HK1091205B - Isethionate salt of a selective cdk4 inhibitor - Google Patents

Description

Isethionate salt of a selective CDK4 inhibitor

Background

Technical Field

The present invention relates to salt forms of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one, which are selective cyclin dependent kinase 4(CDK4) inhibitors useful for the treatment of inflammation and cell proliferative disorders such as cancer and restenosis.

Discussion of related Art

Cyclin-dependent kinases and related serine/threonine protein kinases are cellular enzymes that play an important role in regulating cell differentiation and proliferation. The catalytic unit of cyclin-dependent kinases is activated by a regulatory subunit called a cyclin. At least sixteen mammalian cyclins have been identified (D.G.Johnson and C.L.Walker, Annu.Rev.Pharmacol.Toxicol. (1999) 39: 295-312). Cyclin B/CDK1, cyclin a/CDK2, cyclin E/CDK2, cyclin D/CDK4, cyclin D/CDK6, and possibly other heterodimers including CDK3 and CDK7 are important regulators of cell cycle progression. Other roles for cyclin/CDK heterodimers include regulation of transcription, DNA repair, differentiation, and apoptosis (d.o.morgan, annu.rev.cell.dev.biol. (1997) 13261-13291).

Cyclin-dependent kinase inhibitors have proven useful in the treatment of cancer. It has been shown that enhanced or transient aberrant activation of cyclin-dependent kinase activity leads to the formation of human tumors (C.J. Sherr, Science (1996) 274: 1672-. Indeed, the formation of human tumors is often associated with mutations in the CDK proteins themselves or in their regulators (C.Cordon-Cardo, am.J.Pathol. (1995) 147: 545. multidot. 560; J.Karp and S.Broder, nat. Med. (1995) 1: 309. multidot. 320; M.Hall et al, adv.cancer Res. (1996) 68: 67-108). Naturally occurring protein inhibitors of CDKs such as p16 and p27 cause growth inhibition of lung cancer cell lines in vitro (A. Kamb, curr. Top. Microbiol. Immunol. (1998) 227: 139-148).

Small molecule CDK inhibitors may also be useful in the treatment of cardiovascular diseases such as restenosis and atherosclerosis, as well as other vascular diseases due to abnormal cell proliferation. Vascular smooth muscle proliferation and intimal hyperplasia following balloon angioplasty were inhibited by overexpression of cyclin-dependent kinase inhibitor protein p21 (M.W. Chang et al, J.Clin.Invest. (1995) 96: 2260; Z-Y.Yang et al, Proc.Natl.Acad.Sci. (USA) (1996) 93: 9905). Furthermore, CVT-313(Ki 95nM), a purine CDK2 inhibitor, inhibited neointimal formation by more than 80% in rats (e.e.brooks et al, j.biolchem. (1997) 29207-.

CDK inhibitors are useful in the treatment of diseases caused by various infectious agents, including fungi, protozoan parasites such as Plasmodium falciparum (Plasmodium falciparum), and DNA and RNA viruses. For example, cyclin-dependent kinases are required for viral replication following Herpes Simplex Virus (HSV) infection (L.M.Schang et al, J.Virol. (1998) 72: 5626), and CDK homologs are known to play important roles in yeast (yeast).

Selective CDK inhibitors may be used to ameliorate the effects of various autoimmune diseases. Rheumatoid arthritis, a chronic inflammatory disease, is characterized by excessive proliferation of synovial tissue. Inhibition of synovial tissue proliferation should minimize inflammation while preventing joint destruction. It has been found that expression of the CDK inhibitor protein p16 in synovial fibroblasts inhibits growth (K. Taniguchi et al, nat. Med. (1999) 5: 760-767). Similarly, treatment with adenovirus expressed from p16 substantially inhibited joint swelling in a rat model of arthritis. CDK inhibitors are effective against other cell proliferative disorders including psoriasis (characterized by keratinocyte hyperproliferation), glomerulonephritis and lupus.

Certain CDK inhibitors are useful as chemoprotectants by virtue of their ability to inhibit cell cycle progression in normal, non-deformed cells (Chen et al. J. Natl. cancer Institute (2000) 92: 1999-2008). Pre-treatment of cancer patients with CDK inhibitors prior to the use of cytotoxic agents may reduce the side effects commonly associated with chemotherapy. Normal proliferating tissues are not affected by the cytotoxic effects of the action of selective CDK inhibitors.

Review articles on small molecule cyclin-dependent kinase inhibitors have indicated that identifying compounds that inhibit only specific CDK proteins, but not other enzymes, has difficulties. Thus, although they could potentially be used to treat a variety of diseases, no CDK inhibitors are currently licensed for commercial use (P.M. Fischer, curr. Opin. drug Discovery (2001) 4: 623 634; D.W.Fry and M.D.Garrett, curr. Opin. Oncology, Endocrine & Metabolic Invest (2000) 2: 40-59; K.R.Webster and D.Kimball, Embedded Drugs (2000) 5: 45-59; T.M.Sielcki et al, J.Med.chem. (2000) 43: 1-18).

Despite the above difficulties, recent studies have identified a number of selective CDK4 inhibitors as discussed above that have proven useful in the treatment of cancer (both as anticancer and chemoprotective agents), cardiovascular diseases (such as restenosis and atherosclerosis), diseases caused by various infectious agents, and autoimmune diseases (including rheumatoid arthritis). For disclosure of these selective CDK4 inhibitors, reference may be made to the commonly assigned international patent application PCT/IB03/00059 (the' 059 application) filed on 10.1.2003, which is incorporated herein by reference in its entirety for all purposes.

The' 059 application discloses a particularly potent selective CDK4 inhibitor, 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one:

the compounds of formula 1 have IC against CDK4 and CDK2 inhibition in standard enzymatic assays₅₀The concentrations (at 25 ℃ C.) were 0.011. mu.M and > 5. mu.M, respectively. Related IC₅₀A discussion of the standard CDK4 and CDK2 assays can be found in d.w.fry et al, j.biol.chem. (2001) 16617-.

Although the compound of formula 1 is a potent selective CDK4 inhibitor, its use in pharmaceutical products remains problematic. For example, the free base has poor water solubility (9 μ g/mL) while having low bioavailability in animal studies. The dihydrochloride salt of the compound of formula 1 appears to have sufficient water solubility. However, hygroscopic studies have shown that even at low relative humidity (10% RH), the dihydrochloride still absorbs moisture at levels exceeding 2% of its mass and is therefore unsuitable for use in solid pharmaceutical products. The monohydrochloride salt of the compound of formula 1 has a barely acceptable hygroscopicity, absorbing more than 2% of its mass of water at a relative humidity of greater than 80%. However, the process for preparing the monohydrochloride salt typically results in a partially crystalline solid material, which means potential problems in scale-up. Thus, other salt forms of the compound of formula 1 are desirable.

Summary of The Invention

The present invention provides monohydroxyethanesulfonic acid salts of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one, represented by formula 2:

the isethionate salt may exist in one or more polymorphic forms, including form a, form B and form D. Each polymorph can be distinguished by its powder X-ray diffraction (PXRD) pattern (diffractogram), or raman spectrum, or Differential Scanning Calorimetry (DSC) thermogram, or some combination of PXRD pattern, raman spectrum, and DSC thermogram. The isethionate salt may be anhydrous or may contain varying levels of water or one or more solvents.

Accordingly, in one aspect the present invention provides 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ], designated as form a]Monohydroxyethanesulfonate of pyrimidin-7-one characterized by one or more of the following: powder X-ray diffraction pattern having peaks at 2 θ values of about 8.7, 13.5 and 17.6, or about 1600cm^-1、1290cm^-1、675cm^-1、470cm^-1、450cm^-1And 425cm^-1A raman spectrum of a peak at the position of the raman shift value of (a), or a DSC thermogram having a sharp endothermic peak at 273 ℃.

