CN101155813A - 1-(2-Methylpropyl)-1H-imidazo[4,5-C][1,5]naphthyridine-4-amine ethanesulfonate and 1-(2-methylpropyl )-1H-imidazo[4,5-C][1,5]naphthyridine-4-amine methanesulfonate - Google Patents

Abstract

This invention provides methanesulfonate and ethanesulfonate salts of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridin-4-amine, pharmaceutical compositions containing the salts, methods of making, and methods of use.

Description

1-(2-Methylpropyl)-1H-imidazo[4,5-C][1,5]naphthyridine-4-amine ethanesulfonate and 1-(2-methylpropyl )-1H-imidazo[4,5-C][1,5]naphthyridine-4-amine methanesulfonate

Cross References to Related Applications

This application claims U.S. Provisional Application 60/640490, filed December 30, 2004, U.S. Provisional Application 60/708636, filed August 16, 2005, U.S. Provisional Application 60/649932, filed February 4, 2005, and 2005 Priority to U.S. Provisional Application 60/698416, filed July 12, all of which are hereby incorporated by reference in their entirety.

Background of the invention

The compound 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine has been found to be a useful Immune Response Modifiers (IRMs) (US Patent 6,194,425). However, formulating and manufacturing pharmaceutical products presents many unforeseen challenges.

Summary of the invention

It has now been found that 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine and certain pharmaceutically acceptable salts thereof It is very difficult to formulate for several reasons, especially the low solubility in water. For example, 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]phthalazine, although hydrochloride salts of drugs are often prepared and are often soluble The hydrochloride salt of -4-amine also has very low solubility in water. However, it has also been found that the ethanesulfonate and methanesulfonate salts of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine have Surprisingly desirable properties include good solubility in water and good physical and chemical stability in solid form. These are all beneficial characteristics for formulating and manufacturing useful products.

Accordingly, some aspects of the present invention provide methanesulfonate salts of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine and ethyl Sulfonate.

In one aspect, the present invention provides methanesulfonic acid of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine as shown below salt (I).

In another aspect, the present invention provides ethanesulfonate of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine shown below acid salt (II).

Salt I and Salt II may be in solvated or hydrated form, which may provide improved stability during manufacture.

In some embodiments, the present invention also provides a pharmaceutical composition containing salt I or II or a solvate or hydrate thereof or a combination thereof. Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of salt I or II, or a solvate or hydrate thereof, or a combination thereof. The pharmaceutical composition will generally have the salt of formula I or II in dissolved form.

In some embodiments, the present invention also provides a pharmaceutical composition prepared by the following method, which method comprises a pharmaceutically acceptable carrier and an effective amount of salt I or II, or a solvate or hydrate thereof, or Portfolio merge.

In some embodiments, the present invention also provides methods of use, such as methods of inducing cytokine biosynthesis in animals, methods of treating viral diseases in animals, and/or methods of treating neoplastic diseases in animals, said The method is to administer to said animal an effective amount of salt I or II, or a solvate or hydrate thereof, or a combination thereof, optionally contained in a pharmaceutical composition. In one embodiment, the present invention also provides a method containing dissolved 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine- Methanesulfonate of 4-amine or ethanesulfonate of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine A topical formulation for the treatment of high-risk cervical HPV infection.

In another embodiment, the present invention provides a method for preparing 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine The ethanesulfonate method. The process comprises mixing 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine free base with ethanesulfonic acid and a carrier to form a mixture , wherein the carrier comprises an organic liquid and optionally water; and reacting the components in the mixture under conditions sufficient to form 1-(2-methylpropyl)-1H-imidazo[4,5-c] [1,5]Ethyridine-4-amine ethanesulfonate.

In another embodiment, the present invention provides a method for preparing 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine The mesylate method. The process comprises mixing 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine free base with methanesulfonic acid and a carrier to form a mixture , wherein the carrier comprises an organic liquid and optionally water; and reacting the components of the mixture under sufficient conditions to form 1-(2-methylpropyl)-1H-imidazo[4,5-c][ 1,5] The mesylate salt of naphthyridine-4-amine.

Where the term "comprises" and its various forms appear in the description and claims, it does not have a limiting meaning.

Herein, "a", "an", "the", "at least one", "at least a portion" and "one or more" are used interchangeably. Thus, for example, a carrier containing an organic liquid may be construed to mean that the carrier contains "one or more" organic liquids.

The term "effective amount" (or "therapeutically effective amount") means an amount of a salt sufficient to produce a therapeutic or prophylactic effect, such as cytokine induction, anticancer activity and/or antiviral activity. The precise amount of salt used in the pharmaceutical compositions of this invention will vary according to various factors known to those skilled in the art, such as the physical and chemical properties of the compound, the nature of the carrier and the intended dosage regimen.

The term "solvate" refers to an amorphous or crystalline material (preferably crystalline material) that contains one or more associated solvent molecules, preferably within a crystal lattice. The term "hydrate" refers to a solvate in which the molecule of interest is water. Monohydrates contain 1 molecule of water per molecule of IRM. Water content refers to the weight percent of water determined by the known Karl Fisher method.

Certain compounds can crystallize through more than one type of molecular packing with more than one type of internal lattice. The respective resulting crystal structures may have, for example, different unit cells. This "same chemical structure but different internal structure" phenomenon is called polymorphism, and species with different molecular structures are called polymorphs.

As used herein, the term "polymorph" includes true polymorphs having the same chemical structure as well as pseudopolymorphs containing different levels of hydration and/or solvent within the unit cell.

Herein, the term "about" when referring to a measured quantity refers to a variation in the measured quantity that can be predicted by the skilled artisan making the measurement with a level of care commensurate with the purpose of the measurement and the precision of the measuring equipment used.

Herein, when referring to a spectral line (e.g. NMR, IR) or X-ray diffraction pattern shown in a figure, the term "substantially" means that depending on sample preparation and experimental technique, the positions of peaks may drift up to the error provided , and the intensity of the peaks will also produce changes that can be expected by those skilled in the art.

The ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine and 1-(2- The mesylate salt of methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine may exist in any of its pharmaceutically acceptable forms, including solvates , hydrates, polymorphs, etc., and may be dissolved. It is to be understood that the term "salt" includes any and all of these forms, whether specifically stated or not (although "solvates" and "hydrates" are sometimes specified). The salts described herein may be amorphous or crystalline solids, with or without any relevant solvent molecules or water molecules, and may be fully or partially dissolved (eg, in pharmaceutical compositions). Thus, "salt" may include amorphous salts, crystalline salts, crystalline salt hydrates, crystalline salt solvates, salts in solution, and combinations thereof. For example, unless expressly stated, when referring to the ethanesulfonate salt of "1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine When ", the salt may be crystalline, amorphous, hydrated, solvated or fully or partially dissolved. In topical pharmaceutical compositions it will usually be in dissolved form. Meanwhile, when referring to "1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate or its solvate or hydrate", the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine may be crystalline amorphous, hydrated, solvated or fully or partially dissolved.

The above summary of the invention is not intended to describe each disclosed embodiment or every implementation of the invention. The description that follows more particularly exemplifies exemplary embodiments. Guidelines for various combinations are also provided in the list of examples. In each case, the mentioned list serves only as a representative example and is not to be understood as an exclusive list.

Brief description of the drawings

Figure 1 is a typical X of the crystalline form of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate monohydrate Ray Diffraction Diagram.

Figure 2 is a typical X of the crystalline form of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate Ray Diffraction Diagram.

Figure 3 is a typical solid state diagram of the crystalline form of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate ¹³ C NMR spectrum.

Figure 4 is a typical IR of the crystalline form of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate Spectrum.

Fig. 5 is the curve of pH and solubility of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine determined by potentiometric titration .

Figure 6 is a typical water crystallization form of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate Adsorption isotherm (water adsorption isotherm).

Figure 7 is a typical thermal profile of the crystalline form of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate Analysis plot showing an overlay of data obtained by DSC and TGA.

Figure 8 is a typical water crystallization form of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate monohydrate Adsorption isotherms.

Figure 9 is a typical thermodynamic diagram of the crystalline form of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate monohydrate Analysis plot showing an overlay of data obtained by DSC and TGA.

Detailed Description of Exemplary Embodiments

In some aspects, the present invention provides the mesylate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (I) , or its solvate or hydrate, and ethanesulfonic acid of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine Salt (II), or a solvate or hydrate thereof. Such compounds may be in crystalline form. Generally, these salts have sufficient solubility in water to render them useful in water-based formulations. In fact, their solubility in water is surprisingly better (eg, more than 10 times, even more than 25 times) than other salts of the same compound, such as hydrochloride.

A number of techniques can be employed to characterize crystalline materials including, for example, X-ray powder diffraction (XRPD), solid- ^state13C nuclear magnetic resonance (NMR) spectroscopy, solid-state infrared (IR) spectroscopy, thermal analysis (e.g., thermogravimetric analysis) (TGA), differential thermal analysis (DTA) and differential scanning calorimetry (DSC)). In general, any of the polymorphs mentioned herein can be characterized to particular advantage by these techniques. For example, different polymorphs of the same compound often exhibit different diffraction patterns with distinct sets of diffraction peaks that can be expressed in 2Θ angles, and often have unit cells with different interplanar spacings (Angstroms). Such techniques are well known to those skilled in the art. The data presented here were obtained under the conditions described in the Examples section.

In one embodiment, the present invention provides the mesylate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ( I), or a solvate or hydrate thereof (ie, of the salt).

In one embodiment, the mesylate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is dissolved form.

In one embodiment, the mesylate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is in solid form .

In one embodiment, the mesylate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is amorphous form.

In one embodiment, the mesylate salt of the 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine or its ( That is, the solvate or hydrate of the salt is in crystalline form.

In one embodiment, the mesylate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is solvated form.

In one embodiment, the mesylate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is a hydrate form.

In one embodiment, the mesylate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is monohydrate object form.

In one embodiment, 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate (I) monohydrate The crystalline form of can be characterized by, for example, an X-ray powder diffraction pattern with peaks at 8.51 degrees 2Θ, 14.12 degrees 2Θ, 16.80 degrees 2Θ, 17.88 degrees 2Θ, 21.43 degrees 2Θ, 23.24 degrees 2Θ, and 29.16 degrees 2Θ, each of which is ±0.15 degrees 2 theta.

Alternatively, and preferably, the crystalline monohydrate of (I) can be detected by, for example, an X-ray powder diffraction pattern at 7.15 degrees 2Θ, 8.51 degrees 2Θ, 14.12 degrees 2Θ, 16.80 degrees 2Θ, 17.88 degrees 2Θ, 18.49 degrees 2Θ, 18.88 degrees 2Θ , 21.04 degrees 2Θ, 21.43 degrees 2Θ, 23.24 degrees 2Θ, 25.40 degrees 2Θ, 27.92 degrees 2Θ, 28.77 degrees 2Θ, and 29.16 degrees 2Θ, these values are each ±0.15 degrees 2Θ. The crystalline monohydrate of (I) can also be determined by, for example, the relative peak intensity levels of the above peaks being moderate (7.15 degrees 2θ), moderate (8.51 degrees 2θ), moderate (14.12 degrees 2θ), moderately high (16.80 degrees 2θ), moderately high (16.80 degrees 2θ), moderate (17.88 degrees 2θ), medium (18.49 degrees 2θ), medium (18.88 degrees 2θ), medium-high (21.04 degrees 2θ), medium-high (21.43 degrees 2θ), high (23.24 degrees 2θ), medium-high (25.40 degrees 2θ), medium ( 27.92 degrees 2θ), medium (28.77 degrees 2θ) and medium-high (29.16 degrees 2θ), where the peak intensities are classified according to the following scheme for relative intensities: high is 85.0-100.0%; medium-high is 70.0%-84.9%; medium is 20.0%-69.9%; low-moderate is 5.0%-19.9%; and low is less than 5.0%.

Alternatively, and more preferably, the crystalline monohydrate of (I) can be obtained by, for example, an X-ray powder diffraction pattern at 7.15 degrees 2Θ, 7.55 degrees 2Θ, 8.51 degrees 2Θ, 11.83 degrees 2Θ, 13.65 degrees 2Θ, 14.12 degrees 2Θ, 14.87 degrees 2θ, 16.80 degrees 2θ, 17.88 degrees 2θ, 18.49 degrees 2θ, 18.88 degrees 2θ, 19.92 degrees 2θ, 20.24 degrees 2θ, 21.04 degrees 2θ, 21.43 degrees 2θ, 22.24 degrees 2θ, 23.24 degrees 2θ, 24.64 degrees 2θ, 25.40 degrees 2θ, Peak characterization of 25.71 degrees 2θ, 27.20 degrees 2θ, 27.92 degrees 2θ, 28.77 degrees 2θ, 29.16 degrees 2θ, 30.94 degrees 2θ, 31.29 degrees 2θ, 32.76 degrees 2θ, 33.56 degrees 2θ, 34.04 degrees 2θ, 34.88 degrees 2θ, and 35.40 degrees 2θ , these values are each ±0.15 degrees 2θ.

In another embodiment, 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate (I) mono The crystalline form of the hydrate can be characterized by, for example, a unit cell with a crystal interplanar spacing of about 10.38 angstroms, about 6.27 angstroms, about 5.27 angstroms, about 4.96 angstroms, about 4.14 angstroms, about 3.82 angstroms, and about 3.06 angstroms.

Alternatively, and preferably, the crystalline monohydrate of (I) can be obtained by, for example, a crystal interplanar spacing of about 12.35 angstroms, about 10.38 angstroms, about 6.27 angstroms, about 5.27 angstroms, about 4.96 angstroms, about 4.80 angstroms, about 4.70 angstroms, about 4.22 angstroms, about 4.14 angstroms, about 3.82 angstroms, about 3.50 angstroms, about 3.19 angstroms, about 3.10 angstroms, and about 3.06 angstroms.

In a particularly preferred embodiment, 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate monohydrate The compound can be characterized by an X-ray powder diffraction pattern substantially as shown in FIG. 1 .

In another embodiment, 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate (I) mono The crystalline form of the hydrate can be characterized by, for example, a weight loss of 4.5-5.5% in the temperature range of 60-80° C. as measured by thermogravimetric analysis. Typically, and preferably, this information is combined with X-ray powder diffraction data such as 7.15 degrees 2Theta, 8.51 degrees 2Theta, 14.12 degrees 2Theta, 16.80 degrees 2Theta, 17.88 degrees 2Theta, 18.49 degrees 2Theta, 18.88 degrees 2Theta, 21.04 degrees 2Theta, 21.43 degrees The peaks at 2Θ, 23.24° 2Θ, 25.40° 2Θ, 27.92° 2Θ, 28.77° 2Θ, and 29.16° 2Θ combined, these values were each ±0.15° 2Θ.

