Salts of N-[6-(cis-2,6-dimethylmorpholin-4-yl)pyridine-3-yl]-2-methyl -4'- (trifluoromethoxy) [1,1' -biphenyl] -3 -carboxamide

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] This invention relates to salts of N-[6-(cis-2,6-dimethylmorpholin-4-yl)pyridine-3-yl]- 2-methyl-4'-(trifluoromethoxy)[l,r-biphenyl]-3-carboxamide, as well as to pharmaceutical compositions comprising the same and methods of treatment using the same.

Related Background Art

[0002] The compound N-[6-(cis-2,6-dimethylmorpholin-4-yl)pyridine-3-yl]-2-methyl -4'- (trifluoromethoxy)[l,r-biphenyl]-3-carboxamide has the formula (I):

(I) as described in WO 2007/131201. Valuable pharmacological properties are attributed to this compound; thus, it can be used, for example, as modulating the activity of the hedgehog signaling pathway useful in therapy for diseases which respond to modulating the activity of the hedgehog signaling pathway. WO 2007/131201 does not disclose any specific salts or salt hydrates or solvates of N-[6-(cis-2,6-dimethylmorpholin-4-yl)pyridine-3-yl]-2-methyl -4'- (trifluoromethoxy)[l,r-biphenyl]-3-carboxamide. [0003] It has been found that the salt forms of the present invention shows that in addition to good physico-chemical properties that salts may have high permeability and high bioavailability.

SUMMARY OF THE INVENTION

[0004] The present invention is directed to salts of N-[6-(cis-2,6-dimethylmorpholin-4- yl)pyridine-3 -yl] -2 -methyl -4 ' -(trifluoromethoxy) [1,1' -biphenyl] -3 -carboxamide. Preferred embodiments of the present invention are directed to the hydrochloride, diphosphate and sulfate salts ofN-[6-(cis-2,6-dimethylmoφholin-4-yl)pyridine-3-yl]-2-meth yl-4'- (trifiuoromethoxy)[ 1,1 ' -biphenyl] -3 -carboxamide.

[0005] The invention is further directed to pharmaceutical compositions comprising:

(a) a therapeutically effective amount of an inventive salt of N-[6-(cis-2,6- dimethylmorpholin-4-yl)pyridine-3-yl]-2-methyl-4'-(trifluoro methoxy)[l,r-biphenyl]-3- carboxamide; and

(b) at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient.

[0006] The present invention is also directed to a method of treating a disease which responds to modulating the activity of the hedgehog signaling pathway comprising the step of administering to a subject in need of such treatment a therapeutically effective amount of an inventive salt of N-[6-(cis-2,6-dimethylmorpholin-4-yl)pyridine-3-yl]-2-methyl -4'- (trifiuoromethoxy)[ 1 , 1 '-biphenyl] -3 -carboxamide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 shows the x-ray powder diffraction pattern for N-[6-(cis-2,6- dimethylmorpholin-4-yl)pyridine-3 -yl] -2-methyl-4 ' -(trifluoromethoxy) [1,1 ' -biphenyl] -3 - carboxamide diphosphate salt.

[0008] Figure 2 shows the x-ray powder diffraction pattern for N-[6-(cis-2,6- dimethylmorpholin-4-yl)pyridine-3 -yl] -2-methyl-4 ' -(trifluoromethoxy) [1,1' -biphenyl] -3 - carboxamide monosulfate salt. [0009] Figure 3 shows the x-ray powder diffraction pattern for N-[6-(cis-2,6- dimethylmoφholin-4-yl)pyridine-3-yl]-2-methyl-4'-(trifluoro methoxy)[ 1 , 1 '-biphenyl]-3- carboxamide monohydrochloride salt.

DETAILED DESCRIPTION OF THE INVENTION

[0010] As used herein, "salt" refers to a compound prepared by the reaction of an organic acid or base drug with a pharmaceutically acceptable mineral or organic acid or base; as used herein, "salt" includes hydrates and solvates of salts made in accordance with this invention. Exemplary pharmaceutically acceptable mineral or organic acids or bases are as listed in Tables 1-8 in Handbook of Pharmaceutical Salts, P.H. Stahl and CG. Wermuth (eds.), VHCA, Zurich 2002, pp. 334-345. In particular, salts include, but are not limited to, hydrochloride, phosphate, sulfate, mesylate, esylate and besylate salt forms. As used herein, "polymorph" refers to a distinct "crystal modification" or "polymorphic form" or "crystalline form", which differs from another with respect to x-ray powder diffraction pattern, physico- chemical and/or pharmacokinetic properties, and thermodynamic stability.

[0011] An embodiment of the present invention is directed to salts of N-[6-(cis-2,6- dimethylmorpholin-4-yl)pyridine-3 -yl] -2-methyl-4 ' -(trifluoromethoxy) [1,1 ' -biphenyl] -3 - carboxamide. In preferred embodiments, the salt is selected from the mono hydrochloride, diphosphate and mono sulfate salts of N-[6-(cis-2,6-dimethylmorpholin-4-yl)pyridine-3-yl]-2- methyl-4' -(trifluoromethoxy) [1,1 '-biphenyl] -3 -carboxamide. A particularly preferred embodiment of the of the present invention are the diphosphate and the mono sulfate salts of N-[6-(cis-2,6-dimethylmorpholin-4-yl)pyridine-3-yl]-2-methyl -4'-(trifluoromethoxy)[l,l'- biphenyl] -3 -carboxamide.

[0012] The present invention may be used for treating carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine and endocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin (including squamous cell carcinoma); tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, medulloblastoma and schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors including melanoma, Merkel cell carcinoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer, and teratocarcinoma. The present invention may also be used for treating mastocytosis, germ cell tumors, pediatric sarcomas, and other cancers.

[0013] The present invention is also useful for inhibiting the growth and proliferation of hematopoietic tumors of lymphoid lineage such as leukemia, including acute lymphocytic leukemia (ALL), acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burkitts lymphoma; and hematopoietic tumors of myeloid lineage including acute and chronic myelogenous leukemias (CML), myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia.

Example 1

Preparation of the diphosphate salt

[0014] To a 250 mL, three-necked reaction flask 7.0 g (0.0144 mole) of 2-methyl-4'- trifluoromethoxy-biphenyl-3-carboxylic acid [6-(cis-2,6-dimethyl-morpholin-4-yl)-pyridin-3- yl]-amide free base and acetonitrile (178.5 mL, HPLC grade) was added under nitrogen. The suspension was heated to 58°C under nitrogen over 20 minutes to obtain a clear solution. To the reaction solution 3.403 g of 85% phosphoric acid in water (2 equiv) was added over 18 minutes. Within 5 minutes of the phosphoric acid addition, N-[6-(cis-2,6-dimethylmorpholin- 4-yl)pyridine-3-yl]-2-methyl-4'-(trifluoromethoxy)[ 1 , 1 '-biphenyl]-3-carboxamide diphosphate precipitates out. The white slurry was stirred and cooled to room temp over 100 minutes. The slurry was then cooled to 0 ± 5°C over 5 minutes and stirred for 1 hour. The mixture was filtered under suction and solid was washed with acetonitrile (3 x 9.4 mL). The drug substance was dried under vacuum at 5O ⁰ C for 16 hours to obtain 9.63 g of the phosphate salt (yield: 98%). Example 2

Preparation for the monosulfate salt

[0015] To a 100 mL, three-necked reaction flask was charged 3.0 g (6.18 mmole) of N-[6- (cis-2,6-dimethylmoφholin-4-yl)pyridine-3-yl]-2-methyl-4'-( trifluoromethoxy)[l,r- biphenyl]-3-carboxamide free base and acetonitrile (35 mL, HPLC grade) under nitrogen. The suspension was heated to 50°C under nitrogen over 30 minutes to obtain a clear solution. To the mixture was added a 1.5 mL of 6 M sulfuric acid (1.5 equiv) over 10 minutes. The mixture was stirred at 50°C for 3 hours and allowed to cool to 25°C over 25 minutes. Within 5 minutes the solids came out. The slurry was stirred at 25°C for 16 hours. The mixture was filtered under suction and solid was washed with acetonitrile (10 mL). The drug substance was dried under vacuum at 55°C for 16 hours to obtain 3.0 g of the sulfate salt (yield: 83%).

Example 3

Preparation for the monohydrochloride salt

[0016] To a 100 mL, three-necked reaction flask was charged 3.0 g (6.18 mmole) of N- [6- (cis-2,6-dimethylmoφholin-4-yl)pyridine-3-yl]-2-methyl-4'-( trifluoromethoxy)[ 1,1 '- biphenyl]-3-carboxamide free base and acetone (25 mL, HPLC grade) under nitrogen. The suspension was stirred at 25 ⁰ C under nitrogen for 30 minutes to obtain a clear solution. To the mixture was added a 1.5 mL of 6 M hydrochloric acid (1.5 equiv) over 10 minutes. Within 5 minutes the solids came out. The slurry was stirred at 25 ⁰ C for 16 hours. The mixture was filtered under suction and solid was washed with acetone (10 mL). The drug substance was dried under vacuum at 55°C for 16 hours to obtain 3.0 g of the hydrogen chloride salt (yield: 93%). Example 4

Table 1 below shows stability measured by degradation products (or assay) and appearance color. DPs are analyzed by HPLC (method see Table 3). They are calculated as area-% products. Compositions of the mixtures in mass% are as follows. Mixture 1 : Lactose 200 mesh / maize starch modified 1500 LM / Aerosil 200 / Magnesiumstearate 78.5:20:0.5:1 (m/m/m/m). Mixture 2: Mannitol / Avicel PH 102 / Cutina HR (57:38:5) (m/m/m).

