The invention relates to a new crystalline form of (E) -4- [ [3- [2- (4-cyclobutyl-2-thiazolyl)ethenyl]phenyl]- amino] -2, 2-diethyl-4-oxobutanoic acid, herein designated as "Polymorphic Form B or Polymorph B", and salts thereof with pharmaceutically acceptable bases, which are useful as bronchopulmonary agents, for example, in the relief of asthma and allergic reactions.

In another aspect, the invention relates to pharmaceutical compositions and methods of making and using the referred to Polymorph B of (E) -4- [ [3- [2- (4- cyclobutyl-2-thiazolyl)ethenyl] -phenyl]amino] -2,2- diethyl-4-oxobutanoic acid.

The invention also relates to a process for preparing polymorphic Form B comprising agitating polymorphic Form A in the presence of Polymorphic Form B seed.

A crystalline form of (E) -4- [ [3- [2- (4-cyclobutyl-2- thiazolyl) ethenyl] -phenyl]amino] -2, 2-diethyl-4- oxobutanoic acid, which is herein designated as "Polymorphic Form A or Polymorph A", is described and claimed in U.S. Patent No. 5,001,140. Polymorph A, as described in U.S. Patent No. 5,001,140, is an inhibitor of bronchoconstriction and is therefore useful as a bronchopulmonary agent, for example, in the relief of asthma and allergic reactions.

It has now been discovered that (E) -4-[ [3- [2- (4- cyclobutyl-2-thiazolyl)ethenyl]phenyl]amino] -2,2-diethy1- 4-oxobutanoic acid can be prepared in new crystalline

Pop/Ul 8.12.95

4-oxobutanoic acid can be prepared in new crystalline form, herein designated as "Polymorphic Form B or Polymorph B" , having more advantageous handling properties in the preparation of pharmaceutical compositions. The invention provides (E) -4- [ [3- [2- (4- cyclobutyl-2-thiazolyl)ethenyl] phenyl]amino] -2,2- diethyl-4-oxobutanoic acid in Polymorphic Form B, which is useful as a bronchopulmonary agent, for example, in the relief of asthma and allergic reactions.

Polymorph B possesses better flow characteristics than Polymorph A and is thermodynamically more stable than Polymorph A. Thus, Polymorph B is easier to handle (remove from vessel and transfer to filter) , filter and dry than Polymorph A. Polymorph B is also easier to feed and micronize. Hence, the methods for its manufacture are more easily validated than that of Polymorph A.

Polymorph B may be characterized by its powder X-ray diffraction pattern and/or single crystal X-ray diffraction pattern hereinafter described in greater detail .

PΦ fler X-ray Diffraction

The powder X-ray diffraction pattern of Polymorph A and Polymorph B was obtained by completely and uniformly filling the sample holder of the Scintag XDS2000 with the sample utilizing a spatula. The sample was then irradiated with the Scintag XDS2000 and d spacings were determined based on Debye Scherrer equation under the conditions described in Table I.

Table I. Parameters for Powder X-Ray Diffraction

Instruments and Conditions (Equivalent instrumentation may used. )

Instrument Scintag XDS2000 Theta:Theta system

X-ray Target Copper (λ = 1.54 A)

Voltage 45 kV

Current 40 mA

Detector Germanium solid state cooled by liquid nitrogen detector

Two-Theta Range 54 ^<

Scan Type Continuous

Chopper Increment 0.02°

Divergent Beam Slit 2.0 mm

Divergent Beam Scatter Slit 4.0 mm

Receiving Beam Scatter Sli 0.5 mm

Receiving Beam Slit 0.2 mm

Atmosphere Air

The X-ray powder diffraction pattern of the micronized form of Polymorph A and B are given in Tables II and III which follow. The patterns are given in terms of 'd' spacings and relative intensities.