On the one hand, the present invention also provides the mesylate ( I) method. The process comprises mixing 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine free base with methanesulfonic acid and a carrier to form a mixture , and react the components of the mixture under sufficient conditions to form 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine mesylate.

The carrier comprises an organic liquid and optionally water. In some embodiments, the carrier contains 1 volume percent (volume % or % v/v) to 15 volume percent water in the organic liquid. In some embodiments, the carrier Contains at least 2 moles of water. Examples of suitable organic liquids include isopropanol (i.e., isopropyl alcohol), other lower alcohols (e.g., methanol, ethanol, n-propanol, n-butanol, sec-butanol), toluene, acetone, acetonitrile, methyl acetate , ethyl acetate, isopropyl acetate, isobutyl acetate, and tetrahydrofuran (THF). Other examples of suitable organic liquids include heptane, tert-butyl methyl ether, N,N-dimethylformamide (DMF), 1-methyl-2-pyrrolidone (NMP), dichloromethane and xylene. Mixtures containing any two or more of these organic liquids may also be used.

In some embodiments, the method further comprises heating the free base, methanesulfonic acid, and/or carrier prior to mixing, and/or heating a mixture thereof. In certain embodiments, the method further comprises forming 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine in the mixture The mesylate salt precipitated. In one embodiment, forming the precipitate includes cooling the mixture to form the precipitate.

In some embodiments, the method further comprises: optionally adding another organic liquid to the mixture containing the precipitate; separating at least a portion of the precipitate from at least a portion of the mixture; washing the precipitate; and at least partially drying the precipitate. Suitable other organic liquids include ethers (e.g., tert-butyl methyl ether), acetone, THF, 1,2-dimethoxyethane, diethoxymethane, methyl acetate, ethyl acetate, isopropyl acetate , isobutyl acetate, heptane, toluene and xylene.

Those skilled in the art will appreciate that there are many ways to separate the precipitate from the mixture, such as filtration, decantation and centrifugation. In one embodiment, the precipitate is isolated by filtration. The precipitate may optionally be washed after isolation. Generally, one or more organic liquids (for example, lower alcohols, toluene, acetone, acetonitrile, methyl acetate, ethyl acetate, isopropyl acetate, isobutyl acetate, 1,2-dimethoxyethane, etc. alkanes, diethoxymethane, heptane, xylene and THF) sequentially or in combination to remove impurities. Those skilled in the art will appreciate that there are many methods of drying compounds including, for example, the use of elevated temperatures, drying, reduced pressure, and the like.

In one embodiment, the present invention provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and an effective amount of 1-(2-methylpropyl)-1H-imidazo[4,5-c][ 1,5] Naphthalene-4-amine methanesulfonate (I), or a solvate or hydrate thereof. In another embodiment, the present invention provides a pharmaceutical composition manufactured by a method comprising admixing a pharmaceutically acceptable carrier with an effective amount of (I), or a solvate or hydrate thereof. In one embodiment, the pharmaceutically acceptable carrier includes water.

Additionally, in certain embodiments, methods of inducing cytokine biosynthesis in animals are provided. The method comprises administering to the animal an effective amount of (I), or a solvate or hydrate thereof, or a pharmaceutical composition containing an effective amount of (I) or a solvate or hydrate thereof. In another embodiment, a method of treating a viral disease in an animal is provided. The method comprises administering to said animal a therapeutically effective amount of (I), or a solvate or hydrate thereof, or a pharmaceutical composition comprising a therapeutically effective amount of (I) or a solvate or hydrate thereof. In certain embodiments, the viral disease comprises intracervical human papillomavirus. In another embodiment, a method of treating a neoplastic disease in an animal is provided. The method comprises administering to said animal a therapeutically effective amount of (I), or a solvate or hydrate thereof, or a pharmaceutical composition comprising a therapeutically effective amount of (I) or a solvate or hydrate thereof. In certain embodiments, the neoplastic disease is intracervically located.

In one embodiment, the present invention provides ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ( II), or a solvate or hydrate thereof (ie, of the salt).

In one embodiment, the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is dissolved form.

In one embodiment, the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is in solid form .

In one embodiment, the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is amorphous form.

In one embodiment, the ethanesulfonic acid salt of the 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine or its ( That is, the solvate or hydrate of the salt is in crystalline form.

In one embodiment, the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is solvated form.

In one embodiment, the ethanesulfonic acid salt of the 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is a hydrate form.

In one embodiment, the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is monohydrate object form.

In one embodiment, the ethanesulfonate (II) monohydrate of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine The crystalline form of the compound can be characterized by, for example, an X-ray powder diffraction pattern with peaks at 6.98 degrees 2Θ, 10.50 degrees 2Θ, and 16.70 degrees 2Θ, each of which values are ±0.15 degrees 2Θ.

Alternatively, and preferably, the crystalline monohydrate of (II) can be characterized by, for example, peaks in an X-ray powder diffraction pattern at 6.98 degrees 2Θ, 10.50 degrees 2Θ, 16.70 degrees 2Θ, 18.11 degrees 2Θ, and 26.02 degrees 2Θ, each of which is ±0.15 degrees 2Θ.

Alternatively, and more preferably, the crystalline monohydrate of (II) can be obtained by, for example, X-ray powder diffraction patterns at 6.98 degrees 2Θ, 10.50 degrees 2Θ, 10.70 degrees 2Θ, 16.70 degrees 2Θ, 18.11 degrees 2Θ, 18.88 degrees 2Θ, 21.11 degrees Characterized by peaks at 2Θ, 26.02 degrees 2Θ, and 28.51 degrees 2Θ, these values are each ±0.15 degrees 2Θ. The crystalline monohydrate of (II) can be judged by, for example, the relative peak intensity levels of the above peaks being high (6.98 degrees 2θ), medium low (10.50 degrees 2θ), medium low (10.70 degrees 2θ), medium low (16.70 degrees 2θ) , medium (18.11 degrees 2θ), medium (18.88 degrees 2θ), medium (21.11 degrees 2θ), medium-low (26.02 degrees 2θ) and medium-low (28.51 degrees 2θ), where the peak intensities are compared to relative intensities according to the following scheme Classification: high is 85.0-100.0%; medium-high is 70.0%-84.9%; medium is 20.0%-69.9%; medium-low is 5.0%-19.9%; and low is less than 5.0%.

Alternatively, and even more preferably, the crystalline monohydrate of (II) can be detected by, for example, X-ray powder diffraction patterns at 6.98 degrees 2Θ, 8.96 degrees 2Θ, 10.50 degrees 2Θ, 10.70 degrees 2Θ, 11.60 degrees 2Θ, 14.46 degrees 2Θ, 16.70 degrees 2θ, 17.27 degrees 2θ, 18.11 degrees 2θ, 18.47 degrees 2θ, 18.88 degrees 2θ, 20.57 degrees 2θ, 21.11 degrees 2θ, 21.39 degrees 2θ, 22.52 degrees 2θ, 23.04 degrees 2θ, 23.35 degrees 2θ, 23.84 degrees 2θ, 24.35 degrees 2θ , 26.02 degrees 2θ, 27.33 degrees 2θ, 27.92 degrees 2θ, 28.51 degrees 2θ, 29.42 degrees 2θ, 30.19 degrees 2θ, 31.47 degrees 2θ, 31.80 degrees 2θ, 32.45 degrees 2θ, 33.02 degrees 2θ and 33.73 degrees 2θ. Each is ±0.15 degrees 2Θ.

In another embodiment, the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (II) The crystalline form of the monohydrate can be characterized, for example, by a unit cell with crystal interplanar spacings of about 12.66 Angstroms, about 8.42 Angstroms, and about 5.30 Angstroms.

Alternatively, and preferably, the crystalline monohydrate of (II) can be obtained by, for example, a unit cell with a crystal interplanar spacing of about 12.66 angstroms, about 8.42 angstroms, about 8.26 angstroms, about 5.30 angstroms, about 4.89 angstroms, about 4.70 angstroms, about 4.21 angstroms, about 3.42 angstroms and about 3.13 angstroms to characterize.

In a particularly preferred embodiment, the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ( II) The monohydrate can be characterized by an X-ray powder diffraction pattern substantially as shown in FIG. 2 .

In one embodiment, 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate can be obtained by ¹³ The C NMR spectrum is characterized by peaks at 127.5 ppm (parts per million), 126.0 ppm and 122.9 ppm, each of these values being ±0.3 ppm. Alternatively, and preferably, the crystalline monohydrate of (II) can be characterized by, for example, peaks in the solid state ¹³ CNMR spectrum at 131.9 ppm, 127.5 ppm, 126.0 ppm, 122.9 ppm and 56.2 ppm, each of these values being ±0.3 ppm. Alternatively, and more preferably, the crystalline monohydrate of (II) can be detected by, for example, solid ^state13C NMR spectra 148.0ppm, 134.2ppm, 131.9ppm, 127.5ppm, 126.0ppm, 122.9ppm, 56.2ppm, 45.5ppm, 29.7ppm, Characterized by peaks at 21.8 ppm and 10.5 ppm, these values are each ±0.3 ppm.

In a particularly preferred embodiment, the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ( II) The monohydrate can be characterized by a solid state ¹³ C NMR spectrum substantially as shown in FIG. 3 .

In another embodiment, the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (II) The crystalline monohydrate of can be characterized by, for example, a weight loss of 4.2-5.2% measured by TGA in the temperature range of 55-100°C, preferably 65-100°C. Alternatively, or in addition, solid-state FTIR spectroscopy can be performed, which can provide characterization information. For example, crystalline monohydrate (II) can be characterized by a solid state IR spectrum substantially as shown in FIG. 4 .

Typically, and preferably, TGA and IR data can be combined with, for example, X-ray powder diffraction data. For example, the crystalline monohydrate of (II) can be characterized by the following peaks: 6.98 degrees 2Theta, 10.50 degrees 2Theta, 10.70 degrees 2Theta, 16.70 degrees 2Theta, 18.11 degrees 2Theta, 18.88 degrees 2Theta, 21.11 degrees 2Theta, 26.02 degrees 2Theta, and 28.51 degrees 2Θ, these values are each ±0.15 degrees 2Θ. In addition to X-ray diffraction data, solid ^state13C NMR can be used for characterization along with TGA and IR data. For example, the crystalline monohydrate of (II) can be characterized by the following peaks: 148.0 ppm, 134.2 ppm, 131.9 ppm, 127.5 ppm, 126.0 ppm, 122.9 ppm, 56.2 ppm, 45.5 ppm, 29.7 ppm, 21.8 ppm and 10.5 ppm, these values Each is ±0.3ppm.

In one aspect, the present invention provides a process for preparing the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine . The process comprises mixing 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine free base with ethanesulfonic acid and a carrier to form a mixture , and reacting the components of the mixture under sufficient conditions to form 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine of ethanesulfonate.

The carrier comprises an organic liquid and optionally water. In some embodiments, the carrier contains 1-15% water by volume in the organic liquid. In some embodiments, the carrier Contains at least 2 moles of water. Examples of suitable organic liquids include isopropanol (i.e., isopropyl alcohol), other lower alcohols (e.g., methanol, ethanol, n-propanol, n-butanol, sec-butanol), toluene, acetone, acetonitrile, methyl acetate , ethyl acetate, isopropyl acetate, isobutyl acetate, and tetrahydrofuran (THF). Other examples of suitable organic liquids include heptane, tert-butyl methyl ether, N,N-dimethylformamide (DMF), 1-methyl-2-pyrrolidone (NMP), dichloromethane and xylene. Mixtures containing any two or more of these organic liquids may also be used.

In some embodiments, the method further includes heating the free base, ethanesulfonic acid, and/or carrier prior to mixing, and/or heating a mixture thereof. In certain embodiments, the method further comprises forming 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine in the mixture Precipitation of ethanesulfonate. In one embodiment, forming the precipitate includes cooling the mixture to form the precipitate. Preferably, cooling is performed at a rate of less than 2.0°C/minute.

In some embodiments, the method further comprises: optionally adding another organic liquid to the mixture containing the precipitate; separating at least a portion of the precipitate from at least a portion of the mixture; washing the precipitate; and at least partially drying the precipitate. Suitable other organic liquids include ethers (e.g., tert-butyl methyl ether), acetone, THF, 1,2-dimethoxyethane, diethoxymethane, methyl acetate, ethyl acetate, isopropyl acetate , isobutyl acetate, heptane, toluene and xylene.

As noted above, those skilled in the art will appreciate that there are many methods by which the precipitate can be separated from the mixture, such as filtration, decantation and centrifugation. In one embodiment, the precipitate is isolated by filtration. The precipitate may optionally be washed after isolation. Generally, one or more organic liquids (for example, lower alcohols, toluene, acetone, acetonitrile, methyl acetate, ethyl acetate, isopropyl acetate, isobutyl acetate, 1,2-dimethoxyethane, etc. alkanes, diethoxymethane, heptane, xylene and THF) sequentially or in combination to remove impurities. Those skilled in the art will appreciate that there are many methods of drying compounds including, for example, the use of elevated temperatures, drying, reduced pressure, and the like.

In one embodiment, the present invention provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and an effective amount of 1-(2-methylpropyl)-1H-imidazo[4,5-c][ 1,5] Ethyridine-4-amine ethanesulfonate (II), or a solvate or hydrate thereof. In another embodiment, the present invention provides a pharmaceutical composition manufactured by a process comprising admixing a pharmaceutically acceptable carrier with an effective amount of (II), or a solvate or hydrate thereof. In one embodiment, the pharmaceutically acceptable carrier includes water.

Additionally, in certain embodiments, methods of inducing cytokine biosynthesis in animals are provided. The method comprises administering to the animal an effective amount of (II), or a solvate or hydrate thereof, or a pharmaceutical composition containing an effective amount of (II) or a solvate or hydrate thereof. In another embodiment, a method of treating a viral disease in an animal is provided. The method comprises administering to said animal a therapeutically effective amount of (II), or a solvate or hydrate thereof, or a pharmaceutical composition comprising a therapeutically effective amount of (II) or a solvate or hydrate thereof. In certain embodiments, the viral disease comprises intracervical human papillomavirus. In another embodiment, a method of treating a neoplastic disease in an animal is provided. The method comprises administering to said animal a therapeutically effective amount of (II), or a solvate or hydrate thereof, or a pharmaceutical composition comprising a therapeutically effective amount of (II) or a solvate or hydrate thereof. In certain embodiments, the neoplastic disease is intracervically located.

In another embodiment, there is provided a solution obtained by administering to the cervix containing dissolved 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine A topical formulation of the methanesulfonate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine from Methods of treating high-risk cervical HPV infection.

