WO2009158446A2 - Crystalline forms of ((((4-((5-(cyclopropylcarbamoyl)-2-methylphenyl)amino)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4-(phosphonooxy)phenyl)acetate, method of preparation and use thereof - Google Patents

Abstract

The present invention generally relates to crystalline forms of ( ( ( (4- ( (5- (cyclopropyl carbamoyl) -2-methylphenyl)amino) -5-methylpyrrolo [2,1-f ] [1, 2,4] triazin-6-yl) carbonyl) (propyl) carbamoyl )oxy) methyl (4- (phosphonooxy) phenyl) acetate. The present invention also generally relates to a pharmaceutical composition comprising said crystalline form, as well of methods of obtaining and using the crystalline form for the treatment of inflammatory-associated diseases or disorders.

Description

CRYSTALLINE FORMS OF ((((4-((5-(CYCLOPROPYLCARBAMOYL)-I- METHYLPHENYL)AMINO)-S-METHYLPYRROLOP₉I-F] [I₉I^]TRIAZIN-O-

YL)CARBONYL)(PROPYL)CARBAMOYL)OXY)METHYL (4- (PHOSPHONOOXY)PHENYL)ACETATE₉ METHOD OF PREPARATION AND USE THEREOF

[0001] This application claims priority from U.S. Provisional Application No. 61/075366, filed June 25, 2008, incorporated in its entirety herein by reference.

FIELD OF THE INVENTION [0002] The present invention generally relates to crystalline forms of ((((4-((5-

(cyclopropylcarbamoyl)-2-methylphenyl)amino)-5-methylpyrrolo[2,l-/|[l,2,4]triazin- 6-yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4-(phosphonooxy)phenyl)acetate. The present invention also generally relates to a pharmaceutical composition comprising said crystalline form, as well of methods of obtaining and using the crystalline form for the treatment of inflammatory-associated diseases.

BACKGROUND OF THE INVENTION

[0003] A large number of cytokines participate in the inflammatory response, including IL-I, IL-6, IL-8 and TNF-α. Overproduction of cytokines such as IL-I and TNF-α are implicated in a wide variety of diseases, including inflammatory bowel disease, rheumatoid arthritis, psoriasis, multiple sclerosis, endotoxin shock, osteoporosis, Alzheimer's disease, and congestive heart failure, among others [Henry et al, Drugs FuL, 24: 1345-1354 (1999); Salituro et al, Curr. Med. Chem., 6:807-823 (1999)]. Evidence in human patients indicates that protein antagonists of cytokines are effective in treating chronic inflammatory diseases, such as, for example, monoclonal antibody to TNF-α (Enbrel) [Rankin et al., Br. J. Rheumatol, 34:334-342 (1995)], and soluble TNF-α receptor-Fc fusion protein (Etanercept) [Moreland et al., Ann. Intern. Med., 130:478-486 (1999)]. [0004] The biosynthesis of TNF-α occurs in many cell types in response to an external stimulus, such as, for example, a mitogen, an infectious organism, or trauma. Important mediators of TNF-α production are the mitogen-activated protein (MAP) kinases, and in particular, p38 kinase. These kinases are activated in response to various stress stimuli, including but not limited to proinflammatory cytokines, endotoxin, ultraviolet light, and osmotic shock. Activation of p38 requires dual phosphorylation by upstream MAP kinase kinases (MKK3 and MKK6) on threonine and tyrosine within a Thr-Gly-Tyr motif characteristic of p38 isozymes. [0005] There are four known isoforms of p38, i.e., p38α, p38β, p38γ, and p38δ. The α and β isoforms are expressed in inflammatory cells and are key mediators of TNF-α production. Inhibiting the p38α and β enzymes in cells results in reduced levels of TNF-α expression. Also, administering p38α and β inhibitors in animal models of inflammatory disease has proven that such inhibitors are effective in treating those diseases. Accordingly, the p38 enzymes serve an important role in inflammatory processes mediated by IL-I and TNF-α. Compounds that reportedly inhibit p38 kinase and cytokines such as IL- 1 and TNF-α for use in treating inflammatory diseases are disclosed in U.S. Patent Nos. 6,277,989 and 6,130,235 to Scios, Inc; U.S. Patent. Nos. 6,147,080 and 5,945,418 to Vertex Pharmaceuticals Inc; U.S. Patent Nos. 6,251,914, 5,977,103 and 5,658,903 to Smith-Kline Beecham Corp.; U.S. Patent Nos. 5,932,576 and 6,087,496 to G.D. Searle & Co.; WO 00/56738 and WO 01/27089 to Astra Zeneca; WO 01/34605 to Johnson & Johnson; WO 00/12497 (quinazoline derivatives as p38 kinase inhibitors); WO 00/56738 (pyridine and pyrimidine derivatives for the same purpose); WO 00/12497 (discusses the relationship between p38 kinase inhibitors); and WO 00/12074 (piperazine and piperidine compounds useful as p38 inhibitors).

[0006] Other compounds that inhibit p38 kinase are pyrrolotriazine aniline compounds. Information on these compounds is disclosed in U.S. Patent Nos. 6,670,357; 6,867,300; 7,034,151; 7,160,883; 7,211,666; 7,253,167; and U.S. Application Publication Nos. 2003/0232831 (published Dec. 18, 2003);

2004/0229877 (published Nov. 18, 2004); 2005/0043306 (published Feb. 24, 2005; 2006/0003967 (published Jan. 5, 2006); 2006/0030708 (published Feb. 9, 2006); 2006/0041124 (published Feb. 23, 2006); 2006/0229449 (published Oct. 12, 2006); 2006/0235020 (published Oct. 19, 2006); and 2007/0213300 (published Sept. 13, 2007).

[0007] In particular, U.S. Patent Application Publication No. 2007/0213300A discloses the compound ((((4-((5-(cyclopropylcarbamoyl)-2-methylphenyl)amino)-5- methylpyrrolo[2,l-/|[l,2,4]triazin-6-yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4- (phosphonooxy)phenyl)acetate having the structure of formula I:

or pharmaceutically acceptable salts thereof, which are advantageous as inhibitors of p38 kinase and may be used for treating p38 kinase-associated conditions, including rheumatoid arthritis. The compound of formula I is also referred to herein as "Compound 1". Processes for preparing Compound 1 and methods of treatment employing Compound 1 are also disclosed in U.S. Patent Publication No. 2007/02133OOA. This patent is assigned to the present assignee and is incorporated herein by reference in its entirety.

[0008] In some cases it is desirable to find more optimal forms of such compounds. Properties of more desirable forms of pyrrolotriazine aniline compounds include improved solubility, stability, bioavailability, and/or storage stability. Particle size, particle shape and reproducibility of form may also be important considerations.

SUMMARY OF THE INVENTION

[0009] This invention comprises selected forms of a compound of Formula I (also referred to herein as "Compound 1") especially a first crystalline form having an IUPAC name, ((((4-((5-(cyclopropylcarbamoyl)-2-methylphenyl)amino)-5- methylpyrrolo[2,l-/|[l,2,4]triazin-6-yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4- (phosphonooxy)phenyl)acetate:

Compound 1 .

[0010] In particular, Compound 1 has very limited solubility in common organic solvents presenting a formidable challenge to crystallize suitable forms. This invention comprises a crystalline form selected from the group consisting of Forms HAC-4, P-2, and P-5.

[0011] Also described herein are processes for preparing the various forms. [0012] The first crystalline form of Compound 1 is HAC-4. [0013] A second crystalline form of Compound 1 is P-2. [0014] A third crystalline form of Compound 1 is P-5.

[0015] Further described herein are one or more pharmaceutical compositions comprising at least one crystalline form of Compound 1 and at least one pharmaceutically acceptable carrier and/or diluent. It is also contemplated that other embodiments of the invention will comprise at least one crystalline form of Compound 1, at least one pharmaceutically acceptable carrier and/or diluent, and, optionally, a second active ingredient particularly, for example, a second p38-kinase active agent.

BRIEF DESCRIPTION OF THE DRAWINGS [0016] Figure 1. Shows the simulated and observed powder x-ray diffraction ("PXRD") curves of Form HAC-4. [0017] Figure 2. Shows a differential scanning calorimetry analysis ("DSC") of

Form HAC-4.

[0018] Figure 3. Shows a thermogravimetric analysis ("TGA") of Form HAC-4.

[0019] Figure 4. Shows a SSNMR of Form HAC-4. [0020] Figure 5. Shows a moisture sorption curve of Form HAC-4.

[0021] Figure 6. Shows an observed PXRD curve of Form P-2.

[0022] Figure 7. Shows a DSC of Form P-2.

[0023] Figure 8. Shows a TGA of Form P-2.

[0024] Figure 9. Shows a SSNMR of Forms P-2/P-5. [0025] Figure 10. Shows a simulated and observed PXRD curves of Form P -5.

[0026] Figure 11. Shows a DSC of Form P-5.

[0027] Figure 12. Shows a TGA of Form P-5.

[0028] Figure 13. Shows a moisture uptake profile of Form HAC-4.

[0029] Figure 14. PXRD patterns for the P-2/P-5 system after a change in relative humidity. The bottom pattern is material dried from a propylene glycol slurry (P-5), the middle pattern is the material post 60% RH exposure, and the top pattern is the P-2 pattern reference.

[0030] Figure 15. Inhibition of paw swelling in the rat adjuvant arthritis model by Form HAC-4. [0031] Figure 16: Inhibition of paw swelling in the rat adjuvant arthritis model by Form P-2.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The features and advantages of the invention may be more readily understood by those of ordinary skill in the art upon reading the following detailed description. It is to be appreciated that certain features of the invention that are, for clarity reasons, described above and below in the context of separate embodiments, may also be combined to form a single embodiment. Conversely, various features of the invention that are, for brevity reasons, described in the context of a single embodiment, may also be combined so as to form sub-combinations thereof. [0033] The names used herein to characterize a specific crystalline form, e.g., "HAC-4.", is an identifier that is to be interpreted in accordance with the characterization information presented herein.

[0034] The definitions set forth herein take precedence over definitions set forth in any patent, patent application, and/or patent application publication incorporated herein by reference. All numbers expressing quantities of ingredients, weight percentages, temperatures, and so forth whether or not they are preceded by the word "about" are to be understood as only target approximations so that slight variations above and below the stated number may be used to achieve substantially the same results as the stated number. Accordingly, unless indicated to the contrary, numerical parameters whether or not they are preceded by the word "about" are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0035] All measurements are subject to experimental error and are within the spirit of the invention. [0036] As used herein, "polymorphs" refer to crystalline forms having the same chemical structure but different spatial arrangements of the molecules and/or ions forming the crystals.