Table II. Powder X-Ray Diffraction Data for Micronized

Polymorph A d{A) Intensity d(A) Intensity

2.22 vw

2.17 vw

2.11 vw 2.06 vw

intensity > 75% intensity 25% - 75% intensity 10% - 25%

vw very weak intensity < 10%

Table III. Powder X-Ray Diffraction Data for Micronized Polymorph B

d(A) Intensity

11.68 vw 8.79 vw

6.77 vw

6.62 s 6.49 vw 5.92 vw

5.77 vw 5.63 w 5.31 vw

5.08 vw

4.94 vw

4.68 w

4.49 vw

4.38 vw

4.26 vw

4.12 vw

3.93 vw

3.89 vw

Single Crystal X-rav Diffraction

The intensity data for Polymorph B were measured on an Enraf-Nonius CAD4 diffractometer (graphite- monochromated Cu KCX radiation, G)-2θ scans) . The size of the crystal used for the data collection was approximately 0.06 x 0.10 x 0.25 mm. The data were corrected for absorption. Of the 2212 independent reflections for θ< 50°, 621 were considered observed [Intensity (I) > 3.0σ(I)] .

The structure of Polymorph B was solved by a multiple-solution procedure and was refined by full matrix least squares. In the final refinement, the heteroatoms were refined anisotropically and the carbon atoms were refined isotropically. The hydrogen atoms were included in the structure-factor calculations, but their parameters were not refined. The final discrepancy indices are R=0.080 and R _w =0.080 for the 618 observed reflections. R is the ratio of the sum of the differences between the observed and theoretical structure factors to the sum of the observed structure factors. The final difference map has no peaks greater than +.0.3 e A ^-3 . The X-ray data for both the Polymorph A and Polymorph B Forms is summarized in terms of a unit cell in Table IV.

Table IV. Single Crystal X-ray Data for Polymorph A and B at 295°K

Polymorphic Form A B

Z is the number of molecules in the unit cell.

Polymorph B can be prepared as hereinafter described in Schemes I and II.

SCHEME I

(E) -4- [ [3- [2- (4-cyclobutyl-2- thiazolyl)ethenyl)phenyl] amino] -2, 2-diethyl-4-oxobutanoic acid, the compound of formula I, is made by the reaction of (E)-3-[2- (4-cyclobutyl-2-thiazolyl) ethenyl]benzeneamino, the compound of formula II, with 2,2 diethylsuccinic anhydride (formula III) preferably with agitation until crystallization is complete in a suitable solvent. Suitable solvents include any organic solvent which does not react with or bind chemically to (E) -3- [2- (4-cyclobutyl-2- thiazolyl) ethenyl]benzeneamino, 2,2 diethylsuccinic anhydride or (E) -4- [ [3- [2- (4-cyclobutyl-2- thiazolyl) ethenyl)phenyl] amino] -2 , 2-diethyl-4- oxobutanoic acid, such as, for example, dimethylformamide (DMF) , toluene, DMF/toluene combinations, iso-propyl alcohol (IPA) , tetrahydrofuran (THF) , IPA/THF combinations, preferably, IPA. A temperature in the range of from room temperature to reflux temperature is preferred. The compound of formula I is typically produced as

Polymorph A. However, it can be produced as Polymorph B if seeds of Polymorph B are present.

The conversion of Polymorph A to Polymorph B is set forth in Scheme II below.

SCHEME II

Solvent, Agitation, Temperature Polymorph A » _* - Polymorph B

Seeds of Form B

Polymorph B of (E) -4- [ [3- [2- (4-cyclobutyl-2 thiazolyl) ethenyl)phenyl]amino] -2,2-diethyl-4- oxobutanoic acid can be formed from Polymorph A by agitating Polymorph A in any organic solvent which shows a solubility for (E) -4- [ [3- [2- (4-cyclobutyl-2 thiazolyl) ethenyl)phenyl]amino] -2,2-diethyl-4-oxobutanoic acid and does not react chemically with or bind to (E) -4- [ [3- [2- (4-cyclobutyl-2 thiazolyl)ethenyl)phenyl] amino] -2,2- diethyl-4-oxobutanoic acid, preferably in the presence of Polymorph B seeds. The rate of conversion will depend on the rate of agitation, dilution, the temperature and the amount of Polymorph B seed that is present. Preferably, the rate of agitation should be about 500 rpm. Polymorph B seed is preferably present in an amount of from about 0.5% to 1% of Polymorph A, particularly preferred in an amount of 0.5% of Polymorph A. Examples of suitable solvents include tetrahydrofuran (THF) , isopropyl alcohol (IPA), dimethyl-formamide (DMF) , methyl tertiary butyl ether, acetonitrile, methanol, acetone, miscible combinations of the solvents listed and miscible

combinations of DMF/toluene. The combination of THF/IPA is the preferred solvent, preferably in the ratio of from about 1:20 to about 15:85 THF to IPA, a ratio of 1:20 is particularly preferred.