According to a further aspect of the present invention there is provided 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] designated as form B]Monohydroxyethanesulfonate of pyrimidin-7-one characterized by one or more of the following: powder X-ray diffraction pattern having peaks at 2 θ values of about 5.1, 11.8, 12.1, 12.8, 13.1 and 14.7, or about 1600cm^-1、1290cm^-1、470cm^-1、450cm^-1And 425cm^-1The peak of the Raman shift value of (2), but at 675cm^-1A raman spectrum positioned substantially without peaks, or a DSC thermogram with a sharp endothermic peak at 271 ℃.

In a further aspect the present invention provides 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridine, designated as form DPyridin-2-ylamino) -8H-pyrido [2, 3-d]Monohydroxyethanesulfonate of pyrimidin-7-one characterized by one or more of the following: powder X-ray diffraction pattern having peaks at 2 theta values of about 8.4, 8.9 and 21.9, or at about 463cm^-1Or a DSC thermogram with a sharp endothermic peak at 277 ℃. Powder X-ray diffraction Pattern Using CuK for various salt forms_αRadiation was obtained and a DSC thermogram was obtained using a heating rate of 5 ℃/min.

The invention also provides pharmaceutical dosage forms comprising the isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one and one or more pharmaceutically acceptable excipients. Useful excipients include disintegrants, binders, diluents, surfactants, lubricants, preservatives, antioxidants, fragrances, colorants and the like.

The present invention also provides a process for preparing isethionate salts of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one. One method includes adding a solution of isethionic acid and a first solvent to an aqueous slurry of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one to give a first mixture. The process further comprises lyophilizing the mixture to obtain an amorphous salt, and then mixing it with a second solvent to obtain a second mixture comprising a isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-a ] pyrimidin-7-one.

Another method includes providing seed crystals of a isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one, and then adding the seed crystals to a dispersion of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one and a first solvent to give a first mixture. The method further comprises mixing the first mixture with a solution of isethionic acid and a second solvent to give a second mixture of isethionic acid salts containing 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one.

In both of the above processes, the first and second solvents may be the same or different and may be water miscible solvents including MeOH, EtOH and other alcohols. To increase the yield, the method may comprise heating, cooling or heating and cooling the second mixture to a temperature above or below room temperature. For example, the second mixture may be heated to a temperature of about 30 ℃ to about 60 ℃ and then allowed to cool to room temperature. Alternatively, the second mixture may be left at room temperature and then cooled to a temperature equal to or below about 0 ℃. Similarly, the second mixture may be heated to a temperature of about 30 ℃ to about 60 ℃ and then cooled to a temperature equal to or less than about 0 ℃.

Another process comprises reacting 4- {6- [6- (1-butoxy-vinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydro-pyrido [2, 3-d ] pyrimidin-2-ylamino ] -pyridin-3-yl) -piperazine-1-carboxylic acid tert-butyl ester with isethionic acid in a solvent and water, a mixture containing the bis-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one was obtained. The process optionally comprises adding a hindered base to the reaction mixture to produce a mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one.

The invention further provides a method of treating a disorder or condition caused by abnormal cell proliferation, or viral or fungal infection, or an autoimmune disease in a mammal, including a human. The method comprises administering to the mammal an amount of the isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one effective to treat the disorder or condition. Disorders or conditions caused by abnormal cell proliferation include cancer and vascular smooth muscle proliferation associated with atherosclerosis, postoperative stenosis and restenosis, and endometriosis. Autoimmune diseases include psoriasis, inflammatory-like rheumatoid arthritis, lupus, type 1 diabetes, diabetic nephropathy, multiple sclerosis, glomerulonephritis and organ transplant rejection, including host versus graft disease.

The isethionate salt provides advantages over the free base (formula 1) and other salt forms including the mono-and di-hydrochloric acid addition salts. The water solubility of the isethionate salt is improved by a factor of 20,000 compared to the free base. However, unlike dihydrochloride, the above-mentioned increase in solubility is not accompanied by a substantial increase in hygroscopicity. In addition, the isethionate salt is substantially crystalline and thus free of the scale-up potential problems associated with the monohydrochloride salt. These and other advantages will help overcome various challenges faced in developing pharmaceutical products containing the selective CDK4 inhibitor of formula 1.

Brief Description of Drawings

The various features, advantages and other applications of the present invention will become more apparent by referring to the following description and accompanying drawings.

Figure 1 shows a PXRD pattern for the mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one (form a);

figure 2 shows a PXRD pattern for the mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-D ] pyrimidin-7-one (form B and form D);

figure 3 shows PXRD patterns for the mono-mesylate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-D ] pyrimidin-7-one (form a, form B, form C and form D);

FIG. 4 shows a PXRD pattern for the di-mesylate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one;

FIG. 5 shows a PXRD pattern for the mono-HCl salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one;

FIG. 6 shows a PXRD pattern for the di-HCl salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one;

FIG. 7 shows 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d]Raman spectra of mono-isethionate salts of pyrimidin-7-one (form A, form B and form D) with Raman shifts of 0cm^-1To 1850cm^-1；

FIG. 8 shows 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d]Raman spectra of mono-isethionate salts of pyrimidin-7-one (form A, form B and form D) with a Raman shift of 1350cm^-1To 1800cm^-1；

FIG. 9 shows 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d]Raman spectra of mono-isethionate salts of pyrimidin-7-one (form A, form B and form D) with a Raman shift of 1100cm^-1To 1350cm^-1；

FIG. 10 shows 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d]Raman spectra of mono-isethionate salts of pyrimidin-7-one (form A, form B and form D) with Raman shifts of 500cm^-1To 850cm^-1；

FIG. 11 shows 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d]Raman photophotography of mono-isethionates of pyrimidin-7-ones (form A, form B and form D)Spectrum with Raman shift of 340cm^-1To 550cm^-1；

Figure 12 shows a DSC thermogram of the mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one (form a);

figure 13 shows DSC thermograms of mono-isethionate salts (form B and form D) of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-D ] pyrimidin-7-one;

figure 14 shows DSC thermograms for mono-mesylate salts (form a, form B, form C, and form D) of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-D ] pyrimidin-7-one;

figure 15 shows a DSC thermogram of the di-mesylate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one;

figure 16 shows a DSC thermogram of the di-HCl salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one;

figure 17 shows the water adsorption/desorption isotherms of the free base of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one; and

figure 18 shows water adsorption/desorption isotherms for various salts of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-D ] pyrimidin-7-one including mono-isethionate (form B and form D), mono-and di-HCl, mono-mesylate (form a and form C), di-mesylate and mono-tosylate.

Detailed Description

Definitions and abbreviations

The following definitions are used in this specification unless otherwise stated.

The term "cancer" includes, but is not limited to, the following cancers: breast cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, esophageal cancer, gastric cancer, skin cancer, lung cancer, bone cancer, colon cancer, pancreatic cancer, thyroid cancer, biliary tract cancer, buccal cavity and pharynx (oral), lip cancer, tongue cancer, oral cavity cancer, pharynx cancer, small intestine cancer, colon-rectum cancer, large intestine cancer, rectum cancer, brain and central nervous system cancer, glioblastoma, neuroblastoma, keratoacanthoma, epidermoid carcinoma, large cell carcinoma, adenocarcinoma, adenoma, follicular adenocarcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma, sarcoma, bladder cancer, liver cancer, kidney cancer, myeloid disorders, lymphoid disorders, hodgkin's disease, hairy cell cancer, and leukocytosis.

The phrase "pharmaceutically acceptable" refers to those substances which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without exhibiting excessive toxic effects, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for the intended use.

The term "treating" refers to reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which the term refers, or preventing one or more symptoms of such disorder or condition.

The term "treatment" refers to the act of "treating" as defined above.

Abbreviations used in this specification are listed in table 1.