[0037] As used herein, "amorphous" refers to a solid form of a molecule and/or ion that is not crystalline. An amorphous solid does not display a definitive X-ray diffraction pattern with sharp maxima. [0038] As used herein, the term "substantially pure" means the crystalline form of Compound 1 referred to which contains at least about 90 wt.%, based on the weight of such crystalline form. The term "at least about 90 wt.%," while not intending to limit the applicability of the doctrine of equivalents to the scope of the claims, includes, but is not limited to, for example, about 90, 90, about 91, 91, about 92, 92, about 93, 93, about 94, 94, about 95, 95, about 96, 96, about 97, 97, about 98, 98, about 99, 99, and about 100 wt. %, based on the weight of the crystalline form referred to. The remainder of the crystalline form of Compound 1 may comprise other Form(s) of Compound 1 and/or reaction impurities and/or processing impurities that arise, for example, when the crystalline form is prepared. For example, a crystalline form of Compound 1 may be deemed substantially pure if the crystalline form contains at least 90 wt. %, based on the weight of such crystalline form as measured by means that are at this time known and generally accepted in the art, and less than about 10 wt. %, based on the weight of such crystalline form, of material comprising other form(s) of Compound 1 and/or reaction impurities and/or processing impurities. The presence of reaction impurities and/or processing impurities may be determined by analytical techniques known in the art, such as, for example, chromatography, nuclear magnetic resonance spectroscopy, mass spectrometry, and/or infrared spectroscopy.

[0039] As used herein, the parameter "molecules/asymmetric unit" refers to the number of molecules of Compound 1 in the asymmetric unit. [0040] As used herein, the unit cell parameter "molecules/unit cell" refers to the number of molecules of Compound 1 in the unit cell.

[0041] When dissolved, the crystalline form of Compound 1 loses its crystalline structure, and is therefore referred to as a solution of Compound 1. At least one crystalline form of Compound 1 disclosed herein may be used to prepare at least one liquid formulation in which at least one crystalline form of Compound 1 is dissolved and/or suspended.

[0042] By "therapeutically effective amount" is meant an amount that when administered either alone, or in combination with an additional therapeutic agent is effective to prevent, suppress, and/or ameliorate a disease and/or condition and/or the progression of a disease and/or condition. [0043] Other definitions include the following:

[0044] For ease of reference, the following abbreviations are employed herein, including the methods of preparation and Examples that follow: a, b and c = unit cell lengths in Angstroms α, β, and γ = unit cell angles in degrees(°) Ac = acetic acid aq. = aqueous DSC = differential scanning calorimetry Et = ethyl

EtOAc = ethyl acetate

EtOCHO = ethyl formate

EtOH = ethanol g = gram(s) h = hour(s)

HPLC = high performance liquid chromatography iBuOH = isobutanol iPr or Iso-P or iso-p = isopropyl L or 1 = liter

LC/MS = high performance liquid chromatography/mass spectrometry

Me = methyl

MeOAc = methyl acetate

MeOH = methanol Meq = milliequivalent mg = milligram(s)

MIBK = methylisobutylketone min = minute(s) mL = milliliter mmol = millimole(s) mol = moles mp = melting point

MS or ms = mass spectrometry

MTBE = methyl tert-butyl ether NaH = sodium hydride

NaOH = sodium hydroxide

BuOAc = butyl acetate nBuOAc = n-butyl acetate

PG = propylene glycol nPrOAc = n-propyl acetate

NMR = nuclear magnetic resonance

Ph = phenyl Pr = propyl

PXRD = powder x-ray diffraction

R factor = crystallographic agreement factor which is a measure of the agreement between the crystallographic model and the experimental X-ray diffraction data rh = relative humidity ret. t. = HPLC retention time (minutes)

RP HPLC = reverse phase HPLC

RT or rt = room temperature sat or sat'd = saturated t-Bu = a tertiary butyl group

TFA = trifluoroacetic acid

TFE = 2,2,2 trifluoroethanol

TGA = thermogravimetric analysis

THF = tetrahydrofuran TLC = thin layer chromatography μg = microgram(s) μL or μl = microliter(s) μm = micromole(s)

V/Z = Unit cell volume/number of Compound 1 in the unit cell Z = molecules of Compound 1 in unit cell

Z'= Zprime = molecules of Compound 1 in the asymmetric unit

[0045] Disclosed herein are crystalline forms of Compound 1, including Forms

HAC-4; P-2, and P-5.

FORM EMBODIMENTS

Form HAC-4

[0046] A first crystalline form of Compound 1 comprises an acetic acid form of

Compound 1 referred to herein as "Form HAC-4" or "HAC-4 Form". The HAC-4 Form is the monoacetic acid solvate form of Compound 1. [0047] In one embodiment of Form HAC-4, the form is characterized by the unit cell parameters measured at room temperature (ca. 25"C) described in Table 1 as displayed in Example 1 (infra).

[0048] In a second embodiment of Form HAC-4, the HAC-4 Form is characterized by the fractional atomic coordinates substantially as listed in Table 2 displayed in Example 2 (infra).

[0049] In a third embodiment of Form HAC-4, the HAC-4 Form is characterized by a simulated powder x-ray diffraction (PXRD) pattern substantially in accordance with the observed and/or simulated PXRD pattern shown in Figure 1. [0050] In a fourth embodiment of Form HAC-4, the HAC-4 Form is characterized by a PXRD pattern (CuKa λ= 1.5418 A at a temperature of about 25°C) comprising four or more, preferably five or more, 2 theta ("2Θ") values selected from: 7.3, 9.3,

9.7, 11.6, 12.5, 16.6, 23.3, 24.5, and 25.3 (degrees 2Θ ±0.1).

[0051] In a fifth embodiment of Form HAC-4, the HAC-4 Form is characterized by a differential scanning calorimetry (DSC) thermogram substantially in accordance with that shown in Figure 3 having a melt/decomposition/desolvation onset typically at about 169 ⁰C.

[0052] In a sixth embodiment of Form HAC-4, the HAC-4 Form is characterized by a thermogravimetric analysis (TGA) thermogram, substantially the same as shown in Figure 4, having a negligible weight loss up to about 100 ⁰C, which agreed with the single crystal structure data.

[0053] In a seventh embodiment of Form HAC-4, the HAC-4 Form is characterized by a moisture-sorption isotherm substantially according to Figure 5 characterized by a 0.5% weight gain in the range of about 25% to about 75% RH at 25 ⁰C indicating that the HAC-4 form was non-hygroscopic.

[0054] In another embodiment of Form HAC-4, the HAC-4 Form is characterized by SSNMR substantially according to Figure 4.

Form P-2 [0055] A second crystalline form of Compound 1 comprises a nonstoichiometric hydrate form of Compound 1 referred to herein as "Form "P-2" or "P-2 Form". [0056] In a first embodiment of Form P-2, the P-2 Form is characterized by a simulated powder x-ray diffraction (PXRD) pattern substantially in accordance with the pattern shown in Figure 6.

[0057] In a second embodiment of Form P-2, the P-2 Form is characterized by a PXRD pattern (CuKa λ= 1.5418 at a temperature of about 25°C) comprising four or more, preferably five, 2 theta ("2Θ") values selected from:4.3, 5.2, 10.2, 11.3 and 15.3 (degrees 2Θ ±0.1).

[0058] In a third embodiment of Form P-2, the P-2 Form is characterized by a differential scanning calorimetry (DSC) thermogram substantially in accordance with Figure 7.

[0059] In a fourth embodiment of Form P-2, the P-2 Form is characterized by a thermogravimetric analysis (TGA) thermogram substantially according to Figure 8.

Form P-5 [0060] A third crystalline form of Compound 1 comprises a second nonstoichiometric hydrate form of Compound 1 referred to herein as "Form "P-5" or "P-5 Form".

[0061] In a first embodiment of Form P-5, the P-5 Form is characterized by a simulated powder x-ray diffraction (PXRD) pattern substantially in accordance with the pattern shown in Figure 9.

[0062] In a second embodiment of Form P-5, the P-5 Form is characterized by a PXRD pattern (CuKa λ= 1.5418 at a temperature of about 25°C) comprising four or more, preferably five, 2 theta ("2Θ") values selected from:6.1, 7.9, 8.9, 12.1 and 19.8 (degrees 2Θ ±0.1). [0063] In a third embodiment of Form P-5, the P-5 Form is characterized by a differential scanning calorimetry (DSC) thermogram substantially in accordance with Figure 10.

[0064] In a fourth embodiment of Form P-5, the P-5 Form is characterized by a thermogravimetric analysis (TGA) thermogram substantially in accordance with Figure 11.

PREPARATION EMBODIMENTS [0065] In one embodiment a process is provided for preparing the HAC-4 Form comprising the step of :hydrogenating ((((4-((5-(cyclopropylcarbamoyl)-2- methylphenyl)amino)-5-methylpyrrolo[2, 1 -fj [ 1 ,2,4]triazin-6- yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4- ((bis(benzyloxy)phosphoryl)oxy)phenyl)acetate (see U.S. Patent Application Publication No. 2007/0213300A, example 126, step 4) in acetic acid and water to afford a solid. The resultant solid produced during the hydrogenation and subsequent catalyst filtration is advantageously stable (-2% degradation at 50 ⁰C over 20 hrs) and may be easily filtered. The HAC-4 form can be directly crystallized from the post hydrogenation filtrate through a traditional crystallization procedure with acetate anti solvents, e.g., isopropyl acetate, ethyl acetate and/or butyl acetate, that allows for advantageous powder property control. In addition, the final product drying target is easily achieved. [0066] In another process of this embodiment, a slurry is provided comprising i) particles of the neat and/or solvent-hydrate mixed form, and ii) water to afford Form HAC-4. The water can be added during preparation of the slurry and/or after slurry preparation. The slurry may be prepared by dispersing the neat and/or solvent- hydrate mixed form into a solvent or solvent-water mixture. Suitable solvents include solvents that are miscible with water including, for example, methanol, ethanol, and organic acids such as acetic acid. The slurry may be subjected to mixing, for example, under high shear conditions. Suitable high shear conditions include a shear rate in the range of about 100 to about 300,000 s^"1, preferably from about 1000 to about 200,000 s^"1, and more preferably from about 5000 to about 100,000 s^"1. Other suitable high shear conditions for mixing devices using a rotator-stator configuration, such as a TURRAX® homogenizer (IKA, Wilmington, NC), include shear frequencies in the range of from about 100 to about 500,000 s^"1, preferably from about 1000 to about 300,000 s^"1, and more preferably from about 5000 to about 300,000 s^"1. [0067] In yet another embodiment of the invention, a process is provided for preparing Form P-2 of Compound 1. The P-2 form is prepared by crystallization from racemic propylene glycol (PG)/MeOH/EtOAc (thus generating a P-2 Form material that contains PG by solution NMR) and then re-slurrying this material in water to form a "non-stoichiometric hydrate" which is then dried (at 30% relative humidity ("rh")) to 5-7% w/w water. This produces a hydroscopic solid with 5-7% weight gain in-between 25-75% room humidity at 25°C for the pre-dried solid. Differential Scanning Calorimetry (DSC) shows a broad endotherms at ca. room temperature to ca. 50⁰C and another from ca. 150 ⁰C to ca. 170 ⁰C.