Polymorph B or a salt thereof or a composition containing a therapeutically effective amount of Polymorph B or a salt thereof can be administered by methods well known in the art. More particularly, Polymorph B or a salt thereof can be administered either singly or in combination with other pharmaceutical agents, for example, antihistamines, mediator release inhibitors, methyl xanthines, beta agonists or antiasthmatic steroids such as prednisone and prednisoline. The foregoing pharmaceutical composition can be administered orally, parenterally, rectally or by inhalation, for example, in the form of an aerosol, micropulverized powder or a nebulized solution. For oral administration, the foregoing pharmaceutical composition can be administered in the form of tablets, capsules, for example, in admixture with talc, starch, milk sugar or other inert ingredients, that is, pharmaceutically acceptable carriers, or in the form of aqueous solutions, suspensions, elixirs or aqueous alcoholic solutions, for example, in admixture with sugar or other sweetening agents, flavoring agents or colorants, thickeners and other conventional pharmaceutical excipients.

For parenteral administration, the foregoing pharmaceutical composition can be administered in solutions or suspensions, for example, as an aqueous or peanut oil solution or suspension using excipients and carriers conventional for this mode of administration. For administration as aerosols, the foregoing pharmaceutical composition can be dissolved in a suitable pharmaceutically acceptable solvent, for example, ethyl

alcohol or combinations of miscible solvents, and mixed with a pharmaceutically acceptable propellant. Such aerosol compositions are packaged for use in a pressurized container fitted with an aerosol valve suitable for release of the pressurized compositions.

Preferably, the aerosol valve is a metered valve, that is one which on activation releases a predetermined effective dose of the aerosol composition.

In the practice of the invention, the dose of

Polymorph B or salt thereof to be administered and the frequency of administration will be dependent on the route of administration, as well as on the severity of the condition, age of the mammal to be treated and the like. Oral doses of Polymorph B or a salt thereof contemplated for use by human adults in practicing the invention are in the range of from about 5 to about 1000 mg per day, preferably about 5 to about 250 mg either as a single dose or in divided doses.

The examples which follow further illustrate the invention. The synthesis of Polymorph A of (E) -4- [ [3- [2- (4-cyclobutyl-2-thiazolyl)ethenyl]phenyl]amino] -2,2- diethyl-4-oxobutanoic acid is disclosed in U.S. Patent No. 5,001,140.

EXAMPLE 1

Conversion of Polymorph A to Polymorph B bv Stirring in Isopropyl Alcohol Without Seeding

To a one liter flask was added 700.95 grams of isopropyl alcohol and 10.08 grams of Polymorph A. The batch temperature was set and controlled at 25°C. Agitation was maintained at 450 RPM. Polymorph A had converted to less than 5% Polymorph B after 7 hours of

stirring. All of the Polymorph A had converted to Polymorph B after 24 hours of stirring. The results on the polymorphic form were confirmed by powder X-Ray diffraction.

EXAMPLE 2

Conversion of Polymorph A to Polvmorph B by Stirring in Isopropyl Alcohol With 1 % Polvmorph B Seeds Added

To a one liter flask was added 550 grams of isopropyl alcohol, 50.36 grams of Polymorph A and 0.50 grams of Polymorph B. The batch temperature was set and maintained at 25°C. Agitation was maintained at 600 RPM. All of the Polvmorph A had converted to Polymorph B at the end of 4.2 hours of agitation. The results on the polymorphic form were confirmed by powder X-Ray diffraction.

