DESCRIPTION
CRYSTALS OF GLYCINE DERIVATIVE AND PHARMACEUTICAL USE
THEREOF
Technical Field
[0001]
The present invention relates to a crystal of glycine derivative and a medical
use thereof.
Background Art
[0002]
Pharmaceuticals are required to maintain the quality thereof for a long time
during distribution, storage and the like, and high chemical and physical stability is
demanded for compounds as effective components. Thus, for the effective
components of pharmaceuticals, crystals which are expected to have high stability
compared to amorphous products are commonly employed.
[0003]
In screening the crystals of effective components of pharmaceuticals, it is
difficult to find optimal conditions to obtain the crystals, and also even when the
crystals can be obtained, the existence of crystalline polymorphs is problematic in
many cases. This is because each crystal form has a different molecular packing
arrangement in spite of the same chemical structure in the molecular unit, so that
there are differences in chemical and physical stabilities between the crystal forms.
[0004]
If the crystal form of a compound employed as an effective component of
pharmaceuticals is selected incorrectly, the decrease in purity, change in hydration
degree, change in crystal form and the like occur due to the external environment
during storage, and it is difficult to maintain the quality of the compound, thereby

resulting in unexpected situations such as the reduction in pharmacological effects
and the occurrence of side effect depending on the crystal forms. Therefore, in
cases where the crystals of the compound as an effective component of
pharmaceuticals are successfully obtained, it is necessary to evaluate the crystalline
polymorphs of the compound strictly.
[0005]
On the other hand, (S,E)-2-(2,6-dichlorobenzamido)-5-[4-(methyl-pyrimidin-
2-ylamino)phenyl]pent-4-enoic acid represented by Formula (1) below has been
known to exhibit therapeutic effects on inflammatory bowel disease, allergic
dermatitis, multiple sclerosis and leukemia (Patent Literatures 1 to 4).

Prior Art References
Patent Literatures
[0006]
Patent Literature 1: WO 2006/068213
Patent Literature 2: WO 2007/145282
Patent Literature 3: WO 2007/148648
Patent Literature 4: WO 2007/148676
Summary of the Invention
Problems to be Solved by the Invention
[0007]
However, since it is impossible to predict the existence of crystalline

polymorphs or stable crystal form from the chemical structure of the compound, and
further there may be a compound which cannot form a crystal, it is necessary to
study conditions of forming a crystal for each compound in various ways.
Although excellent effectiveness as an effective component of pharmaceuticals is
confirmed as for (S,E)-2-(2,6-dichlorobenzamido)-5-[4-(methyl-pyrimidin-2-
ylamino)phenyl]pent-4-enoic acid, the existence of the crystalline polymorphs or
even the possibility of crystal formation have not been known at present, and to
obtain the optimal crystal form was an important task for developing the compound
as a pharmaceutical product.
[0008]
An object of the present invention is to provide a crystal of (S,E)-2-(2,6-
dichlorobenzamido)-5-[4-(methyl-pyrimidin-2-ylamino)phenyl]pent-4-enoic acid
having excellent chemical and physical stability, and a medical use thereof.
Means for Solving the Problems
[0009]
To solve the above-described problems, the present inventors intensively
studied to succeed in forming a crystal of (S,E)-2-(2,6-dichlorobenzamido)-5-[4-
(methyl-pyrimidin-2-ylamino)phenyl]pent-4-enoic acid, and to find the existence of
crystalline polymorphs of Form A, Form B, Form C, From D and Form E (hydrate),
and a plurality of solvate crystals, thereby completing the present invention.
Among these, the crystals of Form B, Form C, Form D and Form E (hydrate) have
low moisture absorption and excellent physical stability; the crystals of Form B,
Form C and Form E (hydrate) have excellent chemical and physical stability under
severe storage conditions; and the crystal of Form C has excellent physical stability
even in the external environment in which the crystal is exposed to solvents.
[0010]
That is, the present invention provides a crystal described in the following (1)

to (10) and a medical use thereof.
(1) A crystal of (S,E)-2-(2,6-dichlorobenzamido)-5-[4-(methyl-pyrimidin-2-
ylamino)phenyl]pent-4-enoic acid.
(2) The crystal according to (1), which exhibits peaks at 29 (°) of 17.1, 17.7, 18.7,
19.9 and 21.0° in powder X-ray diffraction.
(3) The crystal according to (2), which exhibits an endothermic peak in the range of
178 to 182°C in thermogravimetric-differential thermal analysis.
(4) The crystal according to (1), which exhibits peaks at 26 (°) of 5.9, 8.3, 11.8, 13.2
and 21.7° in powder X-ray diffraction.
(5) The crystal according to (4), which exhibits an endothermic peak in the range of
167 to 171 °C in thermogravimetric-differential thermal analysis.
(6) The crystal according to (1), which exhibits peaks at 29 (°) of 6.6, 8.3, 11.1, 14.6
and 18.2° in powder X-ray diffraction.
(7) The crystals according to (6), which exhibit an endothermic peak in the range of
100 to 104°C in thermogravimetric-differential thermal analysis.
(8) The crystal according to any one of (1) to (7), which is a non-solvate or a hydrate.
(9) A pharmaceutical comprising as an effective component the crystal according to
any one of (1) to (8).
(10) A therapeutic or prophylactic agent for inflammatory bowel disease, allergic
dermatitis, multiple sclerosis or leukemia, comprising as an effective component the
crystal according to any one of (1) to (8).
Effect of the Invention
[0011]
Since the crystal of the present invention is excellent in chemical and physical
stability compared to the amorphous form, the crystal is preferable as an effective
component of pharmaceuticals, and can contribute to the provision of highly reliable
pharmaceuticals which suppress risks such as the decrease in pharmacological effects