[0068] Materials resulting from the addition of amino acids (L-AIa, L-Ser, L-VaI and L-GIy) compose yet further embodiments of the present invention and are also characterized by the P-2 PXRD pattern. However, the samples contain the amino acids by elemental analysis and NMR. The L-AIa system has been investigated and materials containing 0.7-0.9 moles of L-AIa do not appear to be hygroscopic and have a sharper endotherm in the DSC (~190°C) than other members of the P-2 group. [0069] In yet another embodiment of the invention, a process is provided for preparing Form P-5 of Compound 1 wherein the non-stoichiometric hydrate, Form P- 2, can be dried under high vacuum in a dry atmosphere to produce solids having the P-5 form. Exposure of the solids to 25-75% room humidity at 25°C will result in a weight gain to 5-7% water and a form conversion back to P-2.

OTHER EMBODIMENTS

[0070] For each of the Forms described above, various purities and compositions can be obtained.

[0071] For each of the Forms described above an embodiment may be obtained which is substantially pure.

[0072] For each of the Forms described above another embodiment may be obtained which contains at least about 90 wt.%, preferably at least about 95 wt.%, and more preferably at least about 99 wt.%, based on weight of the particular crystalline form.

[0073] For each of the Forms described above yet another embodiment may be found which is a substantially pure crystalline form with a substantially pure phase homogeneity of less than about 10%, preferably less than about 5%, and more preferably less than about 2% of the total peak area of the experimentally measured

PXRD pattern arising from peaks that are absent from the simulated PXRD pattern.

Most preferably, such a substantially pure crystalline form has substantially pure phase homogeneity with less than about 1% of the total peak area of the experimentally measured PXRD pattern arising from peaks that are absent from the simulated PXRD pattern.

[0074] For each of the Forms described above yet another embodiment may be found which consists essentially of the particular form (for example, Form HAC-4). The particular crystalline form of such an embodiment may comprise at least about 90 wt. %, preferably at least about 95 wt. %, and more preferably at least about 99 wt. %, based on the weight of the particular crystalline form (for example, Form HAC-4). [0075] For each of the Forms described above yet another embodiment may be found which comprises a particular form (for example, Form HAC-4) and at least one pharmaceutically-acceptable carrier and/or diluent.

[0076] For each of the Forms described above yet another embodiment may be found which comprises a substantially pure crystalline form; and at least one pharmaceutically-acceptable carrier and/or diluent. [0077] For each of the Forms described above yet another embodiment may be found in which a particular form (for example, Form HAC-4) is combined with at least one pharmaceutically acceptable carrier and/or diluent to provide at least one pharmaceutical composition. [0078] In still another embodiment is provided a method for treating a inflammatory-associated disease or disorder comprising administering to a patient in need thereof a therapeutically effective amount of Compound 1, wherein Compound 1 is provided in a crystalline form comprising at least one form selected from the group consisting of Form HAC-4, P-2, and P-5. [0079] In one embodiment, the patient is a human. [0080] In another embodiment, the inflammatory-associated disorder or disease is selected from the group consisting of asthma, acute respiratory distress syndrome, chronic obstructive pulmonary disease, chronic pulmonary inflammatory disease, diabetes, inflammatory bowel disease, Alzheimer's disease, osteoporosis, psoriasis, graft vs. host rejection, multiple sclerosis, atherosclerosis, multiple myeloma, pain, myocardial ischemia and arthritis, including rheumatoid arthritis, psoriatic arthritis, traumatic arthritis, rubella arthritis, gouty arthritis and osteoarthritis. [0081] In another embodiment, the disorder or disease is selected from the group consisting of rheumatoid arthritis, psoriasis, asthma, COPD, ARDS, inflammatory bowel disease, multiple myeloma, pain, atherosclerosis, osteoporosis, myocardial ischemia and ischemia, particularly psoriasis, atherosclerosis and rheumatoid arthritis; and more particularly, rheumatoid arthritis.

[0082] In a further embodiment, the method of treating an inflammatory- associated disorder or disease comprises administering a crystalline form of Form HAC-4, P -2, and/or P-5 to a patient in need thereof. [0083] ((((4-((5-(cyclopropylcarbamoyl)-2-methylphenyl)amino)-5- methylpyrrolo[2, 1 -f\ [ 1 ,2,4]triazin-6-yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4- (phosphonooxy)phenyl)acetate has very limited solubility in common organic solvents. However, its remarkable solubility in acetic acid provided the key to the crystallization of a form that is unexpectedly stable, both chemically and physically. [0084] As a phosphate ester prodrug, ((((4-((5-(cyclopropylcarbamoyl)-2- methylphenyl)amino)-5-methylpyrrolo[2, 1 -f\ [ 1 ,2,4]triazin-6- yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4-(phosphonooxy)phenyl)acetate is susceptible to hydrolysis. As discussed in Example 4, infra, within three months of storage under stress conditions, hydrolysis reactions produce two degradation products in unacceptably high amounts: the parent,

and the des-phosphate analog,

[0085] Generally, ester prodrugs demonstrate sensitivity to acid catalyzed hydrolysis. Accordingly, one would expect a reduction in stability and production of higher levels of hydrolysis products would occur in crystals containing acid. However, as discussed in more detail in Example 4, infra, the monoacetic acid form, the HAC-4 form, is significantly more stable across all conditions studied. In fact, as the data demonstrates, the HAC-4 form is even more stable than the hydrated form. [0086] Moreover, as discussed in Example 5, infra, the HAC-4 form is non- hygroscopic and thus does not undergo physical form changes upon exposure to varying relative humidities. As shown in Figure 13, weight changes of less than 1% are observed when exposing HAC-4 to relative humidities of 0 to 90%. Thus, in addition to the desirable chemical stability, this physical stability makes HAC-4 form much more desirable to handle, manufacture, formulate, and store and thus a superior pharmaceutical development candidate. [0087] Examples of other forms of Compound 1 are disclosed in a copending U.S. Patent Application, Attorney Docket No.11288.

UTILITY

[0088] The compounds of the invention are prodrugs that release selective inhibitors of p38 kinase activity, and in particular, isoforms p38α and p38β.

Accordingly, compounds of Formula I have utility in treating conditions associated with p38 kinase activity. Such conditions include diseases in which cytokine levels are modulated as a consequence of intracellular signaling via p38, and in particular, diseases that are associated with an overproduction of cytokines IL-I, IL-4, IL-8, and TNF-α. As used herein, the terms "treating" or "treatment" encompass either or both responsive and prophylaxis measures, e.g., measures designed to inhibit or delay the onset of the disease or disorder, achieve a full or partial reduction of the symptoms or disease state, and/or to alleviate, ameliorate, lessen, or cure the disease or disorder and/or its symptoms. When reference is made herein to inhibition of "p38α/β kinase," this means that either p38α and/or p38β kinase are inhibited. Thus, reference to an IC50 value for inhibiting p38α/β kinase means that the compound has such effectiveness for inhibiting at least one of, or both of, p38α and p38β kinases. [0089] In view of their utility as prodrugs that release inhibitors of p38α/β kinase activity, the compounds of Formula I are useful in treating p38 associated conditions including, but not limited to, inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, angiogenic disorders, infectious diseases, neurodegenerative diseases, and viral diseases.

[0090] More particularly, the specific conditions or diseases that may be treated with the inventive compounds include, without limitation, lupus nephritis, pancreatitis (acute or chronic), asthma, allergies, adult respiratory distress syndrome, chronic obstructive pulmonary disease, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosis, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, atopic dermatitis, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, psoriasis, graft vs. host disease, inflammatory reaction induced by endotoxin, tuberculosis, atherosclerosis, muscle degeneration, cachexia, psoriatic arthritis, Reiter's syndrome, gout, traumatic arthritis, rubella arthritis, acute synovitis, pancreatic β-cell disease; diseases characterized by massive neutrophil infiltration; rheumatoid spondylitis, ankylosing spondylitis, gouty arthritis and other arthritic conditions, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoidosis, bone resorption disease, allograft rejections, fever and myalgias due to infection, cachexia secondary to infection, myeloid formation, scar tissue formation, ulcerative colitis, pyresis, influenza, osteoporosis, osteoarthritis and multiple myeloma-related bone disorder, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, multiple myeloma, sepsis, septic shock, and Shigellosis; Alzheimer's disease, Parkinson's disease, cerebral ischemias or neurodegenerative disease caused by traumatic injury; angiogenic disorders including solid tumors, ocular neovasculization, and infantile haemangiomas; viral diseases including acute hepatitis infection (including hepatitis A, hepatitis B and hepatitis C), HIV infection and CMV retinitis, AIDS, ARC or malignancy, and herpes; stroke, myocardial ischemia, ischemia in stroke heart attacks, organ hyposia, vascular hyperplasia, cardiac and renal reperfusion injury, thrombosis, cardiac hypertrophy, thrombin-induced platelet aggregation, endotoxemia and/or toxic shock syndrome, and conditions associated with prostaglandin endoperoxidase syndase-2. [0091] In addition, p38 inhibitors of this invention inhibit the expression of inducible pro-inflammatory proteins such as prostaglandin endoperoxide synthase-2 (PGHS-2), also referred to as cyclooxygenase-2 (COX-2). Accordingly, additional p38-associated conditions include edema, analgesia, fever and pain, such as neuromuscular pain, headache, pain caused by cancer, dental pain and arthritis pain. The inventive compounds also may be used to treat veterinary viral infections, such as lentivirus infections, including, but not limited to equine infectious anemia virus; or retro virus infections, including feline immunodeficiency virus, bovine immunodeficiency virus, and canine immunodeficiency virus. [0092] When the terms "p38 associated condition" or "p38 associated disease or disorder" are used herein, each is intended to encompass all of the conditions identified above as if repeated at length, as well as any other condition that is affected by p38 kinase activity.

[0093] The present invention thus provides methods for treating such conditions, comprising administering to a subject in need thereof an effective amount of at least one compound of Formula I or a salt thereof. The methods of treating p38 kinase- associated conditions may comprise administering compounds of Formula I alone or in combination with each other and/or other suitable therapeutic agents useful in treating such conditions. Exemplary of such other therapeutic agents include corticosteroids, rolipram, calphostin, CSAIDs, 4-substituted imidazo [1,2- AJquinoxalines as disclosed in U.S. Patent No. 4,200,750; Interleukin-10, glucocorticoids, salicylates, nitric oxide, and other immunosuppressants; nuclear translocation inhibitors, such as deoxyspergualin (DSG); non-steroidal anti- inflammatory drugs (NSAIDs) such as ibuprofen, celecoxib and rofecoxib; steroids such as prednisone or dexamethasone; antiviral agents such as abacavir; antiproliferative agents such as methotrexate, leflunomide, FK506 (tacrolimus, Prograf); cytotoxic drugs such as azathiprine and cyclophosphamide; TNF-α inhibitors such as tenidap, anti-TNF antibodies or soluble TNF receptor, and rapamycin (sirolimus or Rapamune) or derivatives thereof.