EXAMPLE 3

Recrvstallization of Polvmorph A to Polvmorph B from 11.9 weight% Dimethylformamide/Acetone Seeded with Micronized Polvmorph B to Induce Crystallization

To a one liter flask was added 57.3 grams of dimethyl formamide, are added 427.9 grams of acetone and 20.1 grams of Polymorph A. Agitation was maintained at 750 RPM. The batch temperature set-point was set at 25°C and the batch was maintained at this temperature for 40 minutes. The batch was then heated to 53°C over 1 hour. The batch was held at 53°C for approximately 105 minutes and then heated to approximately 59°C over 10 minutes. The batch was in total solution at 58.5°C. The batch was held at 59°C for approximately 30 minutes and then cooled to 40.7°C over 2 hours. It was held at 40.7°C for 1

hour. The batch remained in solution. The batch was then seeded with 0.116 grams of micronized Polymorph B. Immediate crystallization was observed. The batch was cooled at 3°C/minute to -5°C and then filtered. The reactor and filter cake were rinsed with acetone and then dried overnight under vacuum with a slight nitrogen purge at 40°C. The product was analyzed to be Polymorph B by powder X-Ray diffraction.

EXAMPLE 4

Preparation of Polvmorph B in 10.2 wt.% Dimethyl formamide/Toluene. Reaction and Crystallization Procedure

A 10.2 wt% solution of dimethylformamide (DMF) in toluene was made by combining 44.9 grams of anhydrous DMF with 393.0 grams of toluene. To 250 grams of the above solution was added 20.6 grams of 2,2 diethylsuccinic anhydride. The mixture was charged to a 1 liter resin kettle reactor with temperature, agitation and metering control and a nitrogen blanket. The agitation was set at 600 RPM and the reactor temperature was raised to 61°C at a rate of 75°C/hour. To the remaining DMF/toluene was added 25.2 grams of (E) -3- [2- (4-cyclobutyl-2- thiazolyl) ethenyl]benzeneamine. The (E)-3-[2-(4- cyclobutyl-2-thiazolyl)ethenylIbenzeneamine was uniformly metered in over 1 hour to the reactor. The addition vessel was rinsed with approximately 10 mis of anhydrous toluene. At the end of the addition, the batch was still in solution. It was then seeded with 0.1 grams of

Polymorph B. Immediate crystallization was observed. The batch was then cooled at 5°C/hour to -5°C and filtered through a course fritted disc filter. The filter cake was washed with 381.5 grams of methyl tertiary butyl ether. The filter cake was dried overnight in a vacuum over a slight nitrogen purge at

40°C to give 36 grams of Polymorph B (99.8 weight% by HPLC) . The results on the polymorphic form were confirmed by X-Ray diffraction.

EXAMPLE 5

Preparation of Polvmorph B in 1/20 Tetrahvdrofuran/Isopropvl Alcohol. Reaction and Precipitation Procedure

A 1/20 (w/w) tetrahydrofuran/isopropyl alcohol

(THF/IPA) solution was prepared by mixing 501 grams of anhydrous IPA and 25 grams of anhydrous THF and drying the solution over 50 grams of 4 A (angstorm) molecular sieves. A solution of 25 grams of (E)-3-[2-{4- cyclobutyl-2-thiazolyl)ethenyl]benzeneamine in 263 grams of the 1/20 THF/IPA prepared above was added to a 1 liter glass reactor with a temperature, agitation and metering control and a nitrogen blanket. The agitation was set at 750 RPM and the reactor temperature was raised to 45°C at a rate of 50°C/hour. To the reactor was then metered in over 2 hours a solution of 20 grams of 2,2 diethylsuccinic anhydride in 232 grams of 1/20 THF/IPA as prepared above. The batch was seeded with 0.19 grams of Polymorph B, 45 minutes into the metering period. At the end of the 2 hour metering period, the pump and the line were flushed with approximately 10 mis of anhydrous isopropyl alcohol. The batch was then stirred overnight at 45°C. An additional 5.35 grams of 2,2 diethylsuccinic anhydride was added to the reactor via a charging port. The batch was filtered through a fritted disc funnel the filter cake was washed with 163 grams of cold isopropyl alcohol . The filter-cake was dried over two days under house vacuum under a nitrogen purge at 40°C. The product assayed at 99.7 area% by HPLC and was determined to be Polymorph B by powder X-Ray diffraction.

