HK1116485B - Anhydrous crystalline forms of n-[1-(2-ethoxyethyl)-5-(n-ethyl-n-methylamino)-7-(4-methylpyridin-2-yl-amino)-1h-pyrazolo[4,3-d]pyrimidine-3-carbonyl]methanesulfonamide - Google Patents

Description

Anhydrous crystalline forms of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide

Cross Reference to Related Applications

This application claims priority from U.S. provisional application 60/680,445, filed on 12/5/2005 and U.S. provisional application 60/681,711, filed on 17/5/2005, which are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates to crystalline forms of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide. More particularly, the present invention relates to (1) an anhydrous crystalline form of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide;

(2) pharmaceutical compositions containing at least one of the above crystalline forms; (3) methods of using at least one of the above crystalline forms for treating phosphodiesterase-5 mediated diseases; and (4) processes for preparing the above crystalline forms.

Background

The compound N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide has the following structure (1):

example 115 of published PCT application WO2005/049616 ("Compound application") describes the synthesis of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide. The compound application also discloses that N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is a PDE inhibitor useful for the treatment of phosphodiesterase-5 (PDE-5) mediated disorders such as hypertension.

Pharmaceutical compounds of different solid state crystalline forms may have physical properties that differ substantially in nature. Such differences in physical properties can have an effect on, for example, how the pharmaceutical compound is prepared, processed, formulated, or administered. Accordingly, it would be desirable to identify a novel solid crystalline form of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide that is superior to other solid crystalline forms in preparation, processing, formulation, or administration. Three novel anhydrous crystalline forms of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide have been identified, as follows.

Disclosure of Invention

In one embodiment, the present invention relates to anhydrous crystalline forms of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

In another embodiment, the present invention relates to crystalline form a anhydrous crystalline form of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide ("form a").

In another embodiment, the present invention relates to crystalline form B anhydrous crystalline form of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide ("form B").

In another embodiment, the present invention relates to crystalline form C anhydrous crystalline form of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide ("form C").

In another embodiment, the present invention relates to a composition comprising at least two crystalline forms of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide, selected from the group consisting of form a, form B and form C.

In another embodiment, the present invention relates to a pharmaceutical composition comprising at least one crystalline form of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide, which is selected from crystalline form a, crystalline form B, and crystalline form C, and a pharmaceutically acceptable carrier.

In another embodiment, the invention relates to a method for treating a PDE-5 mediated condition, comprising: administering to a patient a therapeutically effective amount of at least one crystalline form of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide selected from the group consisting of form A, form B, and form C.

In another embodiment, the present invention relates to a process for preparing form a, form B and form C.

Other embodiments of the invention are discussed throughout the specification of this application.

Drawings

Figure 1 shows an X-ray powder diffraction pattern of an exemplary form a.

Figure 2 shows the calculated X-ray powder diffraction pattern of form a.

Figure 3 shows an X-ray powder diffraction pattern of an exemplary form B.

Figure 4 shows an X-ray powder diffraction pattern of exemplary form C.

Figure 5 shows a schematic DSC thermogram for form a.

Figure 6 shows a schematic DSC thermogram for form B.

FIG. 7 shows a schematic DSC thermogram of form C.

Figure 8 shows a schematic FT-IR spectrum of form a.

Figure 9 shows a schematic FT-IR spectrum of form B.

Figure 10 shows a schematic FT-IR spectrum of form C.

Figure 11 shows a schematic raman spectrum of form a.

Fig. 12 shows a schematic raman spectrum of form B.

Fig. 13 shows a schematic raman spectrum of form C.

FIG. 14 shows an X-ray powder diffraction pattern of the material prepared in example 1.

FIG. 15 shows a schematic synthetic scheme for the preparation of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

Detailed Description

Solid state crystalline forms of a compound can materially affect the physical properties of the compound, including: (1) filling properties such as molar volume, density and hygroscopicity; (2) thermodynamic properties, such as melting temperature, vapor pressure, and solubility; (3) kinetic properties, such as, for example, rate of decomposition and stability (including stability under ambient conditions, especially under humid and storage conditions); (4) surface properties, e.g., surface area, wettability, interfacial tension, and topography; (5) mechanical properties, such as hardness, tensile strength, compressibility, handleability, flowability, and mixability; or (6) filtration properties. The selection and control of solid state crystalline forms is particularly important for compounds that are pharmaceutical formulations. Careful selection and control of the solid state crystal form can reduce the synthesis, processing, formulation or administration difficulties associated with the compound.

Three novel anhydrous crystalline forms (form a, form B and form C) of the compound N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide were identified. As explained in further detail below, each of form a, form B, and form C has distinct physical properties relative to each other.

In this application, the term "N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide" (and the corresponding "structure 1") includes all tautomers of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide. For example, two tautomers of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide are represented as tautomer (1) and tautomer (2) below (illustrated by the following resonance structures).

Without being bound to any particular theory, it is assumed that form a crystallizes in the form of the above tautomer (1), and form B and form C crystallize in the form of the above tautomer (2).

A.Abbreviations and Definitions

In that¹In H NMR, the symbol "delta" means¹Chemical shift of H NMR.

In that¹In H NMR, the abbreviation "br" means¹H NMR broad peak signal.

In that¹In H NMR, the abbreviation "d" means¹H NMR bimodal.

The abbreviation "m/z" refers to the mass spectral peak.

In that¹In H NMR, the abbreviation "m" means¹H NMR multiplet.

In that¹In H NMR, the abbreviation "q" means¹H NMR quartet.

In that¹In H NMR, the abbreviation "s" means¹H NMR single peak.

In that¹In H NMR, the abbreviation "t" means¹H NMR triplet.

The term "DSC" refers to a differential scanning calorimeter.

The term "HPLC" refers to high pressure liquid chromatography.

The term "PXRD" refers to X-ray powder diffraction.

The terms "PDE-5 mediated disorder" and "phosphodiesterase-5 mediated disorder" refer to any disorder mediated by PDE-5, including diseases that are directly modulated by PDE-5, and also including disorders that are indirectly modulated by PDE-5 as a signaling component.

The term "composition" refers to a product made by mixing or combining more than one ingredient.

The term "crystalline form" as used in "N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide" refers to a solid crystalline form in which the N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide molecules are arranged to form a recognizable crystal lattice, the above-described crystal lattice (i) includes identifiable unit cells, and (ii) generates diffraction peaks when subjected to X-ray radiation.

In this application, the term "crystallization" refers to crystallization and/or recrystallization, which depend on the specific application environment associated with the process for the preparation of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide starting material.

The term "purity" refers to the chemical purity of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide as determined by conventional HPLC assays.

The term "phase purity" refers to the degree of purity for N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide, which has a particular solid crystalline form, solid state purity of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide, determined by the analytical method described herein.

The term "pharmaceutically acceptable carrier" refers to a carrier that is compatible with the other ingredients of the composition and not deleterious to the patient. Such carriers can be pharmaceutically acceptable substances, compositions or vehicles, for example, liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, associated with the carrying or delivery of chemical agents. Preferred compositions depend on the method of administration.

The term "preventing" refers to preventing the onset of a latent dominant disease in a patient, or preventing the onset of a latent dominant phase of a disease in a patient. Prevention includes, but is not limited to, prophylactic treatment of a patient at risk of developing the disease.

The term "relative intensity" refers to the ratio of the intensity of a single diffraction peak (or spectral line) to the intensity of the strongest diffraction peak in the same derivative spectrum. In other words, the intensity of the strongest peak is set to 100, and all other intensities are scaled accordingly.

The term "therapeutically effective amount" refers to the amount of a drug or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, or animal that a researcher or clinician is attempting to find.

The term "treatment" refers to the palliative, restorative, and prophylactic treatment of a patient. The term "palliative treatment" refers to treatment to reduce or lessen the effect or intensity of a condition in a patient, but without curing the condition. The term "prophylactic treatment" refers to a treatment performed to prevent the occurrence of a condition in a subject. The term "restorative therapy" refers to a therapy that interrupts the progression of a disorder in a patient, alleviates a pathological change in a disorder in a patient, or completely eliminates a disorder in a patient.

B.Characterization of the Crystal forms

The crystalline form of a compound can be described by several crystal parameters, including: single crystal structure, X-ray powder diffraction spectrum, melting temperature, infrared absorption spectrum and Raman spectrum.

1.Single crystal X-ray analysis

The crystal structure of form a was determined by single crystal X-ray diffraction analysis. Single Crystal X-Ray diffraction data used in the analysis were collected at room temperature using a Bruker SMART APEX Single Crystal X-Ray diffractometer and Mo Ka radiation. Intensity was integrated over several consecutive exposures (SMART version 5.622 (control) and SAINT version 6.02 (integration) software, Bruker AXS inc., Madison, WI 1994) where each exposure covered 0.3 ° (in ω), exposure time was 30 seconds, and the total dataset was greater than half a sphere. Using a multiple scanning method (SADABS, a program for scaling and correcting region Detector data, G.M. Sheldrag, University of Gttingen, 1997 (based on the r.h. blanking method, Acta cryst.1995, a51, 33-38)) corrected data for absorption. The crystal structure was then modified using SHELXS-97 (a program for crystal structure modification, G.M. Sheldrag, University of G)ttingen, Germany, 1997, version 97-2) by direct method in space group P2₁The interpretation is carried out in/c and the correction is carried out by the least squares method using SHELXL-97. Selected crystal structure data are summarized in table 1A.

The crystal structure of form C was also determined by single crystal X-ray diffraction analysis in the same manner as described above for form a, except that the exposure time was 120 seconds. The crystal structure was explained in space group P-1 by the direct method using SHELXS-97 and modified by the least squares method using SHELXL-97. The crystal structure data for selected form C is summarized in table 1B.

Table 1A: crystal structure data of crystal form a

Table 1B: crystal form C crystal structure data

As mentioned previously, it is assumed that form a is crystallized in the form of tautomer (1) and form C is crystallized in the form of tautomer (2). Single crystal X-ray analysis supports the above hypothesis.

2.Powder X-ray diffraction

The crystal structures of form a, form B and form C were analyzed using X-ray powder diffraction ("PXRD"). X-ray diffraction data were collected at room temperature using a Bruker AXS D4 powder X-ray diffractometer (Cu ka radiation) equipped with an autosampler, theta-theta goniometer, an automatic beam splitting nip, a secondary monochromator, and a scintillation counter. Samples for analysis were prepared by the following method: the powder was charged into a die cavity 12mm in diameter and 0.25mm deep which had been placed on a silicon wafer sample stage. Rotating the sample while using copper kappa alpha₁X-ray (wavelength 1.5406)) Irradiation, wherein the X-ray tube is operated at 40kV/40 mA. The analysis was performed using an goniometer operating in continuous mode set to count 5 seconds per step of 0.02 ° over two θ ranges from 2 ° to 55 °. Each peak of form a was aligned with the calculated spectrum obtained from the single crystal structure. The resulting peaks for form B and form C are aligned with the peaks of the silicon reference standard.

2-theta angles, d-spacings, and relative intensities for form A from a single crystal structure utilizing Accelrymaterials Studio^TMThe 'Reflex Powder Diffraction' module (version 2.2) of (A) performs the calculation. The relevant simulation parameters in each case were: wavelength 1.540562(Cu K-αl)，Polarizing factor is 0.5, Pseudo-Voigt Profile (U is 0.01, V is-0.001, W is 0.002).

As will be appreciated by those skilled in crystallography, the relative intensities of the various peaks reported in the table below and the following figures may vary due to factors such as the orientation of the crystals in the X-ray beam or the purity of the material being analyzed or the crystallinity of the sample. The peak positions may also shift as the sample height changes, but the peak positions remain substantially at the positions defined in tables 2A, 2C and 2D for form a, form B and form C, respectively. Crystallography skilled artisans will also recognize that measurements with different wavelengths will also result in different displacements according to the bragg equation-n λ 2dsin θ. Such PXRD patterns generated using other wavelengths are considered to be other representative PXRD patterns for the crystalline material of the present invention and, as such, are intended to be within the scope of the present invention.

Fig. 1, 3 and 4 show schematic PXRD spectra for form a, form B and form C, respectively. Tables 2A, 2C and 2D list the major diffraction peaks obtained for form a, form B and form C, respectively, according to the 2 θ values and intensities. Table 2A lists the peaks of form a with relative intensities greater than 25%. Table 2C lists the peaks of form B with relative intensity greater than 2%. Table 2D lists the peaks of form C with relative intensities greater than 10%.

Additionally, fig. 2 shows the calculated PXRD pattern for form a. Table 2B lists the corresponding calculated major diffraction peaks obtained from the 2 θ values and intensities of form a. Table 2B lists the calculated form a peaks with relative intensities greater than 10%.

Table 2A: PXRD data of crystal form A

Table 2B: calculated PXRD data of crystal form A

Table 2C: PXRD data of crystal form B

Table 2D: PXRD data for form C

PXRD of crystal form A

The PXRD pattern for form a includes at least one diffraction peak selected from 8.5 ± 0.1, 9.0 ± 0.1, 16.9 ± 0.1, 20.0 ± 0.1, and 22.5 ± 0.1 ° 2 Θ. In one embodiment, the PXRD pattern for form a includes a diffraction peak at 8.5 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form a includes a diffraction peak at 8.5 ± 0.1 ° 2 Θ, and further includes at least one additional diffraction peak selected from 9.0 ± 0.1, 16.9 ± 0.1, 20.0 ± 0.1, and 22.5 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1, 9.0 ± 0.1, and 16.9 ± 0.1. In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1, 9.0 ± 0.1, 16.9 ± 0.1, 20.0 ± 0.1, and 22.5 ± 0.1 ° 2 Θ. In the above embodiments, the derivative peaks at 8.5 ± 0.1, 9.0 ± 0.1, 16.9 ± 0.1, 20.0 ± 0.1 and 22.5 ± 0.1 ° 2 θ generally have a relative intensity of at least about 10%.