[0094] The above other therapeutic agents, when employed in combination with the compounds of the present invention, may be used, for example, in those amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art. In the methods of the present invention, such other therapeutic agent(s) may be administered prior to, simultaneously with, or following the administration of the inventive compounds.

[0095] The present invention also provides pharmaceutical compositions capable of treating p38-kinase associated conditions, including TNF-α, IL-I, and/or IL-8 mediated conditions, as described above. The inventive compositions may contain other therapeutic agents as described above and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (e.g., excipients, binders, preservatives, stabilizers, flavors, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.

[0096] The compounds of Formula I may be administered by any means suitable for the condition to be treated, which may depend on the need for site-specific treatment or quantity of drug to be delivered. Topical administration is generally preferred for skin-related diseases, and systematic treatment preferred for cancerous or pre-cancerous conditions, although other modes of delivery are contemplated. For example, the compounds may be delivered orally, such as in the form of tablets, capsules, granules, powders, or liquid formulations including syrups; topically, such as in the form of solutions, suspensions, gels or ointments; sublingually; bucally; parenterally, such as by subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques (e.g., as sterile injectable aq. or non-aq. solutions or suspensions); nasally such as by inhalation spray; topically, such as in the form of a cream or ointment; rectally such as in the form of suppositories; or liposomally. Dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents may be administered. The compounds may be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved with suitable pharmaceutical compositions or, particularly in the case of extended release, with devices such as subcutaneous implants or osmotic pumps.

[0097] Exemplary compositions for topical administration include a topical carrier such as PLASTIBASE® (mineral oil gelled with polyethylene). [0098] Exemplary compositions for oral administration include suspensions which may contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents such as those known in the art; and immediate release tablets which may contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and/or lactose and/or other excipients, binders, extenders, dis integrants, diluents and lubricants such as those known in the art. The inventive compounds may also be orally delivered by sublingual and/or buccal administration, e.g., with molded, compressed, or freeze- dried tablets. Exemplary compositions may include fast-dissolving diluents such as mannitol, lactose, sucrose, and/or cyclodextrins. Also included in such formulations may be high molecular weight excipients such as celluloses (AVICEL®) or polyethylene glycols (PEG); an excipient to aid mucosal adhesion such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (SCMC), and/or maleic anhydride copolymer (e.g., GANTREZ®); and agents to control release such as polyacrylic copolymer (e.g., CARBOPOL 934®). Lubricants, glidants, flavors, coloring agents and stabilizers may also be added for ease of fabrication and use.

[0099] Exemplary compositions for nasal aerosol or inhalation administration include solutions which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance absorption and/or bioavailability, and/or other solubilizing or dispersing agents such as those known in the art.

[00100] Exemplary compositions for parenteral administration include injectable solutions or suspensions which may contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. [00101] Exemplary compositions for rectal administration include suppositories which may contain, for example, suitable non-irritating excipients, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures but liquefy and/or dissolve in the rectal cavity to release the drug. [00102] The effective amount of a compound of the present invention may be determined by one of ordinary skill in the art, and includes exemplary dosage amounts for a mammal of from about 0.05 to 100 mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. It will be understood that the specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition. Preferred subjects for treatment include animals, most preferably mammalian species such as humans, and domestic animals such as dogs, cats, horses, and the like. Thus, when the term "patient" is used herein, this term is intended to include all subjects, most preferably mammalian species, that are affected by mediation of p38 enzyme levels. [00103] Compound 1 is useful for its p38-kinase activity. Compound 1 may be used to treat a variety of medical conditions and/or disorders associated with inflammation.. Compound 1 can modulate the function of p38 kinases. In one embodiment, Compound 1 inhibits p38α/β enzymes. Medical conditions associated with p38 kinase activity include asthma, adult respiratory distress syndrome, chronic obstructive pulmonary disease, chronic pulmonary inflammatory disease, diabetes, inflammatory bowel disease, Alzheimer's disease, osteoporosis, psoriasis, graft vs. host rejection, atherosclerosis, multiple myeloma, pain, myocardial ischemia and arthritis, including rheumatoid arthritis, psoriatic arthritis, traumatic arthritis, rubella arthritis, gouty arthritis and osteoarthritis. Compound 1 can be used to treat all the foregoing conditions.

[00104] In one embodiment, a method for treating an inflammatory disorder (disorder as used herein is also intended to be used interchangeably with the term "disease") comprises administering to a patient in need of such treatment a therapeutically effective amount of Compound 1, wherein Compound 1 is provided in a crystalline form comprising at least one form selected from Form HAC-4, P-2, and P-5; optionally administering either simultaneously or sequentially at least one other anti-inflammatory agent, and optionally administering either simultaneously or sequentially at least one other anti-inflammatory treatment. Preferably, in the method of this embodiment, Compound 1 is provided in Form HAC-4. Preferably, the inflammatory-associated disease is selected from the group consisting of asthma, adult respiratory distress syndrome, chronic obstructive pulmonary disease, chronic pulmonary inflammatory disease, diabetes, inflammatory bowel disease, Alzheimer's disease, osteoporosis, psoriasis, graft vs. host rejection, atherosclerosis, multiple myeloma, pain, myocardial ischemia and arthritis, including rheumatoid arthritis, psoriatic arthritis, traumatic arthritis, rubella arthritis, gouty arthritis and osteoarthritis; and, more preferably, atherosclerosis, rheumatoid arthritis, and psoriasis. [00105] In a further embodiment, a method for treating at least one inflammatory- associated disease comprises administering to a patient in need of such treatment a therapeutically effective amount of Compound 1, wherein Compound 1 is provided in a crystalline form comprising at least one of Forms HAC-4, P-2, and/or P-5 (preferably Form HAC-4); optionally administering either simultaneously or sequentially at least one other anti-inflammatory agent, and optionally administering either simultaneously or sequentially at least one other anti-inflammatory treatment. Preferably, the inflammatory-associated disease is selected from the group consisting of asthma, adult respiratory distress syndrome, chronic obstructive pulmonary disease, chronic pulmonary inflammatory disease, diabetes, inflammatory bowel disease, Alzheimer's disease, osteoporosis, psoriasis, graft vs. host rejection, atherosclerosis, multiple myeloma, pain, myocardial ischemia and arthritis, including rheumatoid arthritis, psoriatic arthritis, traumatic arthritis, rubella arthritis, gouty arthritis and osteoarthritis, ankylosing spondylitis, multiple sclerosis, lupus nephritis, Crohn's disease and ulcerative colitis. More preferable inflammatory-associated diseases are selected from chronic obstructive pulmonary disease, psoriasis, atherosclerosis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, lupus nephritis, Crohn's disease or ulcerative colitis, and even more preferably, atherosclerosis, rheumatoid arthritis, and psoriasis. [00106] In another embodiment, the method for treating at least one inflammatory- associated disease involves providing Compound 1 in a substantially pure form. [00107] In another embodiment, a pharmaceutical composition comprises at least one crystalline form of Compound 1 comprising Form HAC-4, P-2, and P-5; optionally at least one component selected from excipients and carriers; and optionally at least one other anti-inflammatory agent. Preferably, the pharmaceutical composition of this embodiment comprises Form HAC-4. [00108] The phrase "other anti-inflammatory agent" includes any known agent useful for treating an inflammatory disorder, preferably atherosclerosis, rheumatoid arthritis, and psoriasis. In treating cancer, a combination of therapeutic agents and/or other treatments is often advantageous. The other anti-inflammatory agent may have the same or different mechanism of action than the primary therapeutic agent. It may be especially useful to employ anti-inflammatory drug combinations wherein the two or more drugs being administered act in different manners or in different phases of the cell cycle, and/or where the drugs being combined each has a demonstrated efficacy in treating the particular disease state manifested by the patient. [00109] The invention herein further comprises use of Form HAC-4, P-2, and/or P- 5 in preparing medicaments for the treatment of inflammatory-associated disorders, and/or it comprises the packaging of Form HAC-4, P-2, and/or P-5 herein together with instructions that it is to be used in combination with other anti-inflammatory agents and treatments for the treatment of inflammation-associated diseases. [00110] The present invention further comprises combinations of Form HAC-4, P- 2, and/or P-5 and one or more additional agents in kit form, e.g., where they are packaged together or placed in separate packages to be sold together as a kit, or where they are packaged to be formulated together. [00111] Additionally, forms of Compound 1 can be formulated or co-administered with other therapeutic agents that are selected for their particular usefulness in addressing side effects associated with the aforementioned conditions. [00112] The above other therapeutic agents, when employed in combination with forms of Compound 1, can be used, for example, in those amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

[00113] In one embodiment, Form HAC-4, P-2, and/or P-5, particularly HAC-4, is used to treat rheumatoid arthritis. [00114] In another embodiment, Form HAC-4, P-2, and/or P-5 of Compound 1 is used to treat psoriasis.

[00115] In another embodiment, Form HAC-4, P-2, and/or P-5 of Compound 1 is used to treat atherosclerosis. [00116] In one embodiment, Form HAC-4, P-2, and/or P-5, particularly HAC-4, is used to treat pain.

[00117] In one embodiment, Form HAC-4, P-2, and P-5, particularly HAC-4, of Compound 1 is used to treat multiple sclerosis.

[00118] Any pharmaceutical composition contemplated herein can, for example, be delivered orally via any acceptable and suitable oral preparations. Exemplary oral preparations, include, but are not limited to, for example, tablets; troches; lozenges; aqueous or oily suspensions; dispersible powders or granules; emulsions; hard or soft capsules; syrups; and elixirs. Pharmaceutical compositions intended for oral administration can be prepared according to methods known in the art and can contain at least one agent selected from sweetening agents, flavoring agents, coloring agents, demulcents, antioxidants, and preserving agents.