EXAMP E $

Tablet Formulation (Wet Granulation)

Item

Preparation of Tablets

(1) Mix Items 1, 2, 3 and 4 and granulate with water.

(2) Dry the granulation at 50° C.

(3) Pass the granulation through suitable milling equipment.

(4) Add Item 5 and mix for three minutes; compress on a suitable press.

EXAMPLE 7

Capsule Formulation

Preparation of Tablets

(1) Mix Items 1, 2 and 3 and wet granulate with water. Dry at 45° C overnight.

(2) Mill through suitable screen using appropriate milling equipment.

(3) Add Items 4 and 5 and mix for five minutes.

EXAMPLE 9

Capsule Formulation

Ingredients mq/ςapsu,le

1. Polymorph B 0.01 0.5 5.0 25.0

2. Lactose Hydrous 168.99 168.5 159.0 123.0

3. Corn Starch 20.0 20.0 25.0 35.0

4. Talc 10.0 10.0 10.0 15.0

5. Magnesium Stearate 1.0 1_ 2 1_J2 1.0

Total 200.0 200.0 200.0 200.0

Preparation of Capsules

(1) Mix Items 1, 2 and 3 in a suitable mixer for 30 minutes .

(2) Add Items 4 and 5 and mix for 3 minutes

(3) Fill into suitable capsule.

EXAMPLE 9

Wet Granulation Formulation

Ingredients mg/capsule

1. Polymorph B 0.01 0.5 5.0 25.0

2. Lactose Anhydrous DTG 106.99 106.5 102.0 118.0

3. Avicel PH 102 15.0 15.0 15.0 25.0

4. Modified Starch 7.0 7.0 7.0 10.0

5. Magnesium Stearate 1.0 1.0 1.0 2d

Total 130.0 130.0 130.0 130.0

Preparation of Capsules

(1) Dissolve Item 1 in a suitable solvent such as alcohol.

(2) Spread the solution in step 1 over Item 2, dry.

(3) Add Items 3 and 4 and mix for 10 minutes.

.4) Add Magnesium Stearate and mix for 3 minutes and compress.

EXAMPLE α

Cream 0 ■ 5%

3% excess ^rlacel 165 ² ween 60

Preparation of Cream

(1) Dissolve Polymorph B in propylene glycol, add methyl paraben, propyl paraben and water and heat to 70° C.

(2) Melt petrolatum, glyceryl monostearate S.E. and cetyl alcohol. Heat to 70° C. Add polysorbate 60 and mix.

(3) Add solution in Step 2 to solution in Step 1 at 70° C and cool to room temperature while stirring.

EXAMPLE 11

Inhalation Aerosol Formulation ( Solut ion )

Item Ingredients % w/w

1 . Polymorph B 1 . 0

2. Ethyl Alcohol 30.0

3. Ascorbic Acid 0.5

4. Freon 12 54.8

5. Freon 114 13.7

Total 100%

Preparation of Aerosol

(1) Dissolve Items 1 and 3 in Item 2.

(2) Fill solution from Step 1 into a suitable glass bottle, insert valve and crimp to seal container.

(3) Pressure-fill a 80:20 mixture of Items 4 and 5 into the container.

NOTE: A suitable valve may be used to deliver 25 to 100 microliters in volume.

EXAMPLE 12

Inhalation Aerosol Formulation ( Suspension)

Item Ingredients % w/w

1 . Polymorph B 1 . 0

2. Sorbitan Trioleate 0.5

64.0

18.5

16.0 Total 100%

Preparation of Aerosol

(1) Mix Items 1 and 2 into 4 and homogenize.

(2) Fill the concentrated suspension from Step 1 into a suitable can and place in valve and crimp to seal container.

(3) Pressure-fill a 80:20 mixture of Items 3 and 5

NOTE: A suitable valve may be used to deliver 25 to 100 microliters in volume.