In another embodiment, the PXRD pattern for form a (a) includes at least one diffraction peak selected from 8.5 ± 0.1, 9.0 ± 0.1, 16.9 ± 0.1, 20.0 ± 0.1, and 22.5 ± 0.1 ° 2 Θ, and (b) does not include at least one diffraction peak selected from 3.6 ± 0.1 and 7.2 ± 0.1 ° 2 Θ.

PXRD of crystal form B

The PXRD pattern for form B includes at least one diffraction peak selected from 3.6 ± 0.1, 7.2 ± 0.1, 10.1 ± 0.1, 14.4 ± 0.1, and 23.8 ± 0.1 ° 2 Θ. In one embodiment, the PXRD pattern for form B includes a diffraction peak at 3.6 ± 0.1 ° 2 Θ. In another embodiment. The PXRD pattern for form B includes a diffraction peak at 3.6 ± 0.1 ° 2 Θ, and further includes at least one other diffraction peak selected from 7.2 ± 0.1, 10.1 ± 0.1, 14.4 ± 0.1, and 23.8 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 and 7.2 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1, 7.2 ± 0.1, and 23.8 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1, 7.2 ± 0.1, 10.1 ± 0.1, 14.4 ± 0.1, and 23.8 ± 0.1 ° 2 Θ. In the above embodiments, the derivative peaks at 3.6 ± 0.1 and 7.2 ± 0.1 ° 2 Θ generally have a relative intensity of at least about 10%.

In another embodiment, the PXRD pattern for form B (a) includes at least one diffraction peak selected from 3.6 ± 0.1, 7.2 ± 0.1, 10.1 ± 0.1, 14.4 ± 0.1, and 23.8 ± 0.1 ° 2 Θ, and (B) does not include at least one diffraction peak selected from 8.5 ± 0.1, 6.7 ± 0.1, and 22.5 ± 0.1 ° 2 Θ.

PXRD of crystal form C

The PXRD pattern for form C includes at least one diffraction peak selected from 6.7 ± 0.1, 10.6 ± 0.1, 14.0 ± 0.1, 17.7 ± 0.1, and 20.2 ± 0.1 ° 2 Θ. In one embodiment, the PXRD pattern for form C includes a diffraction peak at 6.7 ± 0.1 ° 2 Θ. In one embodiment, the PXRD pattern for form C includes a diffraction peak at 10.6 ± 0.1 ° 2 Θ. In one embodiment, the PXRD pattern for form C includes a diffraction peak at 14.0 ± 0.1 ° 2 Θ. In one embodiment, the PXRD pattern for form C includes a diffraction peak at 17.7 ± 0.1 ° 2 Θ. In one embodiment, the PXRD pattern for form C includes diffraction peaks at 20.2 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form C includes a diffraction peak at 6.7 ± 0.1 ° 2 Θ, and further includes at least one additional diffraction peak selected from 10.6 ± 0.1, 14.0 ± 0.1, 17.7 ± 0.1, and 20.21 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 and 20.2 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1, 17.7 ± 0.1, and 20.2 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1, 17.7 ± 0.1, 10.6 ± 0.1, and 20.2 ± 0.1 ° 2 Θ. In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1, 10.6 ± 0.1, 14.0 ± 0.1, 17.7 ± 0.1, and 20.2 ± 0.1 ° 2 Θ. In the above embodiments, the diffraction peaks at 6.7 ± 0.1, 10.6 ± 0.1, 14.0 ± 0.1, 17.7 ± 0.1 and 20.2 ± 0.1 ° 2 θ preferably have a relative intensity of at least about 10%.

In another embodiment, the PXRD pattern for form C (a) includes at least one diffraction peak selected from 6.7 ± 0.1, 10.6 ± 0.1, 14.0 ± 0.1, 17.7 ± 0.1, and 20.2 ± 0.1 ° 2 Θ, and (b) does not include at least one diffraction peak selected from 3.6 ± 0.1 and 9.0 ± 0.1.

3.Differential scanning calorimetry

Each of form a, form B and form C was analyzed using a Differential Scanning Calorimeter (DSC). Each crystal form was analyzed using a TA Instruments Q1000 differential scanning calorimeter. Each sample was heated from 25 ℃ to 300 ℃ at a rate of 20 ℃ per minute in an aluminum pan with a lid on top using a nitrogen purge gas. The temperature of the melting endotherm peak is reported as the melting point. The data from DSC analysis depends on many factors, including: heating rate, purity of the sample, crystal size, and sample size. Thus, the following melting points are representative of the melting points of the samples prepared below.

DSC of form A

A sample of 3.171mg form a was analyzed by DSC as described above. FIG. 5 is a DSC thermogram obtained from a sample of form A. Form A exhibited a first endothermic peak at 174 ℃. + -. 3 ℃ followed by exothermic recrystallization at 179 ℃. + -. 3 ℃ and a second endothermic peak at 219 ℃. + -. 3 ℃. The peak at 174 ℃. + -. 3 ℃ corresponds to the melting point of form A. Exothermic recrystallization at 179 ℃. + -. 3 ℃ corresponds to recrystallization of the molten compound in the form of form B. The peak at 219 ℃. + -. 3 ℃ corresponds to the melting point of form B.

DSC of form B

A 1.603mg sample of form B was analyzed by DSC as described above. FIG. 6 is a DSC thermogram obtained from a sample of form B. The endothermic peak of form B at 218 ℃. + -. 3 ℃ corresponds to the melting point of form B.

DSC of form C

A sample of 4.405mg of form C was analyzed by DSC as described above. FIG. 7 is a DSC thermogram obtained from a sample of form C. Form C exhibited a first endothermic peak at 188 ℃. + -. 3 ℃ followed by exothermic recrystallization at 199 ℃. + -. 3 ℃ and a second endothermic peak at 219 ℃. + -. 3 ℃. The peak at 188 ℃. + -. 3 ℃ corresponds to the melting point of form C. Exothermic recrystallization at 199 ℃. + -. 3 ℃ corresponds to recrystallization of the molten compound as form B. The peak at 219 ℃. + -. 3 ℃ corresponds to the melting point of form B.

4.Fourier transform infrared spectroscopy

The crystal structures of form a, form B, and form C were analyzed using fourier transform infrared ("FT-IR") spectroscopy. FT-IR spectra of samples form a, form B and form C were obtained using a ThermoNicolet Avatar 360 spectrometer with a Smart Golden Gate single reflection ATR accessory (diamond top plate and zinc selenide lens). The measurement conditions selected were: 2cm^-1Resolution, 128 scans, and Happ Genzel apodization (apodization). Because the FT-IR spectrum is recorded using single reflection ATR, there is no need to prepare the sample. However, the use of ATR FT-IR can result in a difference in the relative intensity of the infrared band from that typically seen in KBr pellet FT-IR spectra. Due to the nature of ATR FT-IR, the intensity of the spectral band generally increases when scanning from higher wavelength regions to lower wavelength regions. Unless otherwise stated, experimental error is. + -. 2cm^-1。

Fig. 8, 9 and 10 show schematic FR-IR spectra of form a, form B and form C, respectively. Tables 4A, 4B and 4C list the corresponding characteristic, identifiable absorption bands for form a, form B and form C, respectively.

Table 4A: FT-IR spectrum data of form A

¹w: weak; m: medium; ms: medium strength; s: high strength

²The experimental error is +/-3 cm^-1

Table 4B: FT-IR spectral data for form B

¹w: weak; m: medium; ms: medium strength; s: high strength

Table 4C: FT-IR spectral data for form C

¹w: weak; m: medium; ms: medium strength; s: high strength

FT-IR of form A

The FT-IR spectrum of form A comprises at least one of 696 + -2, 1085 + -2, 1188 + -2, 1540 + -2 and 3247 + -3 cm^-1The absorption band of (1). In one embodiment, the FT-IR spectrum of form A comprises 3247 ± 3cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form A comprises 3247 ± 3cm^-1The absorption band of (A) further comprises at least one absorption band selected from 696 + -2, 1085 + -2, 1188 + -2 and 1540 + -2 cm^-1The absorption band of (1). In another embodiment, the FT-IR spectrum of form A comprises 3247 ± 3 and 696 ± 2cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form A comprises 696 + -2, 1188 + -2, and 3247 + -3 cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form A comprises 696 + -2, 1188 + -2, 1540 + -2, and 3247 + -3 cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form A comprises 696 + -2, 1085 + -2, 1188 + -2, 1540 + -2 and 3247 + -3 cm^-1The absorption band of (b).

In another embodiment, form a has an FT-IR spectrum (a) comprising at least one spectrum selected from 696 ± 2, 1085 ± 2, 1188 ± 2, 1540 ± 2 and 3247 ± 3cm^-1And (b) does not comprise an absorption band of 1645. + -.2 cm^-1The absorption band of (b).

FT-IR of form B

The FT-IR spectrum of form B comprises at least one spectrum selected from 722 + -2, 920 + -2, 1211 + -2, 1395 + -2 and 1452 + -2 cm^-1The absorption band of (1). In one embodiment, the FT-IR spectrum of form BThe graph includes 1452 + -2 cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form B comprises 1452 ± 2cm^-1The absorption band of (A) further comprises at least one absorption band selected from the group consisting of 722 + -2, 920 + -2, 1211 + -2 and 1395 + -2 cm^-1The absorption band of (1). In another embodiment, the FT-IR spectrum of form B comprises 1452 + -2 and 1395 + -2 cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form B comprises 1211 + -2, 1395 + -2, and 1452 + -2 cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form B comprises peaks at 722 + -2, 1211 + -2, 1395 + -2, and 1452 + -2 cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form B comprises peaks at 722 + -2, 920 + -2, 1211 + -2, 1395 + -2, and 1452 + -2 cm^-1The absorption band of (b).

In another embodiment, form B has an FT-IR spectrum (a) comprising at least one spectrum selected from 722 + 2, 920 + 2, 1211 + 2, 1395 + 2, and 1452 + 2cm^-1And (b) does not include an absorption band of 962. + -.2 cm^-1The absorption band of (b).

FT-IR of form C

The FT-IR spectrum of form C comprises at least one spectrum selected from 661 + -2, 703 + -2, 797 + -2, 881 + -2, 909 + -2 and 1269 + -2 cm^-1The absorption band of (1). In another embodiment, the FT-IR spectrum of form C comprises 881 ± 2cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form C comprises 881 ± 2cm^-1The absorption band of (A) further comprises at least one absorption band selected from 661 + -2, 703 + -2, 797 + -2, 909 + -2 and 1269 + -2 cm^-1Other absorption bands of (a). In another embodiment, the FT-IR spectrum of form C is comprised between 881. + -. 2 and 661. + -. 2cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form C comprises 661 + -2, 797 + -2, and 881 + -2 cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form C comprises 661 + -2, 703 + -2, 797 + -2, and 881 + -2 cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form C comprises 661 + -2, 703 + -2, 797 + -2, 881 + -22 and 909. + -. 2cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum of form C comprises 661 + -2, 703 + -2, 797 + -2, 881 + -2, 909 + -2, and 1269 + -2 cm^-1The absorption band of (b).

In another embodiment, the FT-IR spectrum (a) of form C comprises at least one spectrum selected from 661. + -.2, 703. + -. 2, 881. + -. 2, 909. + -. 2 and 1269. + -.2 cm^-1And (b) does not include 688. + -. 2cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum (a) of form C comprises at least one spectrum selected from 661. + -. 2, 703. + -. 2, 797. + -. 2, 881. + -. 2, 909. + -. 2 and 1269. + -. 2cm^-1And (b) does not include an absorption band at 696 + -2 cm^-1The absorption band of (b). In another embodiment, the FT-IR spectrum (a) of form C comprises at least one spectrum selected from 661. + -. 2, 703. + -. 2, 797. + -. 2, 881. + -. 2, 909. + -. 2 and 1269. + -. 2cm^-1Does not include at least one absorption band selected from 688 +/-2 or 696 +/-2 cm^-1The absorption band of (1).

As mentioned previously, it is assumed that form a is crystallized in the form of tautomer (1) and form C is crystallized in the form of tautomer (2). FT-IR analysis supports the above assumptions. Specifically, the FT-IR spectrum of the crystal form C is 1644 +/-2 cm^-1Has a medium-strong absorption band, and the FT-IR spectrum of the crystal form B is 1646 +/-2 cm^-1Has a strong absorption band. We believe that these bands correspond to the stretch frequencies of acyclic C ═ N, which is consistent with tautomer (2). In contrast, the FT-IR spectrum of form a has no absorption bands at the corresponding frequencies. We believe that form a has no acyclic C ═ N condensation frequency, since it crystallizes as tautomer (1).

5.Fourier transform Raman spectroscopy

Form a, form B, and form C were analyzed using fourier transform raman ("raman") spectroscopy. Raman spectra of form a, form B and form C were obtained using a ThermoNicolet 960 raman spectrum. Each sample (about 5mg) was placed in a glass vial and exposed to 1064.5nm Nd-YAG laser energy for excitation. Is collected toResolution of the data was 2cm^-1Wherein the raman intensity is a function of the raman shift. The data were fourier transformed using Happ-Genzel apodization. Unless otherwise stated, experimental error is. + -. 2cm^-1。

FIGS. 11, 12 and 13 show schematic Raman spectra of form A (test condition: 2000 scans, laser power: 750mW, laser power on sample: 400mW), form B (test condition: 4000 scans, laser power: 600mW, laser power on sample: 340mW) and form C (test condition: 960 scans, laser power: 600mW, laser power on sample: 340mW), respectively. The X-axis is the Raman shift (cm)^-1) And the Y-axis is intensity. The intensity is the intensity assigned relative to the main absorption band in the spectrum, and not the absolute value relative to the baseline measurement. Tables 5A, 5B and 5C list the characteristic raman bands of the corresponding form a, form B and form C, respectively.