Compound preparation

As used herein, the term "alkyl" and the prefix "alk-" include straight and branched chain groups as well as cyclic groups. Unless otherwise specified, these groups contain 1-20 carbon atoms. In some embodiments, these groups contain up to 10 carbon atoms, up to 8 carbon atoms, up to 6 carbon atoms, or up to 4 carbon atoms in total. Cyclic groups can be monocyclic or polycyclic and preferably contain 3-10 ring carbon atoms. Examples of cyclic groups include cyclopropyl, cyclopropylmethyl, cyclopentyl and cyclohexyl.

Unless otherwise specified, "alkylene" is a divalent form of the above-mentioned "alkyl". The term "alkylenyl" is used when the "alkylene" is substituted. For example, an arylalkylene group contains an alkylene moiety to which is attached an aryl group.

"Lower alcohols" are understood as straight and branched chain alcohols containing 1 to 4 carbon atoms. Examples include methanol, ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, and t-butanol.

The term "aryl" when referring to "arylsulfonyl halides" includes unsubstituted or substituted carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl. Examples of substituents that may occur on aryl include alkyl, alkoxy, haloalkyl, haloalkoxy, halo, nitro, hydroxy, cyano, aryl, aryloxy and arylalkyleneoxy.

1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine can be prepared according to the route shown in Scheme I. Steps (1)-(6) of Reaction Scheme I can be carried out according to the method described in US Pat. No. 6,194,425 (Gerster et al.) or the modified method described in the following examples.

In step (6) of Scheme I, 1-(2-methylpropyl)-5-oxido(oxido)-1H-imidazo[4,5-c][1,5]naphthyridine was amine to give 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine. Step (6) involves converting 1-(2-methylpropyl)-5-oxy-1H-imidazo[4,5-c][1,5]phthalazine into an ester to activate it and then make The ester is reacted with an aminating agent. Suitable activators include alkyl- or arylsulfonyl chlorides, such as benzenesulfonyl chloride, methanesulfonyl chloride or p-toluenesulfonyl chloride. Suitable aminating agents include, for example, ammonia in the form of ammonium hydroxide and ammonium salts such as ammonium carbonate, ammonium bicarbonate and ammonium phosphate. The reaction can be carried out by adding ammonium hydroxide to 1-(2-methylpropyl)-5-oxygen-1H-imidazo[4,5-c][1,5 ] Naphthalene solution, and then add p-toluenesulfonyl chloride. The reaction can be carried out at room temperature, and the product can be isolated from the reaction mixture by conventional methods.

Preferably, when carrying out the reaction of reaction scheme I step (6), ammonium hydroxide, ammonia or other suitable amination agents mentioned above can be dissolved in a suitable medium such as lower alcohol or 1-(2-methyl Propyl)-5-oxo-1H-imidazo[4,5-c][1,5]naphthyridine mixed. The resulting mixture is then combined with an arylsulfonyl halide to provide 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4 in the mixture -amine, and then combine the mixture with an aqueous alkaline solution. The alkaline aqueous solution is preferably an aqueous solution of an alkali metal hydride such as sodium hydroxide or potassium hydroxide. Alternatively, an aqueous solution of potassium carbonate or sodium carbonate may also be used. The reaction can be achieved by adding an alcoholic solution of ammonium hydroxide or ammonia to 1-(2-methylpropyl)-5-oxygen-1H-imidazo[4,5-c in methanol, ethanol or isopropanol ][1,5]Naphthyridine solution, and then add benzenesulfonyl chloride or p-toluenesulfonyl chloride and conveniently carry out. The reaction can be performed at room temperature. Aqueous sodium hydroxide solution is then added and the product isolated using conventional techniques. The product usually precipitates under the reaction conditions and can be isolated from the reaction mixture by conventional methods.

Option I

Preparation of the ethanesulfonic acid salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine or 1-(2-methylpropane base)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate, including its solvates or hydrates, 1-(2-methyl Propyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine is mixed with ethanesulfonic acid or methanesulfonic acid and carrier respectively, wherein said carrier contains organic liquid and optionally water, and react the components of the mixture under sufficient conditions to form the desired salt. Preferably, and especially for the crystalline solid form, ethanesulfonic acid of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine Salts and mesylate salts of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine are generally prepared according to Scheme II. In steps (1) and (2) of Scheme II, 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine The free base of is mixed with one or more organic liquids or a mixture of one or more organic liquids and water to prepare a solution or suspension of the free base. The resulting solution or suspension can be heated to an elevated temperature, such as the reflux temperature of the solvent, before being mixed with ethanesulfonic acid or methanesulfonic acid in step (3). The solution or suspension can also be mixed with an acid at room temperature. Solutions of acids or neat compounds may also be used. Several organic liquids can also be used; these include, for example, lower alcohols, toluene, acetone, acetonitrile, methyl acetate, ethyl acetate, isopropyl acetate, isobutyl acetate, and tetrahydrofuran (THF). Other examples of organic liquids that can be used include heptane, tert-butyl methyl ether, N,N-dimethylformamide (DMF), 1-methyl-2-pyrrolidone (NMP), dichloromethane, and xylene. Mixtures containing any two or more of these organic liquids may also be used. Useful organic liquid mixtures include mixtures of heptane and tert-butyl methyl ether, isopropanol, 2-butanol, THF, dichloromethane, ethyl acetate, or toluene; tert-butyl methyl ether and lower alcohols, THF, dichloromethane , a mixture of ethyl acetate or toluene; a mixture of isopropanol and dichloromethane, ethyl acetate or toluene; a mixture of 2-butanol and THF, dichloromethane or toluene; a mixture of THF and dichloromethane, ethyl acetate or toluene mixtures; mixtures of dichloromethane and ethyl acetate; mixtures of dichloromethane and toluene; and mixtures of ethyl acetate and toluene. The organic liquid or mixture of organic liquids can optionally be mixed with water. Useful amounts of water that may be mixed with the organic liquid or mixture of organic liquids include 1-15% v/v (volume percent). Preferably, the amount of water mixed is 1-10% v/v. More preferably, the amount of water is 1-5% v/v, most preferably 1-3% v/v. Many commercially available organic solvents already contain up to 1% v/v water. Preferably, the carrier used in steps (1)-(3) of Scheme II is ethyl acetate with 2% v/v water, isopropanol with 2% v/v water, or isopropyl alcohol. Can also be based on the amount of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine free base present in terms of equivalents (moles per mole) to determine the amount of water that can be mixed with an organic liquid. Preferably, the amount of water present is Not less than 2 equivalents (mol/mol). More preferably, 2-10 moles of water are present per mole of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine. Most preferably, 2-5 moles of water are present per mole of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine. The solution or suspension prepared in step (3) may be heated for several minutes, over 15 minutes, over 1 hour, over 4 hours, or over 24 hours, for example, up to the point when the salt begins to decompose.

In steps (4a) and (4b) of Scheme II, the hot salt solution is cooled to form a precipitate. The cooling in steps (4a) and (4b) can be performed at room temperature or at a temperature below room temperature from -20°C to 25°C. Steps (4a) and (4b) differ in the cooling rates used. A slow cooling rate is employed in step (4b). The slow rate may be less than 2°C/minute; preferably less than 1°C/minute. More preferably, the slow rate is less than 0.75°C/minute. Most preferably, it may be 0.1-0.5°C/min. In some cases, the rate can be as low as 0.05°C/minute. A fast cooling rate is employed in step (4a). A fast cooling rate is characterized by the rate at which a small volume (less than 100 mL) of a solution or suspension can be cooled while standing at room temperature. Preferably, the rapid cooling rate is greater than 2°C/minute; and more preferably greater than 4°C/minute. in 1-15% (v/v) water/isopropanol or in the presence of 1 mole of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5] Preparation of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine in the presence of at least 2 moles of water For ethanesulfonate, a slow cooling rate is preferred. Preferably, the cooling rate is less than 2°C/min. More preferably, it is in the range of 0.1-0.5°C/min.

Other methods of forming the precipitate may also be used and will be understood by those skilled in the art. These methods include, for example, introduction of other organic liquids in which the salt is poorly soluble. For example, tert-butyl methyl ether may be mixed with a solution of the salt in acetonitrile, 2-butanol, dichloromethane or THF, wherein any of these solutions may contain at least 2 moles of water per mole of salt. The salt solution can be added to the tert-butyl methyl ether, or the tert-butyl methyl ether can be added slowly or quickly to the salt solution. Preferably tert-butyl methyl ether is slowly added to the salt solution to form a precipitate.

In step (5a) or (5c) of Scheme II, the precipitated salt is separated from the suspension. Separation is usually by filtration, but other methods such as decanting the liquid are also known to those skilled in the art. In step (5b) of scheme II, other liquids are added to the suspension prior to the separation of step (6b). Other liquids help to remove the suspension from the reaction flask and can be used to help separate the solids. The other liquid may be an ether, such as tert-butyl methyl ether. The other liquids may be acetone, THF, 1,2-dimethoxyethane, diethoxymethane, methyl acetate, ethyl acetate, isopropyl acetate, isobutyl acetate, heptane, toluene and / or xylene.

The isolated solid is washed and dried in step (6a) or (7). Washing of the solids is usually performed with one or more organic liquids or a mixture of one or more organic liquids and water. The same liquids as those described in steps (1) and (2) or step (5b) can be used. Drying in step (6a) or (7) can be performed by heating, placing the solid under vacuum, with a stream of inert gas, or a combination of these methods. In the drying step, heating is preferably performed in the range of 25-65°C. When drying is performed under at least partial vacuum, the preferred vacuum range is from 2 x ¹⁰² Pascals (Pa) to 1 x ¹⁰⁵ Pa. For the preparation of ethanesulfonate of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine, preferably at 2×10 ² Pa Drying is carried out under vacuum to 1 x ¹⁰⁵ Pa and at a temperature of 35-55°C, more preferably 40-50°C.

The following examples describe the preparation of ethanesulfonic acid, especially in crystalline form, of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine salt and 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate (including its hydrate or solvent compounds) other methods.

Scheme II

Pharmaceutical composition and biological activity

The pharmaceutical composition of the present invention contains the ethanesulfonic acid of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine in a therapeutically effective amount salt or 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate (including its hydrate or solvate substance) and a pharmaceutically acceptable carrier. Certain pharmaceutical compositions may contain the ethanesulfonate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine in crystalline form or 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate (including its hydrate or solvate ).

1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]phthalazin-4-amine ethanesulfonate or 1-(2-methylpropyl )-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine mesylate salt may be administered as a single therapeutic agent of a treatment regimen, or they may be administered in combination with each other, or 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]phthalazin-4-amine ethanesulfonate or 1-(2-methylpropane Administering the methanesulfonate salt of -1H-imidazo[4,5-c][1,5]naphthyridine-4-amine in combination with other active agents including other immune response modifiers , antiviral agents, antibiotics, antibodies, proteins, peptides, oligonucleotides, etc.

Salts of the invention, including hydrates and solvates, have been shown to induce the production of certain cytokines in experiments performed according to the assay described below. These results suggest that the salts are useful immune response modifiers that can modulate immune responses through many different pathways, making them useful in the treatment of various diseases.

Cytokines inducible by administration of the salts of the invention typically include interferon-alpha (IFN-alpha) and/or tumor necrosis factor-alpha (TNF-alpha) as well as certain interleukins (IL). Cytokines whose biosynthesis can be induced by the salts of the invention include IFN-[alpha], TNF-[alpha], IL-1, IL-6, IL-10, and IL-12, as well as many others. Among other things, these cytokines, and others, inhibit virus production and tumor cell growth, making this salt useful in the treatment of viral and neoplastic diseases. Accordingly, the present invention provides a method of inducing cytokine biosynthesis in an animal, the method comprising administering to said animal an effective amount of a salt or composition of the present invention. The animal to which the salt or composition is to be administered to induce cytokine biosynthesis may suffer from a disease such as a viral disease or a neoplastic disease as described above, and administration of the salt may provide therapeutic treatment. Alternatively, the salt can be administered to the animal before the animal becomes ill so that administration of the salt can provide prophylactic treatment.

In addition to being able to induce cytokine production, the salts of the invention can affect other aspects of the innate immune response. For example, natural killer cell activity may be stimulated due to the induction of cytokines. The salts also activate macrophages, which in turn stimulate the secretion of nitric oxide and the production of other cytokines. In addition, this salt can cause the proliferation and differentiation of B lymphocytes.

The salts of the invention also have an effect on the adaptive immune response. For example, administration of the salt indirectly induces the production of the type 1 T helper ( _TH 1 ) cytokine IFN-γ and can inhibit the production of the type 2 T helper ( _TH 2 ) cytokines IL-4, IL-5, and IL-13. produce.

Whether for the prophylaxis or treatment of disease and whether for influencing innate or acquired immunity, the salt or composition may be administered alone or in combination with one or more active ingredients, eg in a vaccine adjuvant. When administered with other components, the salt and one or more other components may be administered separately; administered together but independently, for example, in a solution; or administered together and associated with each other, such as (a) covalently linked or (b) Non-covalent association, for example in a colloidal suspension.

Conditions that may use the IRMs identified herein as therapeutic agents include, but are not limited to:

(a) Viral diseases, such as those caused by infection with adenoviruses, herpesviruses (for example, HSV-I, HSV-II, CMV, or VZV), poxviruses (for example, orthopoxviruses such as smallpox or vaccinia or molluscum contagiosum), picornaviruses (e.g., rhinoviruses or enteroviruses), orthomyxoviruses (e.g., influenza), paramyxoviruses (e.g., parainfluenza, mumps, measles, and respiratory viruses (RSV)), coronaviruses (eg, SARS), papovaviruses (eg, papillomaviruses, such as those that cause genital, common, or plantar warts), hepadnaviruses (eg, hepatitis B virus) , a flavivirus (for example, hepatitis C virus or dengue virus) or a retrovirus (for example, a lentivirus such as HIV);

(b) Bacterial diseases, such as those caused by infection with the following bacteria: Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter Genus, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium , Campylobacter, Vibrio, Serratia marcescens, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella;

(c) Other infectious diseases, such as mycoses or parasitic diseases, mycoses include but not limited to candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, parasitic diseases include but not limited to malaria, Pneumocystis, leishmaniasis, cryptosporidiosis, toxoplasmosis and trypanosome infection;

(d) Neoplastic diseases such as intraepithelial neoplasia, cervical dysplasia, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, Kaposi's sarcoma, melanoma, leukemia including but Not limited to myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, skin T lymphocytoma, B cell lymphoma and hairy cell leukemia, and other cancers;

(e) _TH2 -mediated atopic diseases such as atopic dermatitis or eczema, eosinophilia, asthma, allergy, allergic rhinitis and Ommen syndrome;

(f) Certain autoimmune diseases such as systemic lupus erythematosus, essential thrombocythemia, multiple sclerosis, discoid lupus, alopecia areata; and

(g) Diseases associated with wound repair, such as inhibition of keloid formation and other types of scarring (eg, enhanced wound healing, including chronic wounds).