Unstressed drug substance

Bulk 1.02 0.42 0.30

0.2 % solutions or suspensions, 2 week 50°C

2% (or 5%) solutions or suspensions, 1 day room temperature

Solid state, 2 week 50°C, tight container

Bulk (HPLC) 1.03 A 0.40 0.31

Solid state, 2 week 80 ⁰ C, tight container

Bulk (HPLC) 1.06 A 0.43 A 0.35 A

Bulk (DSC) No - No - No - change change change

2 weeks 50°C, tight container

1% in mixture 1 0 .96 A 0.41 A 0.27 A

1% in mixture 2 1 .11 A 0.44 A 0.39 A

2 weeks 50°C/ 75% r.h.

Xenon light (approx. 1200 1 cLuxh)

Bulk (HPLC) 1.35 A 1.1 A 1.46 B

Bulk (XRPD) No - No - No - change change change

Bulk corrosivity

2 day 80% r.h. with No visible change No visible change No visible change stainless steel coupon in the surface of in the surface of in the surface of the stainless steel the stainless steel the stainless steel coupon coupon coupon

* There is a new peak in the XRPD pattern when compared with the unstressed diphosphate. ** The XRPD pattern is similar to that of phosphate, yet with one extra peak. 4- Suspension * Clear solution after stress test

- Test not performed A No change of color

B Slight discoloration C Medium discoloration

D Strong discoloration

Example 5 Table 2 below shows chemical and physico-chemical characteristics.

Table 2

Salt Form

Phosphate, di Sulfate, mono Hydrochloride,

Parameter mono

Phosphate buffer, pH 6.8 0.001 (Final pH 0.002 (Final pH 0.000 (Final pH 2.86) 1.82) 5.93)

Water 0.009 (Final pH 0.007 (Final pH 0.005 (Final pH 1.94) 1.26) 1.67)

Methanol >40 24.4 >40

Ethanol 19.7 19.7 56.4

2- Propanol 14.3 4 10.3

Acetone 2.8 1.7 3.2

Ethyl Acetate 0.3 0.09 2.5

Acetonitrile 0.4 1.4 4.1

NMR Chemical shift Chemical shift Chemical shift changed changed changed

* Attempts to measure intrinsic dissolution rate were not successful due to low solubility.

Example 6

Table 3 below shows morphic properties.

Table 3

Salt form

Hydrochloride,

Parameter Phosphate, di Sulfate, mono mono

Thermal properties

As is

- DSC 213 ⁰ C N/A N/A

- XRPD (crystallinity) Crystalline Crystalline Crystalline

After heating and cooling-

- DSC N/A N/A N/A

- XRPD N/A N/A N/A

Hygroscopicity

Crystal modification after 72 hours vibration

DSC/XRPD/TG DSC/XRPD/TG DSC/XRPD/TG

Water XRPD pattern Dissociated to free Dissociated to free changed, different base. base than free base. Final pH 1.26 Final pH 1.94 Final pH 1.94 Salt form

Hydrochloride,

Parameter Phosphate, di Sulfate, mono mono pH 6.8 buffer XRPD pattern Dissociated to free No form change changed, different base Final pH 5.93 than free base. Final pH 1.82

Final pH 2.86

pH 3 buffer XRPD pattern Dissociated to free Dissociated to free changed, different base base than free base. Final pH 2.00 Final pH 2.89

Final pH 2.15

0.1N HCl solution XRPD pattern XRPD pattern XRPD pattern changed, different changed, different changed, different than free base. than that of free than that of free base. base.

Methanol - Y, crystalline to - crystalline

Ethanol XRPD pattern N, no form change N, no form change changed, poorer crystalline than the original.

Iso Propanol N, no form change N, no form change N, no form change

Ethyl acetate N, no form change N, no form change N, no form change

Acetone Y, crystalline to

N, no form change crystalline N, no form change

Acetonitrile N, no form change N, no form change N, no form change

Effect of grinding

No change in No change in No change in XRPD XRPD XRPD

Effect of compression

No change in No change in No change in XRPD XRPD XRPD

N - no, Y - yes Example 7

[0017] Rat TK data of N-[6-(cis-2,6-dimethylmoipholin-4-yl)pyridine-3-yl]-2-methyl -4'- (trifluoromethoxy)[l,r-biphenyl]-3-carboxamide diphosphate dosed as a suspension, with a 10-fold increase in dose, showed a 3.1 -fold increase in exposure from 10 to 100 mpk. The inter-subject exposure is slightly variable, especially at high dose (100 mpk). See Table 4.

Table 4

Example 8

[0018] A suspension formulation of the free form and the diphosphate salt of N-[6-(cis-2,6- dimethylmorpholin-4-yl)pyridine-3 -yl] -2-methyl-4 ' -(trifluoromethoxy)[ 1,1 ' -biphenyl] -3 - carboxamide are dosed to rat at 3 mpk and 2.1 mpk, respectively. It was found that the disphosphate salt provided a large increase in exposure (16-fold increase) compared to the free form as outline in Table 5.

Table 5

Example 9 Details about methodology, instruments and standards used in Examples 4-5

1 pH value

The pH was determined by transferring approximately 10 mg of N-[6-(cis-2,6- dimethylmorpholin-4-yl)pyridine-3-yl]-2-methyl-4'-(trifluoro methoxy)[ 1 , 1 '-biphenyl]-3- carboxamide salt to a 20 mL vial and adding 10 mL of the corresponding buffer or water. The solutions were stirred continuously as the pH was measured.

2 Determination of approximate solubility

Excess salts were equilibrated in solvents for 1 day at 25 ± 0.5°C. Slurries were filtered and the filtrate saved for HPLC solubility determination.

3 Hygroscopicity

Sorption/desorption isotherms: Instrument, Surface Measurement System DVS-I temperature at 25± 0.5°C.

4 Polymorphism behavior Slurries ofN-[6-(cis-2,6-dimethylmorpholin-4-yl)pyridine-3-yl]-2-meth yl-4'- (trifluoromethoxy)[l,l '-biphenyl]-3-carboxamide salts were stirred at high speed for 24 hours at 25± 0.5°C. The slurries were filtered and the solids collected for XRPD analyses.

5 HPLC Method

Column: Symmetry Cl 8, 3.5 micrometer particle diameter, 4.6 x 75mm (Waters)

Column temperature: 35 degrees

Flow rate: 1 mL/min

Mobile phase: A = 0.1% TFA in water, and B - acetonitrile

Gradient table shown below in Table 6:

Table 6

Stability sample preparation for HPLC analysis:

For phosphate in pH 1 buffer solution, acetonitrile was added to dilute the 0.2% slurry to 0.1% clear solution. For phosphate in the rest of buffer solutions, tetrahydrofuran was added to dilute the 0.2% slurry to 0.1% clear solution. For salts in water, acetonitrile was added to dilute the 0.2% slurry to 0.1% clear solution. For salts in methanol, acetonitrile or acetonitrile/water (50:50, v/v), the corresponding solvent or solvent mixture was added to dilute the 0.2% slurry to 0.1% clear solution. For salts in 0.5% CMC, HPMC cellulose 4000 0.5%, or Tween 80, 0.8%, tetrahydrofuran and water were added to dilute the 2% slurry to 0.1% clear solution and to reach tetrahydrofuran/water 50:50 (v/v). For bulk stability samples (including the samples in excipient mixtures), acetonitrile/water (80:20, v/v) was added to make 0.1% clear solution. Example 10

The sulfate salt and diphosphate salt of N-[6-(cis-2,6-dimethylmorpholin-4-yl)pyridine-3-yl]- 2-methyl-4'-(trifluoromethoxy)[l,r-biphenyl]-3-carboxamide (Compound A in Table 7 below) are dosed as a suspension at 1 mg/mL in 0.5% Methyl Cellulose/0.5% Tween 80 at a dosing volume of 10 mL/kg to Wistar rats. The diphosphate salt was found to give 1.6x more exposure in terms of AUC (0-24 hours) ng*hr/mL when compared to the sulfate salt. Results are shown below in Table 7.

Table 7

Compound Dose Rat AUC _0-24 AUC _0-24 C _max /Dose

Q

(mg/ (ng*h/mL) /dose (ng*h/mL) / ^max (ng/mL)/ t _max (h)

(ng/mL) kg) (mg/kg/day) (mg/kg/day)

Compound A 10 1b 25900 2590 1900 190 4 Sulfate

2b 28700 2870 1860 190 2

3b 36200 3620 3560 360 1

Compound A 10 001 43300 4330 2820 282 8

Diphosphate

002 52800 5280 3160 316 8

003 49400 4940 3100 310 4

As shown in Table 7, the mean tmax for sulfate salt is shorter (2.3 hours) versus diphosphate salt (6.7 hours). The mean cmax/dose for phosphate is 303 and for sulfate is 244. The mean auc/dose for phosphate is 4850, for sulfate is 3030. Overall, the sulfate salt showed less exposure in-vivo (about 40% less) than the diphosphate salt.

[0019] While the invention has been described above with reference to specific embodiments thereof, it is apparent that many changes, modifications, and variations can be made without departing from the inventive concept disclosed herein. Accordingly, it is intended to embrace all such changes, modifications, and variations that fall within the spirit and broad scope of the appended claims. All patent applications, patents, and other publications cited herein are incorporated by reference in their entirety.