Table 5A: raman spectrum data of crystal form A

¹w: weak; m: medium; s: high strength

²The experimental error is +/-3 cm^-1

Table 5B: raman spectrum data of crystal form B

¹w: weak; m: medium; s: high strength

Table 5C: raman spectrum data of crystal form C

¹w: weak; m: medium; s: high strength

Raman spectrum of crystal form A

The Raman spectrum of the crystal form A comprises at least one selected from 993 +/-2, 1383 +/-2, 1473 +/-2, 1569 +/-2 and 3255 +/-3 cm^-1The band of (2). In another embodiment, the raman spectrum of form a comprises at 3255 ± 3cm^-1The band of (b). In another embodiment, the raman spectrum of form a comprises at 3255 ± 3cm^-1The band of (b), further comprising at least one band selected from the group consisting of 993 + -2, 1383 + -2, 1473 + -2 and 1569 + -2 cm^-1Other bands of (2). In another embodiment, the raman spectrum of form a comprises peaks at 1569 ± 2 and 3255 ± 3cm^-1The band of (b). In another embodiment, the raman spectrum of form a is comprised at 1473 ± 2, 1569 ± 2 and 3255 ± 3cm^-1The band of (b). In another embodiment, the raman bands of form a are comprised at 1383 ± 2, 1473 ± 2, 1569 ± 2 and 3255 ± 3cm^-1The band of (b). In another embodiment, the raman bands of form a are comprised between 993 ± 2, 1383 ± 2, 1473 ± 2, 1569 ± 2 and 3255 ± 3cm^-1The band of (b).

In another embodiment, the raman spectrum of form a (a) comprises at least one raman spectrum selected from 993 ± 2, 1383 ± 2, 1473 ± 2, 1569 ± 2 and 3255 ± 3cm^-1Does not include the band of (b) at 1652. + -.2 cm^-1The band of (b).

Raman spectrum of crystal form B

The Raman spectrum of the crystal form B comprises at least one of 689 +/-2, 1299 +/-2, 1456 +/-2 and 1535 +/-2 cm^-1The band of (2). In another embodiment, raman of form BThe spectrum is included in 1299 +/-2 cm^-1The band of (b). In another embodiment, the raman spectrum of form B is comprised at 1299 ± 2cm^-1The band of (b), further comprising at least one band selected from 689 + -2, 1456 + -2 and 1535 + -2 cm^-1Other bands of (2). In another embodiment, the raman spectrum of form B comprises 689 ± 2 and 1299 ± 2cm^-1The band of (b). In another embodiment, the raman spectrum of form B comprises 689 ± 2, 1299 ± 2 and 1535 ± 2cm^-1The band of (b). In another embodiment, the raman spectrum of form B comprises at 689 ± 2, 1299 ± 2, 1456 ± 2 and 1535 ± 2cm^-1The band of (b).

In another embodiment, the raman spectrum of form B (a) comprises at least one raman spectrum selected from 689 ± 2, 1299 ± 2, 1456 ± 2 and 1535 ± 2cm^-1And (b) does not comprise a band at 1316. + -.2 cm^-1The band of (b).

Raman spectrum of crystal form C

The Raman spectrum of the crystal form C comprises at least one selected from 707 +/-2, 1447 +/-2 and 2988 +/-2 cm^-1The band of (2). In another embodiment, the raman spectrum of form C comprises 2988 ± 2cm^-1The band of (b). In another embodiment, form C has a raman spectrum at 2988 ± 2cm^-1At least one significant band selected from 707 + -2 and 1447 + -2 cm^-1Other bands of (2). In another embodiment, the raman spectrum of form C is comprised between 707 ± 2 and 2988 ± 2cm^-1The band of (b). In another embodiment, the Raman spectrum of form C comprises about 707. + -.2, 1447. + -.2 and 2988. + -.2 cm^-1The band of (b).

In another embodiment, form C raman spectrum (a) comprises at least one selected from 707 ± 2, 1447 ± 2 and 2988 ± 2cm^-1And (b) does not include band 1417. + -.2 cm^-1The band of (b).

As mentioned previously, it is assumed that form a is crystallized in the form of tautomer (1), and form B and form C are each crystallized in the form of tautomer (2). FT-Raman analysis supports the aboveIt is assumed. Specifically, the FT-Raman spectrum of the crystal form C is 1651 +/-2 cm^-1Has a weak Raman band, and the FT-Raman spectrum of the crystal form B is 1652 +/-2 cm^-1With a medium raman band. We believe that these bands correspond to the stretch frequencies of acyclic C ═ N, which is consistent with tautomer (2). In contrast, the FT-raman spectrum of form a has no raman band at the corresponding frequency. We believe that form a does not have an acyclic C ═ N stretch frequency, since form a crystallizes as tautomer (1).

C.Properties of form A, form B and form C

1.Thermodynamic stability

Form a, form B and form C have different thermodynamic stabilities. Form B is thermodynamically more stable at ambient as well as elevated temperatures than form a (see example 13 below). However, form B and form C are tautomeric relationships. The thermodynamic stabilities of form B and form C cross at a temperature of about 40 ℃ to about 60 ℃ (see example 14 below). In another embodiment, the thermodynamic stabilities of form B and form C are crossed at a temperature of about 40 ℃ to 50 ℃. Form B is thermodynamically more stable than form C at temperatures above the crossover point. Form C is thermodynamically more stable than form B when stable below the crossover point (including at ambient temperatures).

This difference in thermodynamic stability is important in practical applications. The thermodynamic stability of the crystalline form affects the potential shelf life of a formulated pharmaceutical product containing the crystalline form. Generally, the greater the thermodynamic stability, the longer the shelf life of the formulated pharmaceutical product. In addition, this difference in thermodynamic stability may cause problems in the following cases: the processing results in an increase in temperature (e.g., due to milling compounds), or the processing is conducted over a range of temperatures. During processing, such temperature changes may result in the transformation of one crystalline form to another crystalline form. If the resulting crystalline form is not the desired form, then the processing temperature needs to be more carefully controlled.

2.Form of the composition

Form a and form B also have different crystal morphologies. Although various factors such as temperature, solvents, impurities, and fluid mechanics (vibration) can affect the crystal morphology, form a and form B have distinct crystal morphologies. Form a generally has a tabular morphology. Form B typically has a needle-like morphology. Form C includes mixtures of ribbon, flat and chip shapes having a size (largest dimension) in the range of about 5 microns to about 350 microns, typically 50 to 60 microns.

This difference in morphology can affect the ease with which the compound can be processed to prepare a formulated pharmaceutical product. For example, the needle-like morphology makes filtration and processing more difficult. In addition, the tabular morphology is more uniform in size relative to the needle-like morphology, which results in improved flow and processability of the compound, thereby making the filtration, processing and tableting steps easier.

3.Colour(s)

Form a, form B and form C also have different appearances. Form a is typically yellowish to ivory. Form B is typically yellow. Form C is typically pale yellow. The product specifications for formulating pharmaceutical products typically specify not only the chemical purity of the active ingredient, but also the phase purity of the active ingredient. Batch-to-batch variation in the crystalline form of the active ingredient is often unsatisfactory. In the case of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide, the color of the batch may serve the purpose of quality control, thereby providing a means for qualitatively assessing the product as to whether the phase purity of the batch meets the desired phase purity criteria. In addition, the aesthetics of the product is also important, and it is desirable that the color of the final pharmaceutical product be uniform in appearance. In the case where the color properties of the crystalline form affect the appearance of the formulated product, practice is required to properly control the crystalline form present in the product in order to maintain product color consistency.

D.Other embodiments

The following are other embodiments of form a, form B, and form C.

Other embodiments of form a

In one embodiment, the PXRD pattern for form a includes a diffraction peak at 8.5 ± 0.1 ° 2 Θ and the FT-IR pattern includes 3247 ± 3cm^-1The absorption band of (b). In another embodiment, the PXRD spectrum of form A includes diffraction peaks at 8.5 ± 0.1 ° 2 theta and the FT-IR spectrum includes 3247 ± 3 and 696 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1 and 9.0 ± 0.1 ° 2 Θ and the FT-IR pattern includes 3247 ± 3cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1 and 9.0 ± 0.1 ° 2 Θ and the FT-IR pattern includes 696 ± 2cm^-1And 3247. + -. 3cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1, 9.0 ± 0.1, and 16.9 ± 0.1 ° 2 Θ and the FT-IR pattern includes diffraction peaks at 696 ± 2, 1188 ± 2, and 3247 ± 3cm^-1The absorption band of (b).

In another embodiment, the PXRD pattern for form a includes a diffraction peak at 8.5 ± 0.1 ° 2 Θ and the raman pattern includes 3255 ± 3cm^-1The band of (b). In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1 ° 2 Θ and the raman pattern includes 1569 ± 2 and 3255 ± 3cm^-1The band of (b). In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1 and 9.0 ± 0.1 ° 2 Θ and the raman pattern includes 3255 ± 3cm^-1The band of (b). In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1 and 9.0 ± 0.1 ° 2 Θ and the raman pattern includes 1569 ± 2 and 3255 ± 3cm^-1The band of (b). In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1, 9.0 ± 0.1, and 16.9 ± 0.1 ° 2 Θ and the raman pattern includes 1569 ± 2 and 3255 ± 3cm^-1The band of (b).In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1, 9.0 ± 0.1, and 16.9 ± 0.1 ° 2 Θ and the raman pattern includes diffraction peaks at 1473 ± 2, 1569 ± 2, 3255 ± 3cm^-1The band of (b).

In another embodiment, the PXRD pattern for form a includes a diffraction peak at 8.5 ± 0.1 ° 2 Θ and has a melting point of 174 ℃ ± 3 ℃. In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1 and 9.0 ± 0.1 ° 2 Θ and has a melting point of 174 ℃ ± 3 ℃. In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1, 9.0 ± 0.1, and 16.9 ± 0.1 ° 2 Θ having a melting point of 174 ℃ ± 3 ℃.

In another embodiment, the PXRD pattern for form a includes a diffraction peak at 8.5 ± 0.1 ° 2 Θ and the FT-IR pattern includes 3247 ± 3cm^-1The melting point of the absorption band (b) is 174 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1 and 9.0 ± 0.1 ° 2 Θ and the FT-IR pattern includes 3247 ± 3cm^-1The melting point of the absorption band (b) is 174 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1, 9.0 ± 0.1, and 16.9 ± 0.1 ° 2 Θ and the FT-IR pattern includes 3247 ± 3 and 696 ± 2cm^-1The melting point of the absorption band (b) is 174 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1, 9.0 ± 0.1, and 16.9 ± 0.1 ° 2 Θ and the FT-IR pattern includes diffraction peaks at 696 ± 2, 1188 ± 2, and 3247 ± 3cm^-1The melting point of the absorption band (b) is 174 ℃. + -. 3 ℃.

In another embodiment, the PXRD pattern for form a includes a diffraction peak at 8.5 ± 0.1 ° 2 Θ and the FT-IR pattern includes 3247 ± 3cm^-1The Raman spectrum of the absorption band is included in 3255 + -3 cm^-1The band of (b). In another embodiment, the PXRD spectrum of form A includes diffraction peaks at 8.5 + -0.1 and 9.0 + -0.1 ° 2 theta and the FT-IR spectrum includes 696 + -2 and 3247 + -3 cm^-1The Raman spectrum of the absorption band is included in 1569 +/-2 and 3255 +/-3 cm^-1The band of (b). In another embodiment, the PXRD pattern for form a comprises 8.5 ± 0.1, 16.9 ± 01 and 22.5 + -0.1 deg. 2 theta, FT-IR spectra including 696 + -2, 1188 + -2 and 3247 + -3 cm^-1The Raman spectrum of the absorption band is included in 1569 +/-2 and 3255 +/-3 cm^-1The band of (b).

In another embodiment, the PXRD spectrum of form A includes diffraction peaks at 8.5 + -0.1 and 9.0 + -0.1 ° 2 theta and the FT-IR spectrum includes 696 + -2 and 3247 + -3 cm^-1The Raman spectrum of the absorption band is included in 1569 +/-2 and 3255 +/-3 cm^-1The band of (b) has a melting point of 174 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form a includes diffraction peaks at 8.5 ± 0.1, 16.9 ± 0.1, and 22.5 ± 0.1 ° 2 Θ and the FT-IR pattern includes diffraction peaks at 696 ± 2, 1188 ± 2, and 3247 ± 3cm^-1The Raman spectrum of the absorption band is included in 1569 +/-2 and 3255 +/-3 cm^-1The band of (b) has a melting point of 174 ℃. + -. 3 ℃.

Other embodiments of form B

In one embodiment, the PXRD pattern for form B includes a diffraction peak at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes a diffraction peak at 1452 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes 1395 ± 2 and 1452 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes 1211 ± 2, 1395 ± 2, and 1452 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes diffraction peaks at 722 ± 2, 920 ± 2, 1211 ± 2, 1395 ± 2, and 1452 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 and 7.2 ± 0.1 ° 2 Θ and the FT-IR pattern includes 1211 ± 2, 1395 ± 2, and 1452 ± 2cm^-1The absorption band of (b).

In one embodiment, the PXRD pattern for form B includes a diffraction peak at 3.6 ± 0.1 ° 2 Θ having a melting point of 218 ℃ ± 3 ℃. In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 and 7.2 ± 0.1 ° 2 Θ and has a melting point of 218 ℃ ± 3 ℃. In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1, 7.2 ± 0.1, and 23.8 ± 0.1 ° 2 Θ having a melting point of 218 ℃ ± 3 ℃. In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1, 7.2 ± 0.1, 10.1 ± 0.1, 14.4 ± 0.1, and 23.8 ± 0.1 ° 2 Θ having a melting point of 218 ℃ ± 3 ℃.

In another embodiment, the PXRD pattern for form B includes a diffraction peak at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes a diffraction peak at 1452 ± 2cm^-1The melting point of the absorption band at (b) is 218 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes 1395 ± 2 and 1452 ± 2cm^-1The melting point of the absorption band at (b) is 218 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1, 7.2 ± 0.1, 10.1 ± 0.1, 14.4 ± 0.1, and 23.8 ± 0.1 ° 2 Θ and the FT-IR pattern includes diffraction peaks at 722 ± 2, 920 ± 2, 1211 ± 2, 1395 ± 2, and 1452 ± 2cm^-1The melting point of the absorption band at (b) is 218 ℃. + -. 3 ℃.