In addition, the IRM salts of the present invention can be used as vaccine adjuvants in combination with any substance that elevates the humoral and/or cell-mediated immune response, such as: hepatoviral, bacterial or parasitic immunogens; inactivated Viral, tumor-derived, protozoan, biologically-derived, fungal or bacterial immunogens; toxoids; toxins; autoantigens; polysaccharides; proteins; glycoproteins; peptides; cellular vaccines; DNA vaccines; autologous vaccines; recombinant proteins, etc., To treat diseases such as: BCG, Cholera, Plague, Typhoid, Hepatitis A, Hepatitis B, Hepatitis C, Influenza A, Influenza B, Parainfluenza, Polio, Rabies, Measles, Mumps, Rubella, Yellow Fever, Typhoid Colds, diphtheria, Haemophilus influenzae b, tuberculosis, meningococcal and pneumococcal vaccines, adenovirus, HIV, fowl pox, cytomegalovirus, dengue fever, feline leukemia, poultry disease, HSV-1 and HSV-2, hog cholera , Japanese encephalitis, respiratory syncytial virus, rotavirus, papillomavirus, yellow fever and Alzheimer's disease. The salts of the invention, including their topical formulations, may be administered as an adjuvant intramuscularly, intradermally, subcutaneously, mucosally (eg, nasally or vaginally), or by any other route of administration. For example, topical formulations of the salts of the invention may be inoculated through the skin or nasally or vaginally in combination with HPV vaccines such as GARDASIL and CERVARIX.

Certain IRM salts of the invention are especially useful for immunocompromised individuals. For example, certain salts are useful in the treatment of, eg, opportunistic infections in transplant patients, cancer patients, and HIV patients, and tumors that occur after suppression of cell-mediated immunity.

Thus, one or more of the above-mentioned diseases or types of diseases, such as viral diseases or neoplastic diseases, can be treated by administering to an animal in need of treatment (diseased) a therapeutically effective amount of a salt of the invention. The IRM salt of the present invention, ethanesulfonate or 1-( 2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine mesylate, especially useful in the treatment of cervical diseases such as HPV infection and/or or cervical tumors. Certain so-called high-risk HPV subtypes such as HPV 16, 18, etc. are believed to be particularly harmful and can cause cervical cancer. Formulations of the salt compounds of the invention and uses of the salt compounds of the invention are described in co-pending US application 60/698416.

It should also be understood that the salts described herein may be used in combination with any number of other drugs and devices. For example, any other antiviral or anticancer drug, including antibodies, chemotherapy, direct acting antivirals, immunotherapy, as well as chemoablative, laser ablation, cryotherapy, and surgical resection, as is well known in the art . In treating HPV with the salts of the invention, products (e.g., from Digene Corp.) can also be used to detect the presence or absence of various HPV subtypes to determine whether treatment is required or whether treatment has been effective in attenuating or eliminating the virus.

The amount of salt effective to induce cytokine biosynthesis is sufficient to cause one or more cell types, such as monocytes, macrophages, dendritic cells and B cells, to produce an amount of one or more cytokines higher than these Amounts of background levels of cytokines such as IFN-[alpha], TNF-[alpha], IL-1, IL-6, IL-10 and IL-12. The exact amount will vary according to factors known in the art, but is expected to be about 100 nanograms/kg (ng/kg) to about 50 milligrams/kg (mg/kg), preferably about 10 micrograms/kg (μg/kg ) to about 5 mg/kg. The present invention also provides methods for treating viral infection in animals and methods for treating neoplastic diseases in animals, the methods comprising administering to the animals an effective amount of the salt or composition of the present invention. An effective amount to treat or inhibit a viral infection is an amount that will reduce one or more manifestations of a viral infection, such as viral damage, viral load, rate of viral production, and mortality, compared to untreated control animals. The precise amount effective for this treatment will vary according to factors known in the art, but is expected to be from about 100 ng/kg to about 50 mg/kg, preferably from about 10 μg/kg to about 5 mg/kg. The amount of salt effective to treat neoplastic symptoms is that amount that will reduce the size of the tumor or the number of tumor foci. Again, the precise amount will vary according to factors known in the art, but is expected to be from about 100 ng/kg to about 50 mg/kg, preferably from about 10 μg/kg to about 5 mg/kg.

Test Methods

Powder X-ray Diffraction Analysis

Reflection geometry data were collected as (θ/2θ) measurement scans using a Philips (PANalytical, Natick, MA) vertical diffractometer, copper Ka radiation, and a proportional detector recording scattered radiation. The diffractometer is equipped with a variable incident beam slit, a fixed diffraction beam slit and a graphite diffraction beam monochromator. Samples were ground in an agate mortar and added as a dry powder to a background-free sample container constructed of single crystal quartz. Measurement sweeps were performed between 3-55 degrees (2Θ) with a step size of 0.04 degrees and a dwell time of 6 seconds. The X-ray generator was set at 45kV and 35mA. The resulting powder diffraction data were analyzed using the Jade (version 6, Materials Data Inc., Livermore, CA) diffraction software package.

Solid State ¹³ C Nuclear Magnetic Resonance (NMR) Spectroscopy

Solid state ¹³ C NMR spectra were acquired on a Varian 400 MHz wide aperture INOVA spectrometer with variable amplitude cross-polarized pulse sequences. Chemical shifts were obtained with reference to tetramethylsilane using hexamethylbenzene as an external standard. The chemical shifts varied by +/-0.3 ppm due to experimental conditions (magic angle adjustment and lock signal drift).

thermal analysis

Differential scanning calorimetry (DSC) analysis and thermogravimetric analysis (TGA) were performed with TA Instruments MDSC Q1000 and TGA 2950, respectively. The DSC unit was calibrated with indium and tin standards. Temperature calibration was performed on the TGA unit with nickel and alumel standards. The heating rate for DSC and TGA was 10 °C/min with a nitrogen purge at 50 mL/min. For DSC, a hermetically sealed sample pan with small holes is used to reduce measurement variability in dehydration events.

Water Sorption Analysis

Automated water sorption analysis was performed with a VTI Symmetrical Gravimetric Analyzer Model 100 (SGA-100) operating in Step-Isotherm mode. The samples were exposed to cycles of relative humidity (RH) conditions at 25°C. The relative humidity was cycled from 5% to 95% to 5% in 5% increments and the weight of the sample was recorded at each stage after equilibration (less than 0.01% weight change over 5 minutes). The percent weight change of the samples was plotted as a function of relative humidity to obtain adsorption/desorption isotherms.

Fourier Transform Infrared Spectroscopy (FTIR)

Infrared spectra were acquired on a Nicolet Magna-IR Spectrometer 750 with an ENDURANCE single reflection attenuated total reflectance (ATR) module. Neat samples were placed on diamond sampling crystals and spectra were collected from 4000 to 525 wavenumbers (cm ⁻¹ ). Utilize ATR spectral correction software.

Solubility of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine in water

The solubility of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine free base in water was determined potentiometrically as a function of pH. Intrinsic solubility and pH/solubility curves in 0.15M aqueous potassium chloride solution were obtained using a p-SOL microtiter (available from pION, Inc., 5 Constitution Way, Woburn, MA). Titrate samples approximately 3 mg in size. The intrinsic solubility (S _o ) measured by this method was 0.003 mg/mL. In another experiment, the pK a of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine measured by potentiometry _was 6.1 , and use this value to calculate the intrinsic solubility. A curve showing the solubility of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine free base as a function of pH is shown in Figure 5 .

Example

Objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention.

Example 1

Preparation of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate

Part A

Under argon, 1,5-naphthyridine-4-alcohol (1.6 kilograms (kg), 11 moles (mol)) was added fuming nitric acid (16 Liters (L)), while maintaining the temperature of the reactants at 45.5°C or lower. After the addition the reaction was stirred at about 45°C for 23 minutes, heated to reflux over 2.25 hours, heated to reflux (90-95°C) for 5 hours, then cooled to room temperature overnight. The reaction mixture was then cooled to 7.5 °C and water (16 L) was added slowly while maintaining the temperature of the reaction below 25 °C. The resulting mixture was cooled to 9 °C and ammonium hydroxide (20 L) was slowly added to adjust the mixture to pH 6.2 while maintaining the temperature below 15 °C. The resulting mixture was stirred for 10 minutes and cooled to 2.8°C. The resulting solid was isolated by filtration, washed with cold water (2 x 2.2 L, 4 °C), vacuum dried at room temperature and vacuum dried at 75 °C for 47 hours to afford 1.778 kg 3-nitro[1,5]naphthyridine-4 -alcohol.

Part B

Under argon, a solution of 3-nitro[1,5]naphthyridine-4-ol (1.778kg, 9.30mol) in N,N-dimethylformamide (DMF) (16L) was stirred at 17°C 45 minutes. Phosphorus oxychloride (2.095 kg, 13.7 mol) was added slowly while maintaining the temperature at about 20°C, and the reaction was stirred at 20°C for 15.25 hours. The reaction mixture was then added to water (76 L) which had been cooled to 4.5°C over 55 minutes with continuous stirring. During the addition, the temperature of the mixture was not allowed to exceed 10°C, and the temperature at the end of the addition was 9.5°C. The resulting mixture was stirred for 100 minutes while cooling from 9.5°C to 2.5°C. A solid formed and was isolated by filtration, washed with water (2 x 8 L), and dried with suction to provide 3.3 kg of 4-chloro-3-nitro[1,5]phthalazine.

Part C

A solution of the material from Part B in dichloromethane (26 L) was heated to 31 °C and sodium sulfate (2 kg) and magnesium sulfate (500 g) were added. The resulting mixture was stirred for 1 hour then filtered. The filter cake was washed with dichloromethane (5 L) and the filtrate was transferred to another vessel with additional dichloromethane (8 L). Isobutylamine (2.5 L) was added to the filtrate under argon with continuous stirring while maintaining the temperature of the reaction at 17-24°C. The reaction was stirred at a temperature of 17-24°C for 13.5 hours, then concentrated to dryness under reduced pressure at 40°C. The resulting solid was mixed with water (18 L), the resulting mixture was stirred at 20-21 °C for 3 hours and then filtered. The isolated solid was washed with water (3 x 3 L), vacuum dried and further vacuum dried at 75 °C for 16.5 hours to afford 1.98 kg ^N4- (2-methylpropyl)-3-nitro[1,5]diaze Polynaphthyl-4-amine. Divide the product into 5 parts.

Part D

A Parr vessel was charged with toluene (3.86 L), 2-propanol (386 milliliters (mL)), N ⁴ -(2-methylpropyl)-3-nitro[1,5]naphthyridine- 4-Amine (386 g, 1.56 mol) and 5% platinum on carbon (77.2 g, 50% w/w (weight percent) in water). The vessel was sealed and purged 3 times with nitrogen while stirring the reaction mixture. The reaction mixture was then placed under hydrogen pressure (2.1 x ¹⁰⁵ Pascals (Pa) to 4.1 x ¹⁰⁵ Pa, 30 pounds per square inch (psi) to 60 psi) for 130 minutes while maintaining the temperature between 18°C and 22°C . The reaction was repeated 4 more times, the reaction time was from 120 minutes to 215 minutes, and the reaction temperature was 19-24°C. The products of the five reactions were combined, treated with magnesium sulfate (2 kg), allowed to stand for 90 minutes, and filtered through a bed of CELITE filter agent. The filter cake was washed with 1:1 toluene/2-propanol (4 L), then with toluene (16 L), and the filtrate was concentrated in vacuo at about 40°C to provide ^1.59 kg of N4-(2-methylpropyl) as an oil [1,5]naphthyridine-3,4-diamine.

Sections AD were repeated on the same scale to provide an additional 1.236 kg of ^N4- (2-methylpropyl)[1,5]naphthyridine-3,4-diamine as an oil.

Part E

Under argon, diethoxymethyl acetate (2.24 L, 13.7 mol) was added to N ⁴ -(2-methylpropyl)[1,5]naphthyridine-3,4-di A solution of the amine (2.826 kg, 13.07 mol) in toluene (32.5 L) while maintaining the temperature of the reactants at or below 30.3°C. The reaction mixture was stirred at a temperature of 30.1-30.3°C for 40 minutes, heated to reflux over 45 minutes, heated to reflux (92.5-98.5°C) for 185 minutes, and allowed to cool to room temperature overnight. Saturated aqueous potassium carbonate (6 L) was added and the resulting mixture was stirred at 29.3-30.3° C. for 32 minutes, then allowed to stand for 53 minutes. The organic components were separated and concentrated under reduced pressure at a temperature of 55-65°C for 7 hours. The resulting oil was triturated with heptane (3 L) at 20 °C to form a solid which was isolated by stirred filtration, washed with cold heptane (1.5 L, 5 °C) and air dried to provide 2.593 kg of 1-(2- Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine.

Part F

1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthalene (2.593kg, 11.46mol) in chloroform ( 2.8 L) solution was stirred for 1 hour, then 3-chloroperoxybenzoic acid (2.864 g, 70% pure, 11.6 mol) was added in five portions at approximately 5 minute intervals. During the addition, the temperature of the reaction rose from 16.4°C to 26.1°C. The reaction mixture was stirred for 20 hours after the addition as the temperature of the reaction mass dropped from 26.1°C to 17.5°C. The reaction mixture was then stirred with 1% w/w aqueous sodium carbonate (4 x 3.2 L, 30 minutes between washes) for 30 minutes. The organic components were concentrated under reduced pressure at 40°C. The residue (4.6 kg) was triturated with diethyl ether (7.5 L) for 68 minutes and the resulting solid was isolated by agitated filtration, washed with diethyl ether (4.5 L) and suction dried to afford 3.304 kg of 1-(2-methylpropyl )-5-Oxo-1H-imidazo[4,5-c][1,5]naphthyridine.

Part G

To a solution of the material from Part F in dichloromethane (34 L) was added ammonium hydroxide (21 L, 28% weight (w/w)) with continuous stirring while maintaining the temperature of the reaction at or below 11.5°C. With continuous stirring, p-toluenesulfonyl chloride (1.786 kg, 9.368 mol) was added in portions over 1 hour while maintaining the temperature of the reaction mass at 16.4-25°C. The reaction was stirred for 140 minutes, then p-toluenesulfonyl chloride (180 g, 0.94 mol) was added and the reaction stirred for an additional 1 hour. Water (21 L) was added to the reaction mixture, and the resulting mixture was stirred for 30 minutes and left to stand for 14.5 hours. The organic components were separated and concentrated under reduced pressure at 40°C for 8.25 hours. The residue (1.004 kg) was heated to reflux in acetonitrile (10.04 L) for 128 minutes. The suspension was cooled to 20 °C and the resulting solid was isolated by filtration, washed with cold acetonitrile (1.4 L, 4 °C) and air dried to afford 360 g of 1-(2-methylpropyl)-1H-imidazo[4,5 -c] [1,5]naphthyridine-4-amine. A solid was present in the remaining aqueous fraction which was isolated by filtration, washed with water (4 x 2000 mL) and suction dried to afford 1.925 kg of 1-(2-methylpropyl)-1H-imidazo[4 , 5-c][1,5]naphthyridine-4-amine. The 2 solids were combined, heated to reflux in 90% w/w methanol/water (22.85 L) for 310 minutes, and the suspension was allowed to cool to 24.1 °C overnight. The resulting solid was isolated by filtration, washed with cold 90% w/w methanol/water (1.5 L, 5 °C), and dried under vacuum at 75 °C and 1 x ¹⁰⁵ Pa for 5 days to afford 1.368 kg of 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine.

