Technical Field

[0001] The present invention relates to a novel crystal of an indocyanine compound with the property of emitting near-infrared fluorescence, which is a green pigment useful in the fields of medical diagnostic technology, medical surgery technology, scientific measurement and analysis technology, printing technology, writing technology, painting technology, dyeing technology, and staining technology. The present invention relates more specifically to a crystal of a cyclic sugar chain cyclodextrin-linked indocyanine compound with the property of emitting near-infrared fluorescence, a manufacturing method therefor, and a purification method therefor.

Background

[0002] This indocyanine compound is listed as Compound (19) and Compound (20) in US patent 10086090. Compound (20) is obtained as an inclusion compound, which is a green solid. Compound (20) is listed as TK-1 in J. Biomedical Optics (2016) 21 (8), 086009-1-086009-11, which is amorphous.

[0003] Compound (19)

[0004] Compound (20): Inclusion Compound [0005] A counter anion of Compound (19) or Compound (20) was chloride ion in the US patent 10350310.

[0006] The chloride compound (hereinafter Compound A) represented by Formula (I) below.

[0007] Chemical Name

[0008] 3-(3-{[3-(cyclomaltoheptaos-21-0-yl)propyl]amino}-3-oxopropy l)-2-{(1£)-2- [(3£)-3-{(2£)-2-[3-(3-{[3-(cyclomaltoheptaos-21-0-yl)propy l]amino}-3-oxopropyl)-1,1- dimethyl-1,3-dihydro-2/-/-benzo[e]indol-2-ylidene]ethylidene }-2-methoxycyclohex-1-en- 1-yl]ethen-1-yl}-1,1-dimethyl-1/-/-benzo[e]indol-3-ium chloride.

Description of Invention

[0009] As a result of extensive research conducted to obtain crystals of Compound (19) or Compound (20), the present inventors obtained crystals of these compounds with chloride.

[0010] Also the inventors identified a new profile of the crystal and how to obtain the crystals. Furthermore the inventors found that it was very important to control humidity in order to crystalize Compound A. [0011] The inventors also found the state of the crystal form was related to an amount of water (H2O) molecules.

[0012] Specifically, it is the following.

[0013] [1] A crystal of 3-(3-{[3-(cyclomaltoheptaos-21-0-yl)propyl]amino}-3- oxopropyl)-2-{(1E)-2-[(3E)-3-{(2E)-2-[3-(3-{[3-(cyclomaltohe ptaos-21-0- yl)propyl]amino}-3-oxopropyl)-1,1-dimethyl-1,3-dihydro-2H-be nzo[e]indol-2- yl idene]ethylidene}-2-methoxycyclohex- 1 -en- 1 -yl]ethen- 1 -yl}- 1 , 1 -dimethyl- 1 H- benzo[e]indol-3-ium chloride.

[0014] [2] The crystal according to [1], wherein in the powder X-ray analysis using

Cu as a radiation source, 2Q (°) exhibits peaks at around 3.7, around 6.1, around 6.3, around 7.0, around 7.7, around 9.6, around 12.4, around 12.5, around 12.7, around

18.5, and around 18.8.

[0015] [3] The crystal according to [1], wherein in the powder X-ray analysis using

Cu as a radiation source, 2Q (°) exhibits peaks at around 3.9, around 7.3, around 9.6, around 11.0, around 12.5, around 14.6, around 16.5, around 18.5, around 18.7, and around 19.0.

[0016] [4] The crystal according to [1], wherein in the powder X-ray analysis using

Cu as a radiation source, 2Q (°) exhibits peaks at around 7.7, around 9.2, around 9.8, around 10.4, around 11.1, around 12.3, around 12.9, around 13.3, around 14.4, and around 18.7.

[0017] [5] The crystal according to [1], wherein in the powder X-ray analysis using

Cu as a radiation source, 2Q (°) exhibits peaks at around 3.7, around 4.3, around 4.8, around 5.4, around 6.3, around 7.7, around 10.9, around 12.3, around 18.4, and around 19.4.

[0018] [6] The crystal according to [1], wherein in the powder X-ray analysis using

Cu as a radiation source, 2Q (°) exhibits peaks at around 3.7, around 4.3, around 4.8, around 5.4, around 9.5, around 10.9, around 16.2, around 18.4, around 18.6, and around 19.4.

[0019] [7] The crystal according to [1], wherein in the powder X-ray analysis using

Cu as a radiation source, 2Q (°) exhibits peaks at around 3.0, around 3.7, around 4.1, around 4.5, around 5.7, around 6.1, around 7.2, around 7.8, around 17.2, and around

17.6.

[0020] [8] The crystal according to [1], wherein in the powder X-ray analysis using

Cu as a radiation source, 2Q (°) exhibits peaks at around 3.7, around 4.8, around 5.4, around 6.1, around 6.3, around 9.0, around 9.9, around 10.3, around 12.4, around 18.5, and around 19.5.

[0021] [9] A method for preparing a crystal of 3-(3-{[3-(cyclomaltoheptaos-21-0- yl)propyl]amino}-3-oxopropyl)-2-{(1 E)-2-[(3E)-3-{(2E)-2-[3-(3-{[3-(cyclomaltoheptaos- 21 -0-yl)propyl]amino}-3-oxopropyl)-1,1 -dimethyl-1, 3-dihydro-2H-benzo[e]indol-2- yl idene]ethylidene}-2-methoxycyclohex- 1 -en- 1 -yl]ethen- 1 -yl}- 1 , 1 -dimethyl- 1 H- benzo[e]indol-3-ium chloride comprising:

[0022] Adding 40mI to 100mI of water solution of 3-(3-{[3-(cyclomaltoheptaos-21-0- yl)propyl]amino}-3-oxopropyl)-2-{(1 E)-2-[(3E)-3-{(2E)-2-[3-(3-{[3-(cyclomaltoheptaos- 21 -0-yl)propyl]amino}-3-oxopropyl)-1,1 -dimethyl-1, 3-dihydro-2H-benzo[e]indol-2- yl idene]ethylidene}-2-methoxycyclohex- 1 -en- 1 -yl]ethen- 1 -yl}- 1 , 1 -dimethyl- 1 H- benzo[e]indol-3-ium chloride.

[0023] [10] The method according to [9], wherein said volume of water is 40mI to

100mI of water to per 50mg of 3-(3-{[3-(cyclomaltoheptaos-21-0-yl)propyl]amino}-3- oxopropyl)-2-{(1E)-2-[(3E)-3-{(2E)-2-[3-(3-{[3-(cyclomaltohe ptaos-21-0- yl)propyl]amino}-3-oxopropyl)-1 ,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2- yl idene]ethylidene}-2-methoxycyclohex- 1 -en- 1 -yl]ethen- 1 -yl}- 1 , 1 -dimethyl- 1 H- benzo[e]indol-3-ium chloride.

[0024] [11] The method according to [9] or [10], wherein temperature of said method is over 0° C to 5° C.

[0025] [12] The method according to [9] or [10], wherein the temperature of said method is 4° C.

[0026] This data should not strictly be construed because it would depend on measuring conditions. Accordingly this data would be preferably expressed by around 4° C.

[0027] [13] A method for preparing a crystal of 3-(3-{[3-(cyclomaltoheptaos-21-0- yl)propyl]amino}-3-oxopropyl)-2-{(1 E)-2-[(3E)-3-{(2E)-2-[3-(3-{[3-(cyclomaltoheptaos- 21 -0-yl)propyl]amino}-3-oxopropyl)-1,1 -dimethyl-1, 3-dihydro-2H-benzo[e]indol-2- yl idene]ethylidene}-2-methoxycyclohex- 1 -en- 1 -yl]ethen- 1 -yl}- 1 , 1 -dimethyl- 1 H- benzo[e]indol-3-ium chloride under the following circumstance:

[0028] relative humidity 40% to relative humidity 95%.

[0029] [14] The method according to [13], wherein said circumstance is relative humidify 40% to relative humidity 90%. [0030] [15] A crystal of 3-(3-{[3-(cyclomaltoheptaos-21-0-yl)propyl]amino}-3- oxopropyl)-2-{(1E)-2-[(3E)-3-{(2E)-2-[3-(3-{[3-(cyclomaltohe ptaos-21-0- yl)propyl]amino}-3-oxopropyl)-1,1-dimethyl-1,3-dihydro-2H-be nzo[e]indol-2- yl idene]ethylidene}-2-methoxycyclohex- 1 -en- 1 -yl]ethen- 1 -yl}- 1 , 1 -dimethyl- 1 H- benzo[e]indol-3-ium chloride having 10 to 45 mol of water molecule.

[0031] [16] The crystal according to [15] having 17 to 45 mol of water molecule.

[0032] [17] The crystal according to [15] or [16] having 17 to 38 mol of water molecule.

Description of Drawings

[0033] FIG. 1 is an X-ray diffraction diagram of the green solid obtained in Synthesis Example 3.

[0034] FIG. 2 is an X-ray diffraction diagram of Compound A when 50% MeCN aq was used.

[0035] FIG. 3 is an X-ray diffraction diagram of Compound A when 50% EtOH aq was used.

[0036] FIG. 4 is an X-ray diffraction diagram of Compound A when 50% acetone aq was used.

[0037] FIG. 5 shows diffraction spots from an X-ray diffraction measurement of Compound A when 50% propylene glycol aq was used.

[0038] FIG. 6 is a polarized micrograph of Compound A when 50% propylene glycol aq was used.

[0039] FIG. 7 is an X-ray diffraction diagram of Compound A obtained when allowed to stand under saturated steam.

[0040] FIG. 8 is a polarized micrograph of Compound A obtained when allowed to stand under saturated steam.

[0041] FIG. 9 is an X-ray diffraction diagram of Compound A when 20% MeOH aq was used.

[0042] FIG. 10 is an X-ray diffraction diagram of Compound A when 20% EtOH aq was used.

[0043] FIG. 11 is an X-ray diffraction diagram of Compound A when water was used. [0044] FIG. 12 is an X-ray diffraction diagram of Compound A with high humidity treatment.

[0045] FIG. 13 is an X-ray diffraction diagram of Compound A when it was the amorphous form.

[0046] FIG. 14 is an X-ray diffraction diagram of Compound A in 100 pi of purified water.

[0047] FIG. 15 is an X-ray diffraction diagram of Compound A in 40 mI of purified water.

[0048] FIG. 16 is an X-ray diffraction diagram of Compound A under humidity.

[0049] FIG. 17 is Weight change of Compound A under humidity.

Industrial Applicability

[0050] Crystals of Compound A of the present invention would be compounds that are useful as the active ingredient in pharmaceutical products or medicinal products or intermediates in a manufacturing process.

[0051] The crystal of the present invention can be used as a drug substance by combining one or more of the crystal of the present invention with a pharmaceutically acceptable carrier, and excipient, or the like, to prepare the medicament. The preparation of the medicament can be carried out by a method usually employed in this field.

[0052] The medicament containing the crystal of the present invention may take any form of preparation for oral administration, such as tablets, pills, capsules, granules, powders, liquids and solutions, and the like; or preparations for parenteral administration, such as intraarticular, intravenous, or intramuscular injections, and the like, suppositories, percutaneous liquid preparations, ointments, transdermal patches, transmucosal liquid preparations, transmucosal patches, inhalations, and the like.

[0053] The present invention can be used in diagnosis. The diagnostic composition can be substituted for a diagnostic composition including an indocyanine green which has hitherto been used. The application thereof may include, for example, liver function test drugs, circulatory function test drugs, and the like. In addition, the composition can be applied to medical operations and medical diagnosis in which near-infrared fluorescence emitted by administration thereof to a body such as a blood vessel, lymph vessel, brain, eye, stomach, breast, esophagus, skin, kidney, ureter, bladder, urethra, lung, heart, or other moiety is observed. It is considered that the pigment included in the diagnostic composition of the invention has a low binding property to a living body, and it can label a necessary moiety over a long time.

[0054] Synthesis Example 1

[0055] Compound (20) could be obtained according to the synthesis example described in J. Biomedical Optics (2016) 21 (8), 086009-1-086009-11.

[0056] Synthesis Example 2

[0057] Compound (19) or Compound (20) in US patent 10086090 or WO 2011093098 were obtained using the described method.

[0058] A mixture of 0.02 g of the compound represented by Chemical Formula (18), 0.081 g of the compound represented by Chemical Formula 14, 0.016 g of WSC,

0.011 g of HOBt, 0.3 ml_ of pyridine, and 0.2 ml_ of N, /V-dimethylformamide was stirred for six hours at 0°C in a dark place. Next, 5 ml_ of acetone were added, and the precipitate was filtered under reduced pressure, dissolved in a 0.1% aqueous trifluoroacetic acid solution, and subjected to ODS column chromatography. A mixture of water and methanol containing 1 mM hydrochloric acid was used in the eluate, and the compound represented by Chemical Formula (19) was eluted. The eluate was concentrated under reduced pressure to obtain 0.045 g of an inclusion compound represented by Chemical Formula (20) as a green solid. (When the eluate is concentrated under reduced pressure, the water content at the end of the concentration is high and so the resulting compound is an inclusion compound.)

[0059] The following abbreviations were used.

[0060] HOBt: 1-hydroxybenzotriazole, WSC: water soluble carbodiimide(1-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride) i!S

[0061] Synthesis Example 3

[0062] First, 58 kg of methanol, 2.6 kg of dimethyl sulfoxide, 19 kg of water, 5.26 kg of 3-(2-carboxyethyl)-2-{(1E)-2-[(3E)-3-{(2E)-2-[3-(2-carboxyet hyl)-1 ,1-dimethyl-1 ,3- dihydro-2/-/-benzo[e]indol-2-ylidene]ethylidene}-2-methoxycy clohex-1-en-1-yl]ethen-1- yl}-1,1-dimethyl-1/-/-benzo[e]indol-3-ium chloride, and 23.7 kg of 21-0-(3- aminopropyl)cyclomaltoheptaose (pure content 21.5 kg) were mixed together at an internal temperature of about 20°C. Next, 4.68 kg of 4-(4,6-dimethoxy-1,3,5-triazin-2- yl)-4-methylmorpholinium chloride (DMT-MM) (pure content 3.98 kg) were added, and the mixture was washed with an aqueous methanol solution (6.8 kg of methanol and 2.1 kg of water) and stirred at an internal temperature of about 20°C for about two hours. Afterwards, 2.34 kg of DMT-MM (pure content 1.99 kg) and 0.726 kg of N- methylmorpholine were added, and the mixture was stirred at an internal temperature of about 20°C for about two hours. Next, 2.34 kg of DMT-MM (pure content 1.99 kg) was added at an internal temperature of about 20°C. Then, 84 kg of methanol were added dropwise over about one hour, and the mixture was stirred at an internal temperature of about 20°C for about 16 hours. Next, 250 kg of acetone were added dropwise over two hours, and the resulting suspension was stirred at an internal temperature of about 20°C for about 19 hours. Filtration was performed, and the filtrate was washed with an acetone-methanol mixture (58 kg of acetone and 28 kg of methanol) and then washed twice with 84 kg of acetone. Vacuum drying was performed at an external temperature setting of about 30°C to obtain 24.68 kg of a green solid. This was dissolved in 5 mM aqueous hydrochloric acid and purified using ODS column chromatography (ODS 130 kg, mobile phase: 5 mM aqueous hydrochloric acid solution 30% aqueous methanol solution 40% aqueous methanol solution (v/v) 80% aqueous methanol solution (v/v)). The target product was collected and the methanol was distilled off under reduced pressure to obtain a concentrated solution. Next, all of the concentrated liquid was adsorbed in HP20SS column chromatography, and the flushed active component (HP20SS 130 kg, mobile phase: 60% aqueous methanol solution (v/v) 80% aqueous methanol solution (v/v)) was collected. The active component was concentrated under reduced pressure, subjected to clarifying filtration, and then freeze-dried to obtain 8.62 kg of a green solid. Then, 4.9 kg of this was dissolved in 1 mM aqueous hydrochloric acid solution and purified by ODS column chromatography (ODS 150 kg, mobile phase: 1 mM aqueous hydrochloric acid solution 30% aqueous methanol solution (v/v) 40% aqueous methanol solution (v/v)). The target product was collected and the methanol was distilled off under reduced pressure to obtain a concentrated solution. Next, all of the concentrated liquid was adsorbed in HP20SS column chromatography, and the flushed active component (HP20SS 67.8 kg, mobile phase: 60% aqueous methanol solution 80% aqueous methanol solution (v/v)) was collected. Activated carbon was added, and the mixture was stirred at an internal temperature of about 20°C for about two hours. The activated carbon was removed using a filter pre-coated with radiolite, clarifying filtration was performed, and then lyophilization was performed to obtain 1.88 kg of 3-(3-{[3-(cyclomaltoheptaos-21-0-yl)propyl]amino}-3-oxopropy l)-2- {(1£)-2-[(3£)-3-{(2£)-2-[3-(3-{[3-(cyclomaltoheptaos-21-0 -yl)propyl]amino}-3- oxopropyl)-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylide ne]ethylidene}-2- methoxycyclohex-1-en-1-yl]ethen-1-yl}-1,1-dimethyl-1H-benzo[ e]indol-3-ium chloride (“Compound A”) as a green solid.

[0063] The following are the physicochemical properties of the resulting compound. [0064] Instrument Analysis Data ¹ H-NMR [0065] Assignment of ¹ H NMR Spectrum (D2O)

Table 1 s: singlet, d: doublet, m: multiplet a) Assignments may be interchangeable.

[0066] Elemental Analysis Table 2 a) Corrected by the hypothetical water content of 11.5%

[0067] According to the data in FIG. 1, the green solid of Compound A was amorphous.

Examples

[0068] Crystals of Compound A were obtained in the following examples.

[0069] In the crystallization process, crystals could be obtained at room temperature unless otherwise indicated.

[0070] The following abbreviations are used in the text.

[0071] MeOH: methanol, EtOH: ethanol, MeCN: acetonitrile, aq: aqueous solution [0072] Example 1

[0073] After dissolving Compound A (100 mg) in 50% MeCN aq (0.5 ml) and allowing the solution to stand for one day, the resulting solid was sampled in a holder for transmission measurement in which Kapton film had been set and subjected to an X-ray diffraction measurement using PANalytical Empyrean with copper X-ray tube at a tube voltage of 45 kV and a tube current of 40 mA to confirm the diffraction peaks. (FIG. 2)

[0074] The characteristic peaks in the diffraction are as follows.

[0075] In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 3.7, around 4.3, around 4.8, around 5.4, around 9.5, around 10.9, around 16.2, around 18.4, around 18.6, and around 19.4.

[0076] Example 2

[0077] After dissolving Compound A (100 mg) in 50% EtOH aq (0.4 ml) and allowing the solution to stand for one day, the resulting solid was sampled in a holder for transmission measurement in which Kapton film had been set and subjected to an X- ray diffraction measurement using PANalytical Empyrean with copper X-ray tube at a tube voltage of 45 kV and a tube current of 40 mA to confirm the diffraction peaks.

(FIG. 3)

[0078] The characteristic peaks in the diffraction are as follows. [0079] In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 3.7, around 4.3, around 4.8, around 5.4, around 6.3, around 7.7, around 10.9, around 12.3, around 18.4, and around 19.4.

[0080] Example 3

[0081] Five days after dissolving Compound A (100 mg) in 50% acetone aq (0.2 ml) seed crystals of Compound A were added. The solution was allowed to stand for seven days. The resulting solid was sampled in a holder for transmission measurement in which Kapton film had been set and subjected to an X-ray diffraction measurement using PANalytical Empyrean with copper X-ray tube at a tube voltage of 45 kV and a tube current of 40 mA to confirm the diffraction peaks. (FIG. 4)

[0082] The characteristic peaks in the diffraction are as follows.

In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 3.0, around 3.7, around 4.1, around 4.5, around 5.7, around 6.1, around 7.2, around 7.8, around 17.2, and around 17.6.

[0083] Example 4

[0084] Five days after dissolving Compound A (100 mg) in 50% propylene glycol aq (0. 2ml), seed crystals of Compound A were added. The solution was allowed to stand for one day, and then single crystals were taken from the resulting solid and subjected to an X-ray diffraction measurement using Rigaku XtaLAB P200 with copper target at a tube voltage of 40 kV and a tube current of 30 mA to confirm the diffraction spots.

(FIG. 5)

[0085] A polarized micrograph of the solid was also taken. (FIG. 6)

[0086] Example 5

[0087] After allowing Compound A (10 mg) to stand for one day under saturated steam, the resulting solid was sampled in a holder for transmission measurement in which Kapton film had been set and subjected to an X-ray diffraction measurement using PANalytical Empyrean with copper X-ray tube at a tube voltage of 45 kV and a tube current of 40 mA to confirm the diffraction peaks. (FIG. 7)

[0088] The characteristic peaks in the diffraction are as follows.

[0089] In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 3.7, around 4.8, around 5.4, around 6.1, around 6.3, around 9.0, around 9.9, around 10.3, around 12.4, around 18.5, and around 19.5.

[0090] A polarized micrograph of the solid was taken. (FIG. 8) [0091] Example 6

[0092] After dissolving Compound A (39.1 mg) in 20% MeOH aq (0.195 ml) and allowing the solution to stand for 56 days at 4°C, the resulting solid was sampled in a holder for transmission measurement in which Kapton film had been set and subjected to an X-ray diffraction measurement using PANalytical Empyrean with copper X-ray tube at a tube voltage of 45 kV and a tube current of 40 mA to confirm the diffraction peaks. (FIG. 9)

[0093] The characteristic peaks in the diffraction are as follows.

[0094] In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 3.7, around 6.1, around 6.3, around 7.0, around 7.7, around 9.6, around 12.4, around 12.5, around 12.7, around 18.5, and around 18.8.

[0095] Example 7

[0096] After dissolving Compound A (36.3 mg) in 20% EtOH aq (0.181 ml) and allowing the solution to stand for 56 days at 4°C, the resulting solid was sampled in a holder for transmission measurement in which Kapton film had been set and subjected to an X-ray diffraction measurement using PANalytical Empyrean with copper X-ray tube at a tube voltage of 45 kV and a tube current of 40 mA to confirm the diffraction peaks. (FIG. 10)

[0097] The characteristic peaks in the diffraction are as follows.

[0098] In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 3.9, around 7.3, around 9.6, around 11.0, around 12.5, around 14.6, around 16.5, around 18.5, around 18.7, and around 19.0.

[0099] Example 8

[0100] After dissolving Compound A (28.5 mg) in water (0.087 ml) and allowing the solution to stand for 56 days at 4°C, the resulting solid was sampled in a holder for transmission measurement in which Kapton film had been set and subjected to an X- ray diffraction measurement using PANalytical Empyrean with copper X-ray tube at a tube voltage of 45 kV and a tube current of 40 mA to confirm the diffraction peaks. (FIG. 11)

[0101] The characteristic peaks in the diffraction are as follows.

[0102] In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 7.7, around 9.2, around 9.8, around 10.4, around 11.1, around 12.3, around 12.9, around 13.3, around 14.4, and around 18.7. [0103] Example 9 [0104] Crystallization test

[0105] Three vials containing the compound A (see 9-1, 9-2 and 9-3) were stored at room temperature for 9 days, and then the vials were stored at 4 °C for 45-47 days. The resulting solids were subjected to HPLC and X-ray diffraction measurement to confirm the purity and the physical state of compound A. X-ray diffraction measurement was conducted by using PANalytical Empyrean with copper X-ray tube at a tube voltage of 45 kV and a tube current of 40 mA.

[0106] 9-1 High humidity storage: Compound A (52.0 mg) was stored under saturated water vapor at room temperature for 9 days, and then the vial was stored at 4 °C for 45 days.

[0107] 9-2 Aqueous solution storage: Compound A (19.8 mg) was dissolved in 5 ml of purified water at room temperature for 9 days, and then the vial was stored at 4 °C for 45 days.

[0108] 9-3 As powder: Compound A (28.0 mg) was stored at room temperature for 9 days, and then the vial was stored at 4 °C for 47 days.

[0109] The results were shown in Table 3.

[0110] In #9-1 the crystal of Compound A was obtained from Table 3 and Fig. 12.

[0111] The characteristic peaks of #9-1 in the diffraction are as follows.

[0112] In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 3.9, around 5.8, around 7.3, around 9. 5, around 11.1, around 12.4, around 12.5, around 18.7, around 19.0, and around 19.5.

[0113] In #9-3 Compound A was still an amorphous from Table 3 and FIG. 13.

Table 3

[0114] Example 10

[0115] T est to identify a range of crystallization with water [0116] Five vials containing the compound A (see 10-1 to 10-5) were stored at 4 °C for 45 days. The resulting solids were subjected to HPLC and X-ray diffraction measurement to confirm the purity and the physical state of compound A. X-ray diffraction measurement was conducted by using PANalytical Empyrean with copper X-ray tube at a tube voltage of 45 kV and a tube current of 40 mA.

[0117] 10-1 Compound A (50 mg) in purified water 100 pi

[0118] 10-2 Compound A (50 mg) in purified water 10 mI

[0119] 10-3 Compound A (50 mg) in purified water 20 mI

[0120] 10-4 Compound A (50 mg) in purified water 30 mI

[0121] 10-5 Compound A (50 mg) in purified water 40 mI

[0122] The results were shown in Table 4.

[0123] In #10-1 the crystal of Compound A was obtained from Table 4 and FIG. 14.

[0124] The characteristic peaks in the diffraction are as follows:

[0125] In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 3.9, around 7.3, around 9.6, around 11.1, around 12.5, around 18.5, around 18.7, around 19.0, around 19.5, and around 21.2.

[0126] In #10-5 the crystal of Compound A was obtained from Table 4 and FIG. 15.

[0127] The characteristic peaks in the diffraction are as follows:

[0128] In a powder X-ray analysis using Cu as a radiation source, 2Q (°) exhibits peaks at around 4.0, around 5.8, around 6.1, around 7.3, around 9.5, around 10.4, around 12.6, around 18.5, around 18.8, and around 19.0.

[0129] As a result, it was thought that Compound A (50 mg) in purified water of 40 mI to 100 mI formed a crystal.

Table 4

[0130] Example 11 [0131] Observation of crystallinity under humidity controlled condition

[0132] X-ray diffraction measurement at controlled humidity conditions were measured by PANalytical Empyrean attached with Anton Paar MHC-trans chamber. Copper X-ray tube was used and tube voltage and tube current were 45 kV and 40 mA respectively. Compound A was sampled into a special holder for this measurement and stored in 100%RH(relative humidify) at room temperature for one day. The resulting solid was subjected to an X-ray diffraction measurement with relative humidity of sample chamber shifting from 90%RH to 40%RH subsequently 40%RH to 90%RH with 10%RH increments (holding each RH for 15 minutes before measurements), and temperature of chamber was maintained at 25°C.

[0133] The crystallinity was decreasing when the humidity was getting lower.

[0134] And then when the humidity was getting higher again, the crystallinity recovered (FIG.16).

[0135] The range of the humidity to maintain the crystal form was from relative humidity 40% to relative humidity 90% based on a result shown in FIG.16.

[0136] Example 12

[0137] Test of molecular weight of the crystal under humidity

[0138] Water sorption isotherm of Compound A from 5%RH to 95%RH at 25°C was generated by dynamic vapor sorption measurement using TA Instruments G5000SA.

[0139] Deliquescent was not observed, and the weight of Compound A was changed 26wt% from 5%RH to 95%RH at 25°C (FIG.17).

[0140] An absorption amount of H O was about 10wt% and 44wt% at 40%RH and 95%RH respectively, and these sorption amounts were equal to 17 mol and 44 mol of H O molecules based on the molecular weight of Compound A, 3079.44.

[0141] From the results described above and Example 11, more than 17 mol of H O molecules were needed to form the crystal.