95% of the solvent. Acetic acid acetic acid (130 liters) was added with stirring. Then slowly add 37% hydrochloric acid (1 〇 15 15g) below 1 〇. The mixture was stirred for 1 hour at 1 〇 c. The solid was transferred to a plate transition and washed with methyl tert-butyl ether (15 L). The product (wet cake) was transferred to a vacuum desiccator and dried at 23 ± 3 ° C to obtain 4.6 kg of product 4 (60 〇 / 〇 yield). Preparation of 6-tert-butoxycarbonylamino-1H_indole-4-carboxylic acid methyl ester 5 · THF (600 ml) and 4 aqueous NaOH solution (138 ml, 0.55 mol) 20 Feed to reaction Inside the bottle. Then hydrazine hydrochloride 4 (61.0 g of 0·269 mol) was added and the mixture was stirred until the solid was completely dissolved. The solution in THF (50 ml) was slowly added (BOC) 2 〇 (70.43 g, 0.32 mol) 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 completion of the reaction. The aqueous phase is separated. The organic phase was washed with a saturated solution of 2008 200871 and an aqueous solution of ammonium chloride (15 ml). The organic solution was then concentrated to give a paste (176 g). THF (40 ml) and heptane (8 ml) were added to the mixture, and the mixture was stirred for 30 minutes. The solid was collected by filtration to give 69.86 g of product 5 (90% yield). NMR (300 MHz, DMSO, d6) δ 1.50 (s, 5 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-t-butoxymethylamino-3-methylindenyl-1Ηα-inducing yttrium succinic acid AI 6 : N,N-dimethylformamide (85 ml) was fed to 250 ml reaction The bottle was then cooled to -5 °C in an ice-acetone bath. Phosphorus helium (35.15 g, 0.23 10 mol) was injected into the reaction flask at 4 ° C for 10 minutes. After the addition was completed, the mixture was further stirred at 0 ° C for 30 minutes. In a 2 liter three-necked flask equipped with an overhead stirrer and a thermometer, π 丨 丨 5 (60.57 g, 〇·2 ΐ Mo) and THF (600 ml) were added to form a clear solution. The solution was cooled to -7 ° C in an ice-acetone bath. Cold 15 pre-formed Wismar's reagent was added via cannula over 1 □ minutes in a stirred THF solution while maintaining the temperature at _3 °C. Upon completion of the addition, the heavy precipitate formed within a few minutes. The reaction mixture was again stirred at Ot for 45 minutes. EtOAc (600 mL) was added to the reaction mixture, and then a cold aqueous NaOAc solution (3 Μ, 310 mL, 〇·93 Mo) was added with stirring. After that, the temperature rises to 10 C south. Remove the cooling bath. The reaction mixture was warmed to 21 ° C over 2 Torr for 20 hours and stirred at this temperature for 2.5 hours. Completely dissolve solids. HPLC analysis indicated that all intermediates were converted to the product. Suspend the transfer and separate the water layer. The organic layer was concentrated under reduced pressure to remove volatiles. Obtained a paste (226 grams). Water (303⁄4 liters) and EtOAc (70* liters) were added to the paste. The resulting mixture was stirred for 30 minutes. The solid was collected by filtration, washed with EtOAc EtOAc EtOAc (EtOAc) iHnmr(3〇〇MHz, DMSO-d6) δ 1.50(s,9H), 3.86(s,3H), 7.68(d,1H,J=1.8 Hz)? 7.97(s? 1H)5 8.23(s? 1H )? 9.56(s, 1H)? l〇.l〇(s? 1H)? 