In another embodiment, the PXRD pattern for form B includes a diffraction peak at 3.6 ± 0.1 ° 2 Θ and the raman pattern includes a diffraction peak at 1299 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 ° 2 Θ and the raman pattern includes diffraction peaks at 689 ± 2 and 1299 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 ° 2 Θ and the raman pattern includes diffraction peaks at 689 ± 2, 1299 ± 2, and 1535 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 ° 2 Θ and the raman pattern includes diffraction peaks at 689 ± 2, 1299 ± 2, 1456 ± 2, and 1535 ± 2cm^-1The absorption band of (b).

In another embodiment, the PXRD pattern for form B includes a diffraction peak at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes a diffraction peak at 1452 ± 2cm^-1(ii) an absorption band, Raman spectrum comprised between 1299. + -.2 cm^-1The absorption band of (b). In another embodimentWherein the PXRD spectrum of form B comprises diffraction peaks at 3.6 + -0.1 deg. 2 theta and the FT-IR spectrum comprises 1395 + -2 and 1452 + -2 cm^-1The Raman spectrum of the absorption band is included in 1299 +/-2 and 689 +/-2 cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form B includes diffraction peaks at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes diffraction peaks at 1395 ± 2, 1452 ± 2, and 1535 ± 2cm^-1The Raman spectrum of the absorption band is included in 1299 +/-2 and 689 +/-2 cm^-1The absorption band of (b).

In another embodiment, the PXRD pattern for form B includes a diffraction peak at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes a diffraction peak at 1395 ± 2cm^-1And 1452. + -.2 cm^-1(ii) an absorption band, Raman spectrum comprised between 1299. + -.2 cm^-1The melting point of the absorption band at (b) is 218 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form B includes a diffraction peak at 3.6 ± 0.1 ° 2 Θ and the FT-IR pattern includes a diffraction peak at 1395 ± 2cm^-11452 + -2 and 1535 + -2 cm^-1The Raman spectrum of the absorption band is included in 1299 +/-2 and 689 +/-2 cm^-1The melting point of the absorption band at (b) is 218 ℃. + -. 3 ℃.

Other embodiments of form C

In another embodiment, the PXRD pattern for form C includes a diffraction peak at 6.7 ± 0.1 ° 2 Θ and the FT-IR pattern includes 881 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 ° 2 Θ and the FT-IR pattern includes 881 ± 2 and 661 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 ° 2 Θ and the FT-IR pattern includes diffraction peaks at 881 ± 2, 797 ± 2, 703 ± 2, and 661 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 and 26.1 ± 0.1 ° 2 Θ and the FT-IR pattern includes 881 ± 2 and 661 ± 2cm^-1The absorption band of (b).

In another embodiment, the PXRD pattern for form C includes a diffraction peak at 6.7 ± 0.1 ° 2 Θ and has a melting point of 188 ℃ ± 3 ℃. In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 and 26.1 ± 0.1 ° 2 Θ and has a melting point of 188 ℃ ± 3 ℃. In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1, 20.2 ± 0.1, and 17.7 ± 0.1 ° 2 Θ having a melting point of 188 ℃ ± 3 ℃. In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1, 10.6 ± 0.1, 14.0 ± 0.1, 17.7 ± 0.1, and 20.2 ± 0.1 ° 2 Θ and has a melting point of 188 ℃ ± 3 ℃.

In another embodiment, the PXRD pattern for form C includes a diffraction peak at 6.7 ± 0.1 ° 2 Θ and the raman pattern includes a peak at 2988 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 ° 2 Θ and the raman pattern includes patterns at 707 ± 2 and 2988 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 ° 2 Θ and the raman pattern includes diffraction peaks at 707 ± 2, 1447 ± 2, and 2988 ± 2cm^-1The absorption band of (b). In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 and 26.1 ± 0.1 ° 2 Θ and the raman pattern includes 707 ± 2, 1447 ± 2, and 2988 ± 2cm^-1The absorption band of (b).

In another embodiment, the PXRD pattern for form C includes a diffraction peak at 6.7 ± 0.1 ° 2 Θ and the FT-IR pattern includes a peak at 661 ± 2cm^-1881 + -2 cm^-1The melting point of the absorption band (b) is 188 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 and 20.2 ± 0.1 ° 2 Θ and the FT-IR pattern includes 661 ± 2, 881 ± 2, and 797 ± 2cm^-1The melting point of the absorption band (b) is 188 ℃. + -. 3 ℃.

In another embodiment, the PXRD pattern for form C includes a diffraction peak at 6.7 ± 0.1 ° 2 Θ and the FT-IR pattern includes 881 ± 2cm^-1The Raman spectrum of the absorption band is included in 2988 +/-2 cm^-1The melting point of the absorption band (b) is 188 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form C includes a diffraction peak at 6.7 ± 0.1 ° 2 Θ and the FT-IR pattern includes a peak at 661 ± 2cm^-1881 + -2 cm^-1The Raman spectrum of the absorption band is included in 2988 +/-2 cm^-1The melting point of the absorption band (b) is 188 ℃. + -. 3 ℃. In another embodiment, form C has a PXRD pattern including diffraction peaks at 6.7 ± 0.1 and 20.2 ± 0.1 ° 2 Θ and a FT-IR pattern including 661 ± 2cm^-1881 + -2 cm^-1The Raman spectrum of the absorption band is comprised in 707 +/-2 and 2988 +/-2 cm^-1The melting point of the absorption band (b) is 188 ℃. + -. 3 ℃. In another embodiment, the PXRD pattern for form C includes diffraction peaks at 6.7 ± 0.1 and 20.2 ± 0.1 ° 2 Θ and the FT-IR pattern includes 881 ± 2, 797 ± 1, 703 ± 2 and 661 ± 2cm^-1The Raman spectrum of the absorption band is comprised in 707 +/-2, 1447 +/-2 and 2988 +/-2 cm^-1The melting point of the absorption band (b) is 188 ℃. + -. 3 ℃.

Phase-pure forms of form A, form B and form C and combinations thereof

Each of form a, form B, and form C can be obtained in substantially pure phase form. Or each of form a, form B, and form C may coexist in combination with one or more other forms.

In one embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide wherein at least about 50% by weight of the compound is crystalline form a. In another embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide wherein at least about 20%, at least about 30%, or at least about 40% by weight of the compound is crystalline form a. In another embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide wherein at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% by weight of the compound is form a. In another embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is substantially pure crystalline form a.

In one embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide wherein at least about 50% by weight of the compound is crystalline form B. In another embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide wherein at least about 20%, at least about 30%, or at least about 40% by weight of the compound is crystalline form B. In another embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide wherein at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% by weight of the compound is form B. In another embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is substantially pure crystalline form B.

In one embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide wherein at least about 50% by weight of the compound is crystalline form C. In another embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide wherein at least about 20%, at least about 30%, or at least about 40% by weight of the compound is crystalline form C. In another embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide wherein at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% by weight of the compound is form C. In another embodiment, the invention includes N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is substantially pure crystalline form C.

F.Process for preparing form A, form B and form C

The invention also includes processes for preparing form a, form B, and form C. Representative methods are disclosed in the examples of the present application.

The invention also includes each of form a, form B, and form C prepared according to the methods disclosed herein. In one embodiment, the invention includes form a prepared according to this method. In another embodiment, the invention includes form B prepared according to this process. In another embodiment, the invention includes form C prepared according to this method.

G.Pharmaceutical composition

Form a, form B and form C and combinations of the above forms may be administered by any suitable route, preferably in the form of a pharmaceutical composition suitable for the above route and in a therapeutically effective dose. Accordingly, the invention specifically includes pharmaceutical compositions comprising at least one anhydrous crystalline form of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide and one or more pharmaceutically acceptable carriers. The amount of form a, form B, and/or form C to be administered and the dosage regimen for treating a disease or disorder with form a, form B, and/or form C depends on various factors including: age, body weight, sex and physical condition of the patient, the severity of the disease, the route and frequency of administration and the particular compound employed, and thus the above amounts and dosage regimen may vary widely. The pharmaceutical composition comprises form a, form B and/or form C in an amount of about 0.1 to 2000mg, preferably about 0.5 to 500mg, most preferably about 1 to 200 mg. Daily dosages of about 0.01 to 100mg/kg body weight, preferably about 0.5 to about 20mg/kg body weight, most preferably about 0.1 to 10mg/kg body weight are suitable. Daily doses may be taken from one to four times a day.

In one embodiment, a pharmaceutical composition comprises form a and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide in substantially phase-pure crystalline form a and a pharmaceutically-acceptable carrier. In another embodiment, a pharmaceutical composition comprises form B and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide in substantially phase-pure crystalline form B and a pharmaceutically-acceptable carrier. In another embodiment, a pharmaceutical composition comprises form C and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide in substantially phase-pure crystalline form C and a pharmaceutically-acceptable carrier.

In another embodiment, a pharmaceutical composition comprises a combination of at least two crystalline forms of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide selected from the group consisting of form a, form B, and form C, and a pharmaceutically acceptable carrier. In one embodiment, the weight ratio of the first crystalline form to the second crystalline form is at least about 1: 1. In another embodiment, the ratio is at least about 3: 2, at least about 7: 3, at least about 4: 1, at least about 9: 1, at least about 95: 5, at least about 96: 4, at least about 97: 3, at least about 98: 2, or at least about 99: 1. In another embodiment, a pharmaceutical composition comprises three crystalline forms of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide selected from crystalline form a, crystalline form B, and crystalline form C, and a pharmaceutically acceptable carrier.

H.Method of treatment

The invention also includes methods for treating a disorder in a patient suffering from or susceptible to the disorder by administering to the patient a therapeutically effective amount of one or more mixtures of form a, form B, form C, or a combination thereof. In one embodiment, the treatment is prophylactic treatment. In another embodiment, the treatment is palliative. In another embodiment, the treatment is a restorative treatment.

The conditions which can be treated according to the invention are PDE-5 mediated conditions. The disorders include cardiovascular diseases, metabolic diseases, central nervous system diseases, pulmonary diseases, sexual dysfunction and renal dysfunction.

In one embodiment, the condition comprises a cardiovascular disease, in particular a cardiovascular disease selected from the group consisting of: hypertension (including essential hypertension, pulmonary hypertension, secondary hypertension, isolated systolic hypertension, hypertension induced by diabetes, hypertension induced by arteriosclerosis, and renal vascular hypertension), hypertension complications (e.g., vascular organ damage, congestive heart failure, angina pectoris, stroke, glaucoma and renal function weakness), valvular insufficiency, stable, unstable and variant (Prinzmetal) angina, peripheral vascular disease, myocardial infarction, stroke, thrombosis, restenosis, arteriosclerosis, atherosclerosis, pulmonary hypertension, post-bypass vascular stenosis, angioplasty-treated diseases (e.g., diseases treated by percutaneous transluminal angioplasty or diseases treated by percutaneous transluminal coronary angioplasty), hyperlipidemia, hypoxic vascular constriction, vasculitis (e.g., kawasaki syndrome), heart failure (e.g., hyperemia, metabolic disorders, systole, diastole and left ventricular heart failure, right ventricular heart failure, and left ventricular hypertrophy), Raynaud's disease, preeclampsia, gestational hypertension, cardiomyopathy, and atherosclerotic occlusive disease.

In another embodiment, the disorder is hypertension. In another embodiment, the disorder is pulmonary hypertension. In another embodiment, the disorder is heart failure. In another embodiment, the disorder is diastolic heart failure. In another embodiment, the disorder is systolic heart failure. In another embodiment, the disorder is angina pectoris. In another embodiment, the disorder is thrombosis. In another embodiment, the disorder is stroke.

In another embodiment, the disorder is a metabolic disease, in particular a metabolic disease selected from the group consisting of: syndrome X, impaired insulin resistance or glucose tolerance, diabetes (e.g., type I and type II diabetes), insulin resistance syndromes (e.g., insulin receptor dysregulation, Rabson-Mendenhall syndrome, leprechaunism syndrome, Kobberling-Dunnigan syndrome, Seip syndrome, Lawrence syndrome, Cushing syndrome, acromegaly, pheochromocytoma, hyperglycomatous tumors, primary aldosteronism, somatostatin tumors, Lipotatropic diabetes, beta-cytotoxin-induced diabetes, Grave disease, Hashimoto thyroiditis, and congenital Addison disease), diabetic complications (e.g., diabetic gangrene, diabetic arthritis, diabetic nephropathy, diabetic glomerulosclerosis, diabetic deramatopathy, diabetic neuropathy, peripheral diabetic neuropathy, diabetic cataract and diabetic retinopathy), hyperglycemia, and obesity.

In another embodiment, the disorder is insulin resistance. In another embodiment, the disorder is a renal disorder.

In another embodiment, the disorder is a central nervous system disease, in particular a central nervous system disease selected from the group consisting of: vascular dementia, head injury, cerebral infarction, dementia, concentration disorder, Alzheimer's disease, Parkinson's disease, motoneuron disease (ALS), Huntington's disease, multiple sclerosis, Creutzfeld-Jacob, anxiety, depression, sleep disorders, and migraine. In one embodiment, the disorder is Alzheimer's disease. In another embodiment, the disorder is Parkinson's disease. In one embodiment, the disorder is ALS. In another embodiment, the disorder is a concentration disorder.

In one embodiment, the disorder is a lung disorder, in particular a lung disorder selected from the group consisting of: asthma, acute respiratory distress, cystic fibrosis, Chronic Obstructive Pulmonary Disease (COPD), bronchitis, and chronic reversible pulmonary obstruction (chronic reversible pulmonary obstruction).

In one embodiment, the condition is a sexual dysfunction, in particular a sexual dysfunction selected from the group consisting of: impotence (organic or mental), male erectile dysfunction, clitoral dysfunction, sexual dysfunction following spinal cord injury, female sexual arousal disorder, female sexual organ dysfunction, female dyspareunia dysfunction and female hypoactive sexual dysfunction. In another embodiment, the disorder is erectile dysfunction.