Part H

While stirring, 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (300 g, 1.24 mol) was added into a mixture containing 2% ( The vessel was v/v) water/isopropanol (2000 mL), then rinsed with 2% (v/v) water/isopropanol (100 mL), and the resulting mixture was heated to 81 °C. A solution of ethanesulfonic acid (151 g, 95%, 1.37 mol) in 2% (v/v) water/isopropanol (600 mL) was slowly added to the above reaction mixture over about 20 minutes. During the addition the mixture became a clear solution with a temperature of 81-82°C. The addition funnel was rinsed with 2% v/v water/isopropanol (320 mL); the temperature decreased during the addition but returned to reflux temperature. The resulting solution was heated to reflux for 8 minutes and then slowly cooled to room temperature at a rate of 0.2°C/min (57°C for 234 minutes). The resulting slurry was then cooled to 0-5°C at a rate of 0.2°C/min (ie, the slurry was cooled to 21°C over 130 minutes). The solid was isolated by filtration, rinsed and aided in transfer with cold 2% (v/v) water/isopropanol (400 mL, 3.5°C). The solid was washed with cold 2% (v/v) water/isopropanol (300 mL, 3.5 °C) at about 43 °C and 1.69 x ¹⁰⁴ Pa to 1.70 x ¹⁰⁴ Pa (169 mbar to 170 mbar) Drying for 23 hours and 40 minutes afforded 455 g of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate . This material was processed in a Hamilton Beach 14 speed blender to provide a white "floc" solid, mp 221.5°C - 223.4°C. Anal _. Calcd. for _C13H15N5 - _C2H6O3S - _H2O : C, 48.77; H _{, 6.28} ; _N , 18.96. Found: C, 48.70; H, 6.25; N, 18.96. The material was characterized by powder X-ray diffraction analysis, ¹³ C NMR spectroscopy, water adsorption analysis, thermogravimetric analysis and differential scanning calorimetry. The powder X-ray diffraction pattern and ¹³ C NMR spectrum are shown in Figs. 2 and 3 . The DSC/TGA overlay is shown in Figure 7, and the water adsorption isotherm is shown in Figure 6.

Example 2

Preparation of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate Part A

Under argon, a solution of 3-nitro[1,5]naphthyridine-4-ol (0.800 kg, 4.19 mol) in DMF (8 L) was cooled to 19°C. Phosphorus oxychloride (803.6 g, 5.24 mol) was added over 70 minutes while maintaining the temperature at 19-20°C. A precipitate formed and the reaction was stirred overnight at room temperature. The reaction mixture was divided into 2 portions and each portion was added to ice water (13-15 L) with stirring while maintaining the temperature below 15°C. The resulting mixture was stirred for 30 minutes and the resulting solids were isolated by filtration, washed with cold water (2 L) and combined. The solid was then mixed with dichloromethane (ca. 9 L) and heated to 30°C. The organic components were removed and the remaining mixture was filtered through a bed of CELITE filter agent. The filter cake was washed with dichloromethane (2 L). The filtrate was combined with the organic fraction and the resulting solution was left over sodium sulfate until it was used in part B.

Part B

The mixture from Part A was filtered. Under nitrogen, isobutylamine (766.2 g, 10.48 millimoles (mmol)) was added to the filtrate with stirring over 95 minutes while maintaining the temperature of the reaction at 19-25° C. The reaction was stirred at room temperature overnight, then concentrated under reduced pressure to dryness. The resulting solid was stirred overnight at room temperature with deionized water (16 L), isolated by filtration and dried under vacuum at 62 °C for 26.5 hours to afford 0.92 kg of ^N4- (2-methylpropyl)-3-nitrate as a yellow solid Base [1,5]naphthyridine-4-amine. The product was divided into 3 parts.

Part C

A Parr vessel was charged with toluene (2.3 L), 2-propanol (230 mL), ^N4- (2-methylpropyl)-3-nitro[1,5]naphthyridine-4-amine ( 230 g, 0.924 mol) and 5% platinum on carbon (23.0 g). The vessel was sealed and purged 3 times with nitrogen while shaking the reaction mixture. The reaction mixture was then placed under hydrogen pressure (3.4×10 ⁵ Pa, 50 psi) overnight. Additional 5% platinum on carbon (7 g) was added while the reaction was placed under hydrogen pressure (3.4 x ¹⁰⁵ Pa, 50 psi) overnight. The reaction mixture was filtered through a layer of CELITE filter agent. The filter cake was washed with 10:1 toluene/2-propanol and the filtrate was concentrated under reduced pressure to afford 201.7 g of ^N4- (2-methylpropyl)[1,5]naphthyridine-3 as a dark oil, 4-diamine. This material was mixed with material from two other runs and used in Part D.

Part D

Under nitrogen, pyridine hydrochloride (6.94 g, 56.5 mmol) was added to N ⁴ -(2-methylpropyl)[1,5]naphthyridine-3,4-diamine (642.0 g, 2.97 mol) in toluene solution (3.3 L), and the resulting mixture was stirred until a homogeneous solution was obtained. The solution was heated to 90°C and triethyl orthoformate (484.6 g, 3.27 mol) was added. The addition was stopped when the temperature of the reaction mass reached 100°C and pyridine hydrochloride (6.94 g, 56.5 mmol) was added. The addition was continued for 65 minutes while maintaining the temperature of the reactants in the range of 93-105°C and removing volatiles formed during the reaction by distillation. The reaction was heated at about 90°C for 25 minutes, allowed to cool to room temperature and stirred overnight. Additional pyridine hydrochloride (2 g) was then added and the reaction was heated to 112°C until 2.4 L of distillate was collected. The reaction was then cooled to room temperature, diluted with toluene, stirred for 2 days and washed with aqueous sodium carbonate (3 L, 5%). The organic components were separated and dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting dark oil was triturated with hexane (1.5 L) to form a solid which was collected by filtration, washed with hexane (250 mL), and dried under vacuum at 50-52 °C to afford 440.4 g of 1- (2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine. A portion of this material was mixed with material from another run and used in Part E.

Part E

A solution of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]phthalazine (554.3 g, 2.45 mol) in dichloromethane (5.54 L) was cooled to 6°C, and added in portions (75g each time) 3-chloroperoxybenzoic acid (665g, 70% pure, 2.70mmol) every 10 minutes while maintaining the temperature of the reactant at or below 20°C to provide 1-( 2-Methylpropyl)-5-oxy-1H-imidazo[4,5-c][1,5]naphthalene in dichloromethane.

Part F

To the solution obtained in Part E was added ammonium hydroxide (5 L, 28% w/w) with continuous stirring and the mixture was cooled to 8-10°C. A solution of p-toluenesulfonyl chloride (506 g, 2.70 mol) in dichloromethane (2 L) filtered through a 10-20 micron porous sintered glass filter was added slowly over 55 minutes while maintaining the temperature of the reaction mass at 14 -16°C. The reaction was stirred for about 48 hours; while allowing it to return to 20°C during this time. The reaction mixture was cooled to 3.5 °C and the resulting solid was isolated by filtration and washed successively with cold dichloromethane (500 mL) and water (2 L). The solid was stirred in an aqueous solution (3 L) of sodium carbonate (125 g) and sodium thiosulfate (35 g) for 5 hours, isolated by filtration and washed with deionized water. The solid was stirred overnight in deionized water (2 L), isolated by filtration, washed with deionized water (200 mL) and dried under vacuum at 58-64 °C for 24 hours to afford 384.8 g of 1-(2-methylpropyl)- 1H-Imidazolo[4,5-c][1,5]naphthyridine-4-amine, which was combined with material from another run to afford 721 g of product. The product was heated to reflux in 90% w/w methanol/water (3.605 L) over 30 minutes, heated to reflux for 150 minutes, then allowed to cool slowly to 20° C. overnight. The resulting solid was isolated by filtration, washed with 90% w/w methanol/water (300 mL) and dried under vacuum at 70 °C for 24 hours to afford 705.6 g of 1-(2-methylpropyl)-1H-imidazo as a beige solid [4,5-c][1,5]naphthyridine-4-amine.

Part G

1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (100.0g, 0.414mol) and 2% (v/v) A mixture of deionized water/isopropanol (700 mL) was heated to reflux and ethanesulfonic acid (95% pure, 52.8 g, 0.456 mol) was added over 10 minutes using an addition funnel. The funnel was rinsed with 2% (v/v) deionized water/isopropanol (300 mL), and the resulting solution was heated to reflux for 30 minutes. The solution was stirred and allowed to cool overnight to 28°C at a rate of 0.5°C/min. The resulting viscous suspension was allowed to cool to 21°C over an additional 50 minutes and the solid was collected by vacuum filtration, washed with 2% (v/v) deionized water/isopropanol (300 mL) and vacuum dried at 46-47°C for 21.5 hours To provide 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate as a white solid , mp 220.0-221.0°C. Anal _. Calcd. for _C13H15N5 - _C2H6O3S - _H2O : C, 48.77; H _{, 6.28} ; _N , 18.96. Found: C, 48.76; H, 6.53; N, 19.01. The material was characterized by powder X-ray diffraction analysis and the data were found to be consistent with Figure 2.

Example 3

Preparation of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate

1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine prepared in Part AG of Example 1 was used for this implementation Example raw materials. 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (2.0g, 8.3mmol) and 2% A (v/v) mixture of deionized water/isopropanol (20 mL) was heated to reflux, ethanesulfonic acid (1.0 g, 9.1 mmol) was added, and then washed with 2% (v/v) deionized water/isopropanol ( 6 mL), and the resulting solution was heated to reflux for 15 minutes. The solution was stirred and allowed to cool at a rate of 0.5°C/minute for 1 hour, 0.33°C/minute for the second hour, and 0.1°C/minute for the third hour. Stirring was continued for 22 hours until the temperature reached 20°C. The resulting solid was collected by vacuum filtration, washed with 2% (v/v) deionized water/isopropanol (15 mL) and dried under vacuum at 45-46°C (1.6×10 ³ -2.1×10 ³ Pa) (12-16 mmHg ) for 20 hours to provide 2.79 g of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate as a white solid monohydrate. Anal _. Calcd. for _C13H15N5 - _C2H6O3S - _H2O : C, 48.76; H, _{6.30 ; N} , 18.96. Found: C, 48.76; H, 6.43; N, 18.99. The material was characterized by powder X-ray diffraction analysis, DSC, TGA and water adsorption analysis, and the data were found to be consistent with those shown in Figures 2, 7 and 6, respectively.

Example 4

Preparation of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate

1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine prepared in Part AG of Example 1 was used for this implementation Example raw materials. 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (2.0g, 8.3mmol) and 2% A (v/v) mixture of deionized water/isopropanol (20 mL) was heated to reflux, ethanesulfonic acid (1.0 g, 9.1 mmol) was added, and then washed with 2% (v/v) deionized water/isopropanol ( 6 mL), and the resulting solution was heated to reflux for 15 minutes. The solution was stirred and allowed to cool at a rate of 0.25°C/min for 2 hours, 0.23°C/min for the 3rd hour and 0.13°C/min for the 4th hour. Stirring was continued for 45 hours until the temperature reached 24°C. The resulting solid was collected by vacuum filtration, washed with 2% (v/v) deionized water/isopropanol (15 mL) and dried under vacuum at 45-46 °C (2.6×10 ² Pa to 2.1×10 ³ Pa) (2- 16 mmHg) for 20 hours to provide 2.74 g of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine as a white solid. Sulfonate monohydrate. The material was characterized by powder X-ray diffraction analysis, DSC, TGA and water adsorption analysis and the data were found to be consistent with those shown in Figures 2, 7 and 6, respectively.

Example 5

Preparation of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate

1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine prepared in Parts A-G of Example 1 was used for this implementation Example raw materials. 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (2.0g, 8.3mmol) and 2% A (v/v) mixture of deionized water/ethyl acetate (14 mL) was heated to reflux, ethanesulfonic acid (1.0 g, 9.1 mmol) was added, and then washed with 2% (v/v) deionized water/ethyl acetate ( 6 mL), the resulting solution was heated to reflux for 15 minutes and allowed to cool overnight.

The resulting solid was collected by vacuum filtration, washed with 2% (v/v) deionized water/ethyl acetate (15 mL) and dried under vacuum at 45-46 °C (2.6×10 ² Pa to 2.1×10 ³ Pa) (2- 16 mmHg) for 21 hours to provide 2.91 g of white needles of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amineethanesulfonic acid salt monohydrate. Calcd for _C13H15N5 - _C2H6O3S - _H2O : C _, 48.76; H _, 6.30 _{; N} , 18.96. Found: C, 48.48; H, 6.47; N, 18.93. The material was characterized by powder X-ray diffraction analysis, DSC, TGA and water adsorption analysis, and the data were found to be consistent with those shown in Figures 2, 7 and 6, respectively.

Example 6

Preparation of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate

Part A

Under nitrogen, a suspension of 3-nitro-1,5-naphthyridine-4-ol (12.00 kg, 67.78 mol) in DMF (49 L) was stirred at a temperature of 20-24° C. for 30 minutes. Phosphorus oxychloride (10.6 kg, 69.1 mol) was added slowly over 53 minutes while maintaining the temperature at 20.6-25.6°C. The addition vessel was rinsed again with DMF (5 L) and added to the reaction. The reactant was stirred at a temperature of 20-24° C. for 19 hours and 17 minutes, and then rapidly added to pure water (275 L) which had been cooled to 8.4° C. over 4 minutes. During the addition, the temperature of the mixture did not exceed 18°C. The original container was rinsed again with water (80 L) and added rapidly to the resulting mixture, the temperature of the mixture being 16.6-17.2°C during the addition. The resulting mixture was stirred for 30 minutes and cooled to about 10°C. A solid formed and was isolated by filtration, washed with cold water (6 x 33 L, 10°C) to provide 20.55 kg of 4-chloro-3-nitro[1,5]naphthyridine, which contained some water and after filtration Use in Part B within 2.75 hours.

