HK40108321A - Compositions and dosage forms for treatment of hpv infection and hpv-induced neoplasia - Google Patents

Description

Compositions and dosage forms for treating HPV infection and HPV-induced neoplasia.

Cross-references to related applications

This application claims the benefit of U.S. Provisional Application No. 63/391,283, filed July 21, 2022; U.S. Provisional Application No. 63/400,661, filed August 24, 2022; Chinese Patent Application No. 202211206517.7, filed September 30, 2022; and U.S. Provisional Application No. 63/412,143, filed September 30, 2022. These applications are incorporated herein by reference for all purposes.

Technical Field

This invention provides compositions, advantageous salts, prodrugs, stereoisomers, crystalline forms, dosage forms, and uses thereof for treating human papillomavirus (HPV) infection or related disorders (such as HPV-induced neoplasia) in hosts in need.

Background Technology

According to the U.S. Centers for Disease Control and Prevention, there is currently no direct cure for human papillomavirus (HPV). In 2018, more than 43 million people were infected, with more than 13 million new infections.

Currently, treatment options for HPV infection are only adjunctive and limited. They all have significant drawbacks. Commonly used drug therapies include salicylic acid, trichloroacetic acid, imiquimod, and condylox. Trichloroacetic acid and salicylic acid chemically burn wart tissue as a means of removing the virus, often causing skin irritation, ulceration, and pain in the process. Furthermore, salicylic acid is not used to treat HPV infections in the anogenital area. Imiquimod (Aldara ^™ , Zyclara ^™ ) stimulates the immune system to clear the infection through Toll-like receptor signaling, causing redness and swelling. Condylox (Condylox ^™ ) destroys tissue through microtubule destabilization to prevent host cell replication.

More problematic is that HPV infection has caused cellular transformation in human patients, cells that have not yet developed into cancer but have reached the neoplastic stage. HPV-induced neoplastic forms include cervical intraepithelial neoplasia (“CIN”), anal intraepithelial neoplasia (“AIN”), perianal intraepithelial neoplasia (“PAIN”), vulvar intraepithelial neoplasia (“VIN”), penile intraepithelial neoplasia (“PIN”), and vaginal intraepithelial neoplasia (“VAIN”). Cancers caused by HPV include cervical cancer, anal cancer, perianal cancer, penile cancer, vaginal cancer, vulvar cancer, and oropharyngeal cancer.

Identifying and treating HPV-induced lesions before they develop into potentially untreatable cancers is crucial. Almost all cases of cervical cancer are caused by sexually transmitted infection with carcinogenic HPV types. The primary goal of early screening (e.g., a Pap smear) is to identify abnormal cervical cells with severe cellular changes so that they can be removed or destroyed.

Cervical intraepithelial neoplasia is most commonly treated with observation (wait-and-see approach) or by excision or ablation of the cervical transformation zone. Techniques include cryotherapy, laser therapy, loop electrosurgical excision procedure (LEEP), and cone biopsy. All of these surgical procedures can damage the affected area and may result in scarring. LEEP, the most common intervention, is effective in 60%–90% of cases; however, it is associated with a significantly increased risk of miscarriage, ectopic pregnancy, and negative psychological outcomes. Despite extensive research, no medication has been approved to replace or be used in combination with these surgical methods.

Human papillomaviruses (HPVs) are a group of non-enveloped DNA viruses that infect keratinocytes of the skin and mucous membranes, including the anogenital region, in humans. They are known to cause skin warts, genital warts, respiratory papillomatosis, and cancer. Several species of the alpha-papillomavirus genus contain high-risk types of HPV, which are more likely to cause tumors and then cancer in humans. Most carcinogenic HPV types come from alpha-7 and alpha-9, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. The most common carcinogenic HPV types are 16 and 18. HPV-16 and -18 are the cause of most cervical cancers. Most genital warts are caused by low-risk HPV types 6 and 11. Vaccines have been developed targeting HPV 6, 11, 16, and 18, and may be effective if administered before first sexual intercourse. However, the HPV vaccine may offer very little benefit to sexually active women who are already infected with HPV.

Available specific prophylactic vaccines include Gardasil 9 (a 9-valent HPV vaccine; covering HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58), Gardasil 4 (quadrivalent), and Cervarix (bivalent). These are useful if the person is vaccinated before exposure to the virus (which typically means before sexual activity). Prophylactic vaccines are designed to produce neutralizing antibodies that clear the virus before it infects cells. In contrast, therapeutic vaccines are designed to produce a CD4+ and/or CD8+ T-cell-based response to clear HPV-infected cells. Exemplary antigens used in therapeutic vaccines include E6 and E7. There are currently no approved therapeutic vaccines. Non-limiting examples of therapeutic vaccines being investigated in clinical trials include VGX-3100 (INOVIO), GGX-188E (Genexine, Inc.), and ADXS11-001 (Advaxis, Inc.).

Cervical intraepithelial neoplasia (CIN) is a precursor to cervical cancer. Up to 20% of women infected with HPV develop CIN (Rozenda et al., "PCR-based high-risk HPV test in cervical cancer screening gives objective risk assessment of women with cytomorphologically normal cervical smears" 1996, Int J Cancer, 68, 766-769). CIN is graded from mild (Grade 1) to severe (Grade 3) on the Bethesda Scale. When a woman is diagnosed with Grade 1 CIN, a "wait and see" approach is usually adopted. Due to the adverse side effects of surgical methods, treatment is only recommended for CIN grades 2-3.

The cervical epithelium consists of several layers of tissue, collectively known as stratified squamous epithelium. These layers are the superficial cell layer, intermediate cell layer, parabasal cell layer, and basal cell layer. Topical medications used to treat cervical intraepithelial neoplasia must be able to penetrate these multiple layers of tissue to adequately reach and treat the transformed cells. This is a challenging task because the cells are tightly bound and lack blood vessels.

In 1996, the National Cancer Institute Consensus Group identified the need for non-surgical intervention for cervical intraepithelial neoplasia (CIN). National Institutes of Health Consensus Development Conference statement on cervical cancer. April 1-3, 1996. J Women’s Health, 1996, 1, 1-38. Since the publication of this guideline, many different approaches to treating HPV and CIN have been explored, including immunomodulators, antiproliferative drugs, antiviral drugs, and hormones. However, there is still no FDA-approved treatment that has proven effective in clinical trials for HPV infection or CIN (Desravines, N. et al. “Topical therapies for the treatment of cervical intraepithelial neoplasia (CIN) 2-3: A narrative review” Gynecol Oncol Rep. 2020, 33, 100608).

The University of California Board of Trustees, along with Karl Hostetler et al., as designated inventors, has filed a series of patents for various acyclic nucleotide derivatives for the treatment of papilloma infection, including (i) U.S. Patent Nos. 8,835,603; 9,629,860; 9,156,867; 10,449,207; 10,195,222; 10,076,533; 10,076,532; (i) U.S. Patent Nos. 9,775,852; 9,387,217, with a priority date of March 15, 2013; (ii) U.S. Patent Nos. 10,702,532; 10,213,430; 9,493,493; and 9,801,884, with a priority date of September 15, 2014; and (iii) U.S. Patent Nos. 11,014,950 and 10,377,782, with a priority date of September 15, 2015. Some of these patents have been granted to Antiva Biosciences, Inc., which is developing new therapies to treat precancerous lesions caused by HPV.

Antiva Biosciences conducted human clinical trials with the phosphonate ABI-1968 to evaluate its ability to adequately penetrate all layers of the cervical epithelium and release the antiviral agent PMEG ((9-[2-phosphonomethoxy)ethyl)guanine]). PMEG was then phosphorylated to the active compound PMEGpp (PMEG polyphosphate). It was determined that ABI-1968 did not reach a tissue concentration of 15 ng/mg (see columns F and G in Figure 116) when used at doses up to 3%, and therefore was unsuitable as a topical treatment for cervical intraepithelial neoplasia. Effectively administering medication topically to epithelial layers infected with HPV to destroy neoplastic cells in multiple epithelial layers is a significant challenge. The drug must possess sufficient lipophilicity to penetrate tissue layers and be metabolized to sufficient concentrations of the active agent to kill pathogenic cells when needed.

Published articles discuss various local drug delivery strategies, including semi-solid dosage forms, gels, tablets, films, and vaginal suppositories. See, for example, Keshari Sahoo, C. et al., “Intravaginal Drug Delivery System: An Overview”, 2013, American Journal of Advanced Drug Delivery, 1, 43-55; da Neves, J. et al., “Gels as vaginal drug delivery systems”, 2006, International Journal of Pharmaceutics, 318(2)1-14; Cencia Rohan, L. et al., “Vaginal Drug Delivery Systems for HIV Prevention”, 2009, AAPS, 11(78); Kast, C.E. et al., “Design and in vitro drug delivery system for HIV prevention”, 2009, AAPS, 11(78); Kast, C.E. et al., “Design and in vitro drug delivery system for HIV prevention”, 2009, AAPS, 11(78). ation of a novel bioadhesive vaginal drug delivery system forclotrimazole”Journal of Controlled Release,2002,81(3)347-354; Acarturk, F. “Mucoadhesive vaginal drug delivery systems ", Recent Pat Drug Deliv Formul., 2009, 3 (3) 193-205; and Sonal, G. et al. "Exploring Novel Approaches to Vaginal Drug Delivery", Recent Patents on Drug Delivery and Formulation, 2011, 5 (2) 82-94.

The purpose of this invention is to provide effective pharmaceutical compositions and treatments for HPV infection and related conditions in hosts in need, such as HPV-induced neoplasia, including but not limited to cervical intraepithelial neoplasia (CIN), anal intraepithelial neoplasia (AIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), perianal intraepithelial neoplasia (PAIN), and vaginal intraepithelial neoplasia (VAIN).

Summary of the Invention

Effective compositions for treating HPV infection and related diseases (including HPV-induced intraepithelial neoplasia, such as cervical intraepithelial neoplasia, anal intraepithelial neoplasia, perianal intraepithelial neoplasia, penile intraepithelial neoplasia, vulvar intraepithelial neoplasia, and vaginal intraepithelial neoplasia) require selection and combination with multiple factors to work synergistically to achieve the long-sought-after ability to penetrate epithelial layers of tissue in an effective amount to deliver the active agent. Numerous failures have been encountered, and years of research are needed to address this issue, thereby benefiting patients worldwide with potentially cancerous intraepithelial neoplasia.

In particular, the key compounds were found to be the following specific salts:

Compound I is (ethyl(((2-(2-amino-6-methoxy-9H-purine-9-yl)ethoxy)methyl)(benzyloxy)-phosphoryl)-L-alanine ester). U.S. Patent Nos. 9,801,884 and 11,344,555, assigned to the Board of Trustees of the University of California, generally claim protection for Compound I and pharmaceutically acceptable salts, as well as methods of using said compound and salts for the treatment of human papillomavirus infection. Compound I is an acyclic nucleotide phosphonate that can be metabolized to a known potent antiviral compound (PMEG; ((9-[2-phosphonomethoxy)ethyl)guanine])), but PMEG has poor cellular penetration and systemic toxicity limiting its use. The assignee discovered how to improve the locally delivered prodrug to allow for rapid absorption by epithelial cells, a challenging task to date, which ABI-1968 failed to achieve.

Compound I (ethyl(-((2-(2-amino-6-methoxy-9H-purine-9-yl)ethoxy)methyl)-(benzyloxy)phosphoryl)-L-alanine ester) has two chiral centers, one on the phosphorus atom and one on the amino acid moiety, either of which can be either R or S stereoconfiguration. Therefore, compound I exists as four stereoisomers or two diastereomeric pairs: ( _RP , _SC )/( _SP , _SC ) and ( _RP , _RC )/( _SP , _RC ). While U.S. Patent Nos. 9,801,884 and 11,344,555 describe compound I in general, these patents do not address the potential stereochemistry of the phosphorus atom. As discussed further herein, stereoisomers of compound I having R-stereochemistry on the phosphorus and S-stereochemistry on the amino acid carbon have been found to have superior properties compared to the other three stereoisomers.

In non-limiting embodiments, a favorable salt of compound I (e.g., fumarate) is used as a mixture of (R,S) and (S,S) diastereomers, wherein the first R/S specifies the stereochemistry at the phosphorus atom, and the second S is the stereochemistry of the carbon in the amino acid moiety (corresponding to an L-alanine residue having the S-configuration). While any ratio of diastereomers providing the desired results can be used, the (R,S) diastereomer is most prominent. In other embodiments, the ratio of the R to S enantiomers at the phosphorus atom is about 1:1. In some aspects, the compound is enriched at the phosphorus atom as the R chiral enantiomer, wherein the amount of R by weight is, for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or higher.

Corresponding to the natural amino acid configuration, the S-stereoconfiguration on the chiral carbon is advantageous in this invention. In some aspects, the amount of S by weight is, for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or higher. In alternative embodiments, the compound is used with the R-stereoconfiguration at the chiral carbon, and wherein the R-stereoconfiguration is greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or higher.

The enantiomerically pure ( _Rp , _Sc , or simply "R,S") form of Compound I is the primary embodiment. Unless otherwise stated, the enantiomerically pure Compound II is at least 90% free of the opposite enantiomer. Surprisingly, the compounds are oils rather than solids and are therefore not selected as active ingredients for topical formulations. This is especially true because racemic mixtures or enantiomerically enriched R,S and S,S as free bases are solids. Furthermore, as shown in Figure 120, the S,S isomer has moderate crystallinity. However, when formed as a fumarate, the R,S enantiomerically pure Compound I becomes a highly crystalline material and is most advantageous for intraepithelial topical application. Thus, the monofumarate of Compound I exhibits unexpected stability and processability, and therefore offers therapeutic advantages compared to the free base Compound I.

The R,S isomer of the monofumarate can be readily crystallized from isopropanol and heptane. This crystalline form is an anhydrous compound with a melting point of approximately 140°C (Example 15). This crystalline form was reproduced not only at the milligram level but also at the multigram level.

Although the S,S isomer is more readily treated as a free base, the monofumarate of the S,S isomer exhibits polymorphism and a low melting point of approximately 105 °C. Four crystalline forms of the S,S monofumarate were identified (Example 15). In some experiments, the S,S monofumarate was observed to dissociate into a hemifumarate. This pattern of synthesis is not reproducible when carried out on a large scale.

Surprisingly, certain pharmaceutical composition dosage forms prepared from compound I monofumarate and its crystal form mode 1 have been found to possess advantageous properties. Tablets prepared from the free base of compound I degrade significantly within one month at 40°C and 75% RH, but in contrast, tablets prepared from compound I monofumarate exhibit much lower degradation (Example 25) and significantly extended shelf life.

Compound II, mentioned herein and described below, is an example of an enantiomer enriched or pure form having R-stereochemistry primarily at the phosphorus atom and S-stereochemistry at the amino acid carbon atom. In its enantiomeric pure form, Compound II exhibits superior stability compared to its stereoisomer, ethyl((S)-((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)(benzyloxy)phosphoryl)-L-alanine ester monofumarate (Compound III). This is crucial for successful topical application to the cervix, vagina, vulva, perianal region, anus, or penis.

Other advantageous salts of compound I that have been discovered include hemifumarates: ethyl((R)-((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)(benzyloxy)phosphoryl)-L-alanine ester hemifumarate (compound IV) and ethyl((S)-((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)(benzyloxy)phosphoryl)-L-alanine ester hemifumarate (compound V).

Compound II has been found to have high tissue penetration and is surprisingly stable, crystalline, and non-hygroscopic. Compound II and its favorable crystalline form mode 1 can be used to treat HPV infection or HPV-related diseases, such as intraepithelial neoplasia, including but not limited to cervical intraepithelial neoplasia, anal intraepithelial neoplasia, vulvar intraepithelial neoplasia, penile intraepithelial neoplasia, perianal intraepithelial neoplasia, and vaginal intraepithelial neoplasia, to prevent transformation into cancer.

HPV has many strains, some of which are closely associated with cancer development and are referred to as high-risk strains. Compound I fumarate or Compound II can be used to treat high-risk HPV, including HPV-16 and HPV-18. Therefore, in some aspects, the present invention provides Compound II and isolated crystalline form of Compound II, Mod 1; pharmaceutical compositions containing such compounds; methods for treating HPV infection or HPV-related intraepithelial neoplasia using the selected crystalline form described herein; and methods for preparing such compounds and crystalline forms.

In particular, it was surprisingly found that the monofumarate of ethyl((R)-((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)(benzyloxy)phosphoryl)-L-alanine (compound II) exhibits very high tissue penetration when applied topically to the target tissue. Topical administration avoids the toxicity associated with systemic drug administration. Since HPV-infected precancerous and/or cancerous cells exist in multiple layers of epithelium, this compound must possess high penetrability to reach and treat these affected cells.

Compared to ABI-1968, compound I monofumarate exhibits superior tissue penetration and permeability in porcine and human vaginal tissues, despite ABI-1968 also being a phosphate ester of an acyclic purine nucleoside, which failed in clinical trials. At a 0.1% dose, compound I monofumarate achieved concentrations of 40–85 ng/mL in vaginal tissues. ABI-1968, even at a 3% dose, did not reach concentrations of even 15 ng/mL (see Figure 116). This significant improvement in tissue penetration, especially considering the reduced dose, was unexpected.

Compound II is surprisingly stable compared to its corresponding _SP isomer (compound III). As shown in Example 7 and Table 9, compound II has a melting point of about 140°C ± 10°C, for example, 141.5°C, while compound III has a melting point of about 100°C ± 10°C, for example, 106.4°C. The XRPD data comparing the monofumarates of the two compounds also show that compound II is more crystalline than compound III (see Example 13, Table 37 and Figure 71; Comparative Example 15, Table 40 and Figure 77).

Careful selection of the various aspects of this invention is essential to achieving the desired results. One important aspect is the formulation. Topical formulations, as used herein, include semi-solid dosage forms such as gels, creams, ointments, liquids, or solid dosage forms. Non-limiting examples of solid dosage forms include tablets that can be inserted into the affected area.

Compound I (monofumarate), compound II, or compound III have been found to be formulated into solid dosage forms for topical administration. In some embodiments, the tablet formulation provides similar tissue penetration as the gel formulation (55-85 ng/mg for gel, 44-79 ng/mg for tablet, Figure 121).

High crystallinity can facilitate the separation and processing of pharmaceutical compounds. Compared to compound III, compound II exhibits surprisingly low hygroscopicity. When exposed to a cycle of 40%–0–95%–0–45% relative humidity, compound II retains approximately 0.25% water content, and the XRPD pattern remains unchanged (Example 21, Table 46). Under the same conditions, compound III retains approximately 10% water content, a 40-fold increase. These conditions also cause the loss of one peak in the XRPD pattern, indicating that compound III changes its morphology with humidity variations. The hygroscopic and stability advantages of compound II relative to compound III are surprising and unexpected.

For example, compound II, pattern 1, can be prepared by recrystallizing compound II (Example 13, Table 37) and equilibrating it in a suitable solvent (Example 22). In some embodiments, compound II can be dissolved in an alcoholic solvent (e.g., isopropanol) and crystallized to pattern 1 by adding an aliphatic solvent (e.g., heptane). In some embodiments, compound II can be dissolved in an alcoholic solvent (e.g., ethanol) and crystallized to pattern 1 by adding an aliphatic solvent (e.g., heptane). Compound II, pattern 1, can also be prepared by equilibrating it in isopropanol, heptane, water, acetone, isopropanol:heptane (3:10), isopropanol:MTBE (1:3), and ethyl acetate:toluene (1:3).

Compound III, mode I, can be prepared in several steps (see Example 15). First, the free base of compound III is dissolved in isopropanol. One equivalent of fumaric acid is added, causing precipitation. After the addition of heptane, the mixture is stirred at an elevated temperature (e.g., 50°C) for 20 hours, and then cooled. An additional 0.2 equivalent of fumaric acid is added together with heptane, and the mixture is stirred at an elevated temperature for at least about 13 hours. The suspension is then slowly cooled until it reaches below about 5°C, and stirred at that temperature for at least about 2 days. The resulting solid compound III, mode I, is collected by filtration.

Compound I monofumarate (i.e., a mixture of the R and S enantiomers at the phosphorus atom and the S stereoisomer at the amino acid carbon) can be prepared by recrystallizing compound I monofumarate (Example 12, Table 31), equilibrating compound I monofumarate in a suitable solvent, or by crystallization through slow evaporation of the solvent (Example 12, Table 32). In some embodiments, compound I monofumarate can be dissolved in an alcoholic solvent (e.g., isopropanol) and crystallized to form pattern 1 by adding an aliphatic solvent (e.g., heptane). In some embodiments, compound I monofumarate can be dissolved in an alcoholic solvent (e.g., isopropanol) and crystallized to form pattern 1 by adding an ether solvent (e.g., methyl tert-butyl ether). In some embodiments, compound I monofumarate pattern 1 can be produced by equilibrating in a mixture of an ether solvent (e.g., tetrahydrofuran) and an aliphatic solvent (e.g., heptane). In some embodiments, compound I monofumarate pattern 1 can be produced by crystallization through slow evaporation of the solvent at room temperature. Suitable solvents for the slow evaporation crystallization of compound I monofumarate (mode 1) include, but are not limited to, acetone, methyl ethyl ketone, ethyl acetate, methanol, ethanol, isopropanol, and tetrahydrofuran. In some embodiments, compound I monofumarate (mode 1) is characterized by comprising at least three XRPD spectra with 2θ values selected from the following: 6.0 ± 0.2°, 8.9 ± 0.2°, 9.6 ± 0.2°, 11.1 ± 0.2°, 11.9 ± 0.2°, 14.8 ± 0.2°, 15.3 ± 0.2°, 18.1 ± 0.2°, 20.2 ± 0.2°, 23.1 ± 0.2°, 25.2 ± 0.2°, and 27.0 ± 0.2° (see Example 7).

Other crystalline forms of compound I monofumarate were prepared, including modes 2, 3, and 4. However, these crystalline forms are sometimes unstable and even when prepared from monofumarate, hemifumarate (a mixture of compounds IV and V) is formed.

Recrystallization of compound I monofumarate in methyl ethyl ketone; acetone; acetone and heptane; methyl ethyl ketone and heptane; and ethanol and methyl tert-butyl ether all yield hemifumarate mode 2. The ratio of compound I as a free base to fumarate, as measured by ^1H NMR, is about 1:0.5, for example 1:0.52. In one embodiment, mode 2 is characterized by comprising at least three XRPD spectra selected from the following 2θ values: 4.3 ± 0.2°, 6.2 ± 0.2°, 9.0 ± 0.2°, 13.0 ± 0.2°, 17.7 ± 0.2°, 18.7 ± 0.2°, and 25.3 ± 0.2° (see Example 12).

Recrystallization of compound I monofumarate in acetonitrile or acetonitrile and water yields hemifumarate pattern 3. After filtration separation, the ratio of compound I as free base to fumarate, measured by ^1H NMR, is approximately 1:0.95, but this ratio decreases to approximately 1:0.76 after washing with water. In one embodiment, pattern 3 is characterized by comprising at least three XRPD spectra selected from the following 2θ values: 3.5 ± 0.2°, 5.1 ± 0.2°, 6.2 ± 0.2°, 6.9 ± 0.2°, 10.2 ± 0.2°, 15.3 ± 0.2°, 17.6 ± 0.2°, 21.2 ± 0.2°, and 28.9 ± 0.2° (see Example 12). Recrystallization of compound I monofumarate in acetone and toluene yields hemifumarate pattern 4. The ratio of compound I as free base to fumarate, measured by ^1H NMR, is approximately 1:0.7, for example, 1:0.69. In one embodiment, mode 4 is characterized by including at least three XRPD spectra selected from the following 2θ values: 4.0±0.2°, 6.0±0.2°, 11.8±0.2°, 13.2±0.2°, 14.8±0.2°, 17.7±0.2°, 20.4±0.2°, and 25.2±0.2° (see Example 12). Due to the superior properties of monofumarates compared to hemifumarates, mode 1 of compound I monofumarate was chosen for further investigation because of its surprising stability and crystallinity.

In an exemplary non-limiting embodiment, a method for treating HPV-induced intraepithelial neoplasia is provided, the method comprising administering an effective amount of one or a combination of active compounds as described herein in a topical formulation sufficient to treat the neoplasia.

In an exemplary embodiment, the formulation for treating intraepithelial neoplasia is a dosage form containing 0.005 mg to 50 mg, 0.05 mg to 40 mg, 0.1 mg to 30 mg, 0.5 mg to 20 mg, 1 mg to 20 mg, 1 mg to 15 mg, or 1 mg to 10 mg of compound I monofumarate, compound II, or compound III.

In some embodiments, the formulation for treating intraepithelial neoplasia is a dosage form containing a monofumarate of compound I, compound II, or compound III in amounts ranging from about 0.001 to about 20 mg, from about 0.005 to about 10 mg, from about 0.01 mg to about 5 mg, from about 0.03 mg to about 1 mg, from about 0.05 mg to about 0.3 mg, from about 0.03 mg to about 0.07 mg, from about 0.05 mg to about 0.15 mg, or from about 0.15 mg to about 0.45 mg.

In some embodiments, the formulation for treating intraepithelial neoplasia is a dosage form containing from about 0.001 mg to about 0.005 mg, from about 0.005 mg to about 0.01 mg, from about 0.01 mg to about 0.03 mg, from about 0.03 mg to about 0.25 mg, from about 0.20 mg to about 0.5 mg, from about 0.4 mg to about 1 mg, from about 0.75 mg to about 3 mg, from about 1 mg to about 10 mg, and from about 5 mg to about 20 mg. In some embodiments, the formulation for treating intraepithelial neoplasia is a dosage form comprising about or at least 0.005, 0.01, 0.03, 0.05, 0.1 mg, 0.3 mg, 0.5 mg, 0.7 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg of compound I monofumarate, compound II, or compound III.

Specific doses are 0.05 mg, 0.1 mg, 0.2 mg, or 0.3 mg of compound I monofumarate, compound II, or compound III. In some embodiments, doses of 0.05 mg, 0.1 mg, 0.2 mg, or 0.3 mg of compound I monofumarate, compound II, or compound III are administered once, twice, or three times a week as needed. In some embodiments, a 0.05 mg dose of compound I monofumarate, compound II, or compound III is administered at a time specified by a healthcare provider, including daily dosing.

In some embodiments, the topical formulation is applied twice daily, once daily, or several days a week (e.g., 2 or 3 days a week), as long as necessary to achieve the desired result. In some embodiments, the topical formulation is applied on a weekly schedule for one, two, three, four, five, six, or more weeks. In some aspects, the topical formulation is applied on a schedule of three doses per week for two, three, four, five, or six weeks.

In some embodiments, the compound may be administered over one or more treatment cycles, each cycle comprising a treatment period and a withdrawal period, wherein the treatment period comprises administration of the compound as described herein, followed by a withdrawal period (including a period of no treatment), and then the next treatment cycle. In some embodiments, the withdrawal period is from about one day to about six months. In some embodiments, the withdrawal period is one, two, three, four, five, six, seven, eight weeks, or longer before the next treatment cycle. In some embodiments, multiple treatment cycles are administered, such as one, two, three, four, five, or six treatment cycles.

Dosage forms that do not adhere well to the target site may detach, interfering with treatment. Dosage forms that adhere to the target site and dissolve rapidly in low fluid volumes have been found. Adhesion to the target site also prevents exposure to non-target tissues, which may limit toxicity, produce undesirable systemic exposure, and cause side effects. Dosage forms that rapidly soften, decompose, and/or disintegrate in low fluid volumes facilitate the rapid release of the active compound to the target tissue. Dosage forms that disintegrate, for example, in fluids of less than about 50 μL, less than about 100 μL, less than about 125 μL, less than about 150 μL, less than about 175 μL, less than about 200 μL, less than about 250 μL, less than about 500 μL, less than about 1 mL, or less than about 2 mL facilitate drug penetration into the target site.

In some embodiments, the dosage form is a semi-solid, such as a gel or cream. In some embodiments, the dosage form is a tablet. In some embodiments, the dosage form disintegrates within about one to about ten seconds. In some embodiments, the dosage form disintegrates within about ten seconds to one minute; in some embodiments, the dosage form disintegrates within about one minute to about one hour. In some embodiments, the dosage form disintegrates within one to six hours.

The physical dimensions of a dosage form affect its effectiveness. Thinner tablets offer a larger surface area to volume ratio and can degrade more quickly and cover the target area better. In some embodiments, the minimum size of the dosage form is a thickness of less than 3 mm.

Dosage form is crucial for the adequate application of active agents to intraepithelial tissue. For example, formulations can be prepared as tablets, reconstituted powders, dry powders, semi-solid dosage forms, films, or vaginal suppositories (i.e., vaginal suppositories).

Tablet formulations should exhibit mucosal adhesion and substrateivity, and include excipients with solubilizing, erosive (for disintegration), porous (for water absorption), and thickening (to keep the drug at the target site) properties.

Examples of excipients that cause rapid disintegration of solid dosage forms to cover the cervix, anus, penis, perianal area, vulva, or vaginal region include, but are not limited to, mannitol, microcrystalline cellulose, lactose, sucrose, calcium phosphate, sodium phosphate, sodium bicarbonate, citric acid, maleic acid, adipic acid, or fumaric acid. Examples of excipients that can enhance disintegration and coverage of the affected area include, but are not limited to, sodium glycolate starch, pregelatinized starch, crospovidone, and croscarmellose sodium. Mucosal adhesion excipients that can be used in this invention include, but are not limited to, microcrystalline cellulose, polycarboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, and PVP.

Non-limiting examples of tablet formulations include, but are not limited to, microcrystalline cellulose, cropovidone, magnesium stearate, silica, polyethylene oxide, and mannitol. Another non-limiting example of a tablet formulation contains microcrystalline cellulose, magnesium stearate, and mannitol.

Alternative formulations are reconstituted powders or dry powders. These formulations may include the excipients described above, and in some embodiments xanthan gum may be added. As a non-limiting example, dry powder formulations may include, but are not limited to, xanthan gum, mannitol, silica, and sodium benzoate.

Semi-solid dosage forms may include, for example, mucosa-adhesive polymers, solubility/penetration enhancers, lipophilic solubilizers, and penetration enhancers. Mucosa-adhesive polymers may be, for example, but not limited to, carbomer, polyethylene glycol, cyclopovidone, hydroxypropyl methylcellulose, polycarbofil, and/or hydroxyethyl cellulose. Solubility/penetration enhancers may be, for example, but not limited to, polyethylene glycol 6 stearate type I, mixtures of polyethylene glycol stearate and polyethylene glycol 32 stearate type I, cetyl alcohol, stearyl alcohol, polysorbate 80, sodium lauryl sulfate, monoglycerides and diglycerides, sorbitan monostearate, glyceryl isostearate, polyethylene glycol 15-hydroxystearate, poly(15-hydroxystearate), polyethylene glycol 40 hydrogenated castor oil, octyl dodecyl alcohol, and/or soybean lecithin. Lipophilic solubilizers include, but are not limited to, light mineral oil, mineral oil, paraffin wax, and silicone fluid. Penetration enhancers include, but are not limited to, propylene glycol, diethylene glycol monoethyl ether (transcutol), oleic acid, isopropyl myristate, propylene glycol glyceryl monooleate, propylene glycol monocaprylate, PEG-8 beeswax, cetyl alcohol, stearic acid, cetyl palmitate and/or cetearyl alcohol.

Non-limiting examples of semi-solid formulations include, for example, carbomer, propylene glycol, sorbic acid, EDTA, and water. Another non-limiting example of a semi-solid formulation includes carbomer; mineral oil; mixtures of polyethylene glycol 6 stearate type I, polyethylene glycol stearate, and polyethylene glycol 32 stearate type I; parabens; propylene glycol, EDTA, and/or water.

Films can be produced using, for example but not limited to, hydroxypropyl methylcellulose, polyethylene glycol, polymethyl methacrylate, microcrystalline cellulose, xanthan gum, guar gum and/or polyvinylpyrrolidone.

Vaginal suppositories (vaginal suppositories) can be formulated using, for example but not limited to, hardened esters (e.g., Ovucire, Wiptsol, Supposi-Base), polyethylene glycol (polyethylene glycol or macