TITLE OF THE INVENTION ANTI-HYPERCHOLESTEROLEMIC COMPOUND BACKGROUND OF THE INVENTION

The instant invention relates to a crystalline form of N-[3-(4-{(25',3i?)-2-{4-[3,4- dihydroxy-3 -(hydroxymethyl)butyl]phenyl } -3 - [(35)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4- oxoazetidin-l-yl}phenyl)propyl]methanesulfonamide, and its use alone or in combination with other active agents to treat hypercholesterolemia and for preventing, halting or slowing the progression of atherosclerosis and related conditions and disease events.

It has been clear for several decades that elevated blood cholesterol is a major risk factor for coronary heart disease (CHD), and many studies have shown that the risk of CHD events can be reduced by lipid-lowering therapy. Prior to 1987, the lipid-lowering armamentarium was limited essentially to a low saturated fat and cholesterol diet, the bile acid sequestrants (cholestyramine and colestipol), nicotinic acid (niacin), the fibrates and probucol. Unfortunately, all of these treatments have limited efficacy or tolerability, or both. Substantial reductions in LDL (low density lipoprotein) cholesterol accompanied by increases in HDL (high density lipoprotein) cholesterol could be achieved by the combination of a lipid-lowering diet and a bile acid sequestrant, with or without the addition of nicotinic acid. However, this therapy is not easy to administer or tolerate and was therefore often unsuccessful except in specialist lipid clinics. The fibrates produce a moderate reduction in LDL cholesterol accompanied by increased HDL cholesterol and a substantial reduction in triglycerides, and because they are well tolerated these drugs have been more widely used. Probucol produces only a small reduction in LDL cholesterol and also reduces HDL cholesterol, which, because of the strong inverse relationship between HDL cholesterol level and CHD risk, is generally considered undesirable. With the introduction of lovastatin, the first inhibitor of HMG-CoA reductase to become available for prescription in 1987, for the first time physicians were able to obtain large reductions in plasma cholesterol with very few adverse effects.

Studies have unequivocally demonstrated that lovastatin, simvastatin and pravastatin, all members of the HMG-CoA reductase inhibitor class, slow the progression of atherosclerotic lesions in the coronary and carotid arteries. Simvastatin and pravastatin have also been shown to reduce the risk of coronary heart disease events, and in the case of simvastatin a highly significant reduction in the risk of coronary death and total mortality has been shown by the Scandinavian Simvastatin Survival Study. This study also provided some evidence for a reduction in cerebrovascular events. Despite the substantial reduction in the risk of coronary morbidity and mortality achieved by simvastatin, the risk is still substantial in the treated patients. For example, in the Scandinavian Simvastatin Survival Study, the 42% reduction in the risk of coronary death still left 5% of the treated patients to die of their disease over the course of this 5 year study. Further reduction of risk is clearly needed.

A more recent class of anti-hyperlipidemic agents that has emerged includes inhibitors of cholesterol absorption. Ezetimibe, the first compound to receive regulatory approval in this class, is currently marketed in the U.S. under the tradename ZETIA®. Ezetimibe has the following chemical structure and is described in U.S. Patent No.'s Re. 37721 and 5,846,966:

Sugar-substituted 2-azetidinones, including glucuronidated analogs of the following general structure:

and methods for making them are disclosed in U.S. Patent No. 5,756,470, wherein ArI and Ar2 are unsubstituted or substituted aryl groups.

Additional cholesterol absorption inhibitors are described in WO2002/066464 Al (applied for by Kotobuki Pharmaceutical Co.), and US2002/0137689 Al (Glombik et al.). WO2002/066464 Al discloses hypolipidemic compounds of general formula

wherein, among other definitions, Ai, A3 and A4 can be

and wherein R.2 is -CH2OH, -CH2θC(O)-Ri, or -CO2R1; R3 is -OH or -OC(O)Ri, and R4 is -(CH2)kR5(CH2)i- where k and i are zero or integers of one or more, and k+i is an integer of 10 or less; and R5 is a single bond, -CH=CH-, -OCH2-, carbonyl or -CH(OH).

US2002/0137689 Al discloses hypolipidemic compounds of general formula

wherein, among other definitions, Rl, R2, R3, R4 _? R5 ₅ R6 independently of one another can be (C o-C3θ)-alkylene-(LAG), where one or more carbon atoms of the alkylene radical may be replaced by -0-, -(C=O)-, -CH= CH-, -C≡C-, -N((Ci-C6)-alkyl)-, -N((Ci-C6)-alkylphenyl) or -NH-; and (LAG) is a sugar residue, disugar residue, trisugar residue, tetrasugar residue; a sugar acid, or an amino sugar.

In the ongoing effort to discover novel treatments for hyperlipidemia and atherosclerotic process, the instant invention provides a novel crystalline form of the cholesterol absorption inhibitor N- [3 -(4- {(2S,3R)-2- { 4- [3 ,4-dihydroxy-3 -(hydroxymethyl)butyl]phenyl } -3 - [(3S)-3-(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - yl } phenyl)propyl] methanesulfonamide .

DESCRIPTION OF THE FIGURES

FIG. 1 is a characteristic X-ray powder diffraction (XRPD) pattern of the crystalline Form I of N-[3-(4-{(25',3i?)-2-{4-[3,4-dihydroxy-3-(hydroxymethyl)buty l]phenyl}-3- [(35)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - yl } phenyl)propyl] methanesulfonamide .

FIG. 2 is a carbon- 13 cross-polarization magic-angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectrum (13C-SSNMR) of crystalline Form I of N-[3-(4-{(2S,3R)- 2- {4-[3 ,4-dihydroxy-3 -(hydroxymethyl)butyl]phenyl } -3 - [(3S)-3 -(4-fluorophenyl)-3 - hydroxypropyl] -4-oxoazetidin- 1 -yl}phenyl)propyl]methanesulfonamide.

SUMMARY OF THE INVENTION

One object of the instant invention is to provide crystalline Form I of the cholesterol absorption inhibitor N-[3-(4-{(2S',37?)-2-{4-[3,4-dihydroxy-3- (hydroxymethyl)butyl] phenyl } -3 -[(35)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - yl}phenyl)propyl]methanesulfonamide which has the following chemical structural formula:

Compound A

A second object of the instant invention is to provide a method for inhibiting cholesterol absorption comprising administering a therapeutically effective amount of Form I to a patient in need of such treatment. Another object is to provide a method for reducing plasma cholesterol levels, especially LDL-cholesterol, and treating hypercholesterolemia comprising administering a therapeutically effective amount of Form I.

As a further object, methods are provided for preventing or reducing the risk of developing atherosclerosis, as well as for halting or slowing the progression of atherosclerotic disease once it has become clinically evident, comprising the administration of a prophylactically or therapeutically effective amount, as appropriate, of Form I to a patient who is at risk of developing atherosclerosis or who already has atherosclerotic disease. Another object of the present invention is the use of Form I for the manufacture of a medicament useful in treating, preventing or reducing the risk of developing these conditions. Other objects of this invention are to provide processes for making Form I and to provide novel pharmaceutical compositions comprising Form I.

Additional objects will be evident from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

For brevity, the compound N-[3-(4-{(25',3^)-2-{4-[3,4-dihydroxy-3-

(hydroxymethyl)butyl]phenyl-3-[(35)-3-(4-fluorophenyl)-3- hydroxypropyl]-4-oxoazetidin-l- yl}phenyl)propyl]methanesulfonamide may also be referred to herein as "Compound A." The anhydrous crystalline form of N-[3-(4-{(25,3i?)-2-{4-[3,4-dihydroxy-3- (hydroxymethyl)butyljphenyl } -3 - [(35)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - yl}phenyl)propyl]-methanesulfonamide of the instant invention is referred to herein as "Form I."

Compound A

Crystalline Form I was characterized by X-Ray Powder Diffraction (XRPD) and

13c Solid State Nuclear Magnetic Resonance (13c SSNMR), described as follows.

X-Ray Powder Diffraction Characterization of Form I:

X-ray diffraction patterns of Form I were measured using a Panalytical X'Pert Pro with a Cu LFF source (Cu K-alpha - wavelength = 1.54187) at a generator power of 4OkV and 50 mA from 2-40 degrees 2-theta. Major peaks from FIG. 1 that characterize Form I are (wavelength CuKa): °2-theta 18.7, 19.3, 17.1, 22, 22.6, 6.2, 11.8, 12.4 and 13.4. In particular, Form I can be characterized by having an XRPD pattern obtained using CuKa radiation containing at least one °2-theta value selected from the group consisting of 18.7, 19.3, 17.1, 22, 22.6, 6.2, 11.8, 12.4, and 13.4. More particularly, Form I can be characterized by having an XRPD pattern obtained using CuKa radiation containing at least two °2-theta values selected from the group consisting of 18.7, 19.3, 17.1, 22, 22.6, 6.2, 11.8, 12.4, and 13.4. Even more particularly, Form I can be characterized by having an XRPD pattern obtained using CuKa radiation containing at least three °2-theta values selected from the group consisting of 18.7, 19.3, 17.1, 22, 22.6, 6.2, 11.8, 12.4, and 13.4. Furthermore, Form I can be characterized by having an XRPD pattern obtained using CuKa radiation containing °2-theta values 18.7, 19.3 and l7.1; or 22, 22.6 and 6.2; or 11.8, 12.4 and 13.4; or a combination of two or three these groupings. As used herein, °2-theta values are accurate within ±0.1, based on the experiments described in the Examples provided. Additionally a listing of peaks below 28 degrees 2-theta are listed below in Table

1 with heights, d-spacings and relative intensity (ReI. Int.):

TABLE 1

No. Pos. [°2Th.] eight [cts] d-spacing [A] ReI. Int. [%]

1 1.9 837.12 45.684 16.34

2 6.2 275.91 14.184 5.39

3 10.6 190.88 8.314 3.73

4 11.8 232.03 7.533 4.53

5 12.4 360.17 7.113 7.03

6 13.4 974.11 6.629 19.02

7 14.9 267.22 5.965 5.22

8 17.1 1965.4 5.185 38.37

9 17.2 1643.25 5.150 32.08

10 18.3 722.45 4.846 14.1

11 18.7 5122.09 4.745 100

12 19.0 772.64 4.678 15.08

13 19.3 2059.88 4.594 40.22

14 20.1 643.54 4.427 12.56

15 20.4 1005.27 4.351 19.63

16 22.0 1575.3 4.034 30.75

17 22.6 1262.84 3.938 24.65

18 25.0 681.32 3.561 13.3

19 25.8 203.65 3.449 3.98

20 26.3 1281.01 3.388 25.01

21 26.6 818.66 3.351 15.98

22 27.6 377.75 3.234 7.37

Crystalline Form I of N-[3-(4-{(25,3i?)-2-{4-[3,4-dihydroxy-3- (hydroxymethyl)butyl]phenyl } -3 - [(35)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - yl}phenyl)propyl]methanesulfonamide can also be characterized by the X-ray powder diffraction pattern of Figure 1.

Additionally, a sample of crystalline Form I was ground in a mortar and pestle for 1 minute with the application of slight pressure. This sample was sieved through a 200 mesh screen. The sample was then subjected to x-ray diffraction and the peaks recorded. The grinding of a sample will somewhat help alleviate the problem of inaccurate relative intensities due to preferred orientation. The positions of peaks with relative intensities above 14% (°2-theta) with and without grinding are listed below in Table 2.

TABLE 2

U ng round Ground

Pos. [°2Theta] ReI. Int. [%] ReI. Int. [%]

13.4 19.02 48.42

17.1 38.37 64.89

18.3 14.1 34.24

18.7 100 100

19.0 15.08 35.32

19.3 40.22 98.12

20.4 19.63 34.59

22.0 30.75 44.74

22.6 24.65 48.36

26.3 25.01 28.02

26.6 15.98 30.74

Accordingly, Form I can also be characterized by having an XRPD pattern obtained from ground Form I using CuKa radiation containing at least one °2-theta value selected from those listed in Table 2. More particularly, Form I can be characterized by having an XRPD pattern obtained from ground Form I using CuKa radiation containing at least two °2- theta values selected from those listed in Table 2. Even more particularly, Form I can be characterized by having an XRPD pattern obtained from ground Form I using CuKa radiation containing at least three °2-theta values selected from those listed in Table 2. Furthermore, Form I can be characterized by having an XRPD pattern obtained from ground Form I using CuKa radiation containing °2-theta values 18.7, 19.3 and 17.1; or 22, 22.6 and 13.4; or 18.3, 19.0 and 20.4; or a combination of two or three of these groupings. d-Spacings, distances between peaks and distances between d-spacings are also listed below in Table 3 for Form I. The peak at 18.7° 2-theta is taken as reference for these calculations.

TABLE 3

Accordingly, Form I can also be characterized by having an XRPD pattern obtained using CuKa radiation having at least one distance between d-spacings listed in Table 3 using 18.7° 2-theta as reference. More particularly, Form I can be characterized by having an XRPD pattern obtained using CuKa radiation having at least two distances between d-spacings listed in Table 3 using 18.7° 2-theta as reference. Even more particularly, Form I can be characterized by having an XRPD pattern obtained using CuKa radiation having at least three distances between d-spacings listed in Table 3 using 18.7° 2-theta as reference. Furthermore, Form I can alternatively be characterized by having an XRPD pattern obtained using CuKa radiation having the following distances between d-spacings using 18.7° 2-theta as reference: 0.000, -0.151 and 0.439; or -0.712, -0.808 and 1.883; or 0.101, -0.068 and -0.394; or a combination of two or three of these groupings.

Form I can also be characterized by having an XRPD pattern obtained using CuKa radiation having at least one distance between peaks listed in Table 3 using 18.7° 2-theta as reference. More particularly, Form I can be characterized by having an XRPD pattern obtained using CuKa radiation having at least two distances between peaks listed in Table 3 using 18.7° 2-theta as reference. Even more particularly, Form I can be characterized by having an XRPD pattern obtained using CuKa radiation having at least three distances between peaks listed in Table 3 using 18.7° 2-theta as reference. Furthermore, Form I can alternatively be characterized by having an XRPD pattern obtained using CuKa radiation having the following distances between peaks using 18.7° 2-theta as reference: 0.0, 0.6 and -1.6; or 3.3, 3.9 and -5.3; or 0.3, 1.7 and -0.4; or a combination of two or three of these groupings. ¹³ C-SSNMR Characterization of Form I:

In addition to the X-ray powder diffraction patterns described above, crystalline Form I was further characterized by solid-state carbon-13 nuclear magnetic resonance (13C- SSNMR) spectra. The solid-state carbon-13 NMR spectra were obtained on a Bruker DSX 500WB NMR system using a Bruker 4 mm H/X/Y CPMAS probe. The carbon-13 NMR spectra utilized proton/carbon- 13 cross-polarization magic-angle spinning with variable-amplitude cross polarization, total sideband suppression, and SPINAL decoupling at 10OkHz. The samples were spun at 10.0 kHz, and a total of 4096 scans were collected with a recycle delay of 10 seconds. A line broadening of 10 Hz was applied to the spectra before FT was performed. Chemical shifts are reported on the TMS scale using the carbonyl carbon of glycine (176.03 p.p.m.) as a secondary reference.

Crystalline Form I exhibited characteristic signals by 1 ³ C-SSNMR with chemical shift values of 28.8, 34.2, 43.4, 58.3, 74.8, 113.9, 129.9, 136.2, 146.5 and 168.3 parts per million

(p.p.m. or ppm). In particular, Form I can be characterized by having at least one chemical shift value obtained by 13C-SSNMR selected from the group consisting of 28.8, 34.2, 43.4, 58.3, 74.8, 113.9, 129.9, 136.2, 146.5 and 168.3 ppm. More particularly, Form I can be characterized by having at least two chemical shift values obtained by 13 C- S SNMR selected from the group consisting of 28.8, 34.2, 43.4, 58.3, 74.8, 113.9, 129.9, 136.2, 146.5 and 168.3 ppm. Even more particularly, Form I can be characterized by having at least three chemical shift values obtained by 13C-SSNMR selected from the group consisting of 28.8, 34.2, 43.4, 58.3, 74.8, 113.9, 129.9, 136.2, 146.5 and 168.3 ppm. Furthermore, Form I can be characterized by 13C-SSNMR signals with chemical shift values of 28.8, 136.2, 43.4 and 74.8p.p.m.; or 129.9, 58.3, and 168.3 p.p.m.; or 34.2, 113.9 and 146.5 p.p.m.; or a combination of two or three of these groupings.

Below is a listing of peaks from 13C-SSNMR for Form I with their relative intensities: P Peeaakk ! [ppm] Intensity 2 288..88 100.00 1 13366..22 85.51 4 433..44 72.48 7 744..88 67.85 1 12299..99 66.20 1 12288..66 63.87 5 588..33 63.61 7 700..99 62.38 3 344..22 59.26 P Peeaakk f [ppm]. con't Intensity, con't 4 422..33 53.80 3 355..00 51.76 1 11133..99 47.94 6 600..77 46.97 2 266..88 46.62 1 14466..55 46.02 3 333..66 44.25 1 13311..55 42.45 5 599..88 41.48 1 11144..55 41.34 1 13377..44 41.04 6 655..22 39.62 1 11188..77 39.58 132.5 38.87

126.7 38.36

122.8 36.62

115.1 33.34 168.3 31.07 134.7 26.87

160.2 20.30

Crystalline Form I of #-[3-(4-{(2S,3λ)-2-{4-[3,4-dihydroxy-3-

(hydroxymethyl)butyl]phenyl}-3-[(3S)-3-(4-fluorophenyl)-3 -hydroxypropyl]-4-oxoazetidin-l- yl}phenyl)propyl]methanesulfonamide can also be characterized by the solid-state 13C-SSNMR CPMAS nuclear magnetic resonance spectrum of FIG. 2.

In addition, crystalline Form I can be characterized by 13C-SSNMR having chemical shift differences between the lowest ppm resonance and other resonances using 26.8 ppm as the lowest reference resonance. For example, Form I can be characterized by 13C- SSNMR having chemical shift differences between the lowest ppm resonance and other resonances as follows: 2.0, 7.4, 16.6, 31.5, 48.0, 87.1, 103.1, 109.4, 119.7, and 141.5. In particular, Form I can be characterized by having at least one chemical shift difference between the lowest ppm resonance and other resonances, using 26.8 ppm as the lowest reference resonance, selected from the group consisting of 2.0, 7.4, 16.6, 31.5, 48.0, 87.1, 103.1, 109.4, 119.7 and 141.5. More particularly, Form I can be characterized by having at least two chemical shift differences between the lowest ppm resonance and other resonances, using 26.8 ppm as the lowest reference resonance, selected from the group consisting of 2.0, 7.4, 16.6, 31.5, 48.0, 87.1, 103.1, 109.4, 119.7 and 141.5. Even more particularly, Form I can be characterized by having at least three chemical shift differences between the lowest ppm resonance and other resonances, using 26.8 ppm as the lowest reference resonance, selected from the group consisting of 2.0, 7.4, 16.6, 31.5, 48.0, 87.1, 103.1, 109.4, 119.7 and 141.5. Furthermore, Form I can be characterized by 13C-SSNMR having chemical shift differences between the lowest ppm resonance and other resonances as follows: 2.0, 109.4, 26.8 and 48.0; or 103.1, 31.5 and 141.5; or 7.4, 87.1 and 119.7; or a combination of two or three of these groupings.

The instant invention is further related to a process for preparing a crystal form of N-[3-(4-{(2,S,3i?)-2-{4-[3,4-dihydroxy-3-(hydroxymethyl)buty l]phenyl}-3-[(3^)-3-(4- fluorophenyl)-3-hydroxypropyl]-4-oxoazetidin-l-yl}phenyl)pro pyl]methanesulfonamide comprising: a) adding a solvent to N-[3-(4-{(2S',3i?)-2-{4-[3,4-dihydroxy-3- (hydroxymethyl)butyl]phenyl}-3-[(35)-3-(4-fluorophenyl)-3-hy droxypropyl]-4-oxoazetidin-l- yl}phenyl)propyl]methanesulfonamide to create a slurry, and

b) filtering the slurry to obtain a crystal form of JV-[3-(4-{(2S ^r ,3λ)-2-{4-[3,4- dihydroxy-3-(hydroxymethyl)butyl]phenyl}-3-[(35)-3-(4-fluoro phenyl)-3-hydroxypropyl]-4- oxoazetidin- 1 -yl } phenyl)propyl]methanesulfonamide.

In a second embodiment of the instant invention, the process further comprises: a) filtering the slurry to obtain solids, and b) drying the solids to obtain a crystal form of _V-[3-(4-{(2S,31?)-2-{4-[3,4- dihydroxy-3 -(hydroxymethyl)butyl]phenyl } -3 -[(35)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4- oxoazetidin- 1 -yl } phenyl)propyl]methanesulfonamide.

In a third embodiment of the instant invention, the process for preparing a crystal form ofN-[3-(4-{(25,3i?)-2-{4-[3,4-dihydroxy-3-(hydroxymethyl)but yl]phenyl}-3-[(35)-3-(4- fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 -yl } phenyl)propyl]methanesulfonamide comprises: a) adding a solvent to N-[3-(4-{(25',3λ)-2-{4-[3,4-dihydroxy-3- (hydroxymethyl)butyl]phenyl } -3 - [(3S)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - yl}phenyl)propyl]methanesulfonamide, b) filtering to obtain filtrate solutions, and c) evaporating the filtrate solutions to obtain a crystal form of N- [3 -(4- {(25,3i?)-2-{4-[3,4-dihydroxy-3-(hydroxymethyl)butyl]phenyl} -3-[(35)-3-(4-fluorophenyl)-3- hydroxypropyl]-4-oxoazetidin-l-yl}phenyl)propyl]methanesulfo namide. In a further embodiment of the third embodiment, the process further comprises, in step b, filtering at about 55-75°C.

In a fourth embodiment, the process comprises: a) adding a solvent to N-[3-(4-{(25',3i?)-2-{4-[3,4-dihydroxy-3- (hydroxymethyl)butyl]phenyl } -3 - [(3S)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - y 1 } pheny l)propy 1] methanesulfonamide, b) filtering to obtain filtrate solutions, c) cooling the filtrate solutions, c) drying the filtrate solutions to obtain a crystal form of N-[3-(4-{(25',3i?)-2- {4-[3,4-dihydroxy-3-(hydroxymethyl)butyl]phenyl}-3-[(35)-3-( 4-fluorophenyl)-3- hydroxypropyl] -4-oxoazetidin- 1 -yl}phenyl)propyl]methanesulfonamide. In a fifth embodiment, the process comprises: a) adding a solvent to N-[3-(4-{(25',3λ)-2-{4-[3,4-dihydroxy-3-

(hydroxymethyl)butyl]phenyl} -3-[(35)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-oxoazetidin- 1 - y 1 } phenyl)propyl] methanesulfonamide, b) filtering to obtain filtrate solutions, c) adding antisolvent to the filtrate solutions,

d) drying to obtain a crystal form of N-[3-(4-{(25,3i?)-2-{4-[3,4-dihydroxy-3- (hydroxymethyl)butyl]phenyl } -3 - [(35)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - yl}phenyl)propyl]methanesulfonamide.

In a further embodiment of the fifth embodiment, after step c, supernatant is removed.

The instant invention is also directed to a process for for preparing a crystal form of7V-[3-(4-{(25,3i?)-2-{4-[3,4-dihydroxy-3-(hydroxymethyl)bu tyl]phenyl}-3-[(35)-3-(4- fluorophenyl)-3-hydroxypropyl]-4-oxoazetidin-l-yl}phenyl)pro pyl]methanesulfonamide comprising: a) adding a transesterification catalyst to a solution of 3-{4-[(2S,3R)-3-[(3S)-3-

(acetyloxy)-3 -(4-fluorophenyl)propyl] - 1 -(4- { 3 -[(methylsulfonyl)amino]propyl } phenyl)-4- oxoazetidin-2-yl]phenyl}-l,l-bis(hydroxymethyl)propyl acetate in an alcohol; b) adding solvent to obtain a slurry; c) filtering to obtain a solution; d) adding seed to obtain the crystal form.

In a further embodiment, the alcohol is methanol or ethanol. In a further embodiment, the solvent is IPA. In a further embodiment of the above process, the transesterification catalyst is TMSOK.

The instant invention is also directed to a pharmaceutical composition comprising about 5 mg to about 150 mg of crystal form of N-[3-(4-{(2S,3tf)-2-{4-[3,4-dihydroxy-3-

(hydroxymethyl)butyljphenyl } -3 - [(35)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - yl}phenyl)propyl]methanesulfonamide, about 30 to about 150 mg of microcrystalline cellulose, about 30 mg to about 150 mg of lactose monohydrate, about 0.5 to about 20 mg of croscarmellose sodium, about 0.1 to about 10 mg of sodium laurgyl sulfate, about 0.1 to about 10 mg of magnesium stearate, and about 0.1 to about 10 mg of sodium stearyl fumarate. In a further embodiment, the composition comprises about 10 mg to about 100 mg of crystal form of N- [3- (4-{(25,3/?)-2-{4-[3,4-dihydroxy-3-(hydroxymethyl)butyl]phen yl}-3-[(35)-3-(4-fluorophenyl)-3- hydroxypropyl]-4-oxoazetidin-l-yl}phenyl)propyl]methanesulfo namide, about 40 to about 140 mg of microcrystalline cellulose, about 40 mg to about 140 mg of lactose monohydrate, about 2 to about 15 mg of croscarmellose sodium, about 0.5 to about 5 mg of sodium laurgyl sulfate, about 0.5 to about 5 mg of magnesium stearate, and about 0.5 to about 5 mg of sodium stearyl fumarate

In the instant invention, the term "solvent" refers to broad range of organic solvents known to those skilled in the art. In one embodiment, the solvent system is selected from the group consisting of alcohols, amines, arenes, and halogenated alkanes. Examples of solvents that may be used in the process of the instant invention include, but are not limited to, 1 ,2- Dichloroethane, Acetonitrile, Nitromethane, iPrOAc in 1 ,2-dimethoxyethane, Cyclohexane

in Ethyl Acetate, Cyclohexane in Ethanol, Cyclohexane in 2-Propanol, Ethyl Acetate: Heptanes, MIBK (methyl iso-butyl ketone), Water in 1 ,2- Dichloroethane, Cyclohexane in 1 ,2- Dimethoxyethane and the like. In an embodiment, the solvent may be selected from Water in 1 ,2- Dichloroethane, 1 ,2- Dichloroethane, Acetonitrile, Nitromethane, iPrO Ac in 1 ,2- dimethoxyethane, Cyclohexane in Ethyl Acetate, Cyclohexane in Ethanol, Cyclohexane in 2- Propanol, Ethyl Acetate: Heptanes, and Cyclohexane in 1 ,2- Dimethoxyethane. In an embodiment, the solvent may be selected from 100% 1,2-dichloroethane.

In the instant application, known antisolvents may be utilized. In an embodiment of the invention, the antisolvent is selected from water or heptanes. In an embodiment, the solvent and antisolvent system may be selected from a) water in 1,2-dichloroethane and water, b) Water in Ethanol and water, c) THF in 1 -Propanol and heptanes, d) THF in Toluene and heptanes, e) THF in Ethanol and heptanes, f) iPrOAc in 2-Propanol and heptanes, g) Butyronitrile and heptanes, h) iPrOAc in Ethanol and heptanes, i) Cyclohexane in Ethyl Acetate and heptanes, j) Cyclohexane in Ethanol and heptanes, or k) Cyclohexane in 2-Propanol and heptanes.

In the instant invention, a transesterification catalyst is used. Transesterification is the process of exchanging the alkoxy group of an ester compound by another alcohol. These reactions are often catalyzed by the addition of an acid or base. As used herein, the term "acid" refers to organic or inorganic acids. Examples of an organic acid include, but are not limited to, carboxylic acids such as stearic acid, acetic acid, formic acid, propionic acid, butyric acid, and the like. Examples of inorganic acid include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, and the like. As used herein, the term "base" refers to an organic base, an inorganic base, and the like. Examples of a base include, but ^{are n} °t limited to, TMSOK, K2CO3, Cs ₂ CC>3, Li ₂ CO ₃ , Na ₂ CO ₃ , KOH, LiOH, NaOH, CsOH, K ₃ PO-^, KF, Et ₃ N and other tertiary amines, diisopropylamine and other secondary amines, and butylamine and other primary amines. In an embodiment of the instant invention, the base is TMSOK.

As used herein, the term "alcohol" is any organic compound in which a hydroxyl group (-OH) is bound to a carbon atom of an alkyl or substituted alkyl group. In an embodiment, the alcohol is selected from methanol, ethanol, propanol, isopropyl alcohol, and tert-butyl alcohol. In another embodiment, the alcohol is methanol or ethanol.

The term "patient" includes mammals, especially humans, who use the instant active agent for the prevention or treatment of a medical condition. Administering of the drug to the patient includes both self-administration and administration to the patient by another person. The patient may be in need of treatment for an existing disease or medical condition, or may desire prophylactic treatment to prevent or reduce the risk for diseases and medical conditions affected by inhibition of cholesterol absorption.

The term "therapeutically effective amount" is intended to mean that amount of a pharmaceutical drug that will elicit the biological or medical response of a tissue, a system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. The term "prophylactically effective amount" is intended to mean that amount of a pharmaceutical drug that will prevent or reduce the risk of occurrence of the biological or medical event that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician. Particularly, the dosage a patient receives can be selected so as to achieve the amount of LDL cholesterol lowering desired; the dosage a patient receives may also be titrated over time in order to reach a target LDL level. The dosage regimen utilizing the compound of the instant invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; and the renal and hepatic function of the patient. A consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically effective or prophylactically effective dosage amount needed to prevent, counter, or arrest the progress of the condition.

The compound of the instant invention is a cholesterol absorption inhibitor and is useful for reducing plasma cholesterol levels, particularly reducing plasma LDL cholesterol levels, when used either alone or in combination with another active agent, such as an anti- atherosclerotic agent, and more particularly a cholesterol biosynthesis inhibitor, for example an HMG-CoA reductase inhibitor. Thus the instant invention provides methods for inhibiting cholesterol absorption and for treating lipid disorders including hypercholesterolemia comprising administering a therapeutically effective amount of Form I to a patient in need of such treatment. The term hypercholesterolemia includes but is not limited to homozygous familial hypercholesterolemia (HoFH) and heterozygous familial hypercholesterolemia (HeFH) and therefore Form I can be used treat HoHF and HeHF patients. Form I can also be used for the treatment of mixed hyperlipidemia which is characterized by an elevated LDL cholesterol level and elevated triglycerides level along with an undesirably low HDL cholesterol level. Form I can also be used to treat or prevent sitosterolemia and/or to lower the concentration of one or more sterols other than cholesterol in the plasma or tissue of a patient.

Further provided are methods for preventing or reducing the risk of developing atherosclerosis, as well as for halting or slowing the progression of atherosclerotic disease once it has become clinically evident, comprising the administration of a prophylactically or therapeutically effective amount, as appropriate, of Form I to a patient who is at risk of developing atherosclerosis or who already has atherosclerotic disease.

Atherosclerosis encompasses vascular diseases and conditions that are recognized and understood by physicians practicing in the relevant fields of medicine. Atherosclerotic cardiovascular disease including restenosis following revascularization procedures, coronary heart disease (also known as coronary artery disease or ischemic heart disease), cerebrovascular disease including multi-infarct dementia, and peripheral vessel disease including erectile dysfunction are all clinical manifestations of atherosclerosis and are therefore encompassed by the terms "atherosclerosis" and "atherosclerotic disease."

Form I may be administered to prevent or reduce the risk of occurrence, or recurrence where the potential exists, of a coronary heart disease event, a cerebrovascular event, and/or intermittent claudication. Coronary heart disease events are intended to include CHD death, myocardial infarction (i.e., a heart attack), and coronary revascularization procedures. Cerebrovascular events are intended to include ischemic or hemorrhagic stroke (also known as cerebrovascular accidents) and transient ischemic attacks. Intermittent claudication is a clinical manifestation of peripheral vessel disease. The term "atherosclerotic disease event" as used herein is intended to encompass coronary heart disease events, cerebrovascular events, and intermittent claudication. It is intended that persons who have previously experienced one or more non-fatal atherosclerotic disease events are those for whom the potential for recurrence of such an event exists.

Accordingly, the instant invention also provides a method for preventing or reducing the risk of a first or subsequent occurrence of an atherosclerotic disease event comprising the administration of a prophylactically effective amount of Form I to a patient at risk for such an event. The patient may or may not have atherosclerotic disease at the time of administration, or may be at risk for developing it.

Persons to be treated with the instant therapy include those at risk of developing atherosclerotic disease and of having an atherosclerotic disease event. Standard atherosclerotic disease risk factors are known to the average physician practicing in the relevant fields of medicine. Such known risk factors include but are not limited to hypertension, smoking, diabetes, low levels of high density lipoprotein (HDL) cholesterol, and a family history of atherosclerotic cardiovascular disease. Published guidelines for determining those who are at risk of developing atherosclerotic disease can be found in: Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III),

JAMA, 2001; 285 pp.2486-2497. People who are identified as having one or more of the above- noted risk factors are intended to be included in the group of people considered at risk for developing atherosclerotic disease. People identified as having one or more of the above-noted risk factors, as well as people who already have atherosclerosis, are intended to be included within the group of people considered to be at risk for having an atherosclerotic disease event. The oral dosage amount of Form I is from about 0.1 to about 30 mg/kg of body weight per day, preferably about 0.1 to about 15 mg/kg of body weight per day. For an average body weight of 70 kg, the dosage level may be from about 1 mg to about 1000 mg of drug per day, and more particularly from about 1 mg to 250 mg per day. However, dosage amounts will vary depending on factors as noted above. Although the active drug of the present invention may be administered in divided doses, for example from two to four times daily, a single daily dose of the active drug is preferred. As examples, the daily dosage amount may be selected from, but not limited to, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 35mg, 40 mg, 45mg, 50 mg, 55mg, 60mg, 65mg, 70mg, 75 mg, 80 mg, 85mg, 90mg, 95mg, 100 mg, 200 mg and 250 mg. The active drug employed in the instant therapy can be administered in such oral forms as tablets, capsules, caplets, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Oral formulations are preferred, and particularly solid oral formulations such as tablets.

Administration of the active drug can be via any pharmaceutically acceptable route and in any pharmaceutically acceptable dosage form. This includes the use of oral conventional rapid-release, time controlled-release and delayed-release (such enteric coated) pharmaceutical dosage forms. Additional suitable pharmaceutical compositions for use with the present invention are known to those of ordinary skill in the pharmaceutical arts; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. In the methods of the present invention, the active drug is typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with a non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, modified sugars, modified starches, methyl cellulose and its derivatives, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and other reducing and non-reducing sugars, magnesium stearate, steric acid, sodium stearyl fumarate, glyceryl behenate, calcium stearate and the like. For oral administration in liquid form, the drug components can be combined with non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders,

lubricants, disintegrating agents and coloring and flavoring agents can also be incorporated into the mixture. Stabilizing agents such as antioxidants, for example butylated hydroxyanisole (BHA), 2,6-di-tert-butyl-4-methylphenol (BHT), propyl gallate, sodium ascorbate, citric acid, calcium metabisulphite, hydroquinone, and 7-hydroxycoumarin, particularly BHA, propyl gallate and combinations thereof, can also be added to stabilize the dosage forms. When Form I is formulated together with an HMG-CoA reductase inhibitor such as simvastatin, the use of at least one stabilizing agent is preferred in the composition, particularly BHA or a combination of BHA with propyl gallate. Other suitable components include gelatin, sweeteners, natural and synthetic gums such as acacia, tragacanth or alginates, carboxymethylcellulose, polyethylene glycol, waxes and the like.

An example of a suitable pharmaceutical composition is one comprised of Form I, microcrystalline cellulose, lactose (particularly lactose monohydrate), croscarmellose sodium, and magnesium stearate, with or without the inclusion of sodium lauryl sulfate. The drug load (% by weight of Form I based on total weight of the tablet without external surface coatings) in a single tablet can range for example from 1% to 35%. Examples of drug load include but are not limited to 1%, 10 %, 25% and 35%.

The instant invention also encompasses a process for preparing a pharmaceutical composition comprising combining Form I with a pharmaceutically acceptable carrier. Also encompassed is the pharmaceutical composition which is made by combining Form I with a pharmaceutically acceptable carrier.

One or more additional active agents may be administered in combination with Form I, and therefore an embodiment of the instant invention encompasses a drug combination. The drug combination encompasses a single dosage formulation comprised of Form I and an additional active agent or agents, as well as administration of each of Form I and the additional active agent or agents in separate dosage formulations, which allows for concurrent or sequential administration of the active agents. The additional active agent or agents can be lipid modifying agents, particularly a cholesterol biosynthesis inhibitor such as an HMG-CoA reductase inhibitor, or agents having other pharmaceutical activities, or agents that have both lipid-modifying effects and other pharmaceutical activities. Examples of HMG-CoA reductase inhibitors useful for this purpose include statins in their lactonized or dihydroxy open acid forms and pharmaceutically acceptable salts and esters thereof, including but not limited to lovastatin (MEV ACOR®; see US Patent No. 4,342,767); simvastatin (ZOCOR®; see US Patent No. 4,444,784); dihydroxy open- acid simvastatin, particularly the ammonium or calcium salts thereof; pravastatin, particularly the sodium salt thereof (PRA V ACOL®; see US Patent No. 4,346,227); fluvastatin particularly the sodium salt thereof (LESCOL®; see US Patent No. 5,354,772); atorvastatin, particularly the calcium salt thereof (LIPITOR®; see US Patent No. 5,273,995); rosuvastatin (CRESTOR®; see US Patent No. 5,260,440); and pitavastatin also referred to as NK- 104 (see PCT international

publication number WO 97/23200). Examples of additional active agents which may be employed include but are not limited to one or more of FLAP inhibitors; 5-lipoxygenase inhibitors; additional cholesterol absorption inhibitors such as ezetimibe (ZETIA®), described in U.S. Patent No.'s Re. 37721 and 5,846,966; cholesterol ester transfer protein (CETP) inhibitors, for example JTT-705; HMG-CoA synthase inhibitors; squalene epoxidase inhibitors; squalene synthetase inhibitors (also known as squalene synthase inhibitors); acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors including selective inhibitors of ACAT-I or ACAT-2 as well as dual inhibitors of ACATl and -2; microsomal triglyceride transfer protein (MTP) inhibitors; niacin; niacin receptor agonists such as acipimox and acifran, as well as niacin receptor partial agonists; niacin in combination with a DP receptor antagonist; LDL (low density lipoprotein) receptor inducers; platelet aggregation inhibitors, for example glycoprotein Ilb/IIIa fibrinogen receptor antagonists and aspirin; human peroxisome proliferator activated receptor gamma (PP ARγ) agonists including the compounds commonly referred to as glitazones for example pioglitazone and rosiglitazone and, including those compounds included within the structural class known as thiazolidinediones as well as those PP ARγ agonists outside the thiazolidinedione structural class; PP ARa agonists such as clofibrate, fenofibrate including micronized fenofibrate, and gemfibrozil; PPAR dual α/γ agonists; vitamin Bβ (also known as pyridoxine) and the pharmaceutically acceptable salts thereof such as the HCl salt; vitamin B 12 (also known as cyanocobalamin); folic acid or a pharmaceutically acceptable salt or ester thereof such as the sodium salt and the methylglucamine salt; anti-oxidant vitamins such as vitamin C and E and beta carotene; beta-blockers; angiotensin II antagonists such as losartan; angiotensin converting enzyme inhibitors such as enalapril and captopril; calcium channel blockers such as nifedipine and diltiazam; endothelian antagonists; agents that enhance ABCl gene expression; FXR ligands including both inhibitors and agonists; and LXR ligands including both inhibitors and agonists of all sub-types of this receptor, e.g. LXRα and LXRβ; bisphosphonate compounds such as alendronate sodium; and cyclooxygenase-2 inhibitors such as celecoxib and valdecoxib. Additioanlly, Form I can be used in combination with PCSK9 antagonists; antisense; Apo-AI, Apo-AI variants such as Apo-AI milano, or Apo-AI mimetics such as D4F; an HDL selective delipidation process; anti-hypertensive agents such as renin inhibitors and angiotensin II antagonists; anti-diabetic agents such as DPP-IV inhibitors, including e.g., JANUVIA and JANUMET; anti-obesity agents such as CBl inverse agonists; and 1 lβHSDl inhibitors.

Form I can also be used in combination with a nucleic acid molecule inhibitor that targets NPClLl or other protein sequences involved in hyperlipidemia. A polynucleotide-based gene expression inhibitor comprises any polynucleotide containing a sequence whose presence or expression in a cell causes the degradation of or inhibits the function, transcription, or translation of a gene in a sequence-specific manner. Polynucleotide-based expression inhibitors may be selected from the group comprising: siRNA, microRNA, interfering RNA or RNAi, dsRNA,

ribozymes, antisense polynucleotides, and DNA expression cassettes encoding siRNA, microRNA, dsRNA, ribozymes or antisense nucleic acids. RNAi molecules are polynucleotides or polynucleotide analogs that, when delivered to a cell, inhibit RNA function through RNA interference. Small RNAi molecules include RNA molecules less that about 50 nucleotides in length and include siRNA and miRNA. SiRNA comprises a double stranded structure typically containing 15-50 base pairs and preferably 19-25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. MicroRNAs (miRNAs) are small noncoding polynucleotides, about 22 nucleotides long, that direct destruction or translational repression of their mRNA targets. Antisense polynucleotides comprise sequence that is complimentary to a gene or mRNA. Antisense polynucleotides include, but are not limited to: morpholinos, 2'-O-methyl polynucleotides, DNA, RNA and the like. The polynucleotide-based expression inhibitor may be polymerized in vitro, recombinant, contain chimeric sequences, or derivatives of these groups. The polynucleotide- based expression inhibitor may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited.

Form I could also be used in combination with peptide inhibitors and antibody molecule antagonists such as a PCSK9-specific antagonist. A PCSK9-specific antagonist can be any binding molecule with specificity for PCSK9 protein including, but not limited to, antibody molecules as described below, any PCSK9-specific binding structure, any polypeptide or nucleic acid structure that specifically binds PCSK9, and any of the foregoing incorporated into various protein scaffolds; including but not limited to, various non-antibody-based scaffolds, and various structures capable of affording selective binding to PCSK9 including but not limited to small modular immunopharmaceuticals (or "SMIPs"; see, Haan & Maggos, 2004 Biocentury Jan 26); Immunity proteins (see, e.g., Chak et al., 1996 Proc. Natl. Acad. Sci. USA 93:6437-6442); cytochrome b562 (see Ku and Schultz, 1995 Proc. Natl. Acad. Sci. USA 92:6552-6556); the peptide D2p8 (see Barthe et al., 2000 Protein Sci. 9:942-955); avimers (Avidia; see Silverman et al., 2005 Nat. Biotechnol. 23:1556-1561); DARPins (Molecular Partners; see Binz et al., 2003 J. MoI. Biol. 332:489-503; and Forrer et al., 2003 FEBS Lett. 539:2-6); Tetranectins (see, Kastrup et al., 1998 Acta. Crystallogr. D. Biol. Crystallogr. 54:757-766); Adnectins (Adnexus; see, Xu et al., 2002 Chem. Biol. 9:933-942), Anticalins (Pieris; see Vogt & Skerra, 2004 Chemobiochem. 5:191-199; Beste et al., 1999 Proc. Natl. Acad. Sci. USA 96:1898-1903; Lamia & Erdmann, 2003 J. MoI. Biol. 329:381-388; and Lamia & Erdmann, 2004 Protein Expr. Purif. 33:39-47); A- domain proteins (see North & Blacklow, 1999 Biochemistry 38:3926-3935), Lipocalins (see Schlehuber & Skerra, 2005 Drug Discov. Today 10:23-33); Repeat-motif proteins such as Ankyrin repeat proteins (see Sedgwick & Smerdon, 1999 Trends Biochem. Sci. 24:311 -316; Mosavi et al., 2002 Proc. Natl. Acad. Sci. USA 99:16029-16034; and Binz et al., 2004 Nat.

Biotechnol. 22:575-582); Insect Defensin A (see Zhao et al., 2004 Peptides 25:629-635); Kunitz domains (see Roberts et al., 1992 Proc. Natl. Acad. Sci. USA 89:2429-2433; Roberts et al., 1992 Gene 121 :9-15; Dennis & Lazarus, 1994 J. Biol. Chem. 269:22129-22136; and Dennis & Lazarus, 1994 J. Biol. Chem. 269:22137-22144); PDZ-Domains (see Schneider et al., 1999 Nat. Biotechnol. 17:170-175); Scorpion toxins such as Charybdotoxin (see Vita et al., 1998

Biopolymers 47:93-100); 10th fibronectin type III domain (or 10Fn3; see Koide et al., 1998 J. MoI. Biol. 284:1141-1151, and Xu et al., 2002 Chem. Biol. 9:933-942); CTLA-4 (extracellular domain; see Nuttall et al., 1999 Proteins 36:217-227; and Irving et al., 2001 J. Immunol. Methods 248:31-45); Knottins (see Souriau et al., 2005 Biochemistry 44:7143-7155 and Lehtio et al., 2000 Proteins 41 :316-322); Neocarzinostatin (see Heyd et al. 2003 Biochemistry 42:5674- 5683); carbohydrate binding module 4-2 (CBM4-2; see Cicortas et al., 2004 Protein Eng. Des. SeI. 17:213-221); Tendamistat (see McConnell & Hoess, 1995 J. MoI. Biol. 250:460-470, and Li et al., 2003 Protein Eng. 16:65-72); T cell receptor (see Holler et al., 2000 Proc. Natl. Acad. Sci. USA 97:5387-5392; Shusta et al., 2000 Nat. Biotechnol. 18:754-759; and Li et al., 2005 Nat. Biotechnol. 23:349-354); Affibodies (Affibody; see Nord et al., 1995 Protein Eng. 8:601-608; Nord et al., 1997 Nat. Biotechnol. 15:772-777; Gunneriusson et al., 1999 Protein Eng. 12:873- 878); and other selective binding proteins or scaffolds recognized in the literature; see, e.g., Binz & Pluckthun, 2005 Curr. Opin. Biotech. 16:1-1 1; Gill & Damle, 2006 Curr. Opin. Biotechnol. 17:1-6; Hosse et al., 2006 Protein Science 15:14-27; Binz et al., 2005 Nat. Biotechnol. 23:1257- 1268; Hey et al., 2005 Trends in Biotechnol. 23:514-522; Binz & Pluckthun, 2005 Curr. Opin. Biotech. 16:459-469; Nygren & Skerra, 2004 J. Immunolog. Methods 290:3-28; Nygren & Uhlen, 1997 Curr. Opin. Struct. Biol. 7:463-469. Antibodies and the use of antigen-binding fragments is well defined in the literature. The use of alternative scaffolds for protein binding is well appreciated in the scientific literature as well, see, e.g., Binz & Pluckthun, 2005 Curr. Opin. Biotech. 16:1-11; Gill & Damle, 2006 Curr. Opin. Biotechnol. 17:1-6; Hosse et al., 2006 Protein Science 15:14-27; Binz et al., 2005 Nat. Biotechnol. 23:1257-1268; Hey et al., 2005 Trends in Biotechnol. 23:514-522; Binz & Pluckthun, 2005 Curr. Opin. Biotech. 16:459-469; Nygren & Skerra, 2004 J. Immunolog. Methods 290:3-28; Nygren & Uhlen, 1997 Curr. Opin. Struct. Biol. 7:463-469. Accordingly, non-antibody-based scaffolds or antagonist molecules with selectivity for PCSK9 that counteract PCSK9-dependent inhibition of cellular LDL-uptake form important embodiments of the present invention. Aptamers (nucleic acid or peptide molecules capable of selectively binding a target molecule) are one specific example. They can be selected from random sequence pools or identified from natural sources such as riboswitches. Peptide aptamers, nucleic acid aptamers (e.g., structured nucleic acid, including both DNA and RNA- based structures) and nucleic acid decoys can be effective for selectively binding and inhibiting proteins of interest; see, e.g., Hoppe-Seyler & Butz, 2000 J. MoI. Med. 78:426-430; Bock et al.,

1992 Nature 355:564-566; Bunka & Stockley, 2006 Nat. Rev. Microbiol. 4:588-596; Martell et al., 2002 Molec. Ther. 6:30-34; Jayasena, 1999 Clin. Chem. 45:1628-1650.

A therapeutically or prophylactically effective amount, as appropriate, of Form I can be used for the preparation of a medicament useful for inhibiting cholesterol absorption, as well as for treating and/or reducing the risk for diseases and conditions affected by inhibition of cholesterol absorption, such as treating lipid disorders, preventing or reducing the risk of developing atherosclerotic disease, halting or slowing the progression of atherosclerotic disease once it has become clinically manifest, and preventing or reducing the risk of a first or subsequent occurrence of an atherosclerotic disease event. For example, the medicament may be comprised of about 5 mg to about 1000 mg of Form I. The medicament comprised of Form I may also be prepared with one or more additional active agents, such as those described supra. iV-[3-(4-{(2.S,3i?)-2-{4-[3,4-dihydroxy-3-(hydroxymethyl)bur yl]phenyl}-3-[(35)- 3-(4-fluorophenyl)-3-hydroxypropyl]-4-oxoazetidin-l-yl}pheny l)propyl]methanesulfonamide was determined to inhibit cholesterol absorption employing the Cholesterol Absorption Assay in Mice, below. This assay involves comparing a test compound to ezetimibe with respect to their ability to inhibit cholesterol absorption in mice. Both ezetimibe and the tested compound inhibited cholesterol absorption by >90% at the highest dose tested. The tested compound had an ID 50 < lmg/kg.

Cholesterol Absorption Assay in Mice: C57BL/6 male mice (n = 6/group), aged 10 - 14 weeks, were dosed orally with 0.2 ml 0.25 % methyl cellulose solution with or without test compound or ezetimibe (0.12-10 mg/kg). Thirty minutes later all of the mice were dosed orally with 0.2 ml INTRALIPID™ containing 2 μCi [ ³ H] -cholesterol per mouse. Five hours later, the animals were euthanized, and liver and blood were collected. Cholesterol counts in liver and plasma were determined, and percent inhibition of cholesterol absorption was calculated.

A variety of chromatographic techniques may be employed in the preparation of Form I. These techniques include, but are not limited to: High Performance Liquid Chromatography (HPLC) including normal- reversed- and chiral-phase; Medium Pressure Liquid Chromatography (MPLC), Super Critical Fluid Chromatography; preparative Thin Layer Chromatography (prep TLC); Gas Chromatography (GC); flash chromatography with silica gel or reversed-phase silica gel; ion-exchange chromatography; and radial chromatography. All temperatures are degrees Celsius unless otherwise noted. Degrees Celsius may be noted in the examples as "C" without the degree symbol (e.g. 50C) or " ⁰ C" with a degree symbol (e.g. 50°C). Some abbreviations used herein include:

Ac Acyl (CH ₃ C(O)-)

Aq. Aqueous

Bn Benzyl

C. Celsius calc. Calculated

DCM dichloromethane

DIEA N, N-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMF N,N-dimethylformamide equiv. Equivalent(s)

ES-MS Electron Spray Ion-Mass Spectroscopy

EtOAc Ethyl acetate h Hour(s)

HPLC High performance liquid chromatography

IPA Isopropyl Alcohol

IPAC Isopropyl Acetate min Minute(s) mp Melting point

ML mother liquors

MS Mass spectrum

Prep. Preparative r.t. (or rt) Room temperature sat. Saturated

TBAI tetrabutylammonium iodide

TBS Tert-butyl dimethylsilyl

TEA Triethyl amine

TFA Trifluoroacetic acid

THF Tetrahydrofuran

TLC Thin layer chromatography

TMSOK Potassium trimethylsilanolate

Example 1

Preparation of N-prop-2-yn-l-ylmethanesulfonamide (i-1):

^v H O _/ NJ,Me

H °

Methanesulfonylchloride (1.40 mL, 18.1 mmol) was added dropwise to a stirred solution of propargylamine (1.00 g, 18.1 mmol) and dimethylaminopyridine (44.0 mg, 0.36

mmol) in pyridine (10 mL) at 0°C. After aging for approximately 15 h, the reaction mixture was poured into IN HCl and extracted twice with ethyl acetate. The combined organic extracts were washed with saturated aqueous sodium bicarbonate, brine, dried (MgSO ₄ ), filtered and concentrated in vacuo, to afford the title compound i-1. Crude i-1 crystallized on standing and was used without further purification. ¹ HNMR (500 MHz, CDCl ₃ ) δ: 4.92 (br s, IH), 3.99 (dd, J = 2.3, 6.2 Hz, 2H), 3.11 (s, 3H), 2.70 (br t, J = 2.3 Hz).

Example 2

Preparation of 5-ethynyl-2,2-dimethyl-L3-dioxan-5-yl acetate (i-6):

i-6

To a dry 25OmL roundbottom flask was charged with a 0.5M solution of ethynylmagnesium bromide in THF (115mL, 57.7mmol) under nitrogen atmosphere. The resulting solution was cooled to O ⁰ C in an ice bath. To the cooled solution was added slowly a solution of 2,2-dimethyl-l,3-dioxane-5-one (5g, 38.44mmol) in 5OmL dry THF. The ice bath was removed and the resulting reaction mixture was stirred at ambient temperature for 1.5hrs. The reaction mixture was quenched with sat. aq. NH ₄ Cl (5OmL) and then extracted with ethyl acetate (10OmL). The organic layer was dried over Na ₂ SO ₄ , filtered and the solvent removed under vacuum to afford the crude intermediate. The crude intermediate was dissolved in CH ₂ Cl ₂ (10OmL) under nitrogen atmosphere. To the resulting solution was added simultaneously by syringe acetic anhydride (4.34mL, 46mmol) and TEA (6.4mL, 46mmol). To the reaction mixture was added DMAP (0.56g, 4.6mmol). The reaction mixture was stirred for 3hrs at room temperature at which time the reaction was quenched by the addition of IN aq. HCl (10OmL). The reaction mixture was transferred to separately funnel and the organic layer was separated. The organic layer was washed with aq. NaHCO ₃ (10OmL), water (5OmL), brine, dried, filtered and the solvent removed under vacuum to afford the title compound (i-6) which was used without further purification. ¹ HNMR (500 MHz, CDCl ₃ ) δ: 4.14 (d, J = 12.6, 2H) 4.07 (d, J = 12.6 Hz, 2H), 2.65 (s, IH), 2.12 (s, 3H), 1.45 (s, 3H), 1.41 (s, 3H).

Example 3

The compounds (3R,4S)-3 - [(35)-3 -(4-fluorophenyl)-3 -hydroxypropyl] -4-(4- hydroxyphenyl)-l-(4-iodophenyl)azetidin-2-one Q-T) and (l-7a) were prepared according to

Burnett, D. S.; Caplen, M. A.; Domalski, M. S.; Browne, M. E.; Davis, H. R. Jr.; Clader, J. W. Bioorg. Med. Chem. Lett. (2002), 12, 311. Compound t& is the dihydroxy-protected analog of I ₁ 7, where the protecting groups are acetyl.

oxoazetidin-2-yllphenyl acetate (i-8):

To a solution of (l.S)-l-(4-fluorophenyl)-3-[(2 ₁ S ^r ,3i?)-2-(4-hydroxyphenyl)-l-(4- iodophenyl)-4-oxoazetidin-3-yl]propyl acetate (l-7a) (2g, 3.58 mmol) (prepared according to Burnett, D. S.; Caplen, M. A.; Domalski, M. S.; Browne, M. E.; Davis, H. R. Jr.; Clader, J. W. Bioorg. Med. Chem. Lett. (2002), 12, 311) in CH ₂ Cl ₂ (25 mL) under nitrogen atmosphere was added acetic anhydride (0.4 mL, 4.30 mmol), triethylamine (0.75 mL, 5.38 mmol) and DMAP. The reaction mixture was stirred at RT for lhr and the solvent removed under vacuum. The residue was purified by MPLC (silica column) with stepwise gradient elution; (0 - 100% EtOAc/hexanes as eluent) to afford the title compound (i-8). mlz (ES) (M-OAc) ⁺ . ¹ HNMR (500 MHz, CDCl ₃ ) δ: 7.57 (d, J = 8.6, IH) 7.38-7.26 (m, 5H), 7.22 (br d, J = 7.1 H, 2H), 7.14 ( d, J = 8.5 Hz, IH), 7.08-7.02 (m, 3H), 5.74 (t, J = 6.7 Hz, IH), 4.62 (d, J = 2.3 Hz, IH), 3.10 (dt, J = 2.3, 7.8 Hz, IH), 2.34 (s, 3H), 2.08 (s, 3H), 2.09-2.03 (m, 2H), 1.94-1.86 (m, 2H).

Example 4

Preparation of amorphous N-r3-f4-(f2S.3i?V2-(4-r3.4-dihvdroxy-3-

(hydroxymethvDbutyliphenyl } -3 - [(35^-3 -(4-fluorophenylV3 -hydroxypropyll -4-oxoazetidin- 1 - vUphenylipropyllmethanesulfonamide (referred to herein as Compound A)

Step A: Preparation of 4- \(2S. 3R)-3- \(3S)-3 -(acetyloxyV 3 -(4-fluorophenyl)propyll - 1 -(A- (3-r(methylsulfonyl)aminolprop- 1 -yn- 1 -yl)phenyl)-4-oxoazetidin-2-yllphenyl acetate

Dichlorobis(triphenylphosphine)palladium(II) (1.27 g, 1.68 mmol) and copper(I) iodide (632 mg, 3.32 mmol) were added to a solution of i^δ (10.0 g, 16.6 mmol) and M _. (3.34 g, 25.0 mmol) in triethylamine (16.2 mL, 116.34 mmol) and DMF (150 mL). The reaction mixture was saturated with nitrogen and stirred at room temperature. After 2h, the reaction mixture was partitioned between 40OmL EtOAc and 25OmL water. The organic layer was washed with water (15OmL), brine (15OmL), dried (MgSO ₄ ), filtered and concentrated in vacuo. Purification of the crude residue by MPLC (silica column) with stepwise gradient elution; (0 - 100% EtOAc/hexanes as eluent) afforded the title compound, mlz (ES) 629 (M+Na) ⁺ , 547 (M-OAc) ⁺ . ¹ HNMR (500 MHz, CDCl ₃ ) δ: 7.35 (d, J = 8.4 Hz, IH), 7.28 (dd, J = 6.4, 8.4 Hz, IH), 7.19 (d, J = 8.5 Hz, IH), 7.12 (d, J = 8.5 Hz, IH), 7.08 (d, J = 8.3 Hz, IH), 7.02 (dd, J = 6.5, 8.6 Hz, IH), 5.72 (t, 6.6 Hz, IH), 4.60 (d, J = 2.3 Hz, IH), 4.21-4.16 (m, IH), 4.15 (overlapped dd, J = 7.1, 11 Hz, IH), 3.15-3.12 (m, 2H), 3.09-3.04 (m, IH), 2.96 (s, 3H), 2.58 (t, 7.6 Hz, 2H), 2.30 (s, 3H), 2.07 (overlapped s, 3H), 2.09- 2.03 (m, 2H), 1.90-1.83 (m, 4H).

Step B: Preparation of 4-U2S. 3R)-3-\(3S >3-(acetyloxy>3-(4-fluorophenvnpropyll-l-(4- { 3 - lϊmethylsulfonvDaminoi propyl > phenyl)-4-oxoazetidin-2-yllphenyl acetate

A mixture of the intermediate from Step A (8.5 g, 14 mmol) and 10% palladium on activated carbon (2.2 g) in ethanol (10OmL) and EtOAc (150 mL) was hydrogenated at atmospheric pressure. After 15 h, the reaction mixture was filtered through MgSO4 and filter aid and the filtered catalyst washed several times with EtOAc. The filtrate was concentrated in vacuo to afford the title compound which was used without further purification, mlz (ES) 663 (M+Na) ⁺ , 551 (M-OAc) ⁺ .

Step C: Preparation of (15)- 1 -(4-fluorophenyl)-3-IY3i?. 4^-l-r4-{3- [(methylsulfonyl)amino1propyUphenyl)-2-oxo-4-(4-{[( ^' trifluoromethyl)- sulfonyl] oxy> phenyl)azetidin-3 -y 1] propyl acetate

Guanidine hydrochloride (1.34 g, 13.93 mmol) was added to a mixture of the intermediate from Step B, (8.5g, 13.93 mmol) and triethylamine (1.95 mL, 13.93 mmol) in methanol (150 mL). After 3 h, the solvent was removed under vacuum and the residue was dissolved in EtOAc (20OmL) / water (10OmL) and 2N aq. HCl. The mixture was transferred to a separatory funnel and the layers separated. The organic layer was washed with brine (10OmL), dried (MgSO ₄ ), filtered and concentrated in vacuo to afford a clear oil.

The crude intermediate was dissolved in methylene chloride (100 mL) and to the solution was added (bis(trifluoromethylsulfonyl)amino pyridine (8.14g, 13.93 mmol), triethylamine (1.95 mL, 13.93mmol), DMAP (-100 mg, catalytic). The resulting solution was stirred for 2 h at room temperature. The reaction was quenched with IN aq. HCl and the organic

layer was separated. The organic extract was washed with brine, dried (MgSO ₄ ) and concentrated in vacuo. Purification of the crude residue by MPLC (silica column) with stepwise gradient elution (0 - 100% EtO Ac/hexanes as eluent) afforded the title compound, mlz (ES) 723 (M+Na) ⁺ , 641 (M-OAc) ⁺ .

Preparation of (lS)-3-r(2S,3R)-2-f4-([5-(acetyloxyV2.2-dimethyl-1.3-dioxan- 5- yl] ethynyl } phenyl)- 1 -(4- { 3 - [Ymethylsulfonyl)aminolpropyUphenyl)-4- oxoazetidin-3-yll- 1 -(4-1IuOrOPhCnVl)PrOPvI acetate:

To an oven dried flask 25OmL flask was added CuI (300 mg, 1.44 mmol), tetrabutylammonium iodide (TBAI, 1.58g, 4.28 mmol). The charged flask was set under nitrogen atmosphere and a solution of the intermediate from Step C, (3.5 g, 4.28 mmol) in 3OmL anhydrous DMF was added to the flask. A solution of 5-ethynyl-2,2-dimethyl-l,3-dioxan-5-yl acetate (i-6) (1.70 g, 8.56 mmol) in DMF (20 mL) was added to the mixture. The flask was then equipped with a condensor, and the mixture was evacuated and set under nitrogen several times to de-gas the solvent. Solid Pd(PPh ₃ ) ₄ (3.32 g, 3 mmol) was then added to the reaction followed by TEA (4.2 mL, 30 mmol). The reaction mixture was heated to 7O ⁰ C for 2 hours during which time the reaction mixture became dark brown in color. The reaction was removed from the heating bath, cooled and partitioned with EtOAc (25OmL) and IN aq. HCl (100 mL). The organic layer was washed with water (10OmL), brine (75mL), dried over magnesium sulfate, filtered and concentrated under vacuum. The residue was purified by MPLC (silica column) with stepwise gradient elution; (0 - 100% EtO Ac/hexanes as eluent) to afford the title compound, mlz (ES) 689 (M-OAc) ⁺ . ¹ HNMR (500 MHz, CD ₃ OD) δ: 7.44 (d, J = 8.3 Hz, IH), 7.38-7.32 (m, 4H), 7.16 (d, J= 8.5 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 7.06 (t, J = 8.6 Hz, 2H), 5.70 (app t, 6.3 Hz, 1H4.20 (s, 3H), 3.10-3.05 (m, IH), 3.02 (d, J = 7.0 Hz, 2H), 2.89 (s, 3H), 2.60 (t, 7.4 Hz, 2H), 2.10 ( s, 3H), 2.04 (s, 3H), 1.78 (t, J = 7.6, 3H), 1.47 (s, 3H), 1.39 (s, 3H).

Preparation of riSV3-r(2S.3RV2-r4-(2-r5-racetyloxy)-2,2-dimethyl-13-dioxan- 5- yl] ethyl ] phenyl)- 1 -(4- { 3 - [(methyl sulfonyOamino]propyl I phenylV4-oxoazetidin- 3-yll-l-f4-fluorophenyl)propyl acetate:

A roundbottom flask was charged with 10% Pd-C (500mg) and 300mg 20%

Pd(OH) ₂ -C. EtOAc (~2mL) was added to cover the solid catalyst mixture. To this mixture was added a solution of the intermediate from Step D, (1.5g, 2.0 mmol) in ethanol (4OmL) and ethyl acetate (2 mL). The resulting suspension set under hydrogen atmosphere and stirred vigorously for lhr. The catalysts were filtered, solids washed with ethanol and the solvent was removed under vacuum to obtain partially hydrogenated intermediate. The reaction procedure was repeated as above. A roundbottom flask was charged with 10% Pd-C (500mg) and 300mg 20% Pd(OH) ₂ -C. EtOAc (~2mL) was added to cover the solid catalyst mixture. To this mixture was added a solution of the intermediate from above in ethanol (4OmL) and ethyl acetate (2 mL). The resulting suspension set under hydrogen atmosphere and stirred vigorously for 2 hours. The catalyst was filtered through filter aid and MgSO ₄ and washed with EtOH/EtOAc. The filtrate was concentrated in vacuo to afford the title compound which was used without further purification, m/z (ES) 692 (M-OAc) ⁺ . ¹ HNMR (500 MHz, CD ₃ OD) δ: 7.31-7.24 (m, 6H), 7.21- 7.17 (m, 3H), 7.08-7.02 (m, 3H), 5.72 (app t, 6.7 Hz, IH) 4.60 (d, J = 2.1 Hz, IH), 4.20 (app t, J = 6.5, IH), 4.02 (d, J = 12.4 Hz, 2H), 3.90 (d, J = 12.2 Hz, 2H), 3.13 (q, J = 6.7 Hz, 2H), 3.06 (dt, J - 2.2, 7.6 Hz, IH), 2.94 (s, 3H), 2.60 (app q, 7.4 Hz, 4H), 2.35-2.29 (m, 2H), 2.08 (s, 3H), 2.03 (s, 3H), 1.83-1.90 (m, 3H), 1.45 (s, 3H), 1.40 (s, 3H).

Preparation of 3-(4-r(2S.3RV3-rGSV3-('acetyloxy)-3-( ^' 4-fluorophenvnpropyll-l- ( ^" 4- { 3 -|YmethylsulfonvDamino]propyl } phenyl V4-oxoazetidin-2-yl]phenyl I - 1 , 1 - bisOivdroxymethvπpropyl acetate

To a solution of the intermediate of step E (1.5 g, 2 mmol) in THF/water (16mL/4mL) was added TFA (1 mL). The reaction mixture was stirred at RT for 16hr. To the reaction mixture was added 10OmL toluene and the water was removed under vacuum with water bath temperature of 4O ⁰ C. The residue was treated twice with 10OmL toluene followed by azeotropic removal of water. The solvent was completely removed under vacuum. The crude product was purified by MPLC (silica column) with stepwise gradient elution (50 - 100% EtOAc/hexanes as eluent). Mixed fractions were also isolated and were further purified by prep TLC eluting with CH ₂ Cl ₂ /Me0H (95/5). The purified fractions were combined to afford the title compound, mlz (ES) 653 (M-OAc) ⁺ .

Preparation of N-[3-(4-((25.3i?V2-{4-r3,4-dihvdroxy-3- (hydroxymethvDbutyljphenyl } -3 - [(35^-3 -(4-fluorophenv0-3 -hvdroxypropyl]-4- oxoazetidin- 1 -yl 1 phenvDpropylimethanesulfonamide

To a solution of the intermediate from Step F, (1.05g, 1.47 mmol) in methanol (2.5 mL) was added potassium cyanide (100 mg, 1.58 mmol) and the resulting solution stirred at 5O ⁰ C for 2 hours. The solution was concentrated and the residue purified by preparative TLC

plate eluting with methanol/dichloromethane (10/90) to afford the title compound. This product was further purified by MPLC (silica column) with stepwise gradient elution; (5 - 10% EtOH/EtOAc as eluent) to afford the title compound as a white solid, mlz (ES) 611 (M-OAc) ⁺ and 651 (M+Na) ^{+ 1} HNMR (500 MHz, CD ₃ OD) δ: 7.35-7.31 (m, 2H), 7.28-7.234 (m, 4H), 7.18 (d, J = 8.5 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 7.03 (app, t, J = 8.6 Hz, 2H), 4.79 (br d, J = 2.1 Hz, IH), 4.60 (br dd, J = 5.1, 6.60 Hz, IH), 3.53 (s, 4H)), 3.09-3.03 (m, IH), 3.02 (t, J = 6.8 Hz, 2H), 2.88 (s, 3H), 2.73-2.67 (m, 2H), 2.61 (t, 7.6 Hz, 2H), 1.97-1.83 (m, 3H), 1.81-1.73 (m, 3H).

Example 5 Procedures for the crystallization of amorphous iV-[3-(4-{(2 _l S',3i?)-2-{4-[3,4-dihvdroxy-3- ftiydroxymethyPbutyl] phenyl } -3 - 1 ^" (35VS -(4-fluorophenyl)-3 -hydroxypropyl] -4-oxoazetidin- 1 - vUphenvPpropyl]methanesulfonamide without seeding

The starting material Compound A for these experiments was an amorphous solid which had been purified by chromatography over silica gel using mixtures of dichloromethane: Ethanol as eluent and was >99% pure by HPLC assay.

When a % range appears for a solvent system below, it indicates that the experiment was run more than once, employing a 20% incremental v/v change for the solvent system in each experiment. For example, "0-40% (v/v) Water in 1 ,2- Dichloroethane" indicates that three experiments were performed using: 0% water, 100% dichloroethane ; 20% water, 80% dichloroethane; and 40% water, 60% dichloroethane.

A- Slurry process.

Using the Powdernium, a powder dispensing robot, lOmg +/- 1.Omg of

Compound A (amorphous solid) was dispensed into each well of a 96-well plate. A magnetic stir bar was added to each well. 900 μl of solvent was dispensed into each well. The 96-well plate was capped and the system was equilibrated at 65°C for 2 hours. The system was then filtered hot at 65°C. The remaining solids were dried and analyzed by XRPD.

Preferred solvent compositions to obtain the crystalline phase:

100% (v) 1,2- Dichloroethane 100% (v) Acetonitrile

100% (v) Nitromethane 80% (v/v) iPrOAc in 1 ,2-dimethoxyethane 20% (v/v) Cyclohexane in Ethyl Acetate 80% (v/v) Cyclohexane in Ethanol 80% (v/v) Cyclohexane in 2-Propanol

3:1 (v/v) Ethyl Acetate: Heptanes 100% (v/v) MIBK (methyl iso-butyl ketone)

B- Evaporation process.

Using the Powdernium, a powder dispensing robot, lOmg +/- l.Omg of

Compound A (amorphous solid) was dispensed into each well of a 96-well plate. A magnetic stir bar was added to each well. 900 μl of solvent was dispensed into each well. The 96-well plate was capped and the system was equilibrated at 65°C for 2 hours. The system was then filtered hot at 65°C. 200 μl of each of the filtrate solutions were transferred into a new 96-well plate. This plate was left uncapped and allowed to evaporate at room temperature until dry. The remaining solids were dried and analyzed by XRPD. Preferred solvent composition to obtain the crystalline phase:

100% 1,2-dichloroethane.

C- Cooling process.

Using the Powdernium, a powder dispensing robot, lOmg +/- l.Omg of Compound A (amorphous solid) was dispensed into each well of a 96-well plate. A magnetic stir bar was added to each well. 900 μl of solvent was dispensed into each well. The 96-well plate was capped and the system was equilibrated at 65°C for 2 hours. The system was then filtered hot at 65°C. 200 μl of each of the filtrate solutions were transferred into a new 96-well plate. This plate was cooled with a cubic cool down temperature gradient of 65 ⁰ C-IO ⁰ C over 8 hours. The plate was equilibrated at 10 ⁰ C for 2 hours and then the supernatant was removed from the plate. The following day, the plate was wicked to dry the remaining solvent and the solid residues were analyzed by XRPD.

Preferred solvent compositions to obtain the crystalline phase:

0-40% (v/v) Water in 1,2- Dichloroethane. 100% (v) 1 ,2- Dichloroethane

100% (v) Acetonitrile 100% (v) Nitromethane 80% (v/v) iPrOAc in 1,2-dimethoxyethane 40% (v/v) Cyclohexane in Ethyl Acetate 40-80% (v/v) Cyclohexane in Ethanol

60-80% (v/v) Cyclohexane in 2-Propanol

3:1 (v/v) Ethyl Acetate: Heptanes

40% (v/v) Cyclohexane in 1 ,2- Dimethoxyethane

D- Precipitation process:

Using the Powdernium, a powder dispensing robot, lOmg +/- l.Omg of Compound A (amorphous solid) was dispensed into each well of a 96-well plate. A magnetic stir

bar was added to each well. 900 μl of solvent was dispensed into each well. The 96-well plate was capped and the system was equilibrated at 65°C for 2 hours. The system was then filtered hot at 65°C. 100 μl of each of the filtrate solutions were transferred into a new 96-well plate containing 200 μl of antisolvent in each well. The plate was equilibrated for 2 hours and then the supernatant was removed from the plate. The following day, the plate was wicked to dry the remaining solvent and the solid residues were analyzed by XRPD.

Preferred solvent compositions to obtain the crystalline phase: 0-20% (v/v) water in 1,2-dichloroethane (100 μl). Antisolvent: 200 μl of water. 20% (v/v) Water in Ethanol (100 μl). Antisolvent: 200 μl of water. 0-60% (v/v) THF in 1-Propanol (100 μl). Antisolvent: 200 μl of heptanes.

80% (v/v) THF in Toluene (100 μl). Antisolvent: 200 μl of heptanes. 0-60% (v/v) THF in Ethanol (100 μl). Antisolvent: 200 μl of heptanes. 20-100% (v/v) iPrOAc in 2-Propanol (100 μl). Antisolvent: 200 μl of heptanes. 100% (v) Butyronitrile (100 μl). Antisolvent: 200 μl of heptanes. 60% (v) iPrOAc in Ethanol (100 μl). Antisolvent: 200 μl of heptanes.

0-40% (v/v) Cyclohexane in Ethyl Acetate (100 μl). Antisolvent: 200 μl of heptanes. 20-40% (v/v) Cyclohexane in Ethanol (100 μl). Antisolvent: 200 μl of heptanes. 20% (v/v) Cyclohexane in 2-Propanol (100 μl). Antisolvent: 200 μl of heptanes.

Example 6

Procedure for the 60mg scale-Up Experiments for preparing Crystalline Form I

A- Precipitation experiment.

60 mg of the starting material, Compound A amorphous solid, was manually weighed in a glass vial. 2 ml of MIBK were manually dispensed into the vial. The vial was capped and equilibrated for one hour at 65°C.

The vial was removed from the oven and 500 μl of the supernatant was added to 1 ml of room temperature heptanes (anti-solvent). A white precipitate formed. The solids were isolated by vacuum filtration over a glass frit and analyzed.

B- Cooling experiment. 60 mg of the starting material, Compound A amorphous solid, was manually weighed in a glass vial. 2 ml of 1,2-dichloroethane were manually dispensed into the vial. The vial was capped and equilibrated for one hour at 65°C.

A slow cubic cool down from 65-10°C was completed over 10 hours. The mixture was held at 10°C for two hours and then warmed to 20°C. The solids were isolated by vacuum filtration over a glass frit and analyzed.

Results:

The scale-up experiments were analyzed by XRPD. The experiments generated crystalline material of Form I which appeared to be the same crystalline form as each other by XRPD. The scale-up experiments were successful in reproducing a crystalline phase of Compound A identified by the screen.

In the following examples, degrees Celsius may be represented as "C" or " ⁰ C" (for example 50C or 50 ⁰ C).

Example 7 Crystallization with Unmilled Seed

Following the procedures described in Examples 1-4, but in Example 4, Step G, replacing the 1 eq of KCN at 50C, with a catalytic amount of potassium trimethylsilanolate (TMSOK), as the transesterification catalyst, at room temperature, produces Compound A. Simple alcohols were chosen as solvents. Reaction rates are dependent on the size of the alcohol, and thus the use of smaller alcohols, which speed up the reaction, also allows for a reduction in the amount of the reagent. While other smaller alcohols such as EtOH may be used, the reaction is fastest in methanol (5 to 10 volumes) and requires only 10 mol% of TMSOK to drive the reaction to >99% completion in 1 hour at 2OC (2 hours at 10C).

PROCEDURE (DEPROTECTION AND CRUDE CRYSTALLIZATION FROM IP A/HEPTANE).

As described in Example 4, Step G, a solution of diacetate in methanol was filtered through a 1 μm line filter into a 100 L round bottom flask. The solution was cooled to 12C and TMSOK was added in two portions (pH=8). No exotherm was observed. The reaction was monitored by HPLC until <1% of monoacetate was observed by HPLC (2 hours). Assay yield was 91% (305Og). The temperature was kept constant throughout the reaction and the pH stayed also constant. The reaction was quenched by acidification with 50 mL of cone. HCl (pH=5 after addition). The solution was concentrated to 30 L and solvent switched to IPA by flushing a total of 110 L of IPA at constant volume. GC assay showed <0.1% MeOH and

KF=335 ppm. A slurry was obtained after the solvent switch. The batch was heated up to 63C. The thin slurry obtained was line filtered while hot to remove KCl. After the filtration, temperature was brought back to 6OC to obtain a clear solution, cooled down to 58C and seeded at this point with 30g of Form I. The batch was then cooled down at 5C/hour until it reached room temperature and then aged overnight at 2OC. Concentration of Form I in the MLs was 18 mg/g. A total of 15 L of heptane were charged over 1 hour and the batched aged for two more hours. Concentration of Form I in the MLs was 9.2 mg/g (it would reach 5 mg/g if aged longer). The solids were filtered off and washed with 2/1 v/v IP A/heptane (18L total volume). The solids were dried in the filter pot under nitrogen stream.

Solids obtained: 275Og; 90 wt%; 80.7% yield from the diacetate; 90.1 % yield for the crystallization.

PROCEDURE (PURE CRYSTALLIZATION FROM CH3CN/IPAC).

The solids previously isolated were charged into a 75 L round bottom flask, suspended in 11.9 L of acetonitrile and heated up to 56C until a clear solution was obtained. This solution was line filtered through a 1 μm filter into a 50 L round bottom flask. The temperature was brought back up to 52C, cooled down to 49C and seeded with 4Og of Form I. The mixture was cooled down at 3C/hour to room temperature and aged overnight at 2OC. Assay showed 21.8 mg/ml of Form I in the MLs. 7.1 L of IPAc were charged over Ih and the mixture aged for another 2 hours. Assay of the MLs showed 11.6 mg/ml. The slurry was filtered and washed with 4.8 L of CH ₃ CN/IPAc (5/3 v/v) followed by 4.8 L of IPAc. Solids were dried overnight in the filter pot under nitrogen stream.

Solids obtained: 2150g; 90 wt%; 88% yield.

HPLC Conditions

Column: Symmetry C- 18 5μm (Waters)

Flow Rate: 1 ml/min

Run Time: 10 min

Temp: 35 C

Solvents: A: Acetonitrile B: 0.1% H ₃ PO ₄ aq buffer.

Gradient:

Ret times: Form I: 3.3 min min

Monoacetate: 4.1 min Diacetate: 5.1 min.

Methyl ester: 3.7 min

Column: Sunfire Cl 8 5um Flow Rate: 1 ml/min Run Time: 43 min Temp: 5 C

Solvents: A: Acetonitrile B: 0.1% H ₃ PO ₄ aq buffer. Gradient:

Ret times: Form I: 21.4 min

Epi-OH: 22.5 min Olefin: 22.5 min Methyl ester: 32.0 min Monoacetate: 33.5 min Bissulfonamide: 37.1 min

¹ H NMR (400 MHz, CD ₃ OD), δ (ppm): 1.70-2.0 (m, 8H), 2.55-2.65 (m, 2H), 2.65-2.75 (m, 2H), 2.89 (s, 3H), 3.00-3.10 (m, 3H), 3.54 (s, 4H), 4.58-4.65 (m, IH), 4.78 (m, IH), 4.8 (b, OH), 7.03 (d, J= 8.7 Hz, IH), 7.05 (d, J= 8.7 Hz, IH), 7.10 (d, J= 8.3 Hz, 2H), 7.19 (d, J= 8.3 Hz, 2H), 7.25 (d, J= 8.4 Hz, 2H), 7.28 (d, J= 8.4 Hz, 2H), 7.31 (d, J= 7.9 Hz, 2H), 7.33 (d, J= 7.9 Hz, 2H).

¹³ C NMR (100 MHz, CD ₃ OD) (ppm): 26.3, 30.0, 33.1, 33.3, 37.2, 37.7, 39.6, 43.5, 61.1, 62.1, 65.8, 73.8, 75.5, 116.1 (d, J=22.1 Hz), 118.5, 127.4, 128.9 (d, J=8.0 Hz), 130.2, 130.4, 136.8, 137.0, 138.9, 142.4 (d, J=2.5 Hz), 144.9, 163.5 (d, J=237.5 Hz), 169.9 .

Crystallization Of Form I From Etoh- Water

MMee ° ³ ₂ ^e ₀ ^q _C ™ _2Q ^S _h ^0K NHSO ₂ Me

Crude triacetate starting material (10Og, 69.6 mmol) was dissolved in 400 mL of EtOH. To the resulting solution was added TMSOK (2.62g, 20.4 mmol) and the mixture was aged overnight at 21C. Reaction was complete by LC assay afte 16h age. The mixture was concentrated at reduced pressure and the resulting oil was chromatographed on silica gel using mixtures of dichloromethane and IPA as eluent. Chromatographic purification afforded 28.7g of an oily material (66% yield). This material was dissolved in 232 mL of EtOH and the resulting solution was warmed to 35C. 464 mL of water were then added and the solution was warmed to 41C. 150 mg of Form I seed were added. The mixture was cooled to 2OC over 3 hours and aged overnight at 2OC. Solids were filtered and washed with 150 mL of EtOH:H2O 1 :2 v/v. Obtained 25.Og of Form I of 98.7 wt%. Yield for the isolation was 87%.

Example 8

Crude Crystallization

Crude Compound A in dry MeCN (3 vol) was heated to 60°C and cooled to 45°C.

The solution was seeded with 0.5 wt% Form I. Toluene (10 vol) was charged over 4 h at 45°C.

The resulting slurry was cooled down to — 10 ⁰ C at a rate of 15°C/h. The resulting slurry was stirred at — 10°C for 6-8 h. The solid was collected and washed with 3:10 MeCN/toluene and toluene and dried under nitrogen to afford crude Form I.

Example 9

The product, as described in Example 8, was treated with Ecosorb (a kind of charcoal) or silica gel either before or after crude crystallization in order to remove impurities. It was then dissolved in MeCN (5 vol.) at 55-6O ⁰ C. The solution was cooled to 44-48 ⁰ C, and seeded with 2.5 wt% media milled seed (typical size of seed is < 5 urn), charged as a slurry in 5/3

v/v toluene/MeCN. After aging the seed bed for 30-60 minutes, toluene (8.33 vol.) was charged over 10 hrs while maintaining the batch at 44-48°C. Following toluene addition, the batch was cooled slowly to O ⁰ C over 6 hrs and aged at least 1 hr. The crystallized batch was then filtered and washed with 5/3 v/v toluene/MeCN and toluene. The washed cake was then dried under vacuum and nitrogen at 4O ⁰ C.

Example 10

The following are examples of pharmaceutical formulations comprised of Form I.

Form I is formulated as either dry filled capsules or compressed tablets in doses that generally will range from 1 mg to 250 mg of Form I. More generally, the doses will be in the range of 2-100 mg. A typical capsule or tablet formulation contains Form I, microcrystalline cellulose (Avicel), lactose monohydrate, croscarmellose sodium, magnesium stearate and/or sodium stearyl fumarate, and may also contain sodium lauryl sulfate. The capsule formulations are transferred to hard gelatin capsules. Tablet formulations may be coated with a film coat containing lactose, hypromellose, triacetin, titanium dioxide, and ferric oxide.

The formulations are manufactured by first blending Form I with the excipients, then compressing the mixture into ribbons by roller compaction, and then milling the ribbons into granules. The granules are then lubricated and either filled into capsules or compressed into tablets. If tablets are selected, a film coat may be applied to the compressed tablets.

Exemplary formulation compositions that provide lOmg and lOOmg dose of Form I are shown below. The examples provide the composition for uncoated compressed tablets which contain sodium lauryl sulfate as well as both magnesium stearate and sodium stearyl fumarate.

While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various changes,

modifications and substitutions can be made therein without departing from the spirit and scope of the invention. For example, effective dosages other than the particular dosages as set forth herein above may be applicable as a consequence of variations in the responsiveness of the mammal being treated for any of the indications for the active agent used in the instant invention as indicated above. It is intended, therefore, that the invention be defined by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.