TABLE 1 abbreviations

The mono-isethionate salt (formula 2) may exist in one or more polymorphic forms, including form a, form B and form D. As noted above, each polymorph can be distinguished by powder X-ray diffraction (PXRD), or Raman spectroscopy, or Differential Scanning Calorimetry (DSC), or some combination of these characterization methods. The mono-isethionate salt (formula 2) may be anhydrous or may contain variable amounts of water or one or more solvents. In addition, the mono-isethionate salt (formula 2) may be substantially pure, i.e., containing at least about 99% by weight of the particular polymorph, or may be a mixture of two or more polymorphs (e.g., form B and form D, etc.).

The free base (formula 1) is a dibasic compound which can form mono-and di-acid addition salts. pK of the conjugate acid_a7.3 and 4.1, so a relatively strong acid is required to form the disalt. Although di-isethionates of compounds of formula 1 may be formed, mono-isethionates appear to be more suitable because the latter require less counter-ion.

Fig. 1 and 2 provide PXRD diffraction patterns for the mono-isethionate salt form (formula 2) of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one. These forms are defined as form a in fig. 1 and form B and form D in fig. 2. For ease of reading, the diffraction pattern of form D in fig. 2 has been shifted up by 700 units. The significant PXRD peaks (i.e., those having a peak height to noise ratio greater than 3.5) for the mono-isethionate salt polymorphic forms A, B and D are listed in table 2 below, which is underlined, and a subset of these characteristic peaks can be used to distinguish one polymorphic form from another. The list of characteristic peaks provided in table 2 is not the only possible list of characteristic peaks. One of ordinary skill in the art of polymorphic identification can select other combinations of characteristic peaks that can also distinguish one form from another.

For comparison, PXRD diffraction patterns for the mono-, di-, mono-, and di-mesylate, mono-HCl and di-HCl salts of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one are shown in fig. 3-6, respectively. Although these salts may exist in more than one form, only distinct polymorphic forms of the mono-mesylate salt are currently identified, designated form a, form B, form C and form D in fig. 3. To highlight the differences between the mono-mesylate forms, the diffractograms of form B, form C and form D in fig. 3 have been shifted up by different amounts.

Each PXRD pattern shown in FIGS. 1-6 is under the use of CuK_αIrradiated RIGAKUD/Max2200 powder X-ray diffractometer. The diffractometer was equipped with a fine focus X-ray tube. At each operation, the tube voltage and current were set to 40kV and 40mA, respectively, the deflection and dispersion slits were set to 0.5, and the acceptance slit was set to 0.3 mm. The diffracted radiation is detected using a NaI scintillation detector. For each run, a continuous scan of theta-2 theta was made from 3.0 to 40.0 deg. 2 theta at a rate of about 1 deg./minute (3s/0.040 deg. step). The sample for analysis was prepared by placing the sample on a silicon wafer holder. Data was collected using rig aku's rig eas software and then analyzed using a commercial software package developed by the JADE software platform.

For each powder X-ray diffraction measurement, a sample in salt form was placed in a cavity located in the plane of the holder, and the surface of the sample was conditioned with a glass slide. The sample-containing stent is placed in a diffractometer and the sample is initially irradiated with an X-ray electron beam source at a small angle relative to the surface plane of the stent. The X-ray beam is then gradually moved along an arc such that the angle between the incident electron beam and the surface plane of the support gradually increases. At each scan, the amount of diffracted radiation is detected using a scintillation counter, which is recorded as a function of 2 θ. The instrument software represents the scanned diffracted radiation results in intensity versus 2 θ (fig. 1-6).

Differences in PXRD patterns may occur between results obtained from separate measurements of the same polymorph for a variety of reasons. Sources of error include differences in sample preparation (e.g., sample height), instrument errors, scale errors, and operational errors (including errors in determining peak positions). Preferential orientation, i.e., the lack of random orientation in the crystals of PXRD samples, may result in significant differences in relative peak heights. Scale errors and sample height errors typically result in all peaks in the diffraction pattern being displaced by the same amount in the same direction. Small differences in sample height on a planar support may cause large shifts in PXRD peak positions. Systematic studies have shown that sample height differences of 1mm can result in peak shifts as high as 1 ° 2 θ, as seen in Chen et al, j.pharmaceutical and biological Analysis (2001) 26: 63.

in many cases, peak shifts between diffraction patterns due to systematic errors can be eliminated by correcting the shift (e.g., applying a correction factor to all peak values) or by recalibrating the diffractometer. Generally, the same method can be used to compensate for differences between diffractometers, so that the PXRD peak positions obtained by two different instruments have consistency. In addition, when these methods are applied to PXRD measurements from the same or different diffractometers, the peak positions of a particular polymorph typically differ by about ± 0.2 ° 2 θ.

TABLE 2 significant PXRD peaks for mono-isethionate forms A, B and D

FIGS. 7-11 show 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d]Raman spectra of the mono-isethionate salt form of pyrimidin-7-one (formula 2). FIG. 7 shows Raman spectra for mono-isethionate form A, form B and form D with a Raman shift of 0cm^-1To 1850cm^-1FIGS. 8-11 provide Raman spectra for mono-isethionate form A, form B and form D with Raman shifts of 1350cm each^-1To 1800cm^-1、1100cm^-1To 1350cm^-1、500cm^-1To 850cm^-1And 340cm^-1To 550cm^-1. In some of the figures, it isHighlighting the differences that exist between the mono-isethionate salt forms, different ordinate scales (e.g., form a in fig. 7, 8, 10, and 11) or different baselines (e.g., form a and form B in fig. 9) or different ordinate scales and baselines (fig. 10) were used for one or more raman spectra.

Table 3 below lists the characteristic raman spectral peaks used to distinguish one mono-isethionate salt from another. As with PXRD data, the characteristic peak values provided in table 3 are not the only possible list of characteristic peaks, and one of ordinary skill in the art of polymorph identification can select other combinations of characteristic peaks that can also distinguish one polymorph from another.

The raman spectra shown in fig. 7-11 were obtained using a KAISER OPTICAL system scholla raman microscope and spectrograph. The raman spectrometer (spectrometer) used a solid state diode laser operating at 785nm with an output power of about 90 mW. The power delivered to the sample through the microscope objective was approximately 27 mW. The raman signal was detected using a thermoelectric cooled CCD detector. The laser excitation light and the raman scattered light to and from the sample are directed using a fiber optic cable and a spectrograph, respectively, connected to the raman microscope.

To obtain representative raman spectra, samples of each polymorphic form are detected at multiple locations or sites. For each sample, raman spectra were obtained at four or five points, with four sets of dichroms at each point. For a normally solid sample, the data for a given polymorphic form have the greatest difference in peak intensity, but relatively little difference in peak position. For each version, the raman shift values (peak position as a function of wavenumber) differ by less than 1cm, although one can expect^-1The difference in peak position between the different forms is more than 1cm^-1. It is believed that at least some of the differences in peak intensity or peak position are due to the way the laser hits different crystals in the sample.

TABLE 3 characteristic Raman Spectroscopy peaks for the mono-isethionate form A, B, and D

As with the previously described differences, Raman spectra of a particular polymorph obtained using different instruments also appear to have subtle differences in peak position (i.e., 1 cm)^-1Or less), there is a relatively greater difference in peak intensity. If the raman scattering is independent of the excitation wavelength used, the peak position should be equally around the instrument using different excitation sources. Among other things, the peak intensity may vary with the type of detector or optics, the power of the excitation laser, and the sample position.

Figures 12 and 13 show DSC thermograms of the mono-isethionate salt form (formula 2) of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-D ] pyrimidin-7-one, designated as form a (figure 12) and forms B and D (figure 13). In addition, DSC thermograms of mono-mesylate (forms A, B, C and D), bis-mesylate, and bis-HCl salts are shown in fig. 14-16, respectively. DSC data were obtained using a TAINSTRUMENTS 2920 Modulated DSC V2.6. Individual polymorph samples were analyzed in a vented, closed aluminum pan heated to 350 ℃ using a heating rate of 5 ℃/minute and a nitrogen purge stream of 50 mL/minute.