12.25(s, br, 1H). Preparation of 5 I-sideoxy·2,6·dihydro-1H_[1,2]dioxine[4,5,6-cdH丨♦8-ylaminophosphonic acid tert-butyl ester compound 7: Methanol (1200 ml), acetic acid (100 ml) and aldehyde 6 (62.98 g, 〇·2 〇 Mo) were fed to a 2 liter flask. The aqueous hydrazine (35 wt%, 88.8 g, 0.97 mol) was then added with mash. The mixture was heated to 6 (TC and stirred for 3 h. HPLC analysis indicated the reaction was completed. </ RTI> <RTI ID=0.0></RTI> <RTI ID=0.0></RTI> Paste (223 g). Add water slowly (25 ml) with stirring. The mixture was stirred for another 5 minutes to pellet the solid. The solid was collected and dried to give a second product ((10) g). 99%.lH NMR (3〇〇MHz, DMs〇_d^ 15 δ 1.49(s9 9Η)? 7.44(s5 1Η)5 7.51(d? 1Η? J= 2.1 Ηζ)? 7.62(s5 1Η)? 7.76( s? 1H)5 9.45(s5 1H)? 1〇.19(s? 1H)5 11.63(s? br5 1H) 〇5_bromo-1-yloxy-2,6-dihydro-1H_[1, 2] Preparation of diterpene [4,5,6-Μ]$|哚·8_ylaminocarbamic acid tert-butyl ester compound 8: Compound 7 (45. 〇克〇15 Moer) and N N-dimethylformamide (0.45 liters) was fed to a 5 liter reaction flask equipped with an overhead stirrer, a thermometer and a nitrogen gas inlet. This a, two mixed to form a clear solution. Cool the solution under nitrogen protection. Under the protection of nitrogen 5 minutes to divide into three parts to add three desertification mouth than bite 57 (57. 6 grams, 〇 18 moles). The reaction temperature during the addition is controlled below _5 〇. After the addition is completed, the reaction mixture is disturbed ι·5 H. 200806671 HPLC analysis indicated completion of the reaction. Slowly add water (0.225 L) to the reaction mixture while maintaining the temperature below 20 ° C during the addition. At 15. (:, Et〇Ac (〇.225 L) was added to the reaction mixture. Next, add aqueous potassium carbonate (preparation: dissolve 20.73 g of potassium carbonate in 60 ml of water, and cool to l ° ° C for use in 5.) Mix the mixture for 15 minutes to dissolve the solid. Then add aqueous sodium sulfite solution (preparation: 3.78) The sodium sulfite was dissolved in 18 ml of water) and stirred for 10 minutes. Water (0.5 liters) was added over a period of 3 minutes by adding a funnel, and the temperature was maintained at 25-30. (: The solid was slowly precipitated. The mixture was stirred for 20 minutes to make the solid Add water (〇·625 liters) to drive the precipitation to completion. The mixture is stirred for 6 〇 10 minutes. The solid is collected by filtration, washed with water (0.3 liters) and dried in a stream for 90 minutes. Solid at 6 (TC in vacuum) The inside of the oven was dried with nitrogen for 12 hours, and the crude product was suspended in acetone (0.19 L) and stirred for 1 hour. Heptane (〇·19 L) was slowly added to the mixture. The reaction mixture was stirred for 5 hours. It was washed with a mixture of acetone (30 ml) and heptane (30 ml) and dried in a gas stream for 2 hours. The product was further dried in a vacuum oven at 50 ° C for 12 hours. The solid weighed 45.5 g (80% yield, 98.3% purity). NMR (300 MHz, DMSO-d6) 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) , l〇.47(s, 1H), 12.54 (s, br, 1H). 20 5-(l-Methyl-1H-pyrazol-4-yl)small pendant oxy-2,6-dihydro-1H_[1,2] bisindole[4,5,6-cd]吲Preparation of 哚8-butylaminocarbamic acid tert-butyl ester compound 9: Compound 8 (308.24 g, 0.81 mol) and hydrazine, hydrazine dimethyl acetamide (2.5 liters) were added to an overhead stirrer, A thermometer and a nitrogen gas inlet liter flask. A clear solution is formed. Water (0.625 L) was added to the DMA solution while 41 200806671 was stirred and the temperature was controlled at 30 °C. Then, 丨-methyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboron-2-yl)·1Η-pyrazole (186 g, 0.89 mol) was added with stirring. And potassium phosphate (431·3 g, 2.03 mol) was added to the flask with stirring. The mixture was degassed by evacuating the flask followed by nitrogen purge. 2 〇 5 minutes, the degassing procedure was repeated three times. Tritium (triphenylphosphine)! Bar (28.2 g, 〇·〇 24 mol) was added under a nitrogen atmosphere. The reaction mixture was degassed 3 times with a vacuum/nitrogen cycle over 20 minutes. The reaction was heated under nitrogen for 5 hours under the protection of nitrogen. HPLC analysis indicated completion of the reaction. The reaction mixture was cooled to room temperature. N-Ethylcysteine (30 g, 〇18 mol) was added to the reaction flask, and the mixture was mixed for 10 hours. Water (5 liters) was added with stirring for 30 minutes while controlling the temperature at 30C. The solid sinks out. The mixture was further mixed for 2 hours to pellet the solid. The solid was collected by filtration, and washed with a mixture of DMA (0.1 L) and water (0.3 L), water (〇·7 liter), and propylene® g (〇3 liter) and water (〇·3 liter). The filter cake was air-dried overnight in a stream of air and dried in a vacuum oven at (9) for 16 hours. The crude 15 product was dissolved in DMAU·5 liters). N-Ethyl cysteine (3 g, 0.18 mol) was added to the DMA solution, and the solution was stirred for 2 hours. Water (0.8 liters) was added in 1 minute with stirring while controlling the temperature to 3 Torr. The solid precipitated. The mixture was stirred for 20 minutes to pellet the solid. Additional amount of water (2.2 liters) was added over 5 minutes to drive the precipitation to completion. The solid was stirred for a few hours. The mixture was collected by filtration and washed with a mixture of DMA (〇·2 liter) and water (0.4 liter), water (0.8 liter), and C (0.4 liter) and water (〇·4 liter). The compound was dried in a vacuum oven at 60 ° C until the water content fell below 15 wt%. The product weighed 286 g (93% yield, 98% purity). 4 NMR (300 MHz, DMSO-d6) δ MWs, 9 Η), 3.92 (s, 3 Η), 7.57 (s, 1 Η), 7.65 (d, 1 Η, 1-5 Ηζ), 42 200806671 7.68 (d, 1H, J = 1.5 Hz), 7.90 (s, 1H), 8.27 (s, 1H), 9.40 (s, 1H), 10.14 (s, 1H), 11.75 (s, br, 1H). 8-Amino-5-(1-methyl-1H-pyrazol-4-yl)-2H-[l,2]diindole[4,5,6-cd]indole-1(6H)- Preparation of ketohydrochloride 10: 5-(1-methyl-1H-pyridazozol-4-yl) small pendant oxy-2,6-dihydro-1H-[1,2]diindole[ 4,5,6-cd] Tert-butyl-8-ylaminocarbamate 9 (236.06 g, 〇62 mol) was fed to a 10 liter flask equipped with an overhead stirrer, thermometer and air inlet Inside. Dimethyl methane (3.5 liters) was added and the mixture was stirred for 10 minutes. The suspension was then cooled to 10 ° C and then 4 Μ hydrochloric acid was added to dioxane (2.4 10 liters) over 1 Torr. Let the temperature not exceed 25 °C. The reaction mixture was stirred at room temperature for 16 hours. HPLC analysis indicated the consumption of approximately 98% starting material. The solid was collected by centrifugation under nitrogen 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). 