Part B

To a suspension of the material from Part A (20.55 kg) in tetrahydrofuran (67 L) was added isobutylamine (9.4 kg, 12.8 L, 130 mol) with stirring over 77 minutes while maintaining the temperature of the reaction at 20-27°C. Rinse with tetrahydrofuran (5 L) after addition of isobutylamine. The reaction was stirred at a temperature of 20-24°C for 190 minutes, then water (288 L) was added over about 1 hour while maintaining the temperature of the reaction at 21.4-23.8°C. The resulting mixture was stirred at 20-24°C for 75 minutes and then filtered. The separated solid was washed with water (4 x 25 L), which was also used to rinse the reaction vessel, pulled dry and dried under vacuum at a temperature of 45-55 °C for 60 hours to afford 13.7 kg N ⁴ -(2- Methylpropyl)-3-nitro[1,5]naphthyridine-4-amine.

Part C

Add N ⁴ -(2-methylpropyl)-3-nitro[1,5]naphthyridine-4-amine (13.7kg, 55.6mol) and 3% platinum on carbon (0.79kg , 34.61% w/w in water), purged with nitrogen, added toluene (670.0 kg) and 2-propanol (37.0 kg) and purged with nitrogen. The vessel was sealed and purged 3 times with hydrogen. The reaction mixture was then placed under hydrogen pressure (1.2 x ¹⁰⁵ Pa, 17 psi) for 170 minutes while stirring and maintaining the temperature between 18°C and 22°C. The reaction mixture was filtered through a filter pad. The filter cake was washed with toluene (87 kg) and the filtrate was concentrated under reduced pressure at about 25°C to provide a toluene solution of ^N4- (2-methylpropyl)[1,5]naphthyridine-3,4-diamine (about 15 mL/g).

Part D

The solution from Part C (196 L) was cooled to -5°C to 0°C under nitrogen and p-toluenesulfonic acid (0.520 kg, 2.73 mol) was added. The reaction was purged with nitrogen and heated to 88-92°C over 87 minutes. Triethyl orthoformate (9.4 kg, 63 mol) was added slowly with stirring over 64 minutes; the temperature of the reactants rose from 89.4°C to 94°C during the addition. The addition vessel was rinsed with toluene (5 L), which was added to the reaction. The reaction mixture was stirred and heated to reflux (98-100° C.) for 195 minutes; the ethanol distillate was collected. The reaction was purged 3 times with nitrogen, cooled to 00-5°C over 3 hours, purged 3 times with nitrogen and heated to 20-24°C. The reaction mixture was washed sequentially with aqueous sodium carbonate (41 L, 1% w/w) for 1 hour and water (41 L) for 1 hour, and the layers were allowed to separate for 1 hour after each wash. The organic components were then concentrated under reduced pressure at 21.6 °C, cooled to 0-5 °C and purged with nitrogen to provide 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5] Naphthalene solution in toluene (about 7.3 mL/g).

Part E

The solution of Part D (102 L) was heated to 45-50 °C and purged with nitrogen. Under nitrogen, peracetic acid (12.1 kg, 40 weight percent (wt.% or w/w)) was added over 137 minutes while maintaining the temperature of the reactants at 45.6-50° C. and purging the system with nitrogen every 30 minutes. The addition vessel was rinsed with toluene (5 L), which was added to the reaction. The reaction was stirred at a temperature of 47.5-49.7°C for 247 minutes while purging the system with nitrogen every 30 minutes, and then cooled to 0-5°C. The reaction was purged with nitrogen and heated to 43-47°C. While maintaining the temperature, aqueous sodium metabisulfite (51 L, 4% w/w) was added over 28 minutes and aqueous sodium hydroxide (50 L, 10.5% w/w) was added over 28 minutes. Water (7 L) was added, the temperature of the reaction was adjusted to 48-52°C and stirred for 30 minutes. The resulting mixture was cooled to 3-7°C over about 4 hours and the reaction was then maintained at this temperature for 249 minutes. A solid was present and isolated by filtration, washed with cold water (3 x 25 L, 3-7°C), dried by suction and then vacuum dried at 25-35°C for about 137 hours to afford 10.8 kg of 1-(2-formazan as a yellow solid propyl)-5-oxo-1H-imidazo[4,5-c][1,5]naphthyridine.

Part F

Ammonium hydroxide (11.5 kg, 28% w/w) was rapidly added to 1-(2-methylpropyl)-5-oxide-1H-imidazo[4,5-c][ 1,5] Naphthalene (10.8kg, 44.6mol) in methanol (77L) suspension while maintaining the temperature of the reactant at 20.6-21.4°C. The addition vessel was rinsed with methanol (10 L) and added to the reaction. The reaction was stirred for 29 minutes. Sulfonyl chloride (8.2 kg, 46 mol) was added over 50 minutes with continuous stirring while maintaining the temperature of the reaction at 20-29.6°C. The addition vessel was rinsed with methanol (10 L) and added to the reaction. The reaction was stirred at 20.4-22.5°C for 66 minutes and purged with nitrogen three times. Benzenesulfonyl chloride (0.8 kg, 4.5 mol) was added over 12 minutes while maintaining the temperature of the reaction at 22.9-23.7°C, and methanol (5 L) was added. The reaction was then stirred at 21.6-24.1°C for 67 minutes. Aqueous sodium hydroxide (32 L, 10% w/w) was added to the reaction mixture over 30 minutes while maintaining the temperature of the reaction between 22°C and 23°C. Water (10 L) to rinse the addition vessel was added to the reaction mixture. The resulting mixture was cooled to 12°C over 39 minutes and stirred at a temperature of 10.6-12.0°C for 2 hours. The precipitate that appeared was isolated by filtration and washed with cold (8-12°C) 59:41 methanol/water (2 x 11 L), soaking the filter cake for 10 minutes with each wash. The filter cake was washed with cold (8-12 °C) water (4 x 25 L), soaking the filter cake with the washing solution for 10 minutes between each wash, and vacuum drying at 45-55 °C for 24 hours to provide 8.25 kg of light yellow solid 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine. A portion of the solid (2.75 kg) was mixed with 2-butanol (371 L), and the resulting mixture was heated to reflux with stirring over 93 minutes, heated at 99°C for 20 minutes, cooled to 60-65°C over 45 minutes, and filtered to in another warm container. The solution was then cooled to 20-25°C with stirring, concentrated under reduced pressure at 21.4°C to the minimum volume required for stirring and purged with nitrogen. A second portion of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (2.75 kg) was added as a light yellow solid and the second Two volumes of 2-butanol (371 L) were added and the process of reflux, filtration and concentration repeated. A third portion of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (2.60 kg) and the second portion were added as a light yellow solid. Three volumes of 2-butanol (371 L) were added and the reflux and filtration process was repeated. The solution was cooled to 20 °C and 2-butanol (20 L) was added. Repeat the reflux, filtration and concentration process. The resulting mixture (total 198 L) was heated to reflux with stirring over 70 minutes, heated at 97°C for 20 minutes, cooled to 68-72°C at a rate of 0.5°C/min, cooled to 3-5°C over 154 minutes, at 4.3 Stir at -5°C for 2 hours and filter without stirring. The filter cake was washed with the same cold (3-5°C) 2-butanol (50 L) that had been used to wash the crystallization vessel, and dried without agitation under a stream of nitrogen for 1 hour. The solid was dried without agitation on the filter at 46-50°C under vacuum (900-980 mbar, 9.0×10 ⁴ -9.8×10 ⁴ Pa) for 12 hours with a small stream of nitrogen to provide 6.50 kg of 1- (2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine.

Part G

Under nitrogen, add isopropanol (88L) to 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (3.45kg, 14.3 mol), the mixture was stirred and heated to 81°C. Aqueous ethanesulfonic acid (2.5 kg, 70% w/w, 16 mol) was added over 15 minutes and the addition vessel was rinsed with isopropanol (10 L). The mixture was heated to reflux for 30 minutes, cooled to 60-65°C and filtered through a 5 micron filter into another warm container. Rinse the first container with hot isopropanol (60-65°C) and add it to the solution. The filtrate was heated to reflux (81°C) for 37 minutes (min) and then cooled to 45-55°C over 85 minutes (approximately 0.4°C/min). The reaction was cooled to 23-27°C (ca. 0.15-0.25°C/min) over an additional 130 minutes, then stirred at 23-27°C for 100 minutes, after which tert-butyl methyl ether (MTBE) (123 mL) was added over 92 minutes. The resulting mixture was stirred at 24-25°C for 63 minutes, cooled to 4°C over 115 minutes (ca. 0.18°C/min) and stirred at 4-5°C for 125 minutes. The slurry was divided into 2 portions which were transferred to a nitrogen purged filter over 11 minutes using nitrogen pressure to aid filtration. MTBE (88 L) was added to the crystallization vessel, cooled to 1-5°C and transferred to a filter to rinse the product cake. No need to stir while filtering. Without agitation, the solid was dried on the filter under vacuum (900-980 mbar, ^{9.0x104-9.8x104Pa} ) at 35-45°C with a small nitrogen stream for ⁶ hours to afford 3.8kg of solid. The solid was slurried in a water (400 mL)/isopropanol (1 L)/MTBE (15 L) mixture for 24-36 hours. The slurry was filtered and washed with MTBE. The product was dried under vacuum (980 mbar, 9.8 x ¹⁰⁴ Pa) at 40°C to afford 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5] as a white solid Naphthalene-4-amine ethanesulfonate monohydrate. The material was characterized by powder X-ray diffraction analysis and FTIR spectroscopy. It was found that the powder X-ray powder diffraction pattern was consistent with that in Figure 2, and the FTIR spectrum was shown in Figure 4.

Example 7

Preparation of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate monohydrate 1-( 2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (1.0 g, 4.1 mmol) and 2% (v/v) deionized water A mixture of /isopropanol (7 mL) was heated to reflux and a solution of methanesulfonic acid (0.44 g, 4.6 mmol) in 2% (v/v) deionized water/isopropanol (3 mL) was added. The solution was heated rapidly to reflux and then cooled to room temperature. The resulting mixture was then cooled to 0-5 °C and the solid was collected by vacuum filtration and washed with cold isopropanol (5 mL). The isolated solid was a dense mat of needles which were cut into pieces 30 millimeters (mm) wide to 60 mm wide and dried under vacuum (1.3 x ¹⁰³ Pa, 10 mmHg) at 45°C for 24 hours to afford 1.33 g of white needles 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate monohydrate. Anal _. Calcd _. for _{C13H15N5.C2H6O3S.0.6H2O :} C _{, 47.31} ; H, _5.96 ; N, 19.71. Found: C, 48.26; H, 5.76; N, 20.32. The material was characterized by powder X-ray diffraction analysis, DSC, TGA and water adsorption analysis. The powder X-ray powder diffraction pattern is shown in FIG. 1 . The DSC/TGA overlay is shown in Figure 9, and the water adsorption isotherm is shown in Figure 8.

in situ salt choice test

Aqueous solutions (0.5 molar, M) of ethanesulfonic acid, methanesulfonic acid and hydrochloric acid were prepared. For ethanesulfonic acid and methanesulfonic acid, pure acid (0.05 mol) was dissolved in water (100 mL) to prepare a solution. Hydrochloric acid (1 normal, N) was diluted with water to prepare 0.5M hydrochloric acid. Add the free base of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine in the amount shown in Table 1 below and The acid solution (1.05 equiv) shown in Table 1 below was added. The tubes were capped and shaken in a water bath at 25°C for 24 hours. The appearance of the resulting solution was evaluated and a volume of water indicated in Table 1 below was added. The resulting salt mixture or solution was filtered through Whatman No. 5 filter paper, and 1 mL of the filtrate was diluted with methanol (10 mL) to provide an analytical sample. The content of soluble salts was analyzed by reverse phase high performance liquid chromatography (HPLC) using the following method to determine the solubility of the salts of each sample in water.

A dilution was prepared by dissolving phosphoric acid (1 mL, 85%) in HPLC grade water (1 L). The resulting solution was mixed with acetonitrile in a ratio of 60:40. Each sample was diluted to 50.0 mL with this diluent to provide a sample concentration less than or equal to (≤) 50 micrograms per milliliter (μg/mL).

The free base of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (25 mg) was dissolved in methanol with the help of ultrasound (30 mL), then diluted to 50.0 mL with methanol to provide a stock solution, from which an external standard was prepared. Dilute 5 mL of stock solution to a volume of 50.0 mL with diluent.

A Waters LC Module 1 HPLC system equipped with an autosampler, reproducible injector, UV detector (wavelength 326 nm) and Turbochrom data acquisition software was used with a ZORBAXBonus-RP C ₁₄ , 15cm×4.6mm column , 5μm packing. 1-octanesulfonate, sodium salt (2.00 g) and phosphoric acid (1 mL, 85%) were dissolved in HPLC grade water (1 L) and the resulting solution was mixed with HPLC grade acetonitrile in a ratio of 65:35 Mix to make mobile phase. The flow rate employed was 1.0 mL/min.

The column was equilibrated with mobile phase, and injected 6 times with 20 μL of external standard detection system each time to ensure that the relative standard deviation of the peak area was less than or equal to (≤) 1.5%. Each injected sample (20 [mu]L) was tested against a standard. Then use the following formula to calculate the percentage concentration of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine in the solution: {[sample Peak area/weight of sample (mg)]/[peak area of standard product/weight of standard product (mg)] average}×100. The solubility of the salt can then be calculated from the formula provided by Tong, W.Q. and Whitesell, G in Pharmaceutical Development and Technology, 3(2), pp. 215-223 (1998), and the values are shown in Table 1 below .

Table 1

In situ salt selection test in water

Free base g/mmol Acid (volume, mL) Exterior Added H ₂ O (mL), final concentration (M) Solubility (mg/mL) 0.102/0.422 Ethylsulfonic acid (0.886) clarify 3.334, 0.1 24.4 0.101/0.419 Methanesulfonic acid (0.881) translucent 4.000, 0.085 15.2 0.108/0.447 Hydrochloric acid (0.938) milky, slightly viscous 8.002, 0.05 0.39

In situ salt selection assay was also used to determine the ethanesulfonate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine and 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine methanesulfonate in 5% (w/w) propylene glycol Solubility in a mixture of aqueous solution and 5% (w/w) diethylene glycol monoethyl ether aqueous solution. Using the method described above, 5% (w/w) co-solvent in water was added instead of water and the data are provided in Table 2 below. For each sample, a final acid concentration of 0.05M was obtained.