[00119] Exemplary excipients include, but are not limited to, for example, inert diluents, such as, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate; granulating and disintegrating agents, such as, for example, microcrystalline cellulose, sodium croscarmellose, corn starch, and alginic acid; binding agents, such as, for example, starch, gelatin, polyvinyl-pyrrolidone, and acacia; and lubricating agents, such as, for example, magnesium stearate, stearic acid, and talc. [00120] An aqueous suspension can be prepared, for example, by admixing at least one of Form HAC-4, P-2, and/or P-5 with at least one excipient suitable for the manufacture of an aqueous suspension. Exemplary excipients suitable for the manufacture of an aqueous suspension, include, but are not limited to, for example, suspending agents, such as, for example, sodium carboxymethylcellulose or methylcellulose. Oily suspensions can, for example, be prepared by suspending at least one of Form HAC-4, P-2, and/or P-5 in either a vegetable oil, such as, for example, arachis oil; olive oil; sesame oil; and coconut oil; or in mineral oil, such as, for example, liquid paraffin. [00121] Any pharmaceutical composition contemplated herein can, for example, also be delivered intravenously, subcutaneously, and/or intramuscularly via any pharmaceutically acceptable and suitable injectable form. Exemplary injectable forms include, but are not limited to, for example, sterile aqueous solutions comprising acceptable vehicles and solvents, such as, for example, water, Ringer's solution, and isotonic sodium chloride solution; sterile oil-in-water microemulsions; and aqueous or oleaginous suspensions.

[00122] A sterile injectable oil-in-water microemulsion can, for example, be prepared by 1) dissolving at least one crystalline form of Compound 1 in an oily phase, such as, for example, a mixture of soybean oil and lecithin; 2) combining the crystalline form of Compound 1 containing oil phase with a water and glycerol mixture; and 3) processing the combination to form a microemulsion. [00123] Any pharmaceutical composition contemplated herein can, for example, further be administered via any acceptable and suitable rectal preparation, including, but not limited to, for example, a suppository. A suppository can be prepared by mixing at least one crystalline form of Compound 1 with at least one suitable non- irritating excipient that is liquid at rectal temperatures but solid at a temperature below rectal temperature.

[00124] Any pharmaceutical composition contemplated herein can, for example, be administered via any acceptable and suitable topical preparations including, but not limited to, for example, creams; ointments; jellies; solutions; suspensions, transdermal patches; and intranasal inhalers. For purposes of this application, topical preparations include mouth washes and gargles. [00125] Exemplary compositions for nasal aerosol or inhalation administration include solutions that may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance absorption and/or bioavailability, and/or other solubilizing or dispersing agents such as those known in the art. [00126] An "effective amount" of a crystalline form of Compound 1 may be determined by one of ordinary skill in the art, and includes exemplary dosage amounts for a mammal of from about 0.05 to about 300 mg/kg/day, preferably less than about 200 mg/kg/day, in a single dose or in 2 to 4 divided doses. The specific dose level and frequency of dosage for any particular subject, however, may be varied and generally depends on a variety of factors, including, but not limited to, for example, the bioavailability of crystalline form of Compound 1 in the administered form; metabolic stability and length of action of the crystalline form of Compound 1; species, age, body weight, general health, sex, and diet of the subject; mode and time of administration; rate of excretion; drug combination; and severity of the particular condition.

[00127] In one embodiment, the patient is an animal.

[00128] In one embodiment, the patient is a human.

[00129] In yet another embodiment, the patient is a mammalian species including, but not limited to, for example, humans and domestic animals, such as, for example, dogs, cats, and horses.

BIOLOGICAL ASSAYS Generation of p38 Kinases [00130] cDNAs of human p38α, β and γ isozymes were cloned by PCR. These cDNAs were subcloned in the pGEX expression vector (Pharmacia). GST-p38 fusion protein was expressed in E. CoIi and purified from bacterial pellets by affinity chromatography using glutathione agarose. p38 fusion protein was activated by incubating with constitutively active MKK6. Active p38 was separated from MKK6 by affinity chromatography. Constitutively active MKK6 was generated according to Raingeaud et al. [MoL Cell. Biol., 1247-1255 (1996)].

TNF-α Production by LPS-Stimulated PBMCs [00131] Heparinized human whole blood was obtained from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were purified from human whole blood by Ficoll-Hypaque density gradient centrifugation and resuspended at a concentration of 5 x 10⁶/ml in assay medium (RPMI medium containing 10% fetal bovine serum). 50 μl of cell suspension was incubated with 50 μl of test compound (4X concentration in assay medium containing 0.2% DMSO) in 96-well tissue culture plates for 5 minutes at RT. 100 μ of LPS (200 ng/ml stock) was then added to the cell suspension and the plate was incubated for 6 hours at 37°C. Following incubation, the culture medium was collected and stored at -20⁰C. TNF-α concentration in the medium was quantified using a standard ELISA kit (Pharmingen-San Diego, CA). Concentrations of TNF-α and IC50 values for test compounds (concentration of compound that inhibited LPS-stimulated TNF-α production by 50%) were calculated by linear regression analysis.

p38 Assay

[00132] The assays were performed in V-bottomed 96-well plates. The final assay volume was 60 μl prepared from three 20 μl additions of enzyme, substrates (MBP and ATP) and test compounds in assay buffer (50 mM Tris pH 7.5, 10 mM MgCl₂, 50 mM NaCl and 1 mM DTT). Bacterially expressed, activated p38 was pre-incubated with test compounds for 10 min. prior to initiation of reaction with substrates. The reaction was incubated at 25°C for 45 min. and terminated by adding 5 μl of 0.5 M EDTA to each sample. The reaction mixture was aspirated onto a pre-wet filtermat using a Skatron Micro96 Cell Harvester (Skatron, Inc.), then washed with PBS. The filtermat was then dried in a microwave oven for 1 min., treated with MeltilLex A scintillation wax (Wallac), and counted on a Microbeta scintillation counter Model 1450 (Wallac). Inhibition data were analyzed by nonlinear least-squares regression using Prizm (GraphPadSoftware). The final concentration of reagents in the assays are ATP, 1 μM; [γ-³³P]ATP, 3 nM; MBP (Sigma, #M1891), 2μg/well; p38, 10 nM; and DMSO, 0.3%.

TNF-α Production by LPS-Stimulated Mice [00133] Mice (Balb/c female, 6-8 weeks of age, Harlan Labs; n=8/treatment group) were injected intraperitoneally with 50ug/kg lipopolysaccharide (LPS; E coli strain 0111 :B4, Sigma) suspended in sterile saline. Ninety minutes later, mice were sedated by Cθ₂:θ₂ inhalation and a blood sample was obtained. Serum was separated and analyzed for TNF-alpha concentrations by commercial ELISA assay per the manufacturer's instructions (R&D Systems, Minneapolis, MN). [00134] Test compounds were administered orally at various times before LPS injection. The compounds were dosed either as suspensions or as solutions in various vehicles or solubilizing agents.

METHODS OF PREPARATION AND CHARACTERIZATION [00135] Crystalline forms may be prepared by a variety of methods, including, but not limited to, for example, crystallization or recrystallization from a suitable solvent mixture; sublimation; growth from a melt; solid state transformation from another phase; crystallization from a supercritical fluid; and jet spraying. Techniques for crystallization or recrystallization of crystalline forms from a solvent mixture include, but are not limited to, for example, evaporation of the solvent; decreasing the temperature of the solvent mixture; crystal seeding a supersaturated solvent mixture of the compound and/or a salt from thereof; freeze drying the solvent mixture; and adding antisolvents (countersolvents) to the solvent mixture. High throughput crystallization techniques may be employed to prepare crystalline forms including polymorphs. Crystals of drugs, including polymorphs, methods of preparation, and characterization of drug crystals are discussed in Byrn, S. R. et al., SoHd-State Chemistry of Drugs, 2^nd Edition, SSCI, West Lafayette, Indiana (1999). [00136] In a crystallization technique in which solvent is employed, the solvent(s) are typically chosen based on one or more factors including, but not limited to, for example, solubility of the compound; crystallization technique utilized; and vapor pressure of the solvent. Combinations of solvents may be employed. For example, the compound may be solubilized in a first solvent to afford a solution to which antisolvent is then added to decrease the solubility of the Compound In the solution and precipitate the formation of crystals. An antisolvent is a solvent in which a compound has low solubility. [00137] In one method that can be used in preparing crystals, a Compound 1 suspended and/or stirred in a suitable solvent to afford a slurry, which may be heated to promote dissolution. The term "slurry", as used herein, means a saturated solution of the compound, wherein such solution may contain an additional amount of compound to afford a heterogeneous mixture of compound and solvent at a given temperature.

[00138] Seed crystals may be added to any crystallization mixture to promote crystallization. Seeding may be employed to control growth of a particular polymorph and/or to control the particle size distribution of the crystalline product. Accordingly, calculation of the amount of seeds needed depends on the size of the seed available and the desired size of an average product particle as described, for example, in Mullin, J. W. et al., "Programmed Cooling of Batch Crystallizers", Chemical Engineering Science, 26:369-377 (1971). In general, seeds of small size are needed to effectively control the growth of crystals in the batch. Seeds of small size may be generated by sieving, milling, or micronizing large crystals, or by micro- crystallizing a solution. In the milling or micronizing of crystals, care should be taken to avoid changing crystallinity from the desired crystalline form (i.e., changing to an amorphous or other polymorphic form). [00139] A cooled crystallization mixture may be filtered under vacuum and the isolated solid product washed with a suitable solvent, such as, for example, cold recrystallization solvent. After being washed, the product may be dried under a nitrogen purge to afford the desired crystalline form.

[00140] The product may be analyzed by a suitable spectroscopic or analytical technique including, but not limited to, for example, solid state nuclear magnetic resonance; differential scanning calorimetry (DSC); and powder x-ray diffraction (PXRD) to assure the preferred crystalline form of the compound has been formed. The resulting crystalline form may be produced in an amount greater than about 70 wt. % isolated yield, based on the weight of the compound originally employed in the crystallization procedure, and preferably greater than about 90 wt. % isolated yield. Optionally, the product may be delumped by being comilled or passed through a mesh screen. [00141] Crystalline forms of Compound 1 including, but not limited to, for example, the Forms described herein, may be prepared directly from the reaction medium produced via the final process step employed in preparing Compound 1. For example, crystalline form(s) of Compound 1 could be produced by employing a solvent or a mixture of solvents in the final process step employed in preparing Compound 1. Alternatively, crystalline forms of Compound 1 may be obtained by distillation or solvent addition techniques. Suitable solvents for this purpose include, but are not limited to, for example, the aforementioned nonpolar and polar solvents, wherein polar solvents include, but are not limited to, for example, protic polar solvents, such as, for example, alcohols and aprotic polar solvents, such as, for example, ketones.

[00142] The presence of more than one crystalline form and/or polymorph in a sample may be determined by techniques, including, but not limited to, for example, PXRD and solid state nuclear magnetic resonance spectroscopy. For example, the presence of extra peaks when an experimentally measured PXRD pattern is compared to a simulated PXRD pattern may indicate more than one crystalline form and/or polymorph in the sample. The simulated PXRD may be calculated from single crystal x-ray data. See, for example, Smith, D.K., "A FORTRAN Program for Calculating X-Ray Powder Diffraction Patterns" Lawrence Radiation Laboratory, Livermore, California, UCRL-7196 (April 1963).