In another embodiment, the disorder is renal dysfunction, in particular renal dysfunction selected from the group consisting of: acute or chronic renal failure, renal disease (e.g., diabetic nephropathy), glomerulopathy and nephritis.

In another embodiment, the condition is pain. In another embodiment, the condition is acute pain. Examples of acute pain include acute pain associated with injury or surgery. In another embodiment, the disorder is chronic pain. Examples of chronic pain include neuralgia (including postherpetic neuralgia and peripherally-associated pain, cancer or diabetic neuropathy), carpal tunnel syndrome, back pain (including pain associated with herniation or rupture of lumbar intervertebral disc or ossification of the lumbar facet joints, sacral joints, erector spinal muscles or posterior longitudinal ligament), headache, cancer pain (including tumor pain such as bone pain, headache, facial pain or visceral pain) or pain associated with cancer therapy (including postchemotherapy syndrome, chronic post-operative pain syndrome, post-radiation syndrome, pain associated with immunotherapy or pain associated with hormonal therapy), arthritic pain (including osteoarthritis or rheumatoid arthritis), chronic post-operative pain, postherpetic neuralgia, trigeminal neuralgia, HIV neuropathy, amputation post-operative phantom limb pain, post-stroke neuralgia and pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, bone marrow damage, Parkinson's disease, epilepsy and vitamin deficiency. In another embodiment, the condition is nociceptive pain (nociceptive pain), including pain resulting from central nerve injury, tension/sprain, burn, myocardial infarction and acute pancreatitis, post-operative pain (pain caused by any type of post-operative), post-traumatic pain, renal colic, cancer pain and back pain. In another embodiment, the disorder is pain associated with inflammation, including, arthritic pain (e.g., osteoarthritic pain and rheumatoid arthritic pain), ankylosing spondylitis, visceral pain (including inflammatory bowel disease, functional bowel dysfunction, gastroesophageal reflux, dyspepsia, irritable bowel syndrome, functional abdominal pain syndrome, Crohn's disease, ileitis, ulcerative colitis, primary dysmenorrhea, cystitis, pancreatitis, and pelvic pain). In another embodiment, the condition is pain caused by musculoskeletal dysfunction, including myalgia, fibrosgia, spondylitis, hypoimmunity (non-rheumatic) arthritis, fibrositis, muscular dystrophy, glycogen breakdown, polymyositis, and pyomyositis. In another embodiment, the disorder is selected from cardiac pain and vascular pain (including pain caused by angina pectoris, myocardial infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleroredoma, and skeletal muscle ischemia). In another embodiment, the condition is selected from the group consisting of headache (including migraine, e.g., migraine with and without aura), cluster headache, tension-induced headache and headache associated with vascular dysfunction; facial pain, including tenderness, earache, burning mouth syndrome and temporomandibular myofascial pain.

In another embodiment, the disorder is a urinary tract disorder selected from the group consisting of: bladder outlet obstruction, incontinence and benign prostatic hyperplasia.

In another embodiment, the disorder is an ophthalmic disorder selected from the group consisting of: retinal diseases, macular degeneration and glaucoma.

In another embodiment, the disorder is selected from the following diseases: interstitial lesions of the renal tubules, anal fissures, baldness, cancer cachexia, cerebral haemorrhage, gastrointestinal motility dysfunction, intestinal motility dysfunction, dysmenorrhea (primary and secondary), glaucoma, macular degeneration, antiplatelet, hemorrhoids, incontinence, Irritable Bowel Syndrome (IBS), tumour metastasis, multiple sclerosis, neoplasia, nitrate intolerance, nipper esophagus, osteoporosis, infertility, premature birth, psoriasis, retinal disease, skin necrosis and rubella. In another embodiment, the disorder is osteoporosis.

In another embodiment, the disorder is associated with endothelial cell dysfunction, in particular a disorder selected from the group consisting of: atherosclerosis, myocardial ischemia, peripheral ischemia, valve insufficiency, pulmonary hypertension, angina, vascular complications following vascular replacement, vascular dilation, vasular septamenialization, and heart transplantation.

The methods and compositions of the invention are suitable for mammalian use, such as humans, other primates (e.g., monkeys, chimpanzees), companion animals (e.g., dogs, cats, horses), farm animals (e.g., goats, sheep, pigs, cattle), laboratory animals (e.g., mice, rats), and wild and zoo captive animals (e.g., wolves, bears, deer). In another embodiment, the patient is a human.

I.Use in pharmaceutical preparation process

The present invention also includes a process for preparing a pharmaceutical composition (or "medicament") comprising: combining form a, form B, form C, or a combination of the foregoing forms with one or more pharmaceutically acceptable carriers and/or other active ingredients for treating the foregoing conditions.

J.Examples

Example 1: n- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridine-2-) Yl-amino) -1H-pyrazolo [4,3-d]Pyrimidine-3-carbonyl]Process for preparing methanesulfonamides

The preparation of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide was as follows:

step 1

1- (2-ethoxyethyl) -4-nitro-1H-pyrazole-3, 5-dicarboxylic acid dimethyl ester

Dimethyl 4-nitro-1H-pyrazole-3, 5-dicarboxylate (2.0g, 8.83mmol, WO00/24745, p. 48, preparation 2) was added to a solution of 2-ethoxyethyl bromide (1.18mL, 10.45mmol) and potassium carbonate (1.32g, 9.56mmol) in N, N-dimethylformamide (35mL) and the reaction mixture was stirred at room temperature for 48H. The reaction mixture was concentrated in vacuo and the residue partitioned between ethyl acetate (200mL) and water (100 mL). The organic layer was separated, dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by column chromatography on a column of silica eluting with pentane/ethyl acetate (100: 0 to 70: 30) to give the title product, 1.63 g.

¹H NMR(CDCl₃，400MHz)δ：1.07(s，3H)，3.41(q，2H)，3.73(t，2H)，3.89(s，3H)，3.94(s，3H)，4.76(t，2H)。MS APCI+m/z 302，[MH]⁺。

Step 2

4-Nitro-1- (2-ethoxyethyl) -1H-pyrazole-3, 5-dicarboxylic acid 3-methyl ester

The ester of step 1 (1.63g, 5.4mmol) was added to a solution of potassium hydroxide (330mg, 5.9mmol) in methanol (20mL) and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was concentrated in vacuo and the crude product was dissolved in water and washed with ether. The aqueous phase was acidified with 2M hydrochloric acid and extracted with dichloromethane (3X 100 mL). The organic phases were combined, dried over magnesium sulfate and concentrated in vacuo to give the title product.

¹H NMR(CD₃OD，400MHz)δ：1.07(s，3H)，3.47(q，2H)，3.80(t，2H)，3.88(s，3H)，4.77(t，2H)。MS APCI+m/z 288[MH]⁺。

Step 3

5-carbamoyl-1- (2-ethoxyethyl) -4-nitro-1H-pyrazole-3-carboxylic acid methyl ester

Oxalyl chloride (1.2mL, 13.76mmol) and N, N-dimethylformamide (39 μ L) were added to a solution of the carboxylic acid of step 2 (1.33g, 4.63mmol) in dichloromethane (20mL) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo and azeotroped with dichloromethane (3X 50 mL). The product was dissolved in tetrahydrofuran (50mL), cooled in an ice bath, treated with 0.88 aqueous ammonia (10mL), and stirred at room temperature for 18 hours. The mixture was concentrated in vacuo and the residue partitioned between dichloromethane (200mL) and water (50 mL). The organic phase was dried over magnesium sulfate and concentrated in vacuo to give the title product.

¹H NMR(DMSO-D₆，400MHz)δ：1.06(t，3H)，3.40(m，2H)，3.77(m，2H)，3.84(s，3H)，4.38(m，2H)，8.35(m，1H)，8.46(m，1H)。MS APCI+m/z287[MH]⁺。

Step 4

4-amino-5-carbamoyl-1- (2-ethoxyethyl) -1H-pyrazole-3-carboxylic acid methyl ester

Palladium (II) hydroxide was added to a solution of the nitro compound of step 3 (970mg, 3.39mmol) in methanol (20mL) and the mixture was heated to reflux. Ammonium formate (1.07g, 16.97mmol) was added and the reaction mixture was stirred at reflux for 2 hours. By ArbocelThe catalyst was removed by filtration and the reaction mixture was concentrated in vacuo to give the title product.

¹H NMR(DMSO-D₆，400MHz)δ：1.02(t，3H)，3.33(m，2H)，3.66(m，2H)，3.80(s，3H)，4.57(m，2H)，5.11(m，2H)，7.49(m，2H)。MS APCI+m/z257[MH]⁺。

Step 5

1- (2-ethoxyethyl) -5, 7-dioxo-4, 5, 6, 7-tetrahydro-1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid methyl ester

A solution of the step 4 amide (570mg, 3.38mmol) in N, N-dimethylformamide (30mL) was treated with N, N' -carbonyldiimidazole (658mg, 4.06mmol) and the reaction mixture was stirred at room temperature for 1 hour and then at 90 ℃ for 18 hours. The reaction mixture was concentrated in vacuo, the crude product was suspended in acetone and sonicated for 30 min. The solid product was filtered off and dried in vacuo to give the title product.

¹H NMR(DMSO-D₆，400MHz)δ：1.02(t，3H)，3.37(m，2H)，3.77(m，2H)，3.83(s，3H)，4.63(m，2H)，10.75(s，1H)，11.40(s，1H)。MS ES-m/z 281[M-H]^-。

Step 6

5, 7-dichloro-1- (2-ethoxyethyl) -1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid methyl ester

Phosphorus oxychloride (934 μ L, 10.0mmol) and tetraethylammonium chloride (195mg, 1.50mmol) were added to a solution of the step 5 diketone (140mg, 0.50mmol) in propionitrile (5mL) and the reaction mixture was refluxed for 18 hours. The reaction mixture was concentrated in vacuo and the crude product was partitioned between ethyl acetate (50mL) and water (50 mL). The organic layer was dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by column chromatography on a silica gel column eluted with pentane: ethyl acetate (100: 0 to 75: 25) to give the title product.

¹H NMR(CDCl₃，400MHz)δ：1.05(t，3H)，3.41(m，2H)，3.84(m，2H)，4.06(s，3H)，5.00(m，2H)。MS APCI+m/z 319[MH]⁺。

Step 7

5-chloro-1- (2-ethoxyethyl) -7- (4-methylpyridin-2-yl-amino) -1H-pyrido [4,3-d ] pyrimidine-3-carboxylic acid methyl ester

The dichloride obtained in step 6 (1.98g, 6.20mmol) was dissolved in dimethyl sulfoxide (10mL) and the solution was treated with 2-amino-4-methylpyridine (1.34g, 12.4 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was partitioned between dichloromethane (300mL) and water (500mL) and the dichloromethane layer was separated. The organic phase was washed with water (3 × 100mL), dried over magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography on a silica gel column eluting with methylene chloride: methanol (100: 0 to 98: 2). The crude product was triturated with ether (50mL), filtered and concentrated in vacuo to give the title product, 1.2 g.

¹H-NMR(CDCl₃，400MHz)δ：1.06(t，3H)，2.49(s，3H)，3.62(m，2H)，4.00(t，2H)，4.06(s，3H)，5.05(m，2H)，6.98(m，1H)，8.16(m，1H)，8.50(m，1H)。MS APCI+m/z 391[MH]⁺。

Step 8

5-chloro-7- (4-methylpyridin-2-yl-amino) -1- (2-ethoxyethyl) -1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid

The ester of step 7 (4.3g, 11mmol) was dissolved in dioxane (50mL) and the solution was treated with 1M aqueous sodium hydroxide (22.0mL, 22.0 mmol). Then, the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was evaporated to dryness, the residue was dissolved in water (100mL) and washed with dichloromethane (50 mL). The aqueous phase was then acidified with 1M citric acid to a pH of 4-5, forming a yellow precipitate. The mixture was stirred for 15 minutes then filtered and the solid product dried over phosphorus pentoxide in vacuo to give the title product, 3.75 g.

¹H NMR(DMSO-D₆，400MHz)δ：1.00(t，3H)，2.34(s，3H)，3.45(m，2H)，3.81(m，2H)，4.84(m，2H)，6.93(m，1H)，7.89(m，1H)，8.16(m，1H)。

Step 9

N- [ 5-chloro-1- (2-ethoxyethyl) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide

The carboxylic acid of step 8 (1.0g, 2.70mmol), methanesulfonamide (330mg, 3.5mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (660mg, 3.5mmol) and 4-dimethylaminopyridine (390mg, 3.5mmol) were dissolved in N, N-dimethylformamide (10mL) and the reaction mixture was stirred at room temperature for 60 hours. Additional methanesulfonamide (165mg, 1.7mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (330mg, 1.7mmol) and 4-dimethylaminopyridine (195mg, 1.7mmol) were added and the reaction mixture stirred for an additional 20 hours. Additional methanesulfonamide (165mg, 1.7mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (330mg, 1.7mmol) and 4-dimethylaminopyridine (195mg, 1.7mmol) were added and the reaction mixture was stirred for a final 18 hours. The reaction mixture was concentrated in vacuo and the residue partitioned between dichloromethane (25mL) and water (25 mL). The organic phase was separated, washed with water (2X 25mL), dried over magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography on a silica gel column eluting with methylene chloride methanol acetic acid (100: 0 to 96: 3.5: 0.5). The crude product was triturated in warm ethyl acetate (10mL) to give the title product, 290 mg.