Table 2

In situ salt selection assays in water and co-solvents

Free base g/mmol Acid (volume, mL) co-solvent Added H ₂ O (mL), Added co-solvent (mL) Solubility (mg/mL) 0.114/0.472 Ethylsulfonic acid (0.994) Propylene Glycol 8.919, 0.575 Greater than 10.1 0.116/0.481 Methanesulfonic acid (1.010) Propylene Glycol 9.090, 0.547 10.3 0.113/0.469 Ethylsulfonic acid (0.984) Diethylene glycol monoethyl ether 8.865, 0.518 10.5 0.1 20/0.495 Methanesulfonic acid (1.04) Diethylene glycol monoethyl ether 9.36, 0.547 10.5

Cytokine induction in human cells

When tested by the method described below, it was found that the ethanesulfonate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine could Regulates cytokine biosynthesis by inducing the production of interferon alpha and/or tumor necrosis factor alpha.

Cytokine induction was assessed using an in vitro human blood cell system. According to Testerman et al. in "CytokineInduction by the Immunomodulators Imiquimod and S-27609 (immunomodulator imiquimod and S-27609 induction of cytokines)" Journal of Leukocyte Biology, 58, 365-372 (1995-9) Description, based on measuring interferon (alpha) and tumor necrosis factor (alpha) (IFN and TNF, respectively) secreted into the medium to obtain activity.

preparing blood cells for culture

Whole blood from healthy human donors was collected by venipuncture into EDTA vacutainer tubes. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation using HISTOPAQUE-1077. Blood was diluted 1:1 with Dulbecco's phosphate-buffered saline (DPBS) or Hank's balanced salt solution (HBSS). Collect the PBMC layer and wash twice with DPBS or HBSS, and resuspend in RPMI complete medium at 4 × ¹⁰⁶ cells/mL. The PBMC suspension was added to a 48-well flat-bottomed sterile tissue culture plate (Costar, Cambridge, MA or Becton Dickinson Labware, Lincoln Park, NJ) containing an equal volume of RPMI complete medium containing the test compound.

Compound preparation

Compounds were dissolved in dimethyl sulfoxide (DMSO). The concentration of DMSO should not exceed 1% of the final concentration when added to the culture wells. Compounds are typically tested at concentrations ranging from 30-0.014 μM.

to incubate

A 60 μM solution of the test compound was added to the first well containing complete RPMI medium, and 3-fold serial dilutions were performed in subsequent wells. An equal volume of PBMC suspension was then added to the wells to bring the concentration of the test compound to the desired range (30-0.014 [mu]M). The final concentration of the PBMC suspension was 2 x ¹⁰⁶ cells/mL. Cover the plates with sterile plastic lids, mix gently, and incubate at 37°C for 18-24 hours under a 5% carbon dioxide atmosphere.

separate

After incubation the plates were centrifuged at 1000 revolutions per minute (rpm) (approximately 200 xg) for 10 minutes at 4°C. Cell-free culture supernatant was aspirated with a sterile polypropylene pipette and transferred to a sterile polypropylene test tube. Samples were maintained at -30°C to -70°C until analysis. The interferon (α) in the sample is analyzed by ELISA, and the tumor necrosis factor (α) in the sample is analyzed by ELISA or IGEN test.

Analysis of Interferon (α) and Tumor Necrosis Factor (α) by ELISA

Interferon (α) concentrations were determined by ELISA using the Human Multi-Species kit from PBL Biomedical Laboratories (New Brunswick, NJ). Results are expressed in pg/mL.

The concentration of tumor necrosis factor (α) (TNF) was determined using an ELISA kit purchased from Biosource International (Camarillo, CA.). Alternatively, TNF concentrations can be determined by ORIGEN M series immunoassays and read on an IGEN M-8 analyzer from IGEN International (Gaithersburg, MD). Immunoassays employ human TNF capture and detection antibody pairs obtained from Biosource International (Camarillo, CA). Results are expressed in pg/mL.

Induction of cytokines in the vagina

In the following gel formulation examples, serum and intravaginal cytokine data were obtained using the following routine testing methods.

Rats were acclimated to a collar (Lomir Biomedical, Malone, NY) for 2 consecutive days prior to actual dosing. Rats were collared to prevent drug ingestion. Animals were then administered 50 μL of the gel vaginally. Rats received a single intravaginal dose and samples were collected at various times after dosing. Blood was collected by cardiac puncture. The blood was briefly clotted at room temperature and the serum was separated from the clot by centrifugation. Serum was stored at -20°C until analysis of cytokine concentrations.

After blood collection, the rats were euthanized and the vagina including the cervix removed and the tissue weighed, placed in sealed 1.8 mL cryovials and snap frozen in liquid nitrogen. The frozen vaginal tissue samples were then suspended in 1.0 mL of protease inhibitor cocktail set III (Calbiochem, San Diego, CA) containing 10% fetal bovine serum (Atlas, Fort Collins, CO), 2 mM L-glutamine, penicillin/streptomycin and 2-mercaptoethanol in RPMI medium (Celox, St. Paul, MN) (RPMI complete medium). Tissue was homogenized for about 1 minute using Tissue Tearor (Biospec Products, Bartlesville, OK). The tissue suspension was then centrifuged at 2000 rpm for 10 minutes under frozen conditions to pellet the debris, and the supernatant was collected and stored at -20°C until analysis of cytokine concentrations.

The ELISA kit for rat tumor necrosis factor-α (TNF) was purchased from BD PharMingen (San Diego, CA), and the rat monocyte chemoattractant protein-1 (MCP-1) ELISA kit was purchased from BioSource Intl. (Camarillo, CA). Both kits were operated according to the manufacturer's instructions. Results for both TNF and MCP-1 are expressed in pg/mL and normalized per 200 mg of tissue. Based on the minimum values used to form the standard curve, the TNFELISA has a sensitivity of 63 pg/mL and the MCP-1 ELISA has a sensitivity of 12 pg/mL.

Viscosity Test Method

In the following examples, for viscosity versus shear rate, a controlled rate step test was used between k and 80 s ^-1 with 16 s ^-1 as interpolation value with a 35 mm 2° cone fitted Haake RS series rheometer measures the viscosity at 20±0.5°C. The values shown in the examples are those at 16s ⁻¹ .

Example 8-10

The gels shown in Table 3 below were prepared in the following manner.

Step 1: Dissolve the paraben in propylene glycol. 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amineethanesulfonate monohydrate was then dissolved in the solution.

Step 2: Dissolve edetate disodium in water. Disperse Carbomer 974P in the solution.

Step 3: Add the solution of Step 1 to the dispersion of Step 2 and stir. A 20% solution of tromethamine was added to the mixture to adjust the pH.

table 3 Element Gel (% w/w) Example 8 Example 9 Example 10 Drug (ethanesulfonate monohydrate) 0.0153 0.0469 0.153 Carbomer 974P 1.50 1.50 1.70 Propylene Glycol 15.00 15.00 15.00 Methylparaben 0.15 0.15 0.15 Propylparaben 0.03 0.03 0.03 Edetate Disodium 0.05 0.05 0.05 20% tromethamine solution 0.54 0.85 1.25

pure water 82.7147 82.5231 81.667 pH 3.8 4.0 4.0 Viscosity (cps) 6000 7000 7000

Examples 11-18

The gels of Examples 11, 12 and 14 were prepared by the general procedure described in Examples 8-10. The gels of Examples 13, 15, 16, 17 and 18 were prepared in the following manner.

Step 1: Parabens were dissolved in propylene glycol (approximately 66 wt-% of the total amount to achieve the final wt-% of the gel). 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amineethanesulfonate monohydrate was then dissolved in the solution.

Step 2: Dissolve edetate disodium in water. Add remaining propylene glycol. Add Carbomer 974P and continue mixing until the Carbomer is fully hydrated.

Step 3: Add the solution of Step 1 to the dispersion of Step 2 and mix. Measure pH after mixing is complete.

Table 4 Element Gel (% w/w) Example 11 Example 12 Example 13 Example 14 Example 15 Example 16 Example 17 Example 18 Drug (ethanesulfonate monohydrate) 0.153 0.153 0.153 0.459 0.459 0.765 1.531 4.593 Carbomer 974P 2.10 2.10 2.10 2.00 2.10 2.10 2.50 2.30 Propylene Glycol 15.00 30.00 60.00 30.00 60.00 60.00 60.00 60.00 Methylparaben 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 Propylparaben 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 Edetate Disodium 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 20% Tromethamine 0.27 0.27 0.00 0.35 0.00 0.00 0.00 0.00 pure water 82.25 67.25 37.52 66.96 37.21 36.91 35.74 32.88 pH 4.0 4.2 3.8 4.3 3.6 3.2 2.9 2.5

Using the above test method, it was found that the gels of Examples 11-18 could induce cytokines after a single administration, but the above test method was modified as follows: tissue samples were centrifuged at 3000rpm for 10 minutes, and all samples of TNF were mixed at a ratio of 1:2 Dilution, for MCP-1 all samples were diluted 1:4, based on the minimum value used to form the standard curve, the sensitivity of the TNF ELISA was 31 pg/mL. In addition, the gels of Examples 15, 17 and 18 were found to induce cytokines after a single administration using the test method described above.

Examples 19-23

The gels shown in Table 5 below were prepared by the following method.

Step 1: Parabens were dissolved in propylene glycol (approximately 66 wt-% of the total amount to achieve the final wt-% of the gel). 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amineethanesulfonate monohydrate was then dissolved in the solution.

Step 2: Dissolve edetate disodium in water. Add remaining propylene glycol. Add Carbomer 974P followed by Xanthan Gum (if using) and continue mixing until the thickener is fully hydrated.

Step 3: Add the solution of Step 1 to the dispersion of Step 2 and mix. Measure pH after mixing is complete.

table 5 Element Gel (% w/w) Example 19 Example 20 Example 21 Example 22 Example 23 Drug (ethanesulfonate monohydrate) 1.531 1.531 1.531 1.531 1.531 Carbomer 974P 1.25 1.50 1.50 2.00 2.50 xanthan gum 1.75 1.50 1.50 1.00 0.00 Propylene Glycol 50.00 50.00 60.00 60.00 60.00 Methylparaben 0.15 0.15 0.15 0.15 0.15 Propylparaben 0.03 0.03 0.03 0.03 0.03 Edetate Disodium 0.05 0.05 0.05 0.05 0.05 pure water 45.24 45.24 35.24 35.24 35.74 pH 3.0 2.8 3.2 3.1 2.9

The gels of Examples 19-23 were found to induce cytokines after a single administration using the above test method.

Examples 24-27

The gels of Examples 24-26 were prepared by the general method of Examples 19-23. The gel of Example 27 was prepared in the general manner of Examples 8-10, but omitting the trometamol.

Table 6 Element Gel (%w/w) Example 24 Example 25 Example 26 Example 27 Drug (ethanesulfonate monohydrate) 1.531 1.531 1.531 1.531 Carbomer 974P 2.00 1.00 2.00 2.50 Xanthan gum 1.00 2.00 1.00 0.00

Propylene Glycol 50.00 50.00 60.00 60.00 Methylparaben 0.15 0.15 0.15 0.15 Propylparaben 0.03 0.03 0.03 0.03 edetate disodium 0.05 0.05 0.05 0.05 20% tromethamine solution 1.44 1.00 0.59 0.00 pure water 43.80 44.24 34.65 35.74 pH 3.4 3.5 3.4 3.1 Viscosity (cps) 11000 7550 ND ND

ND = not determined

The gels of Examples 24-27 were found to induce cytokines after a single administration using the above test method.

Examples 28-32

The gels of Examples 28-32 were prepared in the general manner of Examples 8-10, but in Step 2 carbomer and xanthan gum were added.

Table 7 Element Gel (%w/w) Example 28 Example 29 Example 30 Example 31 Example 32 Drug (ethanesulfonate monohydrate) 0.15 0.45 0.75 1.50 4.50 Carbomer 974P 2.20 2.33 2.50 2.00 1.75 Xanthan gum 1.10 1.17 1.25 1.00 0.88 Propylene Glycol 15.00 30.00 30.00 40.00 60.00 Methylparaben 0.15 0.15 0.15 0.15 0.15 Propylparaben 0.03 0.03 0.03 0.03 0.03 edetate disodium 0.05 0.05 0.05 0.05 0.05 20% tromethamine solution 0.50 1.25 0.67 1.70 2.00 pure water 80.82 64.57 64.60 53.57 30.64 pH 4.0 4.0 3.5 3.5 3.0 Viscosity 8900 11000 8600 12000 17000

Example 33

The gels shown in Table 8 below were prepared by the following method.

Step 1: Edetate disodium was dissolved in water (approximately 99% of the total amount to achieve the final wt-% of the gel).

Step 2: Dissolve the paraben in propylene glycol. 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amineethanesulfonate monohydrate was then dissolved in the solution.

Step 3: Add Carbomer 974P and Xanthan Gum sequentially to the solution in Step 1 and mix, continue mixing until the thickener is fully hydrated.

Step 4: Add the solution of Step 2 to the dispersion of Step 3 and mix.

Step 5: Tromethamine was dissolved in water (20% trometamol by weight), this solution was added to the gel of step 4 and mixed. Continue mixing until the gel is homogeneous. Measure the pH after mixing is complete.

Table 8 Element Gel of Example 33 (% w/w) Drug (ethanesulfonate monohydrate) 0.15 Carbomer 974P 2.20 Xanthan gum 1.10 Propylene Glycol 15.00 Methylparaben 0.15 Propylparaben 0.03 edetate disodium 0.05 tromethamine 0.20 pure water 81.12 pH 3.8 Viscosity (cps) 10700

Example 34

The gels shown in Table 9 below were prepared by the following method.

Step 1: Dissolve the paraben in propylene glycol. 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amineethanesulfonate monohydrate was then dissolved in the solution.

Step 2: Edetate disodium was dissolved in water (approximately half of the total amount to achieve the final wt-% of the gel).

Step 3: Add Carbomer 974P, Xanthan Gum, Step 2 solution and water (approximately half of the total amount to achieve the final wt-% of the gel) to the solution from Step 1 in sequence and mix, continue mixing until The thickener is fully hydrated.

Step 4: Tromethamine was dissolved in water (20 wt% trometamol), this solution was added to the gel of step 3 and mixed. Continue mixing until the gel is homogeneous. Measure pH after mixing is complete.

Table 9 Element Gel of Example 34 (% w/w) Drug (ethanesulfonate monohydrate) 1.50 Carbomer 974P 2.00 Xanthan gum 1.00 Propylene Glycol 40.00 Methylparaben 0.15 Propylparaben 0.03 edetate disodium 0.05 tromethamine 0.40 pure water 54.87 pH 3.6 Viscosity (cps) 7800

Example 35

The gels shown in Table 10 below were prepared by the following method.

Step 1: Dissolve the paraben in propylene glycol. 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate was added in 3 portions and mixed. Continue mixing until the drug is completely dissolved.

Step 2: Edetate disodium was dissolved in water (approximately 1/3 of the total amount to achieve the final wt-% of the gel).

Step 3: Add Carbomer 974P, Xanthan Gum, Step 2 solution and water (approximately 2/3 of the total amount used to achieve the final wt% of the gel) to the solution in Step 1 and mix, continue mixing until the thickener is fully hydrated.