[00143] Crystalline forms of Compound 1, including, but not limited to, those described herein according to the invention may be characterized using a variety of techniques well known to person(s) of ordinary skill in the art. For example, the single x-ray diffraction technique may, under standardized operating conditions and temperatures, be used to characterize and distinguish crystalline form(s) of Compound 1. Such characterization may, for example, be based on unit cell measurements of a single crystal of the desired form at a fixed analytical temperature. The approximate unit cell dimensions in Angstroms (A), as well as the crystalline cell volume, space group, molecules per cell, and crystal density may be measured, for example, at a sample temperature of 25°C. A detailed description of unit cells is provided in Stout et al., X-Ray Structure Determination: A Practical Guide, Chapter 3, Macmillan Co., New York (1968), which is hereby incorporated herein by reference.

[00144] Additionally, the unique spatial arrangement of atoms in a crystalline lattice may be characterized according to the observed fractional atomic coordinates of such atoms.

[00145] Another means of characterizing the crystalline structure of the subject form is by PXRD analysis, the actual diffraction profile of such form is compared to a simulated profile representing pure powder material. Preferably, the actual and simulated profiles are both run at the same analytical temperature, and the subsequent measurements characterized as a series of 2Θ values (usually four or more).

[00146] Other means of characterizing a crystalline form that may be used include, but are not limited to, for example, solid state nuclear magnetic resonance (NMR) and

DSC.

[00147] At least one crystalline form of Compound 1 described herein was analyzed using at least one of the described testing methods.

EXAMPLES

[00148] The following Examples illustrate embodiments of the inventive compounds and starting materials, and are not intended to limit the scope of the claims.

[00149] Various crystal forms of ((((4-((5-(cyclopropylcarbamoyl)-2- methylphenyl)amino)-5-methylpyrrolo[2, 1 -f\ [ 1 ,2,4]triazin-6- yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4-(phosphonooxy)phenyl)acetate, were prepared and characterized as described below.

PROCEDURES FOR CHARACTERIZING THE FORMS

Single Crystal Data

[00150] Data were collected on a Bruker-Nonius (BRUKER AXS, Inc., 5465 East

Cheryl Parkway Madison, WI 53711 USA) CAD4 serial diffractometer. Unit cell parameters were obtained through least-squares analysis of the experimental diffractometer settings of 25 high-angle reflections. Intensities were measured using

Cu Ka radiation (λ = 1.5418 A) at a constant temperature with the Θ-2Θ variable scan technique and were corrected only for Lorentz-polarization factors. Background counts were collected at the extremes of the scan for half of the time of the scan. Alternately, single crystal data were collected on a Bruker-Nonius Kappa CCD 2000 system using Cu Ka radiation (λ = 1.5418 A). Indexing and processing of the measured intensity data were carried out with the HKL2000 software package

(Otwinowski, Z. et al, Macromolecular Crystallography, Carter, Jr., W.C. et al, eds. (Academic, NY), 276:307-326 (1997)) in the Collect program suite. (Collect Data collection and processing user interface: Collect: Data collection software, R. Hooft, Nonius B.V. (1998)). Alternately, single crystal data were collected on a Bruker- AXS APEX2 CCD system using Cu Ka radiation (λ = 1.5418 A). Indexing and processing of the measured intensity data were carried out with the APEX2 software package/program suite (APEX2 Data collection and processing user interface: APEX2 User Manual, vl.27; BRUKER AXS, Inc., 5465 East Cheryl Parkway Madison, WI 53711 USA). [00151] When indicated, crystals were cooled in the cold stream of an Oxford cryo system (Oxford Cryosystems Cryostream cooler: Cosier, J. et al., J. Appl. Cryst, 19: 105 (1986)) during data collection.

[00152] The structures were solved by direct methods and refined on the basis of observed reflections using either the SDP (SDP, Structure Determination Package, Enraf-Nonius, Bohemia NY 11716. Scattering factors, including/' and/", in the SDP software were taken from the "International Tables for Crystallography", Kynoch Press, Birmingham, England (1974), Vol. IV, Tables 2.2A and 2.3.1) software package with minor local modifications or the crystallographic packages MAXUS (maXus solution and refinement software suite: S. Mackay, CJ. Gilmore, C. Edwards, M. Tremayne, N. Stewart, K. Shankland. maXus: a computer program for the solution and refinement of crystal structures from diffraction data or SHELXTL (APEX2 Data collection and processing user interface: APEX2 User Manual, Vol.27; BRUKER AXS, Inc., 5465 East Cheryl Parkway Madison, WI 53711 USA). The derived atomic parameters (coordinates and temperature factors) were refined through full matrix least-squares. The function minimized in the refinements was

∑wflFol " l^Fcl)² R is defined as Σ ||F₀| - |F_C||/Σ |F₀| while R_w = [∑_w( |F₀| - |F_C|)2/Σ_W 2 1/2

F_o| ] where w is an appropriate weighting function based on errors in the observed intensities. Difference maps were examined at all stages of refinement. Hydrogens were introduced in idealized positions with isotropic temperature factors, but no hydrogen parameters were varied.

X-ray Powder Diffraction Data (PXRD)

[00153] PXRD data were obtained using a Bruker C2 GADDS . The radiation was Cu Ka (40 KV, 50mA). The sample-detector distance was 15 cm. Powder samples were placed in sealed glass capillaries of lmm or less in diameter; the capillary was rotated during data collection. Data were collected for 3<2Θ<35° with a sample exposure time of at least 1000 seconds. The resulting two-dimensional diffraction arcs were integrated to create a traditional 1 -dimensional PXRD pattern with a step size of 0.02 degrees 2Θ in the range of 3 to 35 degrees 2Θ. [00154] Alternately, X-ray powder diffraction (PXRD) data were obtained using a Philips MPD diffractometer. About 200 mg was packed by the backloading method into a Philips powder X-ray diffraction (PXRD) sample holder. The sample was transferred to a Philips MPD unit (45 KV, 40 mA, Cu Ka). Data were collected at room temperature in the 2 to 32 2-theta range (continuous scanning mode, scanning rate 0.03 degrees/sec, auto divergence and anti scatter slits, receiving slit: 0.2 mm, sample spinner : ON).

[00155] Alternately, X-ray powder diffraction (PXRD) data were obtained using a Bruker GADDS (General Area Detector Diffraction System) manual chi platform goniometer. Powder samples were placed in thin walled glass capillaries of lmm or less in diameter; the capillary was rotated during data collection. The sample-detector distance was 17 cm. The radiation was Cu Ka (λ = 1.5418 Ang). Data were collected for 3<2Θ <35° with a sample exposure time of at least 300 seconds.

Hybrid PXRD Patterns (from Low Temp)

[00156] "Hybrid" simulated powder X-ray patterns were generated as described in the literature (Yin, S. et al., American Pharmaceutical Review, 6(2): 80 (2003)). The room temperature cell parameters were obtained by performing a cell refinement using the CellRefine.xls program. Input to the program includes the 2-theta position of ca. 10 reflections, obtained from the experimental room temperature powder pattern; the corresponding Miller indices, hkl, were assigned based on the single- crystal data collected at low temperature. A new (hybrid) PXRD was calculated (by either of the software programs, Alex or LatticeView) by inserting the molecular structure determined at low temperature into the room temperature cell obtained in the first step of the procedure. The molecules are inserted in a manner that retains the size and shape of the molecule and the position of the molecules with respect to the cell origin, but, allows intermolecular distances to expand with the cell.

Hybrid PXRD (from Isostructural Analog)

[00157] "Hybrid" simulated powder X-ray patterns were generated as described in the literature (Yin, S. et al., American Pharmaceutical Review, 6(2): 80 (2003)). The room temperature cell parameters were obtained by performing a cell refinement using the CellRefine.xls program. Input to the program includes the 2-theta position of ca. 10 reflections, obtained from the experimental room temperature powder pattern; the corresponding Miller indices, hkl, were assigned based on the single- crystal data collected for an isostructural analog. A crystal structure for the molecule of interest was generated in a two step process: (1) by replacing the analog molecule in the experimental analog crystal structure with the molecule of interest. This step fixes the orientation and position of the molecule of interest in the unit cell of the analog compound; (2) Inserting the molecule of interest into the room temperature cell obtained from the experimental PXRD of the molecule of interest, as described above. In this step, the molecules are inserted in a manner that retains the size and shape of the molecule and the position of the molecules with respect to the cell origin, but, allows intermolecular distances to expand/contract with the cell. A new (hybrid) PXRD was calculated (by either of the software programs, Alex or LatticeView) based on the crystal structure generated as described above.

DSC (Sealed Pan)

[00158] Differential scanning calorimetry (DSC) experiments were performed in a TA Instruments™ model QlOOO or 2920. The sample (about 2-6 mg) was weighed in an pinpricked hermetically sealed aluminum pan and recorded accurately recorded to a hundredth of a milligram, and transferred to the DSC. The instrument was purged with nitrogen gas at 50mL/min. Data were collected between room temperature and 300⁰C at 10°C/min heating rate. The plot was made with the endothermic peaks pointing down.

TGA (Sealed Pan)

[00159] Thermal gravimetric analysis (TGA) experiments were performed in a TA Instruments™ model Q500 or 2950. The sample (about 10-30 mg) was placed in a pinpricked hermetically sealed aluminum pan on a platinum pan, both previously tared. The weight of the sample was measured accurately and recorded to a thousand of a milligram by the instrument The furnace was purged with nitrogen gas at lOOmL/min. Data were collected between room temperature and 300⁰C at 10°C/min heating rate.

Solid-State Nuclear Magnetic Resonance (SSNMR)

[00160] All solid-state C- 13 NMR measurements were made with a Bruker DSX- 400, 400 MHz NMR spectrometer. High resolution spectra were obtained using high- power proton decoupling and the TPPM pulse sequence and ramp amplitude cross- polarization (RAMP-CP) with magic-angle spinning (MAS) at approximately 12 kHz (Bennett, A.E. et al, J. Chem. Phys., 103:6951 (1995); Metz, G. et al, J. Magn. Reson. A, 110:219-227 (1994)). Approximately 70 mg of sample, packed into a canister-design zirconia rotor was used for each experiment. Chemical shifts (δ) were referenced to external adamantane with the high frequency resonance being set to 38.56 ppm (Earl, W.L. et al., J. Magn. Reson., 48:35-54 (1982)).