4 NMR (300 MHz, DMSO-d6) δ 3.94 (s, 3H), 7.47 (d, 1H, J = 1.8 Hz), 7.58 (d, 1H, 15 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)-l-cyclohexyl-2-(5-(l-methyl-lH-n than saliva)_i_sideoxy-2,6-dihydro-1Η·[1,2]dioxin Preparation of 3,5,6-cd]吲哚-8-ylamino)·2·trioxyethylaminocarbamic acid tert-butyl ester 11 : 8-amino group _5_(1-methyl 20 - 1Η-pyrazol-4-yl)-2Η-[ 1,2]diindole[4,5 6_cd]σ 哚 _ i (6Η) ketone hydrochloride 10 (224.35 g '0.64 mol ion layer The analytical analysis contained 25·7% of the vapor) suspended in anhydrous DMF (2.2 liters). 4-Dimethylaminopyridine (197.08 g ' 1.65 mol) was added and the mixture was stirred for 3 minutes. Then B〇c_D_cyclohexylglycine (179.08 g, 1.65 mol) was added and the mixture was heated to 35 χ:. 43 200806671 1-(3-Dimethylaminopropyl)-3-ethylmethyleneimine (158.5 g, 〇·83 mol) was added in one portion, and the reaction mixture was heated at 50 ° C for 1 hour. HPLC analysis indicated the completion of the reaction. The reaction mixture was allowed to cool to room temperature. Water (2.2 liters) was added over 10 minutes while maintaining the temperature at 30 °C. The mixture was stirred for 20 minutes 5 to pellet the solid. An additional amount of water (4.4 liters) was added over 5 minutes to drive the precipitation to completion and the mixture was stirred for another 30 minutes. The solid was collected by filtration, washed with a mixture of DMF (0.5 L) and water (1.5 L) and then with water (1.0 L). The product was dried in a vacuum oven at 60 ° C for at least 48 hours to obtain 293.91 g of product (94.2% HPLC purity). lH NMR (300 MHz, DMSO-d6) δ 10 ll (m, 5 Η), 1.36 (s, 9 Η), 1.60 (m, 6 Η), 3.93 (s, 3 Η), 3.96 (m, 1H), 7.59 (s, 1H), 7.6 〇 (s, 1H), 7.92 (s, 1H), 8.07 (s, 1H), 8.30 (s, 1H), 10.05 (s, 1H), l〇.2〇 (s, 1H), 11.86 (s, br, 1H). (2R)_2-Amino-2-cyclohexyl_N_(5-(1-methyl-1H-pyrazol-4-yl) small pendant oxy-2,6-dihydro·1Η-[1,2 Preparation of dioxo[4,5,6-cd]吲哚-8-yl)ethyl 15 decylamine 12: methanesulfonic acid (44.4 g, 0.46 mol) was added to THF in a 2-liter flask (690 ML). Add (R)-l-cyclohexyl-2-(5-(1-methyl-1Η·η^sial-4-yl)-1-yloxy-2,6-dihydro-1H- via a powder funnel [1,2] 丫η丫[4,5,6-cd]吲哚-8-ylamino)-2-oxoethylaminocarbamic acid tert-butyl ester 11 (30·0 g, 57.74 milliMoz). The funnel and the sides of the 20 flask were rinsed with THF (60 mL). The flask was purged with nitrogen and the reaction was heated to 65 ° C and stirred for 18-24 hours. HPLC analysis showed the reaction was complete. The reaction was cooled to room temperature using a water bath. 2 M 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 liter separatory funnel and washed with THF. Separate the layers. The aqueous phase was extracted with THF (60 mL). 44 200806671 The organic fractions were combined and washed twice with saturated aqueous sodium chloride (2 x 60 mL). MeOH (8 mL) and MgS 4 (42 g) were added. The mixture was stirred for 75 minutes and then filtered through a Hillite. The filter cake was washed with 9:1 THF / MeOH (3 x 50 mL) and then transferred to a 2 liter distillation flask. The solution was concentrated by distillation at atmospheric pressure to a volume of 3 Torr to 5 liters. Ethanol (450 ml) was added slowly and the solution was cooled to 50. (: to crystallize the product. Once the product has crystallized, the mixture is heated again and evaporated to 450 ml. The reaction solvent ratio is monitored by 1H NMR. The distillation is stopped once the THF content is reduced to 5 mol%. The resulting yellow suspension is 60. It was cooled to 25 C' in a minute time and vacuum filtered on paper. The filter cake was washed with ethanol (2×75 ml). The solid 10 was transferred to a crystallizing dish and dried under vacuum at 55 ° C for 16 hours to obtain 13.55 g of compound 12 (54%). Yellow solid. iHNMR pOOMH^DMSO-dde 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). 15 Example 2: (2R)_2-Amino-2-cyclohexyl-oxime (5-(1-methyl-1H-pyrazole ice)

Preparation of the base M_sideoxy-2,6-dihydro-1H-[1,2]dioxinh[4,5,6-cd]吲哚I group]acetamimid polymorph Form I: Hydrochloride salt of compound 1 : divided into several portions in aqueous sodium hydrogencarbonate solution (5%; 5 equivalent; about 18 ml) and added (2R)-2-amino-2-cyclohexyl 20-based (5) with vigorous stirring -(1-methyl-1H-pyrazol-4-yl)minoxy 2,6-dihydro-1H-[1,2] diindole[4,5,6-cd]吲哚Each group] acetamide hydrochloride (1 g). Add 500 ml of ethyl acetate and 80 ml of water to the mixture. The suspension is stirred vigorously for 10-15 minutes, stirring is stopped. The organic phase is carefully decanted to another cone. The bottle, the treatment sequence was repeated 3 times, each time using 500 ml of ethyl acetate. The combination of 45 200806671 machine phase (about 2000 ml) was washed with brine (100 ml), dehydrated with sodium sulfate and filtered. After removal, the obtained yellow solid was dried overnight to obtain 67 mg of the polymorph of the compound 1 (about 84% yield). The amorphous form of the compound 1: (2R)-2-amino-2-cyclohexyl-yi (5_(Bu 5 methyl-11^pyrazole ice-based side oxy-2,6-dihydro·ιη_[ι,2] 吖 吖 和 [4,5,6-cd]哚-8-yl] acetaminophen amorphous form (1 〇〇 mg) was mixed with ethanol (1.5 liters) to form a suspension. The suspension was heated vigorously with K75_8 (rc heated for 5 days, let it at 22 C) After standing for 12 hours, the solid was subjected to a transition and washed with ml of ethanol, then dried under vacuum for 24 hours, and then dried on a vacuum pump at 10 25-30 ° C for 12 hours to obtain 66 mg of crystalline material. Example 3 · (2R) 2-amino-2-cyclohexyl-N-(5-(l-methyl-iH-tr than quaternary-4-yl)-1-yloxy-2,6-dihydro_1Η·[1 , 2] Preparation of dioxo[4,5,6-cd]吲嗓-8-yl]acetamide polymorph II: polymorphic form I of compound 1, (2R)-2-amino- 2-cyclohexyl-Ν-(5-(1-methyl15-yl-111-.pyran-4-yl)-small-oxy-2,6-dihydro-1H-[1,2]dioxime [4,5,6-cd]吲σ多_8_基]Ethylamine (5〇mg) was adjusted to a slurry in 1 ml of methanol at 4. The slurry was centrifuged at 14, rpm to separate solids. The solid was dried in a low temperature vacuum oven to obtain Form II. Example 4a: (2R)-2-Amino-2-cyclohexyl-indole-(5-(1-methyl-lHlbazole-4-2indolyl) Small pendant oxy-2,6-dihydro-1H-[1,2] octadecyl [4,5,6-cd] ♦ Preparation of the first batch of amorphous form of the guanamine amorphous form: 1.3 g of compound 11 (2 HCl) dissolved in water (200 ml) at 22 ° C, followed by saturated aqueous sodium bicarbonate (50 ml) and 5 〇〇 ML ethyl acetate. Two more tables of sodium bicarbonate and 50 ml of water were added. After vigorous stirring, the mixture was filtered through 46 200806671 and the phases were separated. The aqueous phase was again extracted with ethyl acetate (200 mL twice). The combined organic phases were washed with brine and dried over sodium sulfate. Filtration followed by evaporation of the volatiles <RTI ID=0.0></RTI> to <RTI ID=0.0> The total amount of amorphous material obtained was 690 mg. 5 Example 4b: (2R)-2-Amino-2-cyclohexyl-N-(5-(l-methyl-1H.pyrazol+yl)-small-oxy-2,6-dihydro-1H-[ 1,2] Preparation of a second batch of amorphous form of dioxo[4,5,6-cd]吲哚-8-yl]acetamido amorphous form: 749.2 mg of polymorphic form I of compound 1 Dissolved in 85 ml of USP grade ethanol. The solution was filtered using a Bucherner funnel and a Wattman type 2. Filtered. The solution was transferred to a 190 mm X1 mm crystallizing dish and allowed to evaporate under ambient conditions. Hour. 589.1 mg of orange solid was recovered as the second batch of the amorphous form of the compound. Example 5: Polymorph Form I (second spectrum), π type (second spectrum) and amorphous form of Compound 1 Two batches of 13C solid state NMR. 15 samples were filled into the Zr〇2 rotor. Type I and amorphous samples were filled into a 4 mm rotor, and π-type samples were filled into a 7 mm rotor. Under ambient conditions in Brooke _ The Bmker-Biospin 4 and 7 mm CPMAS probes were placed on a wide-bore Brook-Baby-Avance DSX 500 MHz nmr spectrometer to collect the carbon spectrum. The rotor was placed at a magic angle. 15〇kH z 2〇 (4mm rotor) and 7·〇kHz (7mm rotor) spin. Fast spin speed reduces the strength of the spin sideband. The number of scans is adjusted to obtain the appropriate S/N. Use 1H-13C crossover Polarized magic angle spin (CPMAS) [under the death of type II samples followed by total suppression of spin sidebands (TOSS)] to collect one-dimensional spectra of sputum samples and amorphous samples. Apply TOSS to suppress The spin sideband is optimized for 47 200806671, the cross-polarization contact time is adjusted to 2 〇 milliseconds, the decoupling field is set at about 75 kHz. The 512 scan for the ! type has a cyclic delay of seconds. Type II obtained 256 scans with a cyclic delay of 3 〇 seconds, and amorphous samples obtained 2048 scans with a cyclic delay of 3 seconds. All spectra were analyzed using an external sample of gold pentane and the upper field signal was set at 29·5 ppm as a reference. [Figure is simple to say ^ Ming] Figure 1 is (2R,Z)-2-amino-2-cyclohexyl-indole-(5·(1_methyl-1H-pyridin-4-yl) small X-ray powder diffraction pattern of pendant oxy-2,6-dihydrodioximehedo[4,5,6-cd]Shdolyl acetamide polymorph I. 10 Figure 2 is (2R, Z)-2-Amino-2_cyclohexyl-N-(5- (l-Methyl-1H-pyrazol-4-yl)-small-oxyl.-2,6-dihydro- out-seven,]]diindole[4,5,6-cd]吲参8- X-glycine polycrystalline n-type X-ray powder diffraction pattern. Figure 3a is (2R,Z)-2_amino-2-cyclohexyl (5-(bu-methyl_1Η_pyrazole_4_) Small side oxy-2,6·dihydro·1Η-[1,2]diindole 15 [4,5,6-cd]吲嗓-8_yl]acetamide amorphous (first Once) the matte powder diffraction pattern. Figure 3b is (2R,Z)-2-amino-2-cyclohexyl-N-(5-(l-methyl-1Η-σ-pyrazol-4-yl)-1_sideoxy-2,6_ Dihydro-1 Η-[1,2] 丫0丫ping[4,5,6-cd]吲哚-8_yl]acetamide amorphous (second batch) of χ light powder around 20 shots Fig. 4a is (2R,Z)_2-amino-2-cyclohexyl-indole-(5-(1-methylbazol-4-yl)-1_ oxo-2,6-dihydro- 1Η-[1,2]2丫[丫,[4,5,6-cd]吲哚_8·yl]polyamine I (first spectrum) of the solid C-cross polarization and magic Angular spin (CP/MAS) NMR. 48 200806671 Figure 4b shows (2R,Z)-2-amino-2-cyclohexyl_N-(5_(1_methyl_1H-pyrazol-4-yl) )_1·Sideoxy-2,6-dihydro-1H-[1,2]diindole[4,5,6-cd]吲π朵-8-yl]polyamine I (Second spectrum) solid state 13C cross polarization and magic angle spin (CP/MAS) NMR. 5 Figure 5a is (2R,Z)-2·amino-2-cyclohexyl·Ν-(5-( 1-methyl-1H-pyridinyl-4-yl)-1-yloxy-2,6-di-rhoose·1Η-[1,2]diπ丫丫[4,5,6-cd] Solid-state 13C CP/MAS NMR of polymorphous Form II (first spectrum) of 吲哚-8-yl]acetamide. The picture of 5b is (2R,Z)-2-amino-2-3⁄4 hexyl _(5_(1-methyl-1Η-σ ratio 10 oxazol-4-yl) small pendant oxy-2,6-dihydro_1 Η-[1,2] 吖 吖 [4,5,6- Solid phase 13C CP/MAS NMR of polymorphic Form II (second spectrum) of cdp sulphate-8-yl. oxime 6 is (2R,Z)_2-amino-2·cyclohexyl_N -(5_(1-methyl to 0-position-4-yl)-1-flankyl-2,6-dihydro-1H-[1,2]di-n- 丫 平 并 and 15 弓布-8-yl The solid form of acetamide (second batch) is immobilized by 13C CP/MAS NMR. [Main component symbol description] (none) 49

Claims (1)

200806671 X. Patent scope: 1. A (2R,Z)-2-amino-2-cyclohexyl-indole-(5-(1·methyl-1H-indazol-4-yl) small pendant oxy group a crystalline form of _2,6-dihydro-1 fluorene-[1,2]dioxinh[4,5,6-cd]dec-8-yl)acetamide. 5 2. The crystalline form of claim 1 wherein the crystalline form is polycrystalline Form I. 3. The crystalline form of claim 1, wherein the crystalline form has a powder X-ray diffraction pattern comprising a peak at a diffraction angle (2 Θ) of 23.6 ± 0.1 and 8·5 ± 0.1. 10. The crystalline form of claim 2, wherein the crystalline form has a powder X-ray diffraction pattern comprising a peak of a diffraction angle (2 Θ) substantially the same as that shown in Figure 1. 5. The crystalline form of claim 2, wherein the crystalline form is comprised of a 13C solid state NMR peak pattern at a chemical shift of 175·0±0·2, 15 137·6±0·2, 134·9±0 • 2, 110·3±0·2, 106·3±0·2, 41.1±0·2, and 32.6±0.2 ppm spikes. 6. The crystalline form of claim 2, wherein the crystalline form has a 13C solid state NMR peak pattern comprising chemical shifts of 175.0 ± 0.2, 137.6 ± 0.2, 134·9 ± 0.2, 110·3 ± 0.2, 106·3 At least three, at least four, at least five, or at least six of the seven peaks of ±0.2, 41·1±0·2 20 and 32·6±0·2 ppm. 7. The crystalline form of claim 2, wherein the crystalline form has a 13C solid state NMR peak pattern comprising a peak substantially at the same chemical shift position as shown in Figure 4b. The crystal form of any one of claims 2-7, wherein the crystalline form is a substantially pure polymorphic form I. 9. The crystalline form of claim 1 wherein the crystalline form is polymorph II. 5. The crystalline form of claim 9, wherein the crystalline form has a powder X-ray diffraction pattern comprising a peak at a diffraction angle (2 Θ) of 25.3 ± 0.1 and 16.0 ± 0.1. 11. The crystalline form of claim 9, wherein the crystalline form has a powder X-ray diffraction pattern comprising a peak of a diffraction angle (2 Θ) which is substantially the same as that shown in Fig. 2. 12. The crystalline form of claim 9 wherein the crystalline form is comprised of a 13C solid state NMR peak pattern comprising a chemical shift of 177.7 ± 0.2, 133·2 ± 0·2, 127·8 ± 0·2, 103· Peaks of 8±0·2 and 22·7±0·2 ppm. 13. The crystalline form of claim 9 wherein the crystalline form has a 13C solid state NMR peak pattern comprising chemical shifts of 177.7 ± 0.2, 133.2 ± 0.2, 127.8 ± 0.2, 103.8 ± 0.2, and 22.7 ± 0.2 ppm. At least three or at least four of the five peaks. 14. The crystalline form of claim 9, wherein the crystalline form has a 13C solid state NMR peak pattern comprising a peak substantially at the chemical shift position of 20 as shown in Figure 5b. 15. The crystalline form of any one of claims 9-14, wherein the crystalline form is a substantially pure polymorph II. 16·-(2R,Z)-2-Amino-2-cyclohexyl-N-(5-(l-methyl-1H-pyrazol-4-yl)-1_sideoxy-2,6 -Dihydro-1H_[1,2]dioxine 51 200806671 [4,5,6-cd]吲哚8-yl) Amorphous form of acetamidine. 17. The amorphous form of claim 16, wherein the amorphous form is comprised of a 13C solid state NMR peak pattern at a chemical shift of 163 6 ± 〇 2, 138·9 ± 0·2, 131·4 ± 0·2 , 129·9±0·2 and 30.8±0.2 ppm spikes. 5 I8. The amorphous form of claim 16, wherein the amorphous form has a 13C solid state NMR peak pattern comprising a chemical shift of 163.6 ± 〇 2, 138·9 ± 0·2, 131·4 ± 0·2. At least three or at least four of the five peaks of 129·9±0·2 and 30.8±0.2 ppm. 19. The amorphous form of claim 16, wherein the amorphous form has a 13c solid peak pattern comprising a peak substantially at the same chemical shift position as shown in Figure 。. The amorphous form of any one of claims 16 to 19, wherein the amorphous form is substantially pure. 21·(2R,Z)-2-Amino-2-cyclohexyl-indole·(5-(1-methyl-1ίΐ·pyrazole-4-15yl) small sideoxy-2,6- a solid form of dihydro-1Η-[1,2]di//hhe[4,5,6-cd]吲σ多-8-yl)ethene, comprising a polycrystalline form I, polycrystalline At least two of the Type II and amorphous forms. 22. The solid form of claim 21, comprising at least 8 % polymorph Form I. 2〇23_ A pharmaceutical composition comprising (2R,Z)-2.amino-2_cyclohexyl-indole-(5-(1-methyl-1Η-) as claimed in any one of claims 22 Mouth ratio _4_ base) small side oxy-2,6-dihydro-ιη-[ι,2] 吖 吖 [4,5,6_cd]a 引 朵 _8_ base) A form. 24. A method of treating cancer in a mammal comprising administering to the patient in need thereof a therapeutically effective amount of a pharmaceutical composition according to claim 23, wherein the therapeutically effective amount is selected from the group consisting of an anticancer agent. And an anti-cancer treatment for radiotherapy. 25. A method of treating a mammalian disease 5 mediated by CHK1 protein kinase activity, comprising administering to a mammal in need thereof (2R, Z)-2 as claimed in any one of claims 1-23 -amino-2-cyclohexyl-N-(5-(l-methyl-1H-pyrazol-4-yl)-1-yloxy-2,6-dihydro_1H-[1,2] A combination of bismuth and [4,5,6-cd]吲磉-8-yl)acetamide is a therapeutically effective amount of an anticancer treatment selected from the group consisting of anticancer agents and radiotherapy. 53