As shown in fig. 12-15, the mono-isethionate (forms A, B and D), mono-mesylate (forms A, B, C and D) and di-mesylate salts have distinct melting points with sharp endothermic peaks at approximately 273 ℃, 271 ℃, 277 ℃, 309 ℃, 307 ℃, 302 ℃, 304 ℃ and 289 ℃, respectively. In contrast, the di-HCl salt (fig. 16) has a relatively complex DSC thermogram including broad endothermic peaks between about 40 ℃ and 110 ℃ and between about 160 ℃ and 200 ℃, which may indicate the loss of moisture and lattice water, respectively. The di-HCl salt DSC trace (trace) also has a relatively sharp exothermic peak starting at about 207 ℃ and a broad endothermic peak starting at about 275 ℃, which may indicate a form transition and melting or decomposition, respectively, or both.

Figures 17 and 18 show the water and desorption isotherms (at 25 ℃) of the free base of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-D ] pyrimidin-7-one (formula 1) and its various salts including mono-isethionate (forms B and D), mono-and di-HCl, mono-mesylate (forms a and C), di-mesylate and mono-tosylate. These water and desorption data were obtained using a VTI CORPORATION MODELSSGA-100 symmetrical gravimetric analyzer. To obtain a moisture isotherm, the polymorphic sample was placed on a microbalance in a closed environmental chamber and then heated at a rate of 5 ℃/minute until the temperature in the chamber reached 40 ℃. To obtain the dry sample weight, the polymorph was equilibrated at 40 ℃ until the weight change of the sample over 2 minutes was less than 0.0270 wt%. After drying, the samples were cooled to 25 ℃ and then exposed to different humidity levels (in 10% RH increments) of 5 or 10% RH to 90% RH and 90% RH to 10 or 5% RH. At each humidity level, the polymorph was allowed to equilibrate until the weight change of the sample over 2 minutes was less than 0.0270 wt%. The balance mass at each humidity level was recorded, as well as the dry sample weight, to generate a weight change versus relative humidity curve.

Of the compounds shown in fig. 17 and 18, only the free base, mono-isethionate (forms B and D) and mono-tosylate have a mass change of less than 2% when exposed to humidity levels of 10% RH to 90% RH at 25 ℃.

Table 4 lists the water solubilities of the free base (the most stable crystalline phase according to the slurry test) and isethionate (form B, the most stable form according to the slurry test results). Since form B appears to be the most stable isethionate form, it should have the lowest water solubility of the observed isethionate forms. The metastable solubility of the other isethionate salt forms was not evaluated. The water solubility of isethionate is obtained by: the salt was dissolved in water up to about 300mg/mL, solids were observed after about 48 hours of equilibration, and the concentration of the salt in the aqueous phase was measured using HPLC. See table 5 for HPLC conditions. The water solubility of the free base is obtained by: the solid was equilibrated in water for 14 hours and the concentration of the free base in the aqueous phase was then measured using the semi-automated UV-vis method (using SPECTRMAX PLUS spectrophotometer plate reader).

The data in Table 4 show that the water solubility of the mono-isethionate salt (pH5.4) is 20,000 times higher than the free base (pH 7.9). The above significant difference in water solubility cannot be explained by the existence of a relatively reasonable (modest) difference between the pH of the saturated solution of the free base and the mono-isethionate. In fact, it is calculated by Henrson-Hasselbalch equation (using the free base at pH7.9 and pK)_asolubility at s7.3 and 4.1 of 0.0092mg/mL), the theoretical aqueous solubility of the free base at pH5.4 is only 0.62 mgA/mL. An aqueous solution of the mono-isethionate salt prepared at 117mgA/mL and pH5.4 (supersaturated with respect to the free base) was seeded with crystals of the free base without causing precipitation. Instead, the seed crystals were dissolved, indicating that the isethionate ion has some ability to increase the solubility of the free base in water.

TABLE 4 Water solubility of free base and isethionate (form B)

TABLE 5 conventional HPLC conditions for water solubility measurement

The solubility of isethionate in physiological saline is 0.58mgA/mL, much less than its water solubility, very close to the theoretical value of 0.43mgA/mL (calculated according to the Henry-Ha equation) at the final pH of the solution (pH 5.56). In normal saline, the surprising solubilizing power of the isethionate ion is almost eliminated, and the compound behaves more like a normal basic compound in dissolution.

Isethionate salts may be prepared using various techniques. For example, in one method, a solution of isethionic acid and a first solvent is mixed with an aqueous slurry of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one. The mixture was filtered to remove any solids and the resulting filtrate was freeze-dried (lyophilized) to give amorphous isethionate. The amorphous salt is converted to a crystalline form by dissolving it in a second solvent (while heating may be applied to promote complete dissolution). The resulting solution is then cooled to RT or below to precipitate crystals of the salt, which can be isolated by filtration and then dried in a vacuum oven.

The above processes generally employ stoichiometric (i.e., molar ratios of 1: 1 or 2: 1) or near stoichiometric quantities of isethionic acid and 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one. The first and second solvents may be the same or different, and are typically water miscible solvents, including alcohols such as MeOH and EtOH. The amount of heat required to dissolve the amorphous salt in the second solvent depends on the solvent used, but the temperature of the mixture is generally from about 30 ℃ to about 60 ℃, usually from about 30 ℃ to about 50 ℃. In some cases, the temperature of the mixture is from about 30 ℃ to about 40 ℃ or from about 35 ℃ to about 40 ℃.

In another method, the free base (formula 1) is dispersed (slurried) in a first solvent and seeded with crystalline isethionate salt form. The resulting mixture is mixed with isethionic acid and a second solvent. The isethionic acid solution is usually added in portions over a period of time. The resulting slurry or dispersion is stirred at room temperature or higher, typically at a temperature above about 35 ℃ or 40 ℃. To increase the yield, the resulting mixture may be cooled to a temperature below about 0 ℃ at which additional isethionate crystals precipitate. The crystalline isethionate salt may be isolated by filtration and then dried in a vacuum oven. Similar to the previously described process, this technique also uses stoichiometric or near stoichiometric quantities of isethionic acid and 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one. Additionally, the first and second solvents may be the same or different, and are typically water miscible solvents, including alcohols such as MeOH and EtOH. The process generally yields higher than the previously described process, while achieving better (e.g., larger, more uniform) crystallization.

Another method is to generate the isethionate salt directly from the protected step intermediate with the aid of the free base (formula 1). The method comprises reacting an N-BOC protected compound of formula 3

With about 3.5 equivalents (or more) of isethionic acid in a first solvent and water, thereby removing the BOC protecting group and exposing the acetyl group to yield 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d]Di-isethionate salts of pyrimidin-7-one. The above reaction may be carried out at room temperature or higher, and is usually carried out at a temperature of about 30 ℃ to about 60 ℃. To the reaction mixture is added a sterically hindered base (e.g. Et)₃N) in a second solvent, thereby forming a salt with isethionic acid which is soluble in the reaction mixture. The base is added in an amount sufficient to maintain a slight excess of free isethionic acid in the reaction mixture in the presence of di-isethionate. For example, if 3.5 equivalents of isethionic acid are reacted with the BOC-protected compound of formula 3, then about 1.45 equivalents of hindered base may be used, resulting in an excess of about 0.05 equivalents of free isethionic acid. If desired, the di-hydroxyethyl groupThe sulfonate salt may be isolated by filtration.

To obtain the mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one, additional base is added over a prolonged period of time (e.g., dropwise) to ensure the requisite form of mono-isethionate salt (e.g., form B) is formed. The addition of the sterically hindered base is too rapid and may lead to the formation of other metastable polymorphs. To improve the yield, the resulting slurry may be cooled to a temperature of about 5 ℃ or less, then filtered and dried. As with the previously described methods, the first and second solvents may be the same or different, and are typically water-miscible solvents, including alcohols such as MeOH and EtOH.