Step 4: Tromethamine was dissolved in water (20 wt% tromethamine), this solution was added to the gel of step 3 and mixed. Continue mixing until the gel is homogeneous. Measure pH after mixing is complete.

Table 10 Element Gel (% w/w) of Example 35 Drug (ethanesulfonate monohydrate) 4.50 Carbomer 974P 1.75 Xanthan gum 0.88 Propylene Glycol 50.00

Methylparaben 0.15 Propylparaben 0.03 edetate disodium 0.05 tromethamine 0.65 pure water 41.99 pH 3.0 Viscosity (cps) 7100

Other useful formulations are described in co-pending US application 60/698416.

Example 36

Preparation of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate

Part A

Under nitrogen, a DMF suspension (4.5 L) of 3-nitro-1,5-naphthyridine-4-ol (1.00 kg, 5.23 mol) was cooled in an ice bath. Phosphorus oxychloride (882.5 g, 5.75 mol) was added slowly over 1 hour while maintaining the temperature at 16-20°C. After the addition was complete the reaction was stirred at a temperature of 20-24°C for 3 hours and then quickly added to 2 portions of demineralized water (12.5 L each) at 20-24°C. The temperature of the mixture reached 29.5-30.5°C during the addition. The resulting mixture was cooled to about 10°C over 60 minutes. The solids formed in each mixture were isolated by filtration and the fractions were washed with demineralized water (2 x 2 L and 1 x 1 L) until the pH of the filtrate was equal to that of demineralized water. The product 4-chloro-3-nitro[1,5]phthalazine as a tan solid containing water was used in Part B over 1 hour.

Part B

Isobutylamine (784 g, 10.7 mol) was added to a suspension of the material from Part A in THF (6 L) over 45 minutes while maintaining the temperature of the reaction at 17-27°C. Yellow needles appeared in the solution when the addition was 75% complete. After the addition was complete, the reaction was stirred at a temperature of 21.5-22.5°C for 30 minutes and then added with stirring to 2 portions of demineralized water (12 L each). The resulting mixture was stirred for 30 minutes. The solid formed in each mixture was isolated by filtration and the solids were washed with demineralized water (2 x 2 L) in each portion until the pH of the filtrate was equal to that of demineralized water. The solid was then dried overnight on a filter funnel to afford 1.225 kg of ^N4- (2-methylpropyl)-3-nitro[1,5]naphthyridine-4-amine as a yellow solid, which was mixed with The material from another operation was combined.

Part C

A hydrogenation vessel was charged with a suspension of N ⁴ -(2-methylpropyl)-3-nitro[1,5]naphthyridine-4-amine (0.300 kg, 1.22 mol) in toluene (5 L) and Magnesium sulfate (50 g) was added followed by 5% platinum on carbon (15 g) moistened with toluene (500 mL). The reaction mixture was placed under hydrogen pressure (3.4×10 ⁵ Pa, 50 psi), shaken on a Parr shaker at room temperature for 20 hours and then filtered through a layer of CELITE filter agent. The filter cake was washed with toluene (500 mL), and the filtrate was concentrated to 4.5 L under reduced pressure to provide a toluene solution of ^N4- (2-methylpropyl)[1,5]naphthyridine-3,4-diamine.

Part D

The solution of Part C was combined with p-toluenesulfonic acid monohydrate (11.4 g, 59.9 mmol). The reaction was heated to 90°C and triethyl orthoformate (0.178 kg, 1.2 mol) was added over 60 minutes. After the addition was complete, the reaction mixture was heated at 100 °C for an additional 2 hours and the ethanol distillate (350 mL) was collected. The reaction mixture was cooled to room temperature, stirred overnight, then treated with aqueous sodium carbonate (1 L, 1% w/w) and stored to afford 1-(2-methylpropyl)-1H- Imidazo[4,5-c][1,5]phthalazine. This material was mixed with material from 2 more runs on the same scale.

Part E

The aqueous layer of the Part D material was separated and the toluene solution was washed with deionized water (2.7 L). The toluene layer was separated and concentrated to 7 L under reduced pressure. The toluene solution was then heated to 50°C and peracetic acid (841 mL, 32% w/w in dilute acetic acid) was added over 2 hours while maintaining the temperature of the reaction at 45-55°C. After the addition was complete, the reaction was heated at 50°C overnight and then stirred at 40-50°C while sodium metabisulfite (137 g, dissolved in 3.33 L deionized water) was added over 10 minutes. Aqueous sodium hydroxide solution (0.576 L, 50% w/w in 3.564 L of water) was added over 30 minutes. The resulting mixture was stirred at about 50°C for 30 minutes, then cooled to about 10°C for 1 hour. A yellow precipitate appeared and was isolated by filtration, washed with deionized water (5 L), suction dried overnight, and then dried in a vacuum oven at 40-50° C. for about 24 hours to afford 0.693 kg of 1-(2 -methylpropyl)-5-oxy-1H-imidazo[4,5-c][1,5]naphthyridine.

Part F

Concentrated aqueous ammonium hydroxide solution (660 mL) was added to 1-(2-methylpropyl)-5-oxo-1H-imidazo[4,5-c][1,5]naphthyridine (0.520 kg, 1.93 mol) in methanol (4.7 L) and the temperature of the reaction was adjusted to 20 °C. Benzenesulfonyl chloride (0.716 kg, 4.05 mol) was added slowly over 75 minutes and the temperature of the reaction was maintained below 26°C by external cooling. After the addition was complete, the reaction was stirred at about 25°C for 2 hours. Sodium hydroxide (154 g, dissolved in 2 L of deionized water) was then added to the reaction mixture while maintaining the temperature of the reaction mass at about 25°C by external cooling. The resulting mixture was cooled to a temperature below 10 °C for 2 hours. The formed precipitate was isolated by filtration and washed sequentially with 3:2 methanol/deionized water (2 x 500 mL) and deionized water (7.5 L) until the pH of the solution was neutral. The filter cake was dried overnight on a filter funnel to afford 0.426 kg of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]phthalazine- 4-amine.

Part G

Slowly heat 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine (0.300kg, 1.24mol) in isopropanol (10L ) suspension and water (23 mL, 1.28 mol) was added. Aqueous ethanesulfonic acid solution (214 g 70% w/w, 1.36 mol ethanesulfonic acid, 3.6 mol water) was added over 10 minutes at 60°C and all solids dissolved. After the addition was complete, the reaction was heated to 82°C. The reaction was cooled from 80°C to 50°C (0.17°C/min) over 3 hours, then cooled to room temperature overnight with slow stirring. White crystals appeared. MTBE (10 L) was added and the mixture was cooled to about 5°C for 2 hours. The crystals were collected by filtration, washed with MTBE (10 L), dried on the filter funnel overnight, and then dried in a vacuum oven at 35 °C for 3 days to afford 372 g of 1-(2-methylpropyl)-1H- Imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate monohydrate. The substance was characterized by powder X-ray diffraction analysis, TGA and FTIR spectroscopy, and the data were found to be consistent with those shown in Figures 2, 7 and 4, respectively.

The entire contents of the patents, patent documents, and applications mentioned herein are incorporated by reference in their entirety as if each were individually incorporated. Various modifications and alterations of this invention will be apparent to those skilled in the art without departing from the scope and spirit of this invention. It is to be understood that the present invention is not limited to the exemplary embodiments and examples set forth herein, which are presented by way of example only, and that the scope of the present invention is limited only by the claims set forth below.

Claims (37)

1. A salt selected from the group consisting of ethanesulfonic acid of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine Salt and methanesulfonate of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine, or its solvates or hydrates things. 2. The salt of claim 1, wherein the salt is 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine - ethanesulfonate of 4-amine, or a solvate or hydrate thereof. 3. The salt of claim 1, wherein the salt is 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine - Methanesulfonate of 4-amine, or a solvate or hydrate thereof. 4. The salt of claim 1, wherein said salt is in dissolved form. 5. The salt of claim 1, wherein said salt is in solid form. 6. The salt of claim 5, wherein said salt is in crystalline form. 7. The salt of claim 5, wherein said salt is in an amorphous form. 8. The salt of claim 5, wherein said salt is in solvated form. 9. The salt of claim 5, wherein said salt is in the form of a hydrate. 10. The salt of claim 9, wherein said salt is in the form of a monohydrate. 11. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a salt according to any one of the preceding claims. 12. The pharmaceutical composition of claim 11, wherein the pharmaceutically acceptable carrier comprises water. 13. A method for treating neoplastic diseases in animals, said method comprising administering to said animals a therapeutically effective amount of the salt according to any one of claims 1-10 or the salt according to claim 11 or 12 pharmaceutical composition. 14. A method for treating viral diseases in animals, said method comprising giving said animal a therapeutically effective amount of the salt according to any one of claims 1-10 or the salt according to claim 11 or 12 pharmaceutical composition. 15. A method for inducing cytokine biosynthesis in animals, said method comprising administering to said animals an effective amount of the salt according to any one of claims 1-10 or the salt according to claim 11 or 12 pharmaceutical composition. 16. The method of claim 13, wherein the neoplastic disease is intracervically located. 17. The method of claim 14, wherein the viral disease comprises intracervical human papillomavirus. 18. A process for the preparation of ethanesulfonate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine, said Methods include: 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine free base is mixed with ethanesulfonic acid and carrier to form a mixture, wherein the said carrier comprises an organic liquid and optionally water; and reacting the components of the mixture under conditions sufficient to form 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine of ethanesulfonate. 19. The method of claim 18, further comprising heating the free base, ethanesulfonic acid and/or carrier, and/or heating a mixture thereof prior to mixing. 20. The method of claim 18 or 19, further comprising forming 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]phthalazine in the mixture - Precipitation of the ethanesulfonate salt of 4-amine. 21. The method of claim 20, wherein forming the precipitate comprises cooling the mixture to form the precipitate. 22. The method of claim 21, wherein cooling is performed at a rate of less than 2.0°C/minute. 23. The method according to any one of claims 18-22, wherein for each mole of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1, 5] Naphthalene-4-amine free base, said carrier comprising at least 2 moles of water. 24. The method of claim 20, further comprising: optionally adding other organic liquids to the mixture containing the precipitate; separating at least a portion of the precipitate from at least a portion of the mixture; washing the precipitate; and The precipitate is at least partially dried. 25. The salt of claim 3, wherein the salt is in crystalline form and has an X-ray powder diffraction pattern at 8.51 degrees 2Θ, 14.12 degrees 2Θ, 16.80 degrees 2Θ, 17.88 degrees 2Θ, 21.43 degrees 2Θ, 23.24 degrees 2Θ and 29.16 degrees 2Θ peaked, and these values were each ±0.15 degrees 2Θ. 26. The salt according to claim 25, wherein the salt has an X-ray powder diffraction pattern at 7.15 degrees 2θ, 8.51 degrees 2θ, 14.12 degrees 2θ, 16.80 degrees 2θ, 17.88 degrees 2θ, 18.49 degrees 2θ, 18.88 Peaks appeared at 2Θ, 21.04 2Θ, 21.43 2Θ, 23.24 2Θ, 25.40 2Θ, 27.92 2Θ, 28.77 2Θ, and 29.16 2Θ, each of which was ±0.15 2Θ. 27. The salt according to claim 26, wherein the salt has an X-ray powder diffraction pattern at 7.15 degrees 2θ, 7.55 degrees 2θ, 8.51 degrees 2θ, 11.83 degrees 2θ, 13.65 degrees 2θ, 14.12 degrees 2θ, 14.87 degrees 2θ, 16.80 degrees 2θ, 17.88 degrees 2θ, 18.49 degrees 2θ, 18.88 degrees 2θ, 19.92 degrees 2θ, 20.24 degrees 2θ, 21.04 degrees 2θ, 21.43 degrees 2θ, 22.24 degrees 2θ, 23.24 degrees 2θ, 24.64 degrees 2θ, 25.40 degrees 2θ , 25.71 degrees 2θ, 27.20 degrees 2θ, 27.92 degrees 2θ, 28.77 degrees 2θ, 29.16 degrees 2θ, 30.94 degrees 2θ, 31.29 degrees 2θ, 32.76 degrees 2θ, 33.56 degrees 2θ, 34.04 degrees 2θ, 34.88 degrees 2θ and 35.40 degrees 2θ peaks , these values are each ±0.15 degrees 2θ. 28. The salt of claim 3, wherein the unit cell of the salt has the following crystal plane spacing: about 10.38 angstroms, about 6.27 angstroms, about 5.27 angstroms, about 4.96 angstroms, about 4.14 angstroms, about 3.82 angstroms Angstroms and about 3.06 Angstroms. 29. The salt of claim 3, characterized in that: The X-ray powder diffraction pattern of the salt is at 7.15 degrees 2θ, 8.51 degrees 2θ, 14.12 degrees 2θ, 16.80 degrees 2θ, 17.88 degrees 2θ, 18.49 degrees 2θ, 18.88 degrees 2θ, 21.04 degrees 2θ, 21.43 degrees 2θ, 23.24 degrees 2θ, peaks at 25.40 degrees 2Θ, 27.92 degrees 2Θ, 28.77 degrees 2Θ, and 29.16 degrees 2Θ, each of which is ±0.15 degrees 2Θ; and The weight loss in the temperature range of 60-80° C. was determined to be 4.5%-5.5% by thermogravimetric analysis. 30. 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine having an X-ray powder diffraction pattern substantially as shown in Figure 1 of mesylate. 31. A process for preparing the mesylate salt of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine, said Methods include: 1-(2-Methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine free base is mixed with methanesulfonic acid and carrier to form a mixture, wherein said carrier comprises an organic liquid and optionally water; and reacting the components of the mixture under sufficient conditions to form the methanesulfonate of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine salt. 32. The method of claim 31, further comprising heating the free base, methanesulfonic acid and/or carrier, and/or heating a mixture thereof prior to mixing. 33. The method of claim 31 or 32, further comprising forming 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]phthalazine in the mixture - Precipitation of the methanesulfonate salt of 4-amine. 34. The method of claim 33, wherein forming the precipitate comprises cooling the mixture to form the precipitate. 35. The method according to any one of claims 31-34, wherein for each mole of 1-(2-methylpropyl)-1H-imidazo[4,5-c][1, 5] Naphthalene-4-amine free base, said carrier comprising at least 2 moles of water. 36. The method of claim 33, further comprising: optionally adding other organic liquids to the mixture containing the precipitate; separating at least a portion of the precipitate from at least a portion of the mixture; washing the precipitate; and The precipitate is at least partially dried. 37. A mesylate salt containing dissolved 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine by administration to the cervix or 1-(2-methylpropyl)-1H-imidazo[4,5-c][1,5]naphthyridine-4-amine ethanesulfonate topical formulation for the treatment of high-risk cervical Methods of HPV infection.

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