and occurrence of side effect.
Brief Description of the Drawings
[0012]
Fig. 1 is a powder X-ray diffraction pattern of the Form C crystal of the
subject compound.
Fig. 2 is a differential thermal analysis curve obtained by thermogravimetric-
differential thermal analysis of the Form C crystal of the subject compound.
Fig. 3 is a powder X-ray diffraction pattern of the Form B crystal of the
subject compound.
Fig. 4 is a differential thermal analysis curve obtained by thermogravimetric-
differential thermal analysis of the Form B crystal of the subject compound.
Fig. 5 is a powder X-ray diffraction pattern of the Form E (hydrate) crystal of
the subject compound.
Fig; 6 is a differential thermal analysis curve obtained by thermogravimetric-
differential thermal analysis of the Form E (hydrate) crystal of the subject compound.
Fig. 7 is a powder X-ray diffraction pattern of the Form A crystal of the
subject compound.
Fig. 8 is a differential thermal analysis curve obtained by thermogravimetric-
differential thermal analysis of the Form A crystal of the subject compound.
Fig .9 is a powder X-ray diffraction pattern of the Form D crystal of the
subject compound.
Fig. 10 is a differential thermal analysis curve obtained by
thermogravimetric-differential thermal analysis of the Form D crystal of the subject
compound.
Mode for Carrying out the Invention
[0013]
The crystal of the present invention is a crystal of (S,E)-2-(2,6-

dichlorobenzamido)-5-[4-(methyl-pyrimidin-2-ylamino)phenyl]pent-4-enoicacid,
and is characterized by the existence of crystalline polymorphs of Form A, Form B,
Form C, From D and Form E (hydrate).
[0014]
The crystal forms of (S,E)-2-(2,6-dichlorobenzamido)-5-[4-(methyl-
pyrimidin-2-ylamino)phenyl]pent-4-enoic acid (hereinafter also referred to as "the
subject compound") can be distinguished by characteristic peaks depicted in the
powder X-ray diffraction patterns or endothermic peaks depicted in the differential
thermal analysis curves (hereinafter referred to as "DTA curve") obtained by
thermogravimetric-differential thermal analyses (hereinafter referred to as "TG-
DTA"). The powder X-ray diffraction pattern and DTA curve may somewhat vary
depending on the measurement conditions. For example, as for the diffraction
angle 29 in the powder X-ray diffraction, error of around ±0.2° is generally
acceptable.
[0015]
As shown in Fig. 1, the Form C crystal of the subject compound exhibits
characteristic peaks at diffraction angle 29 (°) of 17.1, 17.7, 18.7, 19.9 and 21.0° in
powder X-ray diffraction. Also, the Form C crystal of the subject compound
provides the DTA curve depicted in Fig. 2, and exhibits an endothermic peak at
180°C, that is in the range of 178 to 182°C.
[0016]
As shown in Fig. 3, the Form B crystal of the subject compound exhibits
characteristic peaks at diffraction angle 29 (°) of 5.9, 8.3, 11.8, 13.2 and 21.7° in
powder X-ray diffraction. Also, the Form B crystal of the subject compound
provides the DTA curve depicted in Fig. 4, and exhibits an endothermic peak at
169°C, that is in the range of 167 to 171 °C.
[0017]

As shown in Fig. 5, the Form E (hydrate) crystal of the subject compound
exhibits characteristic peaks at diffraction angle 29 (°) of 6.6, 8.3, 11.1, 14.6 and
18.2° in powder X-ray diffraction. Also, the Form E (hydrate) crystal of the subject
compound provides the DTA curve depicted in Fig. 6, and exhibits an endothermic
peak at 102°C, that is in the range of 100 to 104°C.
[0018]
As shown in Fig. 7, the Form A crystal of the subject compound exhibits
characteristic peaks at diffraction angle 29 (°) of 5.7, 7.4, 11.3 and 12.0° in powder
X-ray diffraction. Also, the Form A crystal of the subject compound provides the
DTA curve depicted in Fig. 8, and exhibits an endothermic peak at 139°C, that is in
the range of 137 to 14PC.
[0019]
As shown in Fig. 9, the Form D crystal of the subject compound exhibits
characteristic peaks at diffraction angle 29 (°) of 5.8,11.5, 11.8 and 23.0°. Also,
the Form D crystal of the subject compound provides the DTA curve depicted in Fig.
10, and exhibits an endothermic peak at 135°C, that is in the range of 133 to 137°C.
[0020]
The powder X-ray diffraction measurement for obtaining a powder X-ray
diffraction pattern may be carried out under the conditions below by using a powder
X-ray diffractometer. A measurement sample is prepared by filling a sample
material in a sample plate (material: silicon; depth: 0.2 mm), and leveling out the
surface of the sample material.
«Conditions of Powder X-ray Diffraction»
X-ray Source : CuKa radiation
* using a curved crystal monochromator (graphite)
Output : 40 kV/50 mA
Divergence Slit : 1/2°

Vertical limiting Slit : 5 mm
Scattering Slit : 1/2°
Receiving Slit : 0.15 mm
Detector : Scintillation counter
Scan Mode : 2G/0 scan, Continuous scan
Measurement Range (29) : 2 to 60°
Scanning Rate (29) : 47min
Scanning Step (29) : 0.02°
[0021]
The endothermic peak means the temperature of the peak top on a DTA curve.
The TG-DTA measurement used herein for obtaining the DTA curve may be carried
out under conditions below by using a TG-DTA analyzer.
«TG-DTA Conditions»
Heating Rate : 5°C/min.
Atmosphere : nitrogen (flow rate: 50 mL/min)
Sample Cell : aluminum open cell
Sample Weight : 4 to 6 mg
[0022]
The Form C crystal of the subject compound can be obtained by dissolving
the subject compound in any form in an aromatic solvent to a concentration of 0.1 to
5 mg/mL, preferably 1 to 5 mg/mL, and leaving to stand or stirring the resulting
solution at 0 to 30°C for 1 to 30 days.
[0023]
The Form C crystal of the subject compound can be obtained by dissolving
under heat the subject compound in any form in an alcohol solvent at 50 to 80°C to a
concentration of preferably 10 to 100 mg/mL, more preferably 50 to 80 mg/mL,
adding the preliminarily obtained Form C crystal as a seed crystal to the resulting

solution, and then stirring the solution at 50 to 80°C for 1 to 48 hours and further at 0
to 30°C for 1 to 24 hours.
[0024]
The Form B crystal of the subject compound can be obtained by dissolving
the subject compound in any form in an alcohol solvent to a concentration of
preferably 20 to 100 mg/mL, more preferably 25 to 50 mg/mL, and leaving to stand
or stirring the resulting solution at 0 to 30°C for 1 to 30 days.
[0025]
The Form E (hydrate) crystal of the subject compound can be obtained by
adding water in an amount of preferably 10 to 1000 mL, more preferably 3 to 100
mL per 1 g of the amorphous subject compound to obtain suspension, and stirring the
suspension at 0°C to 30°C for 1 to 30 days.
[0026]
The Form A crystal of the subject compound can be obtained by dissolving
the subject compound in any form in toluene to a concentration of preferably 5 to 20
mg/mL, more preferably 10 to 15 mg/mL, and leaving the resulting solution to stand
air-tightly at 0 to 30°C for 1 to 30 days.
[0027]
The Form D crystal of the subject compound can be obtained by dissolving
the subject compound in any form in an alcohol solvent or ester solvent to a
concentration of preferably 5 to 20 mg/mL, more preferably 10 to 15 mg/mL, and
leaving to stand or stirring the resulting solution at 0 to 30°C for 1 to 30 days.
[0028]
Examples of the above-described aromatic solvent include benzene,
chlorobenzene, toluene, xylene and cumene; and toluene or xylene is preferable.
[0029]
Examples of the above-described alcohol solvent include methanol, ethanol,

1-propanol, 2-propanol, 1-butanol and 2-butanol; and methanol, ethanol or 2-
propanol is preferable.
[0030]
Examples of the above-described ester solvent include ethyl formate, methyl
acetate, ethyl acetate, propyl acetate, isopropyl acetate and isobutyl acetate; and
methyl acetate, ethyl acetate or propyl acetate is preferable.
[0031]
The crystal of the subject compound may be used as a pharmaceutical useful
for therapies and/or prophylactics of inflammatory bowel disease, allergic dermatitis,
multiple sclerosis or leukemia in mammals (e.g., mouse, rat, hamster, rabbit, dog,
monkey, bovine, sheep and human). When the crystal of the subject compound is
clinically administered as a pharmaceutical, the dose of the crystal may be
appropriately selected depending on the symptom, age, body weight, sex,
administration method or the like. For example, a dose of 0.01 mg to 5 g per day in
case of an injection solution, and a dose of 0.1 mg to 10 g per day in case of an oral
preparation are preferably administered to an adult in terms of the effective
component, and may be administered at one time or dividedly in several times
respectively.
[0032]
When the crystal of the subject compound is clinically administered as a
pharmaceutical, examples of the formulation include oral preparations such as tablets,
capsules, granules, powders and syrups; and parenteral preparations such as inhalants,
injection solutions, eye drops, nasal sprays, suppositories, ointments, creams, lotions
and patches. These formulations may be prepared according to methods commonly
used in the field of formulation. In this case, additives commonly used in the field
of formulation, such as vehicles, stabilizers, preservatives, buffering agents,
solubilizers, emulsifiers, diluents and isotonic agents may be admixed appropriately,

as necessary. Examples of the pharmaceutically acceptable carrier and diluent used
for preparing the above-described formulations include binders (syrups, gelatin, gum
arabic, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose,
sorbitol, polyvinylpyrrolidone, polyvinyl alcohol, tragacanth and the like), vehicles
(sucrose, lactose, D-mannitol, erythritol, crystalline cellulose, ethyl cellulose, corn
starch, calcium phosphate, sorbitol, glycine and the like), disintegrants (partially
pregelatinized starch, croscarmellose sodium, crospovidone and low substituted
hydroxypropylcellulose), and lubricants (magnesium stearate, polyethylene glycol,
talc, silica, sucrose esters of fatty acid and the like).
[0033]
The Pharmaceutical containing as an effective component the crystals of the
subject compound contains the crystals preferably in an amount of 0.001 to 90% by
weight, more preferably in an amount of 0.01 to 70% by weight per dosage unit.
[0034]
To control the productivity, compatibility or solubility of pharmaceuticals, the
crystal of the subject compound may be blended after being ground. The ground
crystals of the subject compound have a particle diameter distribution in which D90
is preferably not more than 1000 urn, and more preferably not more than 100 um.
The term "D90" herein means the particle diameter at the point where the volume
which is cumulatively measured from smaller particles reaches 90%, that is, the point
where the cumulative frequency of the volume distribution reaches 90%.
[0035]
The methods for grinding the crystals of the subject compound include
grindings with fluid energy mills such as a jet mill; and impact mills such as a
hammer mill, a pin mill and a ball mill.
Examples
[0036]

The present invention will now be described concretely by way of Examples,
but the present invention is not restricted to these Examples.
[0037]
(Reference Example 1): Preparation of Amorphous Subject Compound
The subject compound (1 g) prepared by the method described in Patent
Literature 1 was dissolved in methanol (10 mL), and after concentrating the resulting
solution by an evaporator, the concentrate was dried under reduced pressure by using
a vacuum pump for three days to obtain the amorphous subject compound.
[0038]
(Reference Example 2): Preparation of Amorphous Subject Compound
The subject compound (1 g) prepared by the method described in Patent
Literature 1 was dissolved in tetrahydrofuran (10 mL), and after concentrating the
resulting solution by an evaporator, the concentrate was dried under reduced pressure
by using a vacuum pump for one day to obtain the amorphous subject compound.
[0039]
(Reference Example 3): Production of (S)-2-(2,6-dichlorobenzoylamino)pent-4-enoic
acid ethyl ester (another name: (S)-2-(2,6-dichlorobenzamido)pent-4-enoic acid ethyl
ester)