Other salt forms disclosed herein, such as mono-or di-HCl, methanesulfonic acid, or benzensulfonate salts, can be prepared in a manner similar to that described previously for the preparation of isethionate salts (formula 2).

The compounds (formula 1 and salts) disclosed herein include all pharmaceutically acceptable isotopic variations. Isotopic variations are those compounds in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Useful isotopes include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine. Exemplary isotopes thereof include, but are not limited to²H、³H、¹³C、¹⁴C、¹⁵N、¹⁷O、¹⁸O、³²P、³⁵S、¹⁸F. And³⁶Cl。

isotopes of formula I such as deuterium for the compounds disclosed herein²H substitution may result in certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and thus may be useful in certain situations. In addition, certain isotopic variants, for example those incorporating a radioisotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotope tritium being thus³H. And carbon-14 i.e.¹⁴C isAre particularly useful because they are easily incorporated and are convenient to detect.

Isotopic variations of the compounds disclosed herein can generally be prepared by conventional methods known to those skilled in the art or by processes analogous to those described in the examples below for suitable isotopic variations using suitable reagents. Pharmaceutically acceptable solvates of the compounds disclosed herein include those solvates in which the crystallization solvent may be isotopically substituted, e.g. D₂O、d₆-acetone, d₆-DMSO。

The compounds disclosed herein (formula 1 and salts) may be administered in the form of crystalline or amorphous products. They can be obtained, for example, in the form of solid plugs, powders or films by processes such as precipitation, crystallization, freeze drying, spray drying or evaporation drying. Microwave or radio frequency drying may be used to achieve the above objectives.

The compounds disclosed herein may be administered alone or in combination with other drugs, usually in a formulation in admixture with one or more pharmaceutically acceptable excipients. The term "excipient" refers to any other ingredient except the compound represented by formula 1 and salts thereof. The choice of excipient will depend to a large extent on the particular mode of administration.

The compounds disclosed herein may be administered orally. Oral administration should include swallowing, so that the compound is able to enter the gastrointestinal tract, or buccal or sublingual administration, so that the compound enters the blood stream directly from the mouth.

Formulations suitable for oral administration include solid preparations such as tablets, capsules containing microparticles, liquids or powders, lozenges (including liquid-filled lozenges), chewable tablets (chew), multiparticulates (multiparticulates) and nanoparticles, gels, solid solutions, liposomes, films (including mucoadhesives), ovules (ovule), sprays and liquid preparations. Liquid preparations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard gelatin capsules and typically comprise a carrier such as water, EtOH, polyethylene glycol, propylene glycol, methyl cellulose, or a suitable oil, together with one or more emulsifying and/or suspending agents. Liquid formulations can also be prepared by formulating solids, for example, from sachets (recycling).

The compounds disclosed herein may also be used in fast dissolving, fast disintegrating dosage forms, such as those described by Liang and Chen in Expert Opinion in Therapeutic Patents (2001)11 (6): 981 and 986.

For tablet dosage forms, the dose of drug may be from 1% to 80% by weight of the dosage form, typically from 5% to 60% by weight of the dosage form. In addition to the drug, tablets typically contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch, and sodium alginate. Generally, the disintegrant may comprise from 1 to 25 weight percent, preferably from 5 to 20 weight percent of the dosage form.

Binders are commonly used to impart cohesive properties to the tablet. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose. Tablets may also contain diluents such as lactose (monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch, and dibasic calcium phosphate dihydrate.

The tablets also optionally contain surfactants such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. If present, the surfactant may comprise from 0.2% to 5% by weight of the tablet and the glidant may comprise from 0.2% to 1% by weight of the tablet.

Tablets also typically contain lubricating agents such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate and sodium lauryl sulfate. The lubricant typically comprises from 0.25 to 10% by weight, preferably from 0.5 to 3% by weight of the tablet. Other ingredients may include preservatives, antioxidants, fragrances and colorants.

The tablet mixture may be directly compressed to form a tablet. The tablet mixture or mixture portion may be either wet, dry or melt granulated, or melt condensed or extrusion granulated prior to compression. The final formulation may contain one or more layers, which may or may not be coated. Exemplary tablets contain up to about 80% drug, about 10% to about 90% binder, about 0% to about 85% diluent, about 2% to about 10% disintegrant, and about 0.25% to about 10% lubricant. Additional details regarding tablets are found in h.lieberman and l.lachman, Pharmaceutical DosageForms: tablets, VoL 1 (1980).

Solid formulations suitable for oral administration may be formulated as immediate and/or modified release drugs. Sustained release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release. For a review of suitable sustained release formulations, see U.S. Pat. No. 6,106,864. Details regarding other useful release techniques such as high energy dispersing agents and osmotic and coated granules are described in Verma et al, Pharmaceutical Technology On-line (2001)25 (2): 1-14. For a discussion of the controlled release obtained by chewing gum see WO 00/35298.

The compounds disclosed herein (formula 1 and salts) may also be administered directly into the bloodstream, muscle, or internal organs. Suitable modes of parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous administration. Suitable devices for parenteral administration include needle (including microneedle) syringes, needle-free syringes and infusion devices.

Formulations for parenteral administration are typically aqueous solutions containing excipients such as salts, carbohydrates and buffers (preferably at a pH of 3-9), but for some applications it may be more appropriate to formulate them as sterile anhydrous solutions or in dry form for use in conjunction with a suitable vehicle, e.g., sterile, pyrogen-free water. Preparation of a parenteral formulation may be conveniently accomplished by lyophilization under sterile conditions using conventional pharmaceutical techniques well known to those skilled in the art.

Suitable formulation techniques may be used to improve the solubility of the disclosed compounds for use in the preparation of parenteral solutions, for example, in combination with the incorporation of a solubilizer. Parenteral formulations may be formulated for immediate and/or modified release as described previously. Thus, the compounds disclosed herein can be formulated in more solid forms for administration in the form of an implanted reservoir that ensures long-term release of the active compound.

The compounds of the present invention may also be administered topically to the skin or mucosa, either transdermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, emulsions, ointments, dusting powders, dressings (dressing), foams, films, skin patches, wafers, implants, cotton balls (sponges), fibers, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohols, water, mineral oil, liquid paraffin, white petrolatum, glycerin, and propylene glycol. Typical formulations may also contain penetration enhancers. See, e.g., J Pharm Sci (1999)88(10) by Finnin and Morgan: 955-958.

Other means of topical administration include delivery by iontophoresis, electroporation, sonophoresis, and needle-free (e.g., POWDERJECT) or microneedle injection. The topical formulations may be formulated for immediate and/or modified release as previously described.

The compounds disclosed herein may also be administered intranasally or by inhalation, typically in the form of a dry powder from a dry powder inhaler (either as a separate mixture, e.g. in dry admixture with lactose, or as a particulate admixture, e.g. with a phospholipid) or as a spray from a pressurised container, pump, spray (spray), atomiser (atomizer) (preferably an atomiser which generates a fine mist using electrohydrodynamic forces), or atomiser (nebuliser), with or without the use of a suitable propellant, e.g. dichlorofluoromethane. The pressurized container, pump, sprayer, nebulizer, or atomizer contains a solution or suspension comprising the active compound, a substance for dispersing, dissolving, or delaying release of the active compound (e.g., an aqueous EtOH or EtOH solution), one or more solvents as a propellant, and optionally a surfactant, such as sorbitan trioleate or an oligomeric lactic acid (oligomeric lactic acid).