¹H NMR(DMSO-D₆，400MHz)δ：0.95(t，3H)，2.40(s，3H)，3.40(s，3H)，3.45(d，2H)，3.85(m，2H)，4.95(m，2H)，7.15(d，1H)，7.85(s，1H)，8.25(d，1H)。MS ES-m/z 452[M-H]^-。

Step 10

N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide

The chloride (110mg, 0.24mmol) from step 9, N-methyl-ethylamine (79mg, 1.2mmol), N-ethyldiisopropylamine (210. mu.L, 120mmol) and cesium fluoride (37mg, 0.24mmol) were dissolved in dimethyl sulfoxide (1mL) and the reaction mixture was stirred in ReactiVial^TMHeating to 110 deg.C for 5 hr. The reaction mixture was partitioned between ethyl acetate (10mL) and water (10mL), the organic phase was separated and washed with water (2X 10 mL). The organic phase is then dried over magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography on a silica gel column using methylene chloride: methanol (99: 1 to 97: 3). The purified material was dehydrated and dried to give a pale yellow solid (66 mg). The PXRD pattern of this solid is figure 14.

¹H NMR(DMSO-D₆+CF₃CO₂D，400MHz)δ：0.99(t，3H)，1.17(t，3H)，2.44(s，3H)，3.18(s，3H)，3.41(s，3H)，3.44(d，2H)，3.66(d，2H)，3.88(t，2H)，4.93(t，2H)，7.16(d，1H)，8.09(s，1H)，8.26(d，1H)。MS ES-m/z 475[M-H]^-

Example 2: n- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridine-2-) Yl-amino) -1H-pyrazolo [4,3-d]Pyrimidine-3-carbonyl]Process for preparing methanesulfonamides

FIG. 15 is a synthetic scheme depicting another synthetic scheme for the preparation of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

Example 3: of form APreparation method (recrystallization from ethyl acetate)

A process for preparing N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide crystalline form a as follows:

step 1

1- (2-ethoxyethyl) -4-nitro-1H-pyrazole-3, 5-dicarboxylic acid dimethyl ester

Dimethyl 4-nitro-1H-pyrazole-3, 5-dicarboxylate (page 48, preparation 2, WO 00/24745) was added to a solution of 2-ethoxyethyl bromide (1.18mL, 10.45mmol) and potassium carbonate (1.32g, 9.56mmol) in N, N-dimethylformamide (35mL), and the reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was concentrated in vacuo and the residue partitioned between ethyl acetate (200mL) and water (100 mL). The organic layer was separated, dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by column chromatography on a column of silica eluting with pentane/ethyl acetate (100: 0 to 70: 30) to give the title product, 1.63 g.

¹H NMR(CDCl₃，400MHz)δ：1.07(s，3H)，3.41(q，2H)，3.73(t，2H)，3.89(s，3H)，3.94(s，3H)，4.76(t，2H)。MS APCI+m/z 302，[MH]⁺。

Step 2

4-Nitro-1- (2-ethoxyethyl) -1H-pyrazole-3, 5-dicarboxylic acid 3-methyl ester

The ester of step 1 (1.63g, 5.4mmol) was added to a solution of potassium hydroxide (330mg, 5.9mmol) in methanol (20mL) and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was concentrated in vacuo and the crude product was dissolved in water and washed with ether. The aqueous phase was acidified with 2M hydrochloric acid and extracted with dichloromethane (3X 100 mL). The organic phases were combined, dried over magnesium sulfate and concentrated in vacuo to give the nitro product.

¹H NMR(CD₃OD，400MHz)δ：1.07(s，3H)，3.47(q，2H)，3.80(t，2H)，3.88(s，3H)，4.77(t，2H)。MS APCI+m/z 288[MH]⁺。

Step 3

5-carbamoyl-1- (2-ethoxyethyl) -4-nitro-1H-pyrazole-3-carboxylic acid methyl ester

Oxalyl chloride (1.2mL, 13.76mmol) and N, N-dimethylformamide (39 μ L) were added to a solution of the carboxylic acid of step 2 (1.33g, 4.63mmol) in dichloromethane (20mL) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo and azeotroped with dichloromethane (3X 50 mL). The product was dissolved in tetrahydrofuran (50mL), cooled in an ice bath, treated with 0.88 aqueous ammonia (10mL) and stirred at room temperature for 18 h. The mixture was concentrated in vacuo and the residue partitioned between dichloromethane (200mL) and water (50 mL). The organic phase was dried over magnesium sulfate and concentrated in vacuo to give the nitro product.

¹H NMR(DMSO-D₆，400MHz)δ：1.06(t，3H)，3.40(m，2H)，3.77(m，2H)，3.84(s，3H)，4.38(m，2H)，8.35(m，1H)，8.46(m，1H)。MS APCI+m/z287[MH]⁺。

Step 4

4-amino-5-carbamoyl-1- (2-ethoxyethyl) -1H-pyrazole-3-carboxylic acid methyl ester

Palladium (II) hydroxide was added to a solution of the nitro compound of step 3 (970mg, 3.39mmol) in methanol (20mL) and the mixture was heated to reflux. Ammonium formate (1.07g, 16.97mmol) was added and the reaction mixture was stirred at reflux for 2 hours. By ArbocelThe catalyst was removed by filtration and the reaction mixture was concentrated in vacuo to give the amide product.

¹H NMR(DMSO-D₆，400MHz)δ：1.02(t，3H)，3.33(m，2H)，3.66(m，2H)，3.80(s，3H)，4.57(m，2H)，5.11(m，2H)，7.49(m，2H)。MS APCI+m/z257[MH]⁺。

Step 5

1- (2-ethoxyethyl) -5, 7-dioxo-4, 5, 6, 7-tetrahydro-1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid methyl ester

A solution of the step 4 amide (570mg, 3.38mmol) in N, N-dimethylformamide (30mL) was treated with N, N' -carbonyldiimidazole (658mg, 4.06mmol) and the reaction mixture was stirred at room temperature for 1 hour and then at 90 ℃ for 18 hours. The reaction mixture was concentrated in vacuo, the crude product was suspended in acetone and sonicated for 30 min. The solid product was filtered off and dried in vacuo to give the diketone product.

¹H NMR(DMSO-D₆，400MHz)δ：1.02(t，3H)，3.37(m，2H)，3.77(m，2H)，3.83(s，3H)，4.63(m，2H)，10.75(s，1H)，11.40(s，1H)。MS ES-m/z 281[M-H]^-。

Step 6

5, 7-dichloro-1- (2-ethoxyethyl) -1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid methyl ester

Phosphorus oxychloride (934 μ L, 10.0mmol) and tetraethylammonium chloride (195mg, 1.50mmol) were added to a solution of the step 5 diketone (140mg, 0.50mmol) in propionitrile (5mL) and the reaction mixture was refluxed for 18 hours. The reaction mixture was concentrated in vacuo and the crude product was partitioned between ethyl acetate (50mL) and water (50 mL). The organic layer was dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by column chromatography on a silica gel column eluted with pentane: ethyl acetate (100: 0 to 75: 25) to give the dichloro product.

¹H NMR(CDCl₃，400MHz)δ：1.05(t，3H)，3.41(m，2H)，3.84(m，2H)，4.06(s，3H)，5.00(m，2H)。MS APCI+m/z 319[MH]⁺。

Step 7

5-chloro-1- (2-ethoxyethyl) -7- (4-methylpyridin-2-yl-amino) -1H-pyrido [4,3-d ] pyrimidine-3-carboxylic acid methyl ester

The dichloride from step 6 (1.98g, 6.20mmol) was dissolved in dimethyl sulfoxide (10mL) and the solution was treated with 2-amino-4-methylpyridine (1.34g, 12.4 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was partitioned between dichloromethane (300mL) and water (500mL) and the dichloromethane layer was separated. The organic phase was washed with water (3 × 100mL), dried over magnesium sulfate and concentrated in vacuo. The residue was purified by column chromatography on a silica gel column eluting with methylene chloride: methanol (100: 0 to 98: 2). The crude product was triturated with ether (50mL), filtered and concentrated in vacuo to give the monochloro product, 1.2 g.

¹H-NMR(CDCl₃，400MHz)δ：1.06(t，3H)，2.49(s，3H)，3.62(m，2H)，4.00(t，2H)，4.06(s，3H)，5.05(m，2H)，6.98(m，1H)，8.16(m，1H)，8.50(m，1H)。MS APCI+m/z 391[MH]⁺。

Step 8

1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (6-ethylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid methyl ester

A solution of N-ethylmethylamine (4.6mL, 53.8mmol) in N-methylpyrrolidin-2-one (7mL) was added to a solution of the monochloride of step 7 (7.0g, 17.93mmol) in N-methylpyrrolidin-2-one (28mL) at 110 deg.C. The reaction mixture was heated overnight, after completion, the solution was cooled to room temperature and water (25mL) was added. After stirring at room temperature for 2 hours, the slurry was filtered and washed with 2X 15mL of water. The solid was dried under vacuum at 55 deg.C overnight to give an orange solid (5.988g, 15.0mmol, 84%).

¹H NMR(CD₃OD，400MHz)δ：1.12(m，3H)，1.25(m，3H)，2.40(s，3H)，3.21(m，2H)，3.23(s，3H)，3.60(m，2H)，3.75(m，2H)，3.96(s，3H)，4.80(m，2H)，6.94(m，1H)，8.16(m，1H)，8.34(m，1H)。MS APCI-m/z 412[M-H]^-

Step 9

1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (6-ethylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid

The ester of step 8 (13.57g, 32.83mmol) and 1M aqueous sodium hydroxide (90mL) were dissolved in methanol (10mL) and the reaction mixture was stirred at 85 ℃ for 1 hour. The reaction mixture was cooled to room temperature and acidified with 10% aqueous citric acid (90 mL). The aqueous layer was extracted twice with dichloromethane (36mL and 24 mL). The aqueous layer was acidified again with 10% aqueous citric acid (20mL) and extracted with dichloromethane (24 mL). The dichloromethane extracts were combined and ethanol (13mL) was added. The solution was distilled at ambient pressure, replacing the distilled dichloromethane with ethanol (52 mL). Water (12mL) was added and the mixture was cooled to 5 ℃ and stirred for 1 hour. The slurry was filtered and washed with water (24mL) and dried in vacuo at 55 ℃ to give a yellow solid (8.858g, 22.2mmol, 68%).

¹H NMR(CD₃OD，400MHz)δ：1.10(t，3H)，1.30(t，3H)，2.43(s，3H)，3.24(s，3H)，3.57(m，2H)，3.70(m，2H)，3.93(t，2H)，4.84(m，2H)，7.02(m，1H)，8.13(m，1H)，8.16(m，1H)。MS APCI+m/z 400[M-H]⁺。

Step 10

N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide

The carboxylic acid of step 9 (29.0g, 72.6mmol), methanesulfonamide (8.28g, 87mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (18.0g, 94mmol) and 4-dimethylaminopyridine (10.59g, 94mmol) were dissolved in dichloromethane (385mL) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with dichloromethane (to 1500mL), washed twice with aqueous citric acid (50 g of citric acid in 200mL) and once with an acidic solution of a mixture of citric acid and sodium hydroxide. The dichloromethane phase was dried over magnesium sulfate and concentrated in vacuo. The solid residue was refluxed in isopropanol (1L) for 20 minutes, when it was cooled, and the resulting solid was filtered off. The separated yellow solid was then refluxed in ethyl acetate (2000mL) until a solution formed, in which the volume of ethyl acetate was reduced to 1000 mL. The resulting solution was filtered and allowed to cool to room temperature overnight, then placed in an ice bath and stirred for 1.5 hours. The resulting solid was filtered off and washed with ether (2X 50mL), dried on a filter pad for 3 hours and then dried in vacuo with phosphorus pentoxide to give a white powder (16.7 g). PXRD analysis of the powder indicated that the powder was crystalline form a of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

¹H NMR(CDCl₃，400MHz)δ：1.20(t，3H)，1.29(t，3H)，2.41(s，3H)，3.24(s，3H)，3.45(s，3H)，3.64(q，2H)，3.75(m，2H)，3.99(t，2H)，4.82(m，2H)，6.87 (d，1H)，8.20(d，1H)，8.29(s，1H)，9.87(br，1H)。MS ES+m/z 477[MH]⁺. The test is as follows: c, 50.25: h, 5.90: n, 23.41; the calculation is as follows: C20H28N8O 4S; c, 50.41: h, 5.92: n, 23.51.

Example 4: process for the preparation of form A (recrystallization from isopropanol)

Crystalline form a of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide was prepared as follows:

n- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide

Crude N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide (16.7g) (see example 1) was slurried in dichloromethane (20mL) and isopropanol (70 mL). The slurry was heated to reflux (about 60 ℃) and the solid material appeared to remain substantially insoluble. An additional amount of methylene chloride (40mL) was added to the slurry in 5mL increments. The resulting solution was refluxed for about 1 minute and heating was stopped. At the end of this process, the solid appeared to have dissolved to give a yellow solution. The solution was then cooled to 35 ℃ without signs of crystallization. A small amount (less than 0.5g) of crude N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide was seeded with this solution without signs of crystallization. The solution was cooled to room temperature again and there was no sign of crystallization. When cooled to 5 ℃ the solution became a slurry. The slurry was stirred at a temperature of about 5 ℃ and then filtered, and the material collected on the filter was dried at 50 ℃ to give a solid (7.7 g). PXRD analysis of the solid indicated that the solid was crystalline form a of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

Example 5: preparation method of crystal form B (methanol reflux)

Crystalline form B of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is prepared as follows:

n- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide (13.9g) (see example 3), containing crystalline form a, was dissolved in refluxing dichloromethane (160mL) and methanol (200 mL). Methylene chloride was distilled off (about 110mL of distillate was collected). The mixture was cooled to room temperature, granulated for 30 minutes, and filtered. The solid was washed with methanol (30mL) and dried in vacuo to give a bright yellow solid (10.8 g). PXRd analysis of the solid indicated that the solid was crystalline form B of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

Example 6: preparation method of crystal form B (methanol reflux)

Crystalline form B of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is prepared as follows:

1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid (1.19kg, 2.98 moles) (see example 3, step 9), methanesulfonamide (344g, 3.6 moles), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (810.0g, 4.21 moles) and 4-dimethylaminopyridine (488.8g, 4.01 moles) were dissolved in dichloromethane (12L) under a nitrogen atmosphere and the reaction mixture was stirred at room temperature. After 3 hours, another portion of 4-dimethylaminopyridine (62.0g, 551.7g total, 4.52 moles) was added to the solution and the reaction mixture was stirred at room temperature for an additional 20 hours. The reaction mixture was diluted with 10% aqueous citric acid (12L), and the organic phase was separated, washed with 10% aqueous citric acid (12L), and then with water.

The resulting solution (10L) was filtered and distilled at ambient pressure to about half of the initial volume, the hot solution was diluted in portions with methanol (14L total) while dichloromethane was removed in portions by distillation (11L total of distillate fraction, 13L final volume at reflux at 55 ℃). The yellow slurry was cooled to room temperature, stirred overnight, and then cooled to 5 ℃. The slurry was then filtered and washed with chilled methanol portions (5.8L total). The material collected on the filter was dried in vacuo at 55 ℃ for 3 days to give the product as a bright yellow solid (1.038kg, 73% yield) as crystalline form B of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

Example 7: process for the preparation of form B (deoxygenation and methanol reflux)

The crystalline form B of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is prepared as follows;

dichloromethane (260mL) was refluxed under a stream of nitrogen flowing through the top of the reactor to reduce the volume to 240mL, then cooled to room temperature under a nitrogen atmosphere. 1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (6-ethylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid (24g, 60 mmol) (see example 3, step 9), methanesulfonamide (6.88g, 72 mmol) and 4-dimethylaminopyridine (10.98g, 90 mmol) were dissolved in dichloromethane (240mL) under a nitrogen atmosphere. The solution was stirred for 30 minutes, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (16.13g, 84 mmol) was added and the reaction mixture was stirred at room temperature under nitrogen overnight. The reaction mixture was diluted with 10% aqueous citric acid (240mL), the organic phase was separated, washed with 10% aqueous citric acid (240mL), and then with water (240 mL).

The resulting solution was distilled at ambient pressure to about half the initial volume (about 120 mL). The hot solution was slowly diluted with methanol (240mL) and the mixture was then distilled to about 240mL at ambient pressure. The hot mixture was again diluted with methanol (120mL) and distilled to about 240mL at ambient pressure. The hot mixture was again diluted with methanol (120mL) and distilled to about 240mL at ambient pressure. The mixture was cooled to room temperature while stirring for 1 hour, and then cooled to 0-5 ℃ while stirring for 1 hour. The resulting yellow slurry was then filtered and the solid was washed with ice-cold methanol (96 mL). The solid was dried overnight at 55 ℃ in vacuo to give a bright yellow solid (25.78g, 90% yield) which was crystalline form B of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

Example 8: preparation method of crystal form B (ion exchange resin and methanol reflux)

Crystalline form B of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is prepared as follows.

1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (6-ethylpyridin-2-ylamino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carboxylic acid (24g, 60.1 mmol) (see example 3, step 9), methanesulfonamide (6.88g, 72.4 mmol) and 4-dimethylaminopyridine (10.98g, 90 mmol) were dissolved in dichloromethane (240mL) under a nitrogen atmosphere. The solution was stirred for 30 minutes, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (16.14g, 84.1 mmol) was added and the reaction mixture was stirred at room temperature until after 5 hours the reaction was judged to be substantially complete. The reaction mixture was diluted with 10% aqueous citric acid (240mL), and the organic phase was separated, washed with 10% aqueous citric acid (240mL) and then with water (240 mL).

Amberlite IRN-78(24g), a basic ion exchange resin, was added to the stirred separated organic phase and the mixture was stirred for 3 hours. The resin spheres were filtered off, the filter cake was washed with dichloromethane (48mL), and the combined filtrates were washed with 10% aqueous citric acid (120mL) and then twice with water (240 mL).

The resulting solution was distilled at ambient pressure to about half of the original gas (about 120 mL). The hot solution was slowly diluted with methanol (240mL) to precipitate a yellow solid, and the mixture was then distilled to about 240mL at ambient pressure. The hot mixture was again diluted with methanol (240mL) and distilled to about 240mL at ambient pressure. The yellow slurry was cooled to room temperature while stirring overnight, then cooled in an ice bath for 1 hour (ca. 0-5 ℃). The resulting slurry was then filtered and the solid was washed with methanol (96 mL). The resulting solid was dried overnight at 50 ℃ in vacuo to give a bright yellow solid (21.51g, 75.1% yield) as crystalline form B of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

Example 9: preparation method of form B (seroconversion)

About 25mg of form a (see example 3) was slurried with 1mL of methanol at room temperature. The color of the slurry turned yellow rapidly within 10 minutes. A small sample was taken from the slurry. PXRD analysis of this sample indicated that it was N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide crystalline form B.

Example 10: preparation method of crystal form B (temperature conversion)

A sample of form a (see example 3) was heated to 180 ℃ using DSC. The sample melted and recrystallized as form B. The sample was cooled to room temperature. To confirm that no form a remained and was completely converted to form B, the sample was heated again to 175 ℃ in DSC. No significant thermal change was detected. The sample was cooled to room temperature. The sample was heated again to 250 ℃. Melting of form B was observed at 220 ℃.

Example 11: preparation method of form C (seroconversion)

Crystalline form C of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide was prepared as follows:

samples of form B (see examples 5 to 9) were THF/H at 4 deg.C₂O (50: 50 by volume)Volume) was slurried. After 16 days, a small sample was filtered off and dried at room temperature to give a pale yellow solid. DSC analysis of this sample was consistent with the results for form C. An additional small portion of the sample was taken from the slurry as a tide. PXRD analysis of this sample was consistent with the results for form C. After 31 days of slurrying, an additional small portion of the sample was removed from the slurry in the moist state. PXRD analysis of this sample was consistent with the results for form C.

Example 12: preparation method (seeding method) of crystal form C

Crystalline form C of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide was prepared as follows:

a sample of form B (see examples 5 to 9) (129.6g) was stirred in acetone (1300mL) at 23 ℃ under nitrogen. Seeds of form C (20mg) were added and stirring continued at room temperature for 13 days. The solid was collected by filtration and dried under vacuum at ambient temperature above 20 ℃ to give form C product (119.8g) in 92.4% yield. FIG. 4 shows the powder X-ray diffraction pattern of the product.

Example 13: stability of form a and form B

The thermodynamic stability of form a and form B are compared as follows: first, about 25mg of form a was slurried in about 1mL of methanol. The color of the slurry turned yellow rapidly. A small portion of the sample was removed from the slurry. PXRD analysis confirmed that this sample was form B. Second, form a and form B were analyzed by DSC, respectively, and the schematic data for the form a and form B samples are presented in fig. 5 and 6. DSC data indicate that the melting point of form B is higher than the melting point of form a. Thus, the results of the slurry conversion analysis and DSC analysis confirm that form B is thermodynamically more stable than form a.

Example 14: stability of form B and form C

A connectivity study was performed in the following solvent system to determine the relative thermodynamic stability of form B and form C: (1) THF/H₂O (50: 50 v/v), (2) methyl ethyl ketone ("MEK"), (3) methanol, and (4) methanol/Dichloromethane (DCM) (50: 50 v/v). MEK studies were performed at room temperature and studies in the other three solvent systems were performed at 40 ℃ and 60 ℃. In each study, a suspension of form B in a suitable solvent system was prepared. Then, about 10mg of form C was added to each suspension. Next, the suspension is slurried at an appropriate temperature for a specific time. The solvent system, the temperature of the solvent system and the time for slurrying the suspension in each study were as follows:

(1) study a: slurried in MEK for 3 days at room temperature;

(2) study B: at 40 ℃ THF/H₂Slurrying for 3 days in O (50: 50);

(3) study C: at 60 ℃ THF/H₂Slurrying for 3 days in O (50: 50);

(4) study D: slurried in methanol at 40 ℃ for 21 days;

(5) study E: slurrying in methanol at 60 ℃ for 21 days;

(6) study F: slurried at 40 ℃ in methanol/DCM for 5 days; and

(7) study G: slurry in methanol/DCM at 60 ℃ for 21 days.

After the end of the slurrying, samples were removed from each slurry and analyzed by PXRD. PXRD analysis indicated that all samples prepared at room temperature and at 40 ℃ were form C and all samples prepared at 60 ℃ were form B. As previously mentioned, there is a crossover in the thermodynamic stability of form B and form C between 40 and 60 ℃ (i.e., the two forms are double-denatured crystals). At temperatures below this crossover point, form C is the most thermodynamically stable form. At temperatures above the crossover point, form B is the most thermodynamically stable form.

Example 15: in vitro assay

The method A comprises the following steps: aortic ring test

This protocol describes a procedure for measuring exposure to N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d]Pyrimidine-3-carbonyl]Direct relaxation of rat aortic ring in methanesulfonamide. In this assay, PDE5 inhibiting compounds promote relaxation of the aortic annulus by potentiating cGMP signaling induced by the stable in vitro NO-donor diethyltriamine NONONAte ("DETA-NO") (dinitrogen-1-onium-1, 2-diol ester). EC with 95% confidence interval₅₀Is an indicator of the ability of a compound to initiate relaxation. EC (EC)₅₀Is the concentration of the PDE5 inhibiting compound that produces 50% of the highest possible effective response to the PDE5 inhibiting compound.

With CO₂Male Sprague-Dawley rats (250-350g) were asphyxiated with gas, the thoracic aorta carefully dissected away and placed in Krebs buffer. Then, the aorta without connective tissue was carefully dissected and divided into 8 parts, each 3-4mm long.

The aortic rings were suspended under 1 gram of static tension between parallel stainless steel rings in a 15mL tissue bath jacketed with water (37 ℃). The tension was measured with an isometric tension sensor and recorded with a Ponemah tissue platform system. Compounds prepared by each preparation method were equilibrated for at least 60 minutes before drug testing was performed. During this time, the tissues were also incubated with 200 μ M NG-monomethyl L-arginine ("L-NMMA"), and the culture medium was changed every 15-20 minutes (after each wash, L-NMMA was added to maintain the final concentration in each tissue bath at 200 μ M).

After equilibration, the baseline tension for each tissue was recorded. Vasoconstrictive responses to neophilin (1 μ M) were assessed, and when responses to neophilin reached a maximum, vascular reactivity was subsequently assessed by exposure to acetylcholine (1 μ M). After rinsing again, a second baseline value was recorded, vasoconstrictor noradrenaline (25nM) was added to each bath, and the tissue was incubated for a time sufficient to allow the tissue to stabilize (approximately 15 minutes). The stable NO donor DETA-NO was used to supply NO in vitro. The DETA-NO concentration was titrated (accumulated as half log (half log) increases) to allow approximately 5-15% relaxation of vasoconstriction (precondensation) by norepinephrine. An additive concentration-response curve was obtained under the following conditions: a single ring, typically using 5 doses per ring, 15 minutes between each addition.

The method B comprises the following steps: aortic ring test

The protocol of method a was modified to measure relaxation of rat aortic rings exposed to PDE5 inhibitory compounds. The method differs from method a as follows:

for this method, the endothelium is first removed by carefully wiping the vessel lumen between the fingers before preparing the ring (the de-endothelialization ring). The resting tension was set at 2 grams and the vasoconstrictive response to the highest concentration of neopyrine (1 μ M) was evaluated, followed by two exposures to 300nM of neopyrine after a period of flushing. The concentration-response relationship for norepinephrine in each tissue was constructed over a concentration range of 0.1 to 300 nM. After rinsing again, the tissue was treated with EC₉₀Noradrenaline was contracted at a concentration and used for compound testing.

Example 16: in vivo assay

The method A comprises the following steps: culex^TMTest of

Conscious uninintubated Spontaneous Hypertensive Rats (SHR) were used to evaluate N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d]Pyrimidine-3-carbonyl]The efficacy of methanesulfonamide in lowering systemic arterial blood pressure. The test was performed using an Automatic Blood Sampling (ABS) system. Culex^TMThe ABS System (Bioanalytical System, inc., West Lafayette, IN) includes: laptop, four control units and metabolic cage. The ABS systemThe system allows several blood samples to be collected from a single rat without applying undue pressure to the rat.

In addition, the ABS system allows for the collection of urine samples that can be used for identification of biological indicators. By this approach, therapeutic and standard pharmacokinetic studies were performed in conscious, unrestricted SHR rats to define the relationship between plasma free drug concentration or possible biological indicators and drug efficacy (lowering mean arterial blood pressure).

Surgical cannulation of the jugular vein and right carotid artery was performed on SHR rats 12 to 16 weeks old weighing about 300 g. Following surgical recovery, animals were placed in Culex^TMIn the cage and tethered to a movement responsive arm with a sensor that controls the cage movement as the test animal moves, thereby preventing the catheter from becoming entangled. At the right jugular vein catheter and Culex for blood sampling^TMA connection was made between the sterile tubing set and the left jugular catheter for administration of the compound, and the catheter in the right carotid artery was connected to a pressure transducer to monitor blood pressure. To open the catheter, a Culex is passed^TMThe "tend" function of (1) maintaining the right cervical vein cannula, wherein, Culex^TMThe catheter was flushed with 20 μ L heparin saline (10 units/mL) every 12 minutes or between samples, and the left jugular vein cannula was filled with heparin saline (20 units/mL). The right carotid cannula was left open by: heparin saline was slowly infused directly into the extension line when no blood pressure was recorded, or slowly infused with heparin through a pressure transducer during blood pressure monitoring. Animals were allowed to acclimate for at least two hours before compound evaluation. The PDE5 inhibiting compound may be administered by intravenous or oral gavage. Using Culex^TMThe software sets the blood sampling protocol (sampling time and volume). The total amount of blood drawn from each animal did not exceed 750 μ L/24 hours and 10mL/kg over two weeks. Heart rate, blood pressure and drug concentration were monitored. Systemic arterial blood pressure and heart rate were recorded by a PONEMAH (Gould Instrument System, Valley View, OH) pressure sensor using a data acquisition SystemThe data acquisition system recorded blood pressure and heart rate for 6 to 24 hours according to the protocol. Mean arterial blood pressure (the primary test target) was analyzed to assess the efficacy of the compounds.

Blood samples were analyzed using the LC/MS method described below to measure plasma drug concentrations and evaluate potential biological indicators.

LC/MS/MS method

The sample preparation method comprises the following steps: plasma samples (50 μ L unknown, control or blank) were mixed with 10 μ L acetonitrile: water or a standard solution of a PDE-5 inhibiting compound and 150. mu.L of an internal standard solution (100 ng/mLPDE-5 inhibiting compound in acetonitrile) were mixed. The mixture was centrifuged at 3000rpm for 5 minutes and 125 μ L of the supernatant was transferred to a 96-well plate. The solvent was evaporated off under a stream of nitrogen and the residue was regenerated with 80. mu.L acetonitrile/0.1% aqueous formic acid (20: 80 v/v).

A volume of 20. mu.L of each prepared sample was injected into a Phenomenex Synergi 4. mu. mMAX-RP 2.0X 75mm column and eluted at 0.4mL/min with a gradient of eluent from 0.1 aqueous formic acid (mobile phase A) to acetonitrile (mobile phase B). The gradient program includes: initially 90% mobile phase a was applied, linearly changing from 0.2 to 1.15 minutes to 75% mobile phase B after injection, and 75% mobile phase B was held for 2.0 minutes. The mobile phase changed linearly back to 90% mobile phase a from 2.00 minutes to 2.10 minutes, with the next injection at 3.00 minutes. The shift was monitored for multi-step reactions by mass spectrometry using cation Electrospray (ESI): m/z 454.00(MH + PDE-5 inhibiting compound) → m/z 408.00, m/z 466.24(MH + PDE-5 inhibiting compound) → 409.33, to thereby perform probing. The ion spray voltage was set to 5000. Calibration curves were plotted by the ratio of the peak areas of the analyte to the internal standard. And performing reverse prediction by using the peak area ratio and the calibration curve to determine the concentration of the substance to be detected.

The method B comprises the following steps: implanting radio transmitter into spontaneous hypertensive rat, and obtaining blood pressure by remote sensing method

Spontaneously Hypertensive Rats (SHR) were anesthetized with isoflurane gas via an isoflurane anesthesia machine that delivers a range of percent isoflurane as oxygen passes through the machine lumen. The animal is placed in an inhalation chamber and 4-5% isoflurane is inhaled to achieve the level of anesthesia required for the procedure. The anesthetic is then maintained at 1-2% during the procedure by administration through a nose cone, wherein isoflurane is delivered to the operating table by a small isoflurane anesthetic device.

After inhalation of the anesthetic, a transmitter with a commercially available sterile radio-telemetry unit (Data Sciences, International, Roseville, MN 55113-. Before operation, the operation site is shaved and Dial is used^TMA brand anti-bacterial solution (containing 4% chlorhexidine gluconate and 4% isopropyl alcohol) was wiped, followed by a iodine (10%) spray solution. A laparotomy of 2.5 to 3.0cm was performed and the radio-telemetry unit was implanted in the abdomen, where the catheter tip was inserted into the abdominal artery. The Baby weitlanar retractor is used to retain soft tissue. A 1cm section of the abdominal artery was partially dissected, the section was temporarily blocked by a cross-clamp, punctured with a 21 gauge needle, and the transmitter catheter tip was introduced into the vessel and secured by a single 4.0 wire suture secured to the adjacent psoas muscle. The transmitter body was then inserted into the abdominal cavity while secured to the abdominal muscle wall and closed with a continuous 4.0 wire suture. The skin layer was closed with a subcutaneous continuous 4.0 absorbable suture. During closure, cocaine was administered subcutaneously and iodine was topically applied to the suture, inside and around the suture, respectively. All rats received a subcutaneous buprenorphine 0.05mg/kg post-operative injection before consciousness was restored. A typical dose for a 0.300kg rat is 0.050 ml. Before administering buprenorphine, the rats must recover completely from surgical anesthesia. Animals then received the same dose daily for two days, unless the animals indicated that post-operative pain had been tolerated.

After surgery, the rats were returned to the cages and individually placed on the bottom with a paper pad. The recovery period was not less than 7 days before the start of the experimental procedure. It was observed that rats were usually hypertensive for several days after the operation and returned to "normotensive" levels about the seventh day after the operation. During the experiment, rats were fed standard rat feed and water.

The compounds were administered by gavage (e.g., using stainless steel with a balloon tip, 21/2 inch, 18 gauge gavage needle). For single day administration, the target volume is, for example, 3.33ml/kg (gastric feeding). The carrier of the compound administered may vary depending on the solubility of the compound, but an aqueous solution of methylcellulose (0.5%) will be preferred.

The blood pressure Data is obtained by using a Data acquisition program of Data Sciences International. For the complete study, blood pressure samples were recorded 24 hours a day at 1.5-3 minute intervals for 5 seconds. The Data were processed using Data analysis software from Data Science to obtain the mean of the desired time intervals. In Microsoft Excel^TMAll other data processing is done in the spreadsheet.

The method C comprises the following steps: SHR rat

The experimental protocol was designed to screen N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide for a decrease in blood pressure. Spontaneously Hypertensive Rats (SHR) were cannulated in the jugular vein and carotid artery, respectively, one for compound administration and one for direct blood pressure measurement. After surgery and all trials were completed within one working day, the animals fully recovered consciousness. The main parameter to be evaluated is the blood pressure decrease. However, systolic and diastolic blood pressure, as well as heart rate data, are also collected. Following the above intake, rats are dosed in a gradual or additive fashion to observe the response. This particular method allows for screening of multiple doses within a day using the same animal.

The method comprises the following steps:

anesthesia: rats were anesthetized with 5% isoflurane. The incision is shaved and disinfected for surgery. The rats were then transferred to an operating site with a heating pad, a supply of isoflurane, and maintained at 37 ℃, with the isoflurane acting throughout the procedure.

And (3) operation: arterial and venous cannulae were implanted in the jugular vein and carotid artery, respectively. The cannula passes subcutaneously to the back of the neck where it exits the body percutaneously. Stainless steel staples were used to close the respective incisions. The catheter is then threaded through an elastic cord onto a rotating device that protects the cannula from the animal's chewing during the trial.

And (3) recovering: rats were placed in opaque polycarbonate cages with a counting balance arm that supported the weight of the rope and rotating device. The paper pad material is laid on the bottom of the cage. Rats were recovered from surgery at the bottom of the cage, which was lined with paper pad material, and they received a volume of 2mL early in the recovery phase. The animals were not provided food.

When introducing the present invention or exemplary embodiments thereof, "the" and "said" herein mean one or more of the factors. The term "comprising" is inclusive and means that there may be additional elements other than the listed elements. Although the present invention has been described in terms of specific embodiments, the details of these embodiments are not to be construed in any limiting sense.

Claims (33)

1. An N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide crystal having an X-ray powder diffraction pattern including peaks at 8.5 + -0.1 °, 9.0 + -0.1 °, 14.0 + -0.1 °, 16.9 + -0.1 °, 18.2 + -0.1 °, 19.9 + -0.1 °, 21.0 + -0.1 °, 21.4 + -0.1 °, 21.7 + -0.1 °, 22.5 + -0.1 °, 22.7 + -0.1 °, 23.5 + -0.1 °, 23.9 + -0.1 °, 24.8 + -0.1 °, 25.0 + -0.1 °, 25.4 + -0.1 °, 26.0 + -0.1 °, 26.2 + -0.1 °, 30.3 + -0.1 ° and 33.0 ° θ.

2. The crystalline N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 1 wherein the X-ray powder diffraction pattern does not comprise at least one diffraction peak selected from the group consisting of 3.6 ± 0.1 and 7.2 ± 0.1 ° 2 Θ.

3. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 1]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide having a Fourier transform infrared spectrum comprised at 3247 + -3 cm^-1The absorption band of (b).

4. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 1]Pyrimidine-3-carbonyl]A methanesulfonamide crystal, wherein the Fourier transform infrared spectrum further comprises at least one selected from 696 + -2, 1085 + -2, 1188 + -2 and 1540 + -2 cm^-1The absorption band of (1).

5. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 1]Pyrimidine-3-carbonyl]Methanesulfonamide crystals, wherein the Fourier transform infrared spectrum is not included at 1645 + -2 cm^-1The absorption band of (b).

6. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 1]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide, the Raman spectrum of said crystals comprising at 3255 ± 3cm^-1The band of (b).

7. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) as claimed in claim 1-1H-pyrazolo [4,3-d]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide, wherein the Raman spectrum further comprises at least one selected from the group consisting of 993. + -. 2, 1383. + -. 2, 1473. + -. 2 and 1569. + -.2 cm^-1The band of (2).

8. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 1]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide, wherein the Raman band is not included at 1652. + -.2 cm^-1The band of (b).

9. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 1, and a pharmaceutically-acceptable carrier.

10. A pharmaceutical composition comprising a therapeutically effective amount of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide and a pharmaceutically-acceptable carrier, wherein at least 50 weight percent of the N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is present as N- [1- (2-ethylic-ethyl) -5- (N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 1 In the form of crystals of oxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

11. A crystal of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide having an X-ray powder diffraction pattern comprising diffraction peaks at 3.6 + -0.1 °, 7.2 + -0.1 °, 9.4 + -0.1 °, 10.1 + -0.1 °, 14.4 + -0.1 °, 18.1 + -0.1 °, 18.9 + -0.1 °, 19.3 + -0.1 °, 19.4 + -0.1 °, 21.8 + -0.1 °, 22.9 + -0.1 °, 23.8 + -0.1 °, 27.0 + -0.1 °, 29.1 + -0.1 °, 32.9 + -0.1 ° 2 θ.

12. The crystalline N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 11 wherein the X-ray powder diffraction pattern does not comprise at least one diffraction peak, in terms of 2 Θ, selected from 8.5 ± 0.1, 20.2 ± 0.1, and 22.5 ± 0.1 °.

13. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 11]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide having a Fourier transform infrared spectrum included at 1452 + -2 cm^-1The absorption band of (b).

14. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 11]Pyrimidine-3-carbonyl]A methanesulfonamide crystal, wherein the Fourier transform infrared spectrum further comprises at least one selected from the group consisting of 722 + -2, 920 + -2, 1211 + -2 and 1395 + -2 cm^-1The absorption band of (1).

15. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 11]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide, wherein the Fourier infrared transform spectrum is not included in 962 + -2 cm^-1The absorption spectrum of (b).

16. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 11]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide having a Raman spectrum comprised between 1299. + -.2 cm^-1The band of (b).

17. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methyl) as claimed in claim 11Phenylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d]Pyrimidine-3-carbonyl]The methanesulfonamide crystal, wherein the Raman spectrum further comprises at least one selected from 689 + -2, 1456 + -2 and 1535 + -2 cm^-1The band of (2).

18. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 11]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide, wherein the Raman spectrum is not comprised at 1316 ± 2cm^-1The band of (b).

19. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 11 and a pharmaceutically-acceptable carrier.

20. A pharmaceutical composition comprising a therapeutically effective amount of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide and a pharmaceutically-acceptable carrier, wherein at least 50 weight percent of the N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is present as N- [1- (2-ethylic-ethyl) -5- (N-methyl-amino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 11 In the form of crystals of oxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

21. An N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide crystal having an X-ray powder diffraction pattern including peaks at 6.7 + -0.1 °, 7.1 + -0.1 °, 10.6 + -0.1 °, 12.8 + -0.1 °, 14.0 + -0.1 °, 14.5 + -0.1 °, 14.8 + -0.1 °, 15.9 + -0.1 °, 16.8 + -0.1 °, 17.7 + -0.1 °, 19.1 + -0.1 °, 20.2 + -0.1 °, 21.4 + -0.1 °, 23.1 + -0.1 °, 23.8 + -0.1 °, 25.8 + -0.1 °, 26.1 + -0.1 °, 27.0 + -0.1 °, 27.2 + -0.1 ± 0.3.3 ± 0.3 ± 0.34 ° ± θ.

22. The crystalline N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 21 wherein the X-ray powder diffraction pattern does not comprise at least one diffraction peak selected from the group consisting of 3.6 ± 0.1 and 9.0 ± 0.1 ° 2 Θ.

23. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 21]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide having a Fourier transform infrared spectrum comprised in 881 + -2 cm^-1The absorption band of (b).

24. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 21]Pyrimidine-3-carbonyl]A methanesulfonamide crystal, wherein the Fourier transform infrared spectrum further includes at least one selected from 661 + -2, 703 + -2, 797 + -2, 909 + -2 and 1269 + -2 cm^-1The absorption band of (1).

25. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 21]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide, wherein the Fourier transform infrared spectrum does not include at least one selected from 688 + -2 and 696 + -2 cm^-1The absorption band of (1).

26. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 21]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide, the Raman spectrum of said crystals comprising 2988 ± 2cm^-1The band of (b).

27. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 21]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide, wherein the Raman spectrum further comprises at least one selected from 707 + -2 and 1447 + -2 cm^-1The absorption band of (1).

28. N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d according to claim 21]Pyrimidine-3-carbonyl]Crystals of methanesulfonamide, wherein the Raman spectrum is not included at 1417 + -2 cm^-1The band of (b).

29. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 21 and a pharmaceutically-acceptable carrier.

30. A pharmaceutical composition comprising a therapeutically effective amount of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide and a pharmaceutically-acceptable carrier, wherein at least 50 weight percent of the N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide is present as N- [1- (2-ethylic-ethyl) -5- (N-methyl-amino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 21 In the form of crystals of oxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide.

31. Use of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 1 in the manufacture of a medicament for the treatment of a PDE-5 mediated disorder.

32. Use of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 11 in the manufacture of a medicament for the treatment of a PDE-5 mediated disorder.

33. Use of N- [1- (2-ethoxyethyl) -5- (N-ethyl-N-methylamino) -7- (4-methylpyridin-2-yl-amino) -1H-pyrazolo [4,3-d ] pyrimidine-3-carbonyl ] methanesulfonamide of claim 21 in the manufacture of a medicament for the treatment of a PDE-5-mediated disorder.