Raman Spectroscopy

[00161] Raman spectra were acquired at a resolution of 4 cm^"1 with 128 scans co- added, using a Nicolet 950 FT-Raman spectrophotometer. The wavelength of the laser excitation was 1064 nm. A CaF₂ beam splitter and a high sensitivity InGaS detector were used. IR Spectroscopy

[00162] Infra-red spectra were acquired at a resolution of 4cm^"1 with 32 scans co- added, using a Nicolet 560 FT-IR Spectrophotometer, incorporating a KBr beamsplitter and DTGS detector. Sample preparation was via the attenuated total reflectance method (ATR) using a single-bounce diamond ATR sampling accessory (DurasamplIR) from SensIR. An ATR correction step was included to correct the path length.

VTI (Dry On) [00163] Moisture sorption isotherms were collected in a VTI SGA-100 Symmetric Vapor Analyzer using approximately 10 mg of sample. The sample was dried at 60⁰C until the loss rate of 0.0005 wt %/min was obtained for 10 minutes. The sample was tested at 25°C and 3 or 4, 5, 15, 25, 35, 45, 50, 65, 75, 85, and 95% RH. Equilibration at each RH was reached when the rate of 0.0003 wt%/min for 35 minutes was achieved or a maximum of 600 minutes.

VTI (Dry Off)

[00164] Moisture sorption isotherms were collected in a VTI SGA-100 Symmetric Vapor Analyzer using approximately 10 mg of sample. The sample was tested at 25°C and 3, 5, 25, 35, 45, 50, 65, 75, 85, and 95% RH. Equilibration at each RH was reached when the rate of 0.0003 wt%/min for 35 minutes was achieved or a maximum of 600 minutes.

Hot Stage [00165] Crystals were placed on a glass slide, covered with a cover slip, and heated on a Linkham LTS350 (Linkham Scientific Instruments Ltd.) hot stage mounted on a microscope. The heating rate was controlled at 10°/min for the temperature range , ambient to 300⁰C. The crystals were observed visually for evidence of phase transformation, changes in birefringence, opacity, and melting etc.

EXAMPLE 1 Preparation of Form HAC-4

(Compound 1)

[00166] ((((4-((5-(cyclopropylcarbamoyl)-2-methylphenyl)amino)-5- methylpyrrolo[2,l-f][l,2,4]triazin-6-yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4- ((bis(benzyloxy)phosphoryl)oxy)phenyl)acetate (10.00 g, 11.43 mmol, see procedure in U.S. Patent Application Publication No. 2007/0213300A, example 126, step 4) and 20 wt% palladium hydroxide catalyst (0.3 g, 50 % wet) were suspended in a solution of Acetic Acid (81 mL) and water (9 ml) at 20 ⁰C. The head space was purged with nitrogen and then hydrogen was introduced. The mixture was heated to 50⁰C and stirred under hydrogen for 1 hr. The hydrogen was then removed and replaced with nitrogen. The solids were removed by pressure filtration over a 0.5 um filter at 50 ⁰C The filtrate was mixed with isopropyl acetate (4 ml) at 50 ⁰C and then Compound 1 (solid, 50 mg, see procedure in US Patent Application Publication No. 2007/0213300A, example 126) was added to form a white slurry. Additional isopropyl acetate (104 mL) was then added to the slurry over 30 minutes The slurry was cooled from 50 ⁰C to 20 ⁰C over 1 hour, aged and then filtered by suction filtration. The solids were collected on a Buchner funnel and washed with a mixture of isopropyl acetate (12 mL) and acetic acid (3 mL). The filtrate was discarded as waste. The wet solids were dried in a vacuum oven at 50 ⁰C under high vacuum to afford the HAC-4 Form of ((((4-((5-(cyclopropylcarbamoyl)-2-methylphenyl)amino)- 5-methylpyrrolo[2,l-f][l,2,4]triazin-6-yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4- (phosphonooxy)phenyl)acetate.

Characterization of Form HAC-4 [00167] The HAC-4 Form is the monoacetic acid solvate form of Compound 1 The HAC-4 form is characterized by unit cell parameters approximately equal to the following wherein the unit cell parameters of Form HAC-4 are measured at room temperature (about 25°C):

TABLE 1 Crystal Properties of Form HAC-4 (Monoacetic Acid Solvate)

[00168] The fractional atomic coordinate data of Form HAC-4 were measured according to the procedure described above and are presented in Table 2. Occupancies are 1 unless otherwise indicated.

TABLE 2 Positional Parameters for Form HAC-4 at Room Temperature

[00169] The HAC-4 Form was characterized by the simulated and observed powder x-ray diffraction (PXRD) patterns shown in Figure 1. The PXRD pattern has (CuKa λ= 1.5418 A at a temperature of about 25°C) having 2 theta ("2Θ") values of 7.3, 9.3, 9.7, 11.6, 12.5, 16.6, 23.3, 24.5, and 25.3 (degrees 2Θ ±0.1).

[00170] The HAC-4 Form was characterized by a differential scanning calorimetry (DSC) thermogram shown in Figure 3 having a melt/decomposition/desolvation onset typically at about 169 ⁰C and a thermogravimetric analysis (TGA) thermogram shown in Figure 4, having a negligible weight loss up to about 100 ⁰C. [00171] The HAC-4 Form was characterized by a moisture-sorption isotherm shown in Figure 5 which was characterized by 0.5% weight gain in the range of about 25% to about 75% RH at 25 ⁰C indicating that the HAC-4 form was non-hygroscopic. [00172] The HAC-4 Form was characterized by a SSNMR, substantially according to Figure 5.

EXAMPLE 2 Preparation of Form P-2

[00173] Form P-2 is characterized by a "family" of similar patterns. Crystals of Form P-2 are typically tiny, wispy, intergrown needles. The PXRD simulated from observed single crystal intensities is a good match to the observed PXRD of bulk material mounted on the tip of a nichrome wire. Materials from numerous conditions including but not limited to DMA/iPrOAc, EtOH, propylene glycol (homochiral and racemic); materials that analyze for variable amounts of the amino acids (L-GIy and L-AIa); and materials that analyze for variable amounts of metals (Li, Na, K, Ca, Mg, Zn) all belong to this family. Form P-2 contains variable amounts of solvent and its daughter phase has been assigned to be identified as form P-5. [00174] The P-2 form was prepared by crystallization of Compound 1 (solid, see procedure in U.S. Patent Application Publication No. 2007/0213300A, example 126) from racemic propylene glycol (PG)/MeOH/EtOAc (thus generating a P-2 Form material that contains PG by solution NMR) and then re-slurrying this material in water to form a "non-stoichiometric hydrate" which is then dried (at 30% relative humidity ("rh")) to 5-7% w/w water. This produces a hydroscopic solid with 5-7% weight gain in-between 25-75% room humidity at 25°C for the pre-dried solid. DSC showed a broad endotherms at ca. room temperature to ca. 50⁰C and another from ca. 150 ⁰C to ca. 170 ⁰C. Form P-2 is a non-stoichiometric hydrate of Compound 1. [00175] L-Glycine and L-Serine amino acid P-2 forms were synthesized by slurrying Compound 1 (solid, see procedure in U.S. Patent Application Publication No. 2007/02133OOA, Example 126) in a mixture of THF, Methanol and Ethanol. A solution of the amino acid in water is then added then the slurry is heated to reflux (73 to 75 ⁰C). Water was added until a solution was obtained and then the solution was cooled slowly to room temperature to crystallize the amino acid form. Similarly, L-AIa was synthesized by slurrying Compound 1 solids (see above) in ethanol. A solution of L-alanine in water was added and the slurry was heated to 70 to 75 ⁰C to obtain a clear solution. Cooling to 65 to 70 ⁰C and seeding crystallizes the L-AIa amino acid form. The slurry is cooled to 20-25 ⁰C, the solids are isolated and then dried at 30 ⁰C and high vacuum.

Characterization of Form P-2

[00176] The simulated PXRD curve of the P-2 Form was measured as described in the procedure above and are shown in Figure 25. The PXRD pattern (CuKa λ=1.5418A at a temperature of about 25°C) has 2 theta ("2Θ") values of 4.3, 5.2, 10.2, 11.3 and 15.3 (degrees 2Θ ±0.1), when measured at a temperature of about 25°C.

[00177] The P-2 Form was measured according to the procedures described above and is characterized by a differential scanning calorimetry (DSC) thermogram presented in Figure 26, having a dehydration endotherm onset typically at about RT. [00178] The thermogravimetric analysis (TGA) thermogram was also measured according to the procedures described above and is presented in Figure27. The TGA curve indicates a weight loss of about 2% to about 8%, preferably from about 4 to about 6%, at ca. 190 ⁰C. The weight loss is typical of dehydration.

EXAMPLE 3 Preparation of Form P-5

[00179] The "non-stoichiometric P-2 hydrate (see procedure (A) in Example 2, above) was dried under high vacuum in a dry atmosphere to produce solids with P-5 form. Exposure of the solids to 25-75% room humidity at 25 ⁰C will resulted in a weight gain to 5-7% water and a form conversion back to P-2. Form P-5 is a second non-stoichiometric hydrate form of Compound 1.

Characterization of Form P-5

[00180] The simulated PXRD curve of the P-2 Form was measured as described in the procedures above and are shown in Figure 29. The PXRD patterns (CuKa λ=1.5418A at a temperature of about 25 ⁰C) has 2 theta ("2Θ") values of :6.1, 7.9, 8.9, 12.1 and 19.8 (degrees 2Θ ±0.1) when measured 1 25 ⁰C. [00181] The differential scanning calorimetry (DSC) thermogram was measured on the P-5 Form and is presented in Figure 30. It indicates a compound having a having a small dehydration endotherm onset typically at ca. RT.

[00182] A thermogravimetric analysis (TGA) thermogram performed according to the above procedures on Form P-5 (see Figure 31) indicates a compound having less than ca. 4% weight loss measured at 100⁰C. The weight loss corresponded to dehydration.

EXAMPLE 4 Chemical Stability of the HAC-4 Form Methods

[00183] Presentation of Samples: Each bag (Whirl-Pak, 3-mil, 2-oz, Polyethylene) on stability contained 250 mg (for P-2) or 200 mg (for HAC-4) of drug substance. The drug substance was placed in a single bag and closed with a twist tie. The closed bag was placed inside a second bag that was also be closed with a twist tie. The double-bagged sample will then be placed in a 2 x 2 inch mini Fiber Drum fiber drum that was closed.

[00184] Presentation of Samples at 40°C/75%RH Open: Single bag samples were placed in a plastic container with open ports with the bag in the open position to directly expose the contents to high humidity. The plastic container consisted of a body and a lid, which contained ports to allow for equilibration of the interior humidity condition of the container to the 40°C/75%RH chamber conditions. The plastic container with open ports was wrapped with aluminum foil to protect the drug substance from light.

[00185] Sample Pulling Instructions for 40°C/75%RH Open Samples: The

40°C/75%RH sample bags were removed from the plastic container with open ports and immediately closed with a twist tie. The closed bag was then be placed inside a second bag that was also closed with a twist tie.

[00186] Analysis: Samples were analyzed by a suitable stability indicating validated reverse phase HPLC method. Results are reported as "Assay Corrected" against a reference standard and increase in the impurity/degradation product HPLC area percent over the initial value.

Results/Discussion

[00187] As a phosphate ester prodrug, ((((4-((5-(cyclopropylcarbamoyl)-2- methylphenyl)amino)-5-methylpyrrolo[2, 1 -J] [ 1 ,2,4]triazin-6- yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4-(phosphonooxy)phenyl)acetate is susceptible to hydrolysis to produce two degradation products: the parent,

and the des-phosphate analog,

TABLE 3 The relative stability of Form P-2 and Form HAC-4 after 3 months of storage

* Initial levels of parent & des-phosphate, 0.71% and 0.06% respectively f Initial levels of parent & des-phosphate, 0.11% and 0.03% respectively

[00188] From Table 3 (above), it can bee seen that after storage for 3 months at the conditions studied, the amount of the parent and the des-phosphate analog increase significantly in Form P-2 samples depends greatly on storage conditions used. After just 3 months, the room temperature storage condition of 25°C/60%RH has already produced 1.65% of the parent, and 0.67% of the des-phosphate. Typically, it is pharmaceutically necessary to be able to store drug substances for much longer periods of time beyond just 3 months. After exposure to the stress condition of 40°C/75%RH Open, the amount of degradation is completely unacceptable with formation of over 31% of parent drug and 4% of des-phosphate, and over a 38% loss in potency.

[00189] In contrast, the HAC-4 form is significantly more stable across all conditions studied with acceptable levels of degradation products formed. For example, at room temperature storage, only 0.06% parent and 0.01% des-phosphate are produced. Even at the typically challenging stress condition of 40°C/75%RH Open for ester prodrugs, only 0.64% of the parent and 0.02% of the des-phosphate were found.

[00190] From the P-2 performance at the stress condition of 40 °C/75% RH (closed), it was evident the P-2 form would not be as suitable for typical long term pharmaceutical development as over 26% of the parent drug and 3.3% of the des- phosphate were formed compared to just 0.5% of the parent and 0% of the des- phosphate with the HAC-4 form.

EXAMPLE 5 Physical Stability of the HAC-4 Form

Methods

[00191] Using a Hiden IGASorb Moisture Balance, an approximately 15mg sample was exposed to relative humidities of from 0 to 90% in the absorption phase and 80 to 0% in the desorption phase. Once equilibrium in the weight was established at each RH it is recorded and the RH value is changed in steps of 10%. Results are reported in relative humidity vs. percent weight change normalized on a wet basis. A sample verified by PXRD as P -5 was exposed to 60% RH for 45 minutes using the IGASorb mentioned above and analyzed again by PXRD within minutes of exposure.

Results/Discussion

[00192] Form P-2 Hydrate was found not to be physically stable across all relative humidities (RH). Figure 13 demonstrates the moisture uptake profile for the form P- 2/P-5 system. When dried to 0% RH, the system losses a mass of water and becomes the anhydrous P-5 form. However above 30% RH, the system gains a substantial mass of water and the form converts to the P-2 form. Upon decreasing relative humidity from 90% to 30% the system does not loose extensive moisture, however below 30% RH, a significant loss of moisture occurs along with a transition back to the P-5 form. During potential exposure from 0 to 90% RH, the weight of the sample has been observed to change as much as 8%. This change in weight will adversely affect the potency depending on the atmospheric conditions. Also, it is possible for non-homogeneity in the material upon changes in storage conditions. [00193] For these reasons, the P-2/P-5 system must always be carefully monitored and controlled. Otherwise, the PXRD of the material will change based on potentially minimal and routine changes in the storage condition making it very difficult to establish control of the drug substance and drug substance process. This is experimentally demonstrated by Figure 14, which depicts the PXRD patterns after dried material (P-5) was exposed to 60% RH for 45 minutes. The significant increase in weight after P-5 is exposed to moisture was found to correspond to a change in the PXRD pattern from the initial P-5 (bottom pattern) to P-2 (middle pattern), which matches well with the P-2 reference pattern on top. This physical instability is a potential developmental liability.

[00194] The HAC-4 form is non-hygroscopic and thus does not undergo physical form changes upon exposure to varying relative humidities. In Figure 13, weight changes of less than 1% was observed when exposing HAC-4 to relative humidities of 0 to 90%. The physical stability makes HAC-4 much more desirable to handle, manufacture, formulate, and store and thus a superior pharmaceutical development candidate.

EXAMPLE 6

In Vivo Assays And Efficacy [00195] The HAC-4 form was evaluated in the rat adjuvant arthritis model. Adjuvant arthritis was induced in Lewis rats essentially as described Cannon et al. and Johnston et al., except that the Mycobacterium concentration was adjusted to lOmg/ml. See Cannon G. W. et al, "Adjuvant arthritis in rats: susceptibility to arthritis induced by Mycobacterium butyricum and Mycobacterium tuberculosis, " Transplant Proc. Vol. 3 (1999 May;31) at pp.1590-1; and Johnston B. et al, "Alpha 4 integrin-dependent leukocyte recruitment does not require VCAM-I in a chronic model of inflammation. J Immunol, " Vol.164(6) (2000 Mar 15) at pp. 3337-44. [00196] The animals were dosed orally with the HAC-4 form as an aqueous suspension in 0.75% Methocel, 0.1% Tween. Dosing was initiated on day 10 of the study. As shown in Figure 15, the HAC-4 form was active in the model, displaying a dose dependent inhibition of paw swelling. Statistically significant inhibition was observed as labeled in Figure 15.

[00197] The P-2 form was also tested in the rat adjuvant arthritis model, which was performed in the same manner as for the HAC-4 form. The P-2 form was also dosed as an aqueous suspension in 0.75% Methocel, 0.1% Tween. As shown in Figure 16, the P-2 form was also active in the model.

EXAMPLE 7 Comparative Pharmacological Characteristics

[00198] Analysis of the toxicokinetic parameters of the HAC-4 and P2 Forms was performed in monkey plasma by measurement of the parent, 4-(5- (cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[l,2- f][l,2,4]triazine-6-carboxamide (see example 7, WO 2003/090912, published November 6, 2003, assigned to Bristol Myers Squibb Co.), via liquid chromatography/tandem mass spectrometry (LC/MS/MS). All reported results were generated in runs that met pre-established acceptance criteria. Toxicokinetic parameter values (AUC and Cmax) were calculated using noncompartmental methods by eToolbox/Kinetica (Version 2.4, Thermo Electron Corporation, Philadelphia, Pennsylvania). Values below the lower limit of quantification (LLOQ) were not used in calculations. Area under the curve (AUC) was calculated using the trapezoidal rule. In regard to the data reported in both Tables 4 and 5, below, for AUC(O-T), T = 8 or 24 hours postdose w/ concentration above LLOQ.

TABLE 4

Mean Toxicokinetic Parameters of the HAC-4 Form in the Plasma of Monkeys Receiving a Single Oral Dose of 17 mg/kg of ((((4-((5-(cyclopropylcarbamoyl)-2- methylphenyl)amino)-5-methylpyrrolo[2,l-/| [l,2,4]triazin-6- yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4-(phosphonooxy)phenyl)acetate.

TABLE 5

Mean Toxicokinetic Parameters of the P-2 Form in the Plasma of Monkeys Receiving a Single Oral Dose of 17 mg/kg of ((((4-((5-(cyclopropylcarbamoyl)-2- methylphenyl)amino)-5-methylpyrrolo[2,l-/] [l,2,4]triazin-6- yl)carbonyl)(propyl)carbamoyl)oxy)methyl (4-(phosphonooxy)phenyl)acetate.

Claims

WHAT IS CLAIMED IS:

1. A compound, that is a crystalline form of ((((4-((5- (cyclopropylcarbamoyl)-2-methylphenyl)amino)-5-methylpyrrolo[2,l-/|[l,2,4]triazin- 6-yl)carbonyl)(propyl)carbamoyl)oxy)metriyl (4-(phosphonooxy)phenyl)acetate, or a pharmaceutically acceptable salt thereof.

2. The crystalline form according to claim 1, comprising the HAC -4 form.

3. The crystalline form according to claim 2 characterized by unit cell parameters substantially equal to the following:

Cell dimensions: a = 11.8217(3) b= 12.7432(3) c = 12.5694(3) α = 98.326(1) β = 102.393(1) γ = 95.971(1) Space group Plbar

Molecules/unit cell (Z): 2 wherein said crystal is at a temperature of about +22°C.

4. The crystalline form according to claim 2 characterized by a powder x- ray diffraction pattern comprising three or more of 2Θ values (CuKa λ=1.54lA) selected from 7.3, 9.3, 9.7, 11.6, 12.5, 16.6, 23.3, 24.5, and 25.3 (degrees 2Θ ±0.1), at a temperature of about 25°C.

5. The crystalline form according to claim 4 further characterized by a powder x-ray diffraction pattern comprising four or more of 2Θ values (CuKa λ=1.54lA) selected from the group consisting of 77.3, 9.3, 9.7, 11.6, 12.5, 16.6, 23.3, 24.5, and 25.3 (degrees 2Θ ±0.1), at a temperature of about 25°C.

6. The crystalline form according to claim 2 characterized by fractional atomic coordinates substantially as listed in Table 1.

7. The crystalline form according to claim 2 having a powder x ray diffraction pattern substantially according to Figure 1.

8. A pharmaceutical composition comprising a compound according to claim 1, and a pharmaceutically acceptable carrier or diluent.

9. A method of treating a disease in a mammal comprising administering to the mammal a therapeutically-effective amount of a compound according to claim 1 in which the disease is selected from asthma, adult respiratory distress syndrome, chronic obstructive pulmonary disease, chronic pulmonary inflammatory disease, diabetes, inflammatory bowel disease, Alzheimer's disease, osteoporosis, psoriasis, graft vs. host rejection, atherosclerosis, multiple myeloma, pain, myocardial ischemia and arthritis, including rheumatoid arthritis, psoriatic arthritis, traumatic arthritis, rubella arthritis, gouty arthritis and osteoarthritis, ankylosing spondylitis, multiple sclerosis, lupus nephritis, Crohn's disease or ulcerative colitis.

10. A method of claim 9 wherein the disease is selected from chronic obstructive pulmonary disease, psoriasis, atherosclerosis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, lupus nephritis, Crohn's disease or ulcerative colitis.

11. A method of claim 10 wherein the disease is atherosclerosis.

12. The method of claim 10 in which the disease is psoriasis.

13. The method of claim 10 in which the disease is rheumatoid arthritis.

14. The method of claim 10 in which the disease is multiple schlerosis.

15. The method of claim 10 in which the disease is multiple lupus nephritis