Prior to use of the dry powder or suspension, the drug product is micronized to a size suitable for administration by inhalation (typically less than 5 microns). This can be accomplished by any suitable comminution method, such as rotary jet milling, fluidized bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

Capsules, blisters (blisters) and cartridges (made, for example, from gelatin or hydroxypropylmethylcellulose) for use in an inhaler or insufflator may be formulated containing a powder mix of the active compound, a suitable powder base such as lactose or starch, and a performance-modifying agent such as L-leucine, mannitol or magnesium stearate. Lactose may be anhydrous, preferably monohydrate. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

Suitable formulations of solutions for use in nebulisers (which generate a fine mist using electrohydrodynamics) may contain from 1. mu.g to 20mg of a compound of the invention per spray, and the spray volume may be from 1. mu.l to 100. mu.l. Typical formulations may contain a compound of formula 1 or formula 2, propylene glycol, sterile water, EtOH and NaCl. Solvents that may be used in place of propylene glycol include glycerol and polyethylene glycol.

Formulations for inhalation/intranasal administration may be formulated as immediate and/or modified release medicaments using, for example, poly (DL-lacto-co-glycolic acid) (PGLA). Flavoring agents such as menthol and levomenthol, or sweetening agents such as saccharin or saccharin sodium may be added to the formulation for inhalation/intranasal administration.

In the case of dry powder inhalers and aerosols, the dosage unit is determined by delivering a calculated dose of the valve. The dosage units of the invention are generally arranged to contain from 100 μ g to 1000 μ g of active pharmaceutical ingredient per calculated dose or "puff". The total daily dose is typically from 100 μ g to 10mg, which may be administered in a single dose, or more typically in multiple doses throughout the day.

The active compounds can be administered rectally or vaginally, for example in the form of suppositories, pessaries or enemas. Cocoa butter is a traditional suppository base, but other alternatives may be used if appropriate. Rectal/intravaginal formulations may be formulated for immediate and/or modified release as described above.

The compounds disclosed herein may also be administered directly to the eye or ear, usually in the form of drops of micronized suspensions or solutions in isotonic, well-adjusted pH sterile saline. Other formulations suitable for ophthalmic and otic administration include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and particulate or vesicular systems, such as niosomes or liposomes. Polymers such as crosslinked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, cellulosic polymers (e.g., hydroxypropyl methylcellulose, hydroxyethyl cellulose, or methyl cellulose), or heteropolysaccharide polymers (e.g., agarose gel) may be incorporated with preservatives such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis. Ophthalmic/otic formulations may be formulated for immediate and/or modified release as described above.

The compounds disclosed herein may be conjugated to soluble macromolecular entities such as cyclodextrin or polyethylene glycol containing polymers to improve their solubility, dissolution rate, taste masking, bioavailability and/or stability. It has been found that, for example, drug-cyclodextrin complexes are generally suitable for most dosage forms and routes of administration. Inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, cyclodextrins may be used as an auxiliary additive, i.e. as a carrier, diluent or solubiliser. The most commonly used for this purpose are alpha-, beta-and gamma-cyclodextrins. See, for example, international patent application nos. WO 91/11172, WO 94/02518, and WO 98/55148.

A therapeutically effective dose of a compound of formula 1, formula 2, or other salt may be from about 0.01mg/kg to about 100mg/kg of body weight per day. A typical adult dose may be about 0.1 to about 3000mg per day. The amount of active ingredient in a unit dosage formulation may be varied or adjusted from about 0.1mg to about 500mg, preferably from 0.6mg to 100mg, depending upon the particular application and the potency of the active ingredient. Other compatible therapeutic agents may also be included in the composition, if desired. The patient to be treated may be administered a daily dosage of about 0.6 to about 500mg, either in a single dose or in divided doses over a 24 hour period. If a long treatment is required, the above treatment may be repeated at successive time intervals.

Examples

The following examples are illustrative and non-limiting, and representative, specific embodiments of the present invention.

Example 1

4- [6- (6-bromo-8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydro-pyrido [2, 3-d)] Pyrimidin-2-ylamino) -pyridin-3-yl]Preparation of tert-butyl (E) -piperazine-1-carboxylate

Reacting 6-bromo-8-cyclopentyl-2-methanesulfinyl-5-methyl-8H-pyrido [2, 3-d]Pyrimidin-7-one (10.00g, 0.027mol, prepared as in example 6 of WO 01/707041, incorporated herein by reference) and 10.37g (0.0373mol) of 4- (6-amino-pyridin-3-yl) -piperazine-1-carboxylic acid tert-butyl ester in toluene (100mL)) The suspension in (b) was heated in an oil bath under nitrogen for 7 hours. Thin layer chromatography (SiO)₂10% MeOH/DCM) showed the presence of both starting materials. The suspension was heated to reflux for an additional 18 hours. The resulting suspension was cooled to room temperature and filtered to give 4- [6- (6-bromo-8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydro-pyrido [2, 3-d ]]Pyrimidin-2-ylamino) -pyridin-3-yl]-piperazine-1-carboxylic acid tert-butyl ester (5.93g, 38%). The melting point is more than 250 ℃. MS (APCI) M⁺+1: calculated 584.2, found 584.2.

Example 2

4- {6- [ 8-cyclopentyl-6- (1-ethoxy-vinyl) -5-methyl-7-oxo-7, 8-di Hydro-pyrido [2, 3-d]Pyrimidin-2-ylamino]-pyridin-3-yl } -piperazine-1-carboxylic acid tert-butyl ester Preparation of

Mixing 4- [6- (6-bromo-8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydro-pyrido [2, 3-d ]]Pyrimidin-2-ylamino) -pyridin-3-yl]-piperazine-]-a suspension of tert-butyl formate (5.93g, 0.010mol, prepared as in example 1), tetrakis (triphenylphosphine) palladium (0) (1.40g, 0.00121mol), and tributyl (1-ethoxyvinyl) tin (5.32mL, 0.0157mol) in toluene (30mL) was heated to reflux for 3.5 h. The mixture was cooled and filtered to give a solid. The solid was purified by silica gel chromatography for 15 minutes, eluting with a gradient of 5% to 66% ethyl acetate/hexanes, to give 4- {6- [ 8-cyclopentyl-6- (1-ethoxy-vinyl) -5-methyl-7-oxo-7, 8-dihydro-pyrido [2, 3-d ] as a yellow foam]Pyrimidin-2-ylamino]-pyridin-3-yl } -piperazine-1-carboxylic acid tert-butyl ester (4.50g, 78%). MS (APCI) M⁺+1: calculated 576.2, found 576.3.

Example 3

6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) Yl) -8H-pyrido [2, 3-d]Preparation of pyrimidin-7-one hydrochloride

Hydrogen chloride gas was bubbled through ice-cooled 4- {6- [ 8-cyclopentyl-6- (1-ethoxy-vinyl) -5-methyl-7-oxo-7, 8-dihydro-pyrido [2, 3-d]Pyrimidin-2-ylamino]-pyridin-3-yl } -piperazine-1-carboxylic acid tert-butyl ester (4.50g, 0.00783mol, prepared as in example 2) in DCM (100 mL). The resulting suspension was stoppered, stirred at room temperature overnight, and then diluted with ether (200 mL). The solid was collected by filtration, washed with diethyl ether and then dried to give 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d as a yellow solid]Pyrimidin-7-one hydrochloride (4.01g, 92%). Melting point 200 ℃. HPLC, C18 reverse phase, 10% -95% gradient of 0.1% TFA/CH₃0.1% TFA/H of CN₂O solution, for 22 minutes: 99.0% at 11.04 min. MS (APCI) M⁺+1: calculated 448.2, found 448.3. To C₂₄H₂₉N₇O₂·2.4H₂Elemental analysis calcd for O.1.85 HCl: c, 51.64; h, 6.44; n, 17.56, Cl (all), 11.75. Measured value: c, 51.31; h, 6.41; n, 17.20; cl (total), 12.11.

Example 4

6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) Yl) -8H-pyrido [2.3-d]Preparation of a mono-isethionate salt of a pyrimidin-7-one (form B)

To a slurry of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one (7.0g, 15.64mmol, prepared as in example 3, then contacted with NaOH) dispersed in 250mL water was added 30mL of a 0.52M solution of isethionic acid in MeOH (15.64mmol) dropwise to a pH of 5.2. The solution was filtered (fine) through a glass filter and the clear solution was freeze dried to give 9.4g of amorphous salt. This amorphous salt (3.16g) was mixed with 25mL MeOH and, after almost complete dissolution, a new precipitate formed. An additional 25mL of MeOH was added and the mixture was stirred at 46 deg.C to 49 deg.C for 4 hours. The mixture was slowly cooled to 32 ℃ and then placed in a cold room (+4 ℃) overnight. PXRD analysis of the sample indicated the formation of form B. The mixture was filtered and the precipitate was dried in a vacuum oven at 50 ℃. Thus, 2.92g of the mono-isethionate salt of the compound of formula 1 was prepared with a yield of 92%. HPLC-99.25%, PXRD-form B, CHNS, H-NMR consistent with the structure.

Example 5

6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) Yl) -8H-pyrido [2.3-d]Preparation of a mono-isethionate salt of a pyrimidin-7-one (form B)

MeOH (100mL) was placed in a 250mL flask equipped with a mechanical stirrer, thermocouple/controller, condenser, and heating mantle, and preheated to 35 ℃. Three equal portions of amorphous isethionate (2g, prepared as in example 4) were added slowly with intervals of 25 to 30 minutes between each addition. The reaction mixture was stirred at 35 ℃ overnight and then cooled. Samples were filtered and tested by PXRD. The product was pure form B. In larger scale up experiments, the entire reaction mixture was subsequently used as form B seed crystals.

Example 6

6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) Yl) -8H-pyrido [2.3-d]Preparation of a mono-isethionate salt of a pyrimidin-7-one (form B)

MeOH (50mL) was placed in a 250mL flask equipped with a mechanical stirrer, condenser, thermocouple/controller, and heating mantle, and preheated to 40 ℃. Three equal portions of amorphous isethionate (1g, prepared as in example 4) were added slowly, with 30 minute intervals between each addition, and then stirred overnight at 40 ℃. The reaction was monitored in situ by raman spectroscopy. After the sample was removed, it was filtered and analyzed by PXRD. PXRD and raman spectroscopy showed it to be pure form B. The mixture was cooled to 25 ℃ at a rate of 3 ℃/hour, then cooled to-10 ℃, filtered, and dried under vacuum to give 0.85g of form B crystalline product.

Example 7

6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) Yl) -8H-pyrido [2.3-d]Pyrimidin-7-onesPreparation of mono-isethionate salt of (form B)

The free base (formula 1, 0.895mg, 2mmol) was combined with 10mL MeOH, then seeded with 33mg of the mono-isethionate salt of the compound of formula 1 (form B). Then 10 equal parts of a solution of 5.6mL of 0.375M hydroxyethylsulfonic acid in MeOH (2.1mmol) were added over 75 minutes. The mixture was stirred for an additional 1 hour and a sample was taken for PXRD analysis. It was confirmed that crystalline form B had been formed. The mixture was stirred at room temperature overnight and PXRD was again performed. No change in crystalline form was found. The mixture was cooled in a-8 ℃ refrigerator, filtered and dried in a 50 ℃ vacuum oven to give 1.053g (91.8% of theory) of the title compound (form B). HPLC-99.8%, CHNS, H-HMR, RI, is consistent with the structure of form B, PXRD.

Example 8

6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) Yl) -8H-pyrido [2.3-d]Preparation of a mono-isethionate salt of a pyrimidin-7-one (form A)

Amorphous isethionate (47mg, prepared as in example 4) was mixed with 4mL EtOH in a 15mL flask equipped with magnetic stirrer, thermocouple and condenser. The mixture was heated to reflux to form a near clear solution. After refluxing for 10-15 minutes, the mixture became cloudy. Slowly cooled to 50 ℃ and then seeded with form a at 69 ℃. The mixture was kept at 50 ℃ for 5 hours and then cooled to room temperature overnight. The mixture was then cooled to 1 ℃ with an ice bath for 1.5 hours, filtered and washed with 0.5mL of cold EtOH, air dried, and then dried in a vacuum oven at 70 ℃ overnight to give 38.2mg of a fine crystalline material. The crystalline material was found to be mono-isethionate salt form a by PXRD. H-NMR conformed to the mono-isethionate salt and showed residual EtOH present, about 5.9 mol% or 0.6 wt%.

Example 9

6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) Radical) -8H-pyrido[2.3-d]Preparation of a mono-isethionate salt of a pyrimidin-7-one (form D)

Amorphous isethionate (9.0g, prepared as in example 4) was mixed with 300mL MeOH, stirred and heated to 63.8 ℃ (reflux). To this slightly turbid mixture two 50mL portions of MeOH were added. The hot mixture was filtered in a 2-L flask equipped with a mechanical stirrer. The mixture was briefly heated to reflux and then cooled to 60 ℃. IPA (100mL) was added to the mixture. The mixture was again heated to 60 ℃ and then an additional 110mL IPA was added. Precipitation began at 59.7 ℃. The mixture was heated again to 67.5 ℃, cooled to 50 ℃, and then kept overnight. The next morning samples were taken for PXRD analysis. The mixture was cooled to 25 ℃ at a rate of 3 ℃/hour and additional PXRD samples were taken and the mixture reached 28 ℃. The mixture was cooled to room temperature overnight. The precipitate was collected and dried in a vacuum oven at 65 ℃ and 30 torr. The above procedure yielded 7.45g (82.8% yield) of the crystalline compound (form D by PXRD analysis). The previously analyzed sample is also form D. HPLC showed 98.82% purity, with CHNS microanalysis in the range +/-0.4%. Substantially pure form B was obtained in less than three days from a slurry of isethionate form A, B and D in MeOH.

Example 10

Preparation of hydroxyethylsulfonic acid (2-hydroxy-ethanesulfonic acid)

A5-L four-necked round bottom flask equipped with a mechanical stirrer, thermocouple, gas sparge, and a vent through a water trap was charged with 748g (5.05mol) sodium isethionate (ALDRICH), and 4L IPA. The slurry was stirred at room temperature. An ice bath was used to maintain the internal temperature below 50 ℃ while 925g (25.4mol) of hydrogen chloride gas (ALDRICH) was bubbled through the system, the rate being controlled so that dissolution occurred immediately after addition (no foam was noted at this point through the water trap). Sufficient HCl gas was added until the system was saturated (at which point it was noted that foam began to pass through the dehydrator). During the addition of HCl, the temperature rose to 45 ℃. The slurry was then cooled to room temperature and filtered through a coarse glass filter. The filter cake was washed with 100mL IPA and the resulting cloudy filtrate was filtered through a 10-20 μ filter. The resulting clear, colorless filtrate was concentrated under reduced pressure on a rotary evaporator while maintaining the bath temperature below 50 ℃. The resulting 1.07kg of clear, pale yellow oil was washed with 50mL of tap water and 400mL of toluene, concentrated on a rotary evaporator under reduced pressure for three days while maintaining the bath temperature below 50 ℃. The resulting 800g of a clear, pale yellow oil was washed with 500mL of toluene and 250mL of IPA, concentrated under reduced pressure on a rotary evaporator for 11 days while maintaining the bath temperature below 50 deg.C. 713g of the resulting clear, pale yellow oil was titrated to 81 wt% (580g, 91.1% yield) containing 7.9 wt% water and 7.5 wt% IPA.

Example 11

4- {6- [6- (1-butoxy-vinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-di Hydro-pyrido [2, 3-d]Pyrimidin-2-ylamino]-pyridin-3-yl } -piperazine-1-carboxylic acid tert-butyl ester Preparation of

A5-L three-necked round bottom flask equipped with a mechanical stirrer, thermocouple, and nitrogen inlet/outlet vented through a silicone oil bubbler was placed under a nitrogen atmosphere and tert-butyl 4- [6- (6-bromo-8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydro-pyrido [2, 3-d ] pyrimidin-2-ylamino) -pyridin-3-yl ] -piperazine-1-carboxylate (300g, 0.51mol, as prepared in example 2), butyl vinyl ether (154g, 1.54mol, ALDRICH), n-butanol (1.5L, ALDRICH), and diisopropylethylamine (107mL, 0.62mol, ALDRICH) were added to it. The slurry was placed under a vacuum of about 50 torr and then aerated with nitrogen three times. 8.3g (0.01mol) of bis- (diphenylphosphinoferrocene) palladium dichloride dichloromethane (JOHNSONMATTHEY, product No. 077598001) was added thereto, and the resulting slurry was purified three times as described above. The mixture was then heated to 95 ℃ while stirring for 20 hours. The resulting reddish slurry was diluted with 2L heptane and then cooled to about 5 ℃. At temperature, 400mL of saturated aqueous potassium carbonate solution were added and the mixture was filtered and washed with 250mL of heptane. After drying in an oven at 45 ℃ for 16 h, 231.7g (75% yield) of the title compound are obtained as a yellow solid.

Example 12

6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) Yl) -8H-pyrido [2, 3-d]Preparation of a mono-isethionate salt of a pyrimidin-7-one (form B)

A22-L three-necked round bottom flask equipped with a mechanical stirrer, thermocouple, and nitrogen inlet/outlet vented through a silicone oil bubbler was placed under a nitrogen atmosphere and 4- {6- [6- (1-butoxy-vinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydro-pyrido [2, 3-d ] was added thereto]Pyrimidin-2-ylamino]-pyridin-3-yl } -piperazine-1-carboxylic acid tert-butyl ester (725g, 1.20mol, prepared as in example 11) and MeOH (14L). The slurry was stirred at room temperature while a solution of isethionic acid (530g, 4.20mol, prepared as in example 10), MeOH (1.5L), and water (70mL, 3.89mol) was added. The resulting slurry was heated to 55 ℃ over 30 minutes and then stirred at 55 ℃ for 30 minutes. 175g (1.73mol) Et was added to the slurry₃A solution of N (ALDRICH) in 200mL MeOH with cooling to 30 ℃. The slurry was kept at 30 ℃ while 128g (1.26mol) Et was added dropwise over 6 hours₃A solution of N in 2L MeOH. A sample of the resulting slurry was collected and identified as crystalline form (form B). The slurry was kept at 5 ℃ for 15 minutes after cooling and then filtered through a coarse glass filter. The resulting filter cake was washed several times with 200mL cold MeOH. The solid product was dried under vacuum at 55 ℃ to yield 710g (91% yield) of the title compound as yellow crystals.

It is to be understood that this description is intended to be illustrative, and not restrictive. Various embodiments will be apparent to those of skill in the art upon reading this specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. All papers and references, including patents, patent applications, and patent publications, are incorporated herein by reference in their entirety and for all purposes.

Claims (17)

1. The mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one.
2. 2. The mono-isethionate salt according to claim 1 which is form a of mono-isethionate salt characterised in that: a powder X-ray diffraction pattern having peaks at 2 θ values located at about 8.7, 12.6, 13.5, 17.6, 18.8, 19.6, 19.8, 23.0, and 24.2; having a pitch of about 1600cm^-1、1290cm^-1、675cm^-1、470cm^-1、450cm^-1And 425cm^-1A raman spectrum of a peak at the raman shift value position of (a); and a DSC thermogram with a sharp endotherm at 273 ℃.
3. 3. The mono-isethionate salt according to claim 1 which is form B of mono-isethionate salt characterised in that: a powder X-ray diffraction pattern having peaks at 2 θ values of approximately 5.1, 10.2, 11.8, 12.1, 12.8, 13.1, 14.7, 15.2, 16.0, 16.6, 17.9, 19.2, 19.7, 21.3, 21.9, 22.6, 23.2, 24.6, 25.6, 26.1, 28.9, 30.0, 30.9, 32.5, 33.0, 32.5, 33.0, 34.2, 35.3, and 36.0; having a pitch of about 1600cm^-1、1290cm^-1、470cm^-1、450cm^-1And 425cm^-1The peak of the Raman shift value of (2), but at 675cm^-1A raman spectrum essentially peak-free in position; and a DSC thermogram with a sharp endotherm at 271 ℃.
4. 4. The mono-isethionate salt according to claim 1 which is form D of mono-isethionate salt characterised in that: a powder X-ray diffraction pattern having peaks at 2 θ values of approximately 8.4, 8.9, 10.8, 12.6, 14.2, 16.8, 17.9, 18.4, 19.1, 20.0, 20.4, 21.0, 21.9, 22.6, 23.0, 23.6, 24.6, 26.2, 27.2, 28.7, 29.8, 30.3, 38.4; having a depth of about 463cm^-1A raman spectrum of a peak at the raman shift value position of (a); and a DSC thermogram with a sharp endotherm at 277 ℃.
5. 5. A pharmaceutical dosage form comprising the mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one and one or more pharmaceutically acceptable excipients.
6. 6. A process for preparing a crystalline mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one, said process comprising:

   mixing a solution of isethionic acid and a first solvent with a dispersion of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one and water to give a first mixture;

   freeze drying the first mixture to obtain an amorphous salt;

   mixing the amorphous salt with a second solvent to obtain a second mixture, the second mixture comprising crystalline mono-isethionic acid salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one, the second solvent being the same as or different from the first solvent; and is

   Optionally heating the second mixture, cooling the second mixture, or heating and cooling the second mixture,

   wherein the first solvent and the second solvent are the same or different and are water-miscible solvents.
7. 7. The method of claim 6, wherein the water miscible solvent is an alcohol.
8. 8. The process of claim 7, wherein the alcohol is MeOH or EtOH.
9. 9. The process of claim 6, wherein the first mixture or the second mixture is prepared by mixing an equimolar amount of isethionic acid with 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one, respectively.
10. 10. A process for preparing a mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one, said process comprising:

    reacting 4- {6- [6- (1-butoxy-vinyl) -8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydro-pyrido [2, 3-d ] pyrimidin-2-ylamino ] -pyridin-3-yl } -piperazine-1-carboxylic acid tert-butyl ester with isethionic acid in a first solvent and water to give a mixture comprising bis-isethionic acid salts of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one, said first solvent is as defined in claim 6; and

    addition of a hindered base to the reaction mixture yields the mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one.
11. Use of the mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one for the preparation of a medicament for the treatment of a disease or disorder caused by abnormal cell proliferation in a mammal.
12. 12. The use of claim 11, wherein the disease or condition is vascular smooth muscle proliferation associated with atherosclerosis, post-operative vascular stenosis and restenosis, or endometriosis.
13. 13. The use of claim 11, wherein the abnormal cell proliferation is cancer.
14. Use of the mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one for the manufacture of a medicament for the treatment of a disease or condition in a mammal caused by a viral or fungal infection.
15. Use of the mono-isethionate salt of 6-acetyl-8-cyclopentyl-5-methyl-2- (5-piperazin-1-yl-pyridin-2-ylamino) -8H-pyrido [2, 3-d ] pyrimidin-7-one for the manufacture of a medicament for the treatment of an autoimmune disease in a mammal.
16. 16. The use of claim 15, wherein the autoimmune disease is psoriasis, inflammatory-like rheumatoid arthritis, lupus, type 1 diabetes, diabetic nephropathy, multiple sclerosis, glomerulonephritis or organ transplant rejection.
17. 17. The use of claim 11, 14 or 15, wherein said mammal is a human.