L-allylglycine ethyl ester tosylate (100 g) was weighed and added to a 2000-
mL flask, and after replacing the atmosphere in the flask with argon, toluene (500
mL) and triethylamine (88.5 mL) were added thereto to obtain suspension. The
suspension was cooled to 0°C, and 2,6-dichlorobenzoyl chloride (50.0 mL) was then
added dropwise over 20 minutes, followed by stirring the resulting solution at 0°C
for 45 minutes. To the reaction solution, sodium hydrogen carbonate (56 g)

dissolved in water (850 mL) was added, and the resulting solution was stirred and
then separated into layers. The aqueous layer was extracted with toluene (400 mL),
and organic layers were then combined and washed with water (400 mL). The
organic layer was concentrated to 285 mL under reduced pressure, and the obtained
toluene solution was cooled to 0°C. To the solution, heptane (1000 mL) was added
dropwise, and the resulting solution was stirred at 0°C for two hours. The crystals
were recovered by filtration, and washed with ice-cooled heptane (200 mL), followed
by drying under reduced pressure to obtain 88.3 g of (S)-2-(2,6-
dichlorobenzoylamino)pent-4-enoic acid ethyl ester (Yield: 88%).
1H NMR (400 MHz, CDC13) δ 1.31 (3H, t, J =7.1Hz), 2.66 (1H, m), 2.80 (1H, m),
4.20-4.30 (2H, m), 4.93 (1H, ddd, J =5.4, 5.4, 7.8 Hz), 5.15 (1H, d, J =9.8 Hz), 5.19
(1H, d, J =15.6 Hz), 5.78 (1H, m), 6.41 (1H, br.d, J =5.4 Hz), 7.25-7.34 (3H, m).
[0040]
(Reference Example 4): Production of (S,E)-2-(2,6-dichlorobenzamido)-5-[4-
(methyl-pyrimidin-2-ylamino)phenyl]pent-4-enoic acid ethyl ester

After replacing the atmosphere in a 500-mL flask with argon, N,N-
dimethylformamide (144 mL) and ethanol (16 mL) were added to thereto. Then,
(S)-2-(2,6-dichlorobenzoylamino)pent-4-enoic acid ethyl ester (16.0 g) produced in
Reference Example 3, N-(4-iodophenyl)-N-methyl-2-pyrimidinylamine (another
name: N-(4-iodophenyl)-N-methylpyrimidin-2-amine) (16.5 g) described in Patent
Literature 1, tetra-n-butylarnmonium bromide (16.3 g), potassium carbonate (14.0 g)
and palladium acetate (230 mg) were successively added to the flask, and after

replacing the atmosphere in the system with argon, the resulting solution was stirred
at 50°C for 18 hours. The reaction solution was cooled to room temperature, and
water (320 mL) was then added thereto, followed by extraction with ethyl acetate
(265 mL). The extract was washed twice with 5% aqueous sodium thiosulfate
solution (80 mL), then twice with 5% aqueous sodium chloride solution (80mL), and
then the extract was concentrated under reduced pressure to about 75 g of the
solution weight. The procedure, in which ethanol (100 mL) was added to the
concentrate and the resulting solution was concentrated under reduced pressure to
about 75 g, was repeated three times, and active carbon (8.0 g) was added to the
obtained ethanol solution, followed by stirring at room temperature for 30 minutes.
The active carbon was removed by filtration, and washed with ethanol (10 mL). To
the filtrate, active carbon (4.0 g) was added, and after stirring the resultant at room
temperature for 30 minutes, the active carbon was removed by filtration, and washed
with ethanol (10 mL). The filtrate was concentrated under reduced pressure to 78 g,
and the obtained ethanol solution was heated to 50°C. After adding distilled water
(30 mL) dropwise to the solution, the resulting solution was cooled to room
temperature and further cooled to 0°C, followed by stirring the solution for one hour.
The crystals was removed by filtration, washed with ice-cooled mixture of ethanol
and water at a ratio of 2:1 (20 mL), and then dried under reduced pressure to obtain
18.3 g of (S,E)-2-(2,6-dichlorobenzamido)-5-[4-(methyl-pyrimidin-2-
ylamino)phenyl]pent-4-enoic acid ethyl ester (Yield: 73%).
1H NMR(400 MHZ, CDC13) δ 1.32 (3H, t, J = 7.1 Hz), 2.84 (1H, m), 2.97 (1H, m),
3.51 (3H, s), 4.25 (1H, m), 4.28 (1H, m), 5.01 (1H, m), 6.11 (1H, dt, J = 15.6, 7.6
Hz), 6.48 (1H, m), 6.52 (1H, d, J = 15.6 Hz), 6.58 (1H, t, J = 4.6 Hz), 7.24-7.37 (7H,
m), 8.33 (2H, d, J = 4.6 Hz).
[0041]
(Example 1): Production of Form C Crystal of Subject Compound

The amorphous subject compound (30 mg) prepared in Reference Example 1
was weighted and added to a vial made of borosilicate glass, and toluene (15 mL)
was added thereto, followed by stirring the resultant at room temperature to dissolve
the compound. The resulting solution was left to stand at room temperature under
open condition. After confirming the precipitates, solvent was removed with a
Pasteur pipette, and the precipitates were dried under reduced pressure by using a
vacuum pump for 30 minutes to obtain white powders of the captioned crystal. For
the obtained crystals, powder X-ray diffraction measurement by using powder X-ray
diffractometer (Rigaku; 2200/RINT ultima+PC) and TG-DTA by using TG-DTA
analyzer (Rigaku; TG810D) were carried out. The results of these measurements
are shown in Fig. 1 ans Fig. 2.
Diffraction angle 26 : 17.1, 17.7, 18.7, 19.9, 21.0°
Endothermic peak : 180°C
[0042]
(Example2): Production of Form C Crystal of Subject Compound
To a 500 mL flask, (S,E)-2-(2,6-dichlorobenzamido)-5-[4-(methyl-pyrimidin-
2-ylamino)phenyl]pent-4-enoic acid ethyl ester (5.50 g) prepared in Reference
Example 4 was weighted and added, and 4 mol/L of hydrochloric acid (110 mL) was
added thereto, followed by stirring the obtained suspension at 50°C for 6 hours.
After cooling the reaction solution to 0°C, ethanol (50 mL) was added, and sodium
hydroxide (18.5 g) dissolved in water (50 mL) was further added thereto, followed
by stirring the resulting reaction solution at room temperature for 20 minutes. The
reaction solution was cooled to 0°C, and after adding 4 mol/L of hydrochloric acid
thereto to adjust pH of the solution to about 3, the resulting solution was extracted
with ethyl acetate (150 mL). The extract was washed with water (100 mL) and
concentrated under reduced pressure. To the concentrate, ethanol (100 mL) was
added, and after concentrating the resultant to 18 g of the solution weight, heptane

(15 mL) was added, followed by stirring the solution at room temperature. The
crystals were removed by filtration and dried under reduced pressure to obtain 4.62 g
of the crude crystals (Yield: 89%).
[0043]
The obtained crude crystals (4.20 g) was weighted and added to a 200 mL
flask, and 2-propanol (56 mL) was added thereto, followed by heating the resulting
solution under stirring. After confirming the dissolution at 73 °C, cooling of the
solution was started. Seed crystals (190 mg) were added thereto at 70°C, and the
solution was cooled to 55°C and stirred at 55°C for 12 hours. Then, the solution
was cooled to 0°C and stirred at 0°C for 18 hours. The crystals were removed by
filtration, and washed with 2-propanol and dried to obtain 3.85 g of Form C crystals
of the subject compound (Yield: 92%). For the obtained crystals, powder X-ray
diffraction measurement and TG-DTA were carried out, and it was confirmed that
the results were consistent with Fig. 1 and Fig. 2.
[0044]
(Example 3): Production of Form B Crystal of Subject Compound
The amorphous subject compound (30 mg) prepared in Reference Example 2
was weighted and added to a vial made of borosilicate glass, and methanol (1.1 mL)
was added thereto, followed by stirring the resulting mixture at room temperature to
dissolve the compound. The mixture was left to stand at room temperature under
open condition. After confirming the precipitates, solvent was removed with a
Pasteur pipette, and the precipitates were dried under reduced pressure by using a
vacuum pump for 30 minutes to obtain white powders of Form B of the subject
compound. For the obtained crystals, powder X-ray diffraction measurement and
TG-DTA were carried out. The results of these measurements are shown in Fig. 3
ans Fig. 4.
Diffraction angle 29 : 5.9, 8.3, 11.8, 13.2, 21.7°

Endothermic peak : 169°C
[0045]
(Example 4): Production of Form E (hydrate) Crystal of Subject Compound
The amorphous subject compound (30 mg) prepared in Reference Example 1
was weighted and added to a vial made of borosilicate glass, and water (10 mL) was
added thereto, followed by stirring the resulting suspension overnight. The solids
were removed by filtration from the suspension to obtain white powders of Form E
(hydrate) of the subject compound. For the obtained crystals, powder X-ray
diffraction measurement and TG-DTA were carried out. The results of these
measurements are shown in Fig. 5 ans Fig. 6.
Diffraction angle 29 : 6.6, 8.3, 11.1, 14.6, 18.2°
Endothermic peak : 102°C
[0046]
(Example 5): Production of Form A Crystal of Subject Compound
The amorphous subject compound (30 mg) prepared in Reference Example 2
was weighted and added to a vial made of silicate glass, and toluene (3 mL) was
added thereto, followed by stirring the resulting mixture at room temperature to
dissolve the compound. Then, the vial made of borosilicate glass was capped and
left to stand air-tightly at room temperature. After confirming the precipitates,
solvent was removed with a Pasteur pipette, and the precipitates were dried under
reduced pressure by using a vacuum pump for 30 minutes to obtain white powders of
Form A of the subject compound. For the obtained crystals, powder X-ray
diffraction measurement and TG-DTA were carried out. The results of these
measurements are shown in Fig. 7 and Fig. 8.
Diffraction angle 29 : 5.7, 7.4,11.3, 12.0°
Endothermic peak : 139°C
[0047]

(Example 6): Production of Form D Crystal of Subject Compound
The amorphous subject compound (30 mg) prepared in Reference Example 1
was weighted and added to a vial made of borosilicate glass, and ethanol (2 mL) was
added thereto, followed by stirring the resulting mixture at room temperature to
dissolve the compound. The mixture was left to stand at room temperature under
open condition. After confirming the precipitates, solvent was removed with a
Pasteur pipette, and the precipitates were dried under reduced pressure by using a
vacuum pump for 30 minutes to obtain white powders of Form D of the subject
compound. For the obtained crystals, powder X-ray diffraction measurement and
TG-DTA were carried out. The results of these measurements are shown in Fig. 9
and Fig. 10.
Diffraction angle 29 : 5.8, 11.5, 11.8,23.0°
Endothermic peak : 135°C
[0048]
(Test Example 1): Evaluation of Moisture Absorption
«Measurement Conditions of Equilibrium Moisture Regain»
Sample Amount : 5 to 12 mg
Measuring Temperature : 25°C
Equilibrium Weight/time : 0.01 wt%/5 minutes
Maximum Equilibration Time : 180 minutes
Measurement Range : 5% relative humidity - 95% relative humidity -
5% relative humidity
Measurement Interval : 5% relative humidity
[0049]
In order to evaluate also whether the crystal form changes or not, the powder
X-ray diffraction measurement was carried out for each crystal of Form A, Form B,
Form C, Form D and Form E (hydrate) obtained after the evaluation test of moisture

absorption. The results are shown in Table 1. Only for the Form A crystal, the
measurement range (29) of the powder X-ray diffraction measurement was changed
to 2 to 35°, and the measurement was carried out at 25°C, 90% relative humidity.
For controlling humidity, a humidity generator (HUM-1 A; manufactured by Rigaku)
was used.
[0050]

1): The weight increase means one observed when the relative humidity was increased from 5%
to 95%.
2): The crystal was transferred to a hydrate different from the Form E (hydrate) crystal when the
relative humidity was increased from 5% to 95%.
[0051]
As shown in Table 1, as for crystals of the Form B, Form C, Form D and
Form E (hydrate) of the subject compound, the weight increases due to
humidification were not substantially changed, and the crystal forms were not
changed. These results revealed that the crystals of Form B, Form C, Form D and
Form E (hydrate) of the subject compound are excellent in physical stability.
[0052]
(Test Example 2): Evaluation of Solid-State Stability
The amorphous form, and the crystals of Form A, Form B, Form C, Form D
and Form E (hydrate) of the subject compound were stored air-tightly at 60°C for
four weeks, and the purities thereof before and after the storage were measured by
high performance liquid chromatography (hereinafter referred to as "HPLC") under

the following conditions. The powder X-ray diffraction measurement and TG-DTA
were carried out to evaluate whether the crystal forms were changed or not due to the
storage. The results are shown in Table 2. Aqueous sodium dihydrogen
phosphate solution in a concentration of 20 mmol/L (hereinafter referred to as "
aqueous SDP solution") used for preparing a mobile phase of HPLC was prepared by
adding distilled water (3 L) to weighed sodium dihydrogen phosphate dihydrate
(9.36 g) and stirring the resulting solution to dissolve the dihydrate. An analytical
sample of HPLC was prepared by weighing and adding each crystal (1.75 mg) of the
subject compound to a 10-mL measuring flask respectively; adding acetonitrile (2
mL) thereto to dissolve the crystal; and then adding aqueous SDP solution thereto to
a total volume of 10 mL.
«HPLC Conditions»
Detection Wavelength :210nm
Column :YMC-Pack Pro C18 AS-303
Mobile Phase A : aqueous SDP solution/acetonitrile = 80:20
(v/v)
Mobile Phase B : acetonitrile/aqueous SDP solution = 70:30
(v/v)
Composition of Mobile Phase B : 0 to 60 minutes: 0 → 100%
60 to 65 minutes: 100%,
65 to 66 minutes: 100 → 0%,
66 to 75 minutes: 0%
Flow rate :1.0mL/min
Column Temperature : 40°C
Amount of Injected Sample : 20 µL


1): The increased amount of each decomposition product increased by not less than 0.15% was
0.49% for a decomposition product at 0.9 of the relative retention time (hereinafter referred to as
"RRT"), 0,20% for a decomposition product at RRT 1.0, 0.23% for a decomposition product at
RRT 1.1, 0.20% for a decomposition product at RRT 1.2, 0.15% for a decomposition product at
RRT 1,6, 1.30% for a decomposition product at RRT 1.8, and 0.22% and 0.20% for two
decomposition products at RRT 2.3. The "RRT" is calculated by dividing the retention time of
the decomposition product in HPLC chromatogram by the retention time of the subject
compound in HPLC chromatogram.
2): Mixed crystals of Form B, Form C, Form D and Form E.
[0054]
As shown in Table 2, the purities of the crystals of Form A, Form B, Form C,
Form D and Form E (hydrate) of the subject compound were not substantially
changed. These results revealed that the crystals of Form A, Form B, Form C,
Form D and Form E (hydrate) of the subject compound are excellent in chemical
stability compared to the amorphous form. The crystal forms of the Form B, Form
C and Form E (hydrate) of the subject compound were not changed due to storage.
These results revealed that the crystals of Form B, Form C and Form E (hydrate) of
the subject compound are excellent also in physical stability.

[0055]
(Test Example 3): Storage Stability Test (Accelerated testing) of Form C Crystal
The Form C crystals of the subject compound were stored under acceleration
condition (40°C, 75% relative humidity) air-tightly or under open condition for six
months, and the purities of the crystals before and after the storage were measured by
HPLC under conditions below. The powder X-ray diffraction measurement and
TG-DTA were carried out to evaluate whether the crystal form was changed or not
due to the storage.
[0056]
Phosphate buffer in a concentration of 20 mmol/L (pH 3.9) (hereinafter
referred to as " Buffer X") used for preparing a mobile phase of HPLC was prepared
by adding 20 mmol/L of aqueous phosphoric acid solution to 20 mmol/L of aqueous
potassium dihydrogen phosphate solution. Here, 20 mmol/L of the aqueous
potassium dihydrogen phosphate solution was prepared by adding the weighed
potassium dihydrogen phosphate (8.2 g) to distilled water (3 L), and stirring the
resulting solution to dissolve the phosphate, and 20 mmol/L of the aqueous
phosphoric acid solution was prepared by adding phosphoric acid (1.4 mL) to
distilled water (1 L), and mixing the resulting solution under stirring.
[0057]
Phosphate buffer in a concentration of 20 mmol/L (pH 7.0) (hereinafter
referred to as "Buffer Y") used for preparing an analytical sample of HPLC was
prepared by adding 20 mmol/L of aqueous phosphoric acid solution to 20 mmol/L of
aqueous dipotassium hydrogen phosphate solution. Here, 20 mmol/L of aqueous
dipotassium hydrogen phosphate solution was prepared by adding the weighed
dipotassium hydrogen phosphate (3.5 g) to distilled water (1 L), and stirring the
resulting solution.
[0058]

Further, the analytical sample of HPLC was prepared by weighing and adding
the Form C crystals of the subject compound (10 mg) to a 50-mL measuring flask,
and adding a mixture of Buffer Y and acetonitrile at a ratio of 80:20 to a total amount
of 50 mL.
[0059]
In "the Guideline on Stability Testing" based on the agreements in
International Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH), for evaluating the stability of
the quality required for maintaining the effectiveness and safety of the
pharmaceutical, the accelerated testing (40°C, 75% relative humidity, six months) is
set as a test to predict chemical influences when stored for a long time under
conventional storage conditions (for example, 25°C), and simultaneously to confirm
the effect of short term excursions outside the storage condition which excursions
may be occurred during shipping and the like. The data obtained by the accelerated
testing are indispensable in the application for approval of pharmaceuticals.
[0060]
«HPLC Conditions»
Detection Wavelength : 210 nm
Column : YMC-Pack Pro C18
AS12S05-2546WT
Mobile Phase A : buffer X/acetonitrile = 80:20 (v/v)
Mobile Phase B : acetonitrile/buffer X = 70:30 (v/v)
Composition of Mobile Phase B : 0 to 80 minutes: 0 → 100%
80 to 85 minutes: 100%
85 to 86 minutes: 100 → 0%
86 to 95 minutes: 0%
Flow Rate : 1.0 mL/min

Column Temperature : 40°C
Amount of Injected Sample : 20 µL
[0061]

[0062]
As a result, as shown in Table 3, the purity of the subject compound after the
Form C crystals were stored under accelerate conditions air-tightly or under open
condition for six months was not substantially changed compared to the initial purity.
The crystal form of the Form C crystals of the subject compound was not changed
due to the storage. Since the quality of the Form C crystal was not clearly changed
chemically and physically in the accelerated testing, the Form C crystal was revealed
to be very stable and excellent as an effective component of pharmaceuticals in view
of storage and distribution. Further, it was revealed that the Form C crystal was
excellent in physical stability in that the crystal form thereof was not transferred to
the another form even in solvents (e.g., toluene, methanol, 2-propanol usable in the
crystallization of the subject compound).
[0063]
(Example 7): Production of Quick Release Tablets using Form C Crystal
The pin milled product of the Form C crystals of the subject compound
(14.17 g), D-mannitol (35.98 g; Roquette Japan K.K.; PEARLITOL (registered

trademark) 50C), crystalline cellulose (42.5 g; Asahi Kasei Corp.; CEOLUS
(registered trademark) grade PH-101), partially pregelatinized starch (42.5 g; Asahi
Kasei Corp.; grade PCS), hydroxypropyl cellulose (8.5 g; Nippon Soda; grade L),
meglumine (8.5 g; Merck) and magnesium oxide (8.5 g; Tomita Pharmaceutical
Co.,Ltd.) were fed to a mixing granulator (Nara Machinery Co., Ltd.; NMG-1L), and
after mixing the resultant, water (32.2 g) was sprayed to carry out stirring
granulation. The granulated powders were dried in an oven at 40°C for two hours
to obtain granules. The obtained granules were subjected to sieving by COMIL
(Powrex Corporation; QC-197S; mesh 1143 µm; speed of rotation 2000 rpm) to
obtain size-selected granules. To the obtained size-selected granules,
croscarmellose sodium (8.5 g; FMC Biopolymer; Ac-Di-Sol (registered trademark))
and magnesium stearate (0.85 g; Taihei Chemical Industrial) were added, and mixed
by using V-blender (Tsutsui Scientific Instruments Co., Ltd., S-3) to obtain granules
for tableting. The obtained granules for tableting were tabletted with a rotary
tableting machine (KIKUSUI SEISAKUSHO LTD.; Correct 19) using a round-
shaped punch and die with a diameter of 8 mm to obtain 180 mg of plain tablets
containing 15 mg of the subject compound, 38.1 mg of D-mannitol, 45 mg of
crystalline cellulose, 45 mg of partially pregelatinized starch, 9 mg of
hydroxypropylcellulose, 9 mg of meglumine, 9 mg of magnesium oxide, 9 mg of
croscarmellose sodium, and 0.9 mg of magnesium stearate per a tablet.
[0064]
(Example 8): Production of Quick Release Tablets using Form C Crystal
The jet milled product of the Form C crystals of the subject compound (41.67
g), lactose (230.83 g; DMV International; Pharmatose (registered trademark) 200M),
crystalline cellulose (175 g; Asahi Kasei Corp.; CEOLUS (registered trademark)
grade PH-101) and hydroxypropyl cellulose (25 g; Nippon Soda; grade L) were fed
to a mixing granulator (Nara Machinery Co., Ltd.; NMG-3L), and after mixing the

resultant, water (95 g) was sprayed to carry out stirring granulation. The
granulated powders were dried in an oven at 40°C for two hours to obtain granules.
The obtained granules were sieved with mesh size of 1 mm, croscarmellose sodium
(25 g; FMC Biopolymer; Ac-Di-Sol (registered trademark)) and magnesium stearate
(2.5 g; Taihei Chemical Industrial) were added thereto, and mixed by using V-
blender (Tsutsui Scientific Instruments Co., Ltd.; S-3) to obtain granules for tableting.
The obtained granules for tableting were tabletted with a rotary tableting machine
(KIKUSUI SEISAKUSHO LTD.; Correct 19) using a round-shaped punch and die
with a diameter of 8 mm to obtain 180 mg of plain tablets containing 15 mg of the
subject compound, 83.1 mg of lactose, 63 mg of crystalline cellulose, 9 mg of
hydroxypropyl cellulose, 9 mg of croscarmellose sodium and 0.9 mg of magnesium
stearate per a tablet.
[0065]
(Example 9): Production of Quick Release Tablets using Form C Crystal
The jet milled product of the Form C crystals of the subject compound
(107.14 g), lactose (165.36 g; DMV International; Pharmatose (registered trademark)
200M), crystalline cellulose (175 g; Asahi Kasei Corp.; CEOLUS (registered
trademark) grade PH-101) and hydroxypropyl cellulose (25 g; Nippon Soda; grade
L) were fed to a mixing granulator (Nara Machinery Co., Ltd.; NMG-3L), and after
mixing, water (100 g) was sprayed to carry out stirring granulation. The
granulated powders were dried in an oven at 40°C for two hours to obtain granules.
The obtained granules were sieved with mesh size of 1 mm, croscarmellose sodium
(25 g; FMC Biopolymer; Ac-Di-Sol (registered trademark)) and magnesium stearate
(5 g; Taihei Chemical Industrial) were added thereto, and mixed by using V-blender
(Tsutsui Scientific Instruments Co., Ltd.; S-3) to obtain granules for tableting. The
obtained granules for tableting were tabletted with a rotary tableting machine
(KIKUSUI SEISAKUSHO LTD.; Correct 19) using a round-shaped punch and die

with a diameter of 7 mm to obtain 140.7 mg of plain tablets. The obtained plain
tablets were placed in a film coating machine (Freund Corporation; HICOATER
MINI), and sprayed with a liquid in which OPADRY (registered trademark) OY-
7300 (Japan Colorcon; mixture of hydroxypropylmethylcellulose 2910, titanium
oxide and polyethylene glycol 400), red ferric oxide (KISHI KASEI CO., LTD.) and
yellow ferric oxide (KISHI KASEI CO., LTD.) were dispersed, thereby obtaining
film-coated tablets containing 30 mg of the subject compound, 46.3 mg of lactose, 49
mg of crystalline cellulose, 7 mg of hydroxypropylcellulose, 7 mg of croscarmellose
sodium, 1.4 mg of magnesium stearate, 3.88 mg of OY-7300, 0.08 mg of red ferric
oxide and 0.03 mg of yellow ferric oxide per a tablet.
[0066]
(Example 10): Production of Quick Release Tablets using Form C Crystal
The jet milled product of the Form C crystals of the subject compound (2.86
g), lactose (2662.9 g; DMV International; Pharmatose (registered trademark) 200M)
and crystalline cellulose (1000 g; Asahi Kasei Corp.; CEOLUS (registered
trademark) grade PH-101) were placed in a fluid bed granulator/dryer (Freund
Corporation; FLO-5), and granulated by spraying an aqueous solution of 7% (wt./v)
hydroxypropylcellulose (1633 g; Nippon Soda; grade L) under fluidization, and
thereafter dried to obtain granules. The obtained granules were subjected to sieving
by COMIL (Powrex Corporation; QC-197S; mesh 1575 µm; speed of rotation 2000
rpm) to obtain size-selected granules. To the obtained size-selected granules,
croscarmellose sodium (200 g; FMC Biopolymer; Ac-Di-Sol (registered trademark))
and magnesium stearate (20 g; Taihei Chemical Industrial) were added, and mixed
by using V-blender (Dalton Co., Ltd, DV-1-10) to obtain granules for tableting.
The obtained granules for tableting were tabletted with a rotary tableting machine
(KIKUSUI SEISAKUSHO LTD.; Correct 19) using a round-shaped punch and die
with a diameter of 7 mm to obtain 140 mg of plain tablets. The obtained plain

tablets were placed in a film coating machine (Freund Corporation; HICOATER
MINI), and sprayed with a liquid in which OPADRY (registered trademark) OY-
7300, red ferric oxide (KISHI KASEI CO., LTD.) and yellow ferric oxide (KISHI
KASEI CO., LTD.) were dispersed, thereby obtaining film-coated tablets containing
0.1 mg of the subject compound, 93.2 mg of lactose, 35 mg of crystalline cellulose, 4
mg of hydroxypropylcellulose, 7 mg of croscarmellose sodium, 0.7 mg of
magnesium stearate, 3.88 mg of OY-7300, 0.08 mg of red ferric oxide and 0.03 mg
of yellow ferric oxide per a tablet.
[0067]
The particle sizes of the milled Form C crystals of the subject compound used
in Examples 7 to 10 were measured by using "Microtrac particle size analyzer
(Nikkiso Co., Ltd.; 9220FRA; wet process). The particle size distributions of the
milled Form C crystals of the subject compound were D10: 7.0 µm, D50: 21.9 µm,
D90: 56.4 µm for apin milled product; and D10: 1.7 µm, D50: 3.4 µm, D90: 5.8 µm,
or D10: 2.1 µm, D50: 3.9 µm, D90: 6.6 µm for a jet milled product. It was
confirmed by powder X-ray diffraction measurement and TG-DTA that no change in
the crystal form due to the above milling occurred.
[0068]
(Test Example 4): Evaluation of Storage Stability of Formulations
Tablets obtained in Examples 7 to 10 were stored at 40°C and 75% relative
humidity air-tightly for three months, and the purities thereof before and after the
storage were measured by HPLC under the conditions below. The results are
shown in Table 4. As the analytical sample for HPLC, a supernatant was used
which was obtained by adding 10 tablets or one tablet to a mixture of buffer Y and
acetonitrile at a mixing ratio of 80:20 to a concentration of the subject compound of
50 or 300 ug/mL, and stirring and centrifuging the resultant.
<>

Detection Wavelength : 210 nm
Column : YMC-Pack Pro C18
AS12S05-2546WT
Mobile Phase A : buffer X/acetonitrile = 80:20 (v/v)
Mobile Phase B : acetonitrile/buffer X = 70:30 (v/v)
Composition of Mobile Phase B : 0 to 80 minutes: 0 → 100%
80 to 85 minutes: 100%
85 to 86 minutes: 100 → 0%
86 to 95 minutes: 0%
Flow Rate :1.0mL/min
Column Temperature : 40°C
Amount of Injected Sample : 20 µL (concentration of the subject compound
is 300 µg/mL), or 80 µL (concentration of the subject compound is 50 µg/mL)
[0069]

[0070]
Even though some crystals are chemically stable as effective components of
pharmaceuticals, the crystals may be destabilized due to the contact with various
additives in a final form of pharmaceuticals such as tablets, and may adversely affect
the keeping of effectiveness and safety of the pharmaceuticals. However, as shown
in Table 4, for all the tablets containing the Form C crystals of the subject compound
as effective components, the purities of the crystals after stored air-tightly under
accelerate conditions for three months were not substantially changed compared to
the initial purities. These results revealed that the Form C crystal of the subject
compound is excellent in chemical stability even after the crystals are formulated via

a fine grinding step.
Industrial Availability
[0071]
The crystal of the subject compound may be used as a pharmaceutical, in
particular, a therapeutic or prophylactic agent for inflammatory bowel disease,
allergic dermatitis, multiple sclerosis or leukemia in the medical field.

We Claim:
1. A crystal of (S,E)-2-(2,6-dichlorobenzamido)-5-[4-(methyl-pyrimidin-2-
ylamino)phenyl]pent-4-enoic acid.
2. The crystal according to claim 1, which exhibits peaks at 20 (°) of 17.1, 17.7,
18.7, 19.9 and 21.0° in powder X-ray diffraction.
3. The crystal according to claim 2, which exhibits an endothermic peak in the
range of 178 to 182°C in thermogravimetric-differential thermal analysis.
4. The crystal according to claim 1, which exhibits peaks at 29 (°) of 5.9, 8.3,
11.8, 13.2 and21.7° in powder X-ray diffraction.
5. The crystal according to claim 4, which exhibits an endothermic peak in the
range of 167 to 171°C in thermogravimetric-differential thermal analysis.
6. The crystal according to claim 1, which exhibits peaks at 29 (°) of 6.6, 8.3,
11.1, 14.6 and 18.2° in powder X-ray diffraction.
7. The crystal according to claim 6, which exhibits an endothermic peak in the
range of 100 to 104°C in thermogravimetric-differential thermal analysis.
8. The crystal according to any one of claims 1 to 7, which is a non-solvate or a
hydrate.
9. A pharmaceutical comprising as an effective component said crystal
according to any one of claims 1 to 8.
10. A therapeutic or prophylactic agent for inflammatory bowel disease, allergic
dermatitis, multiple sclerosis or leukemia, comprising as an effective component said
crystal according to any one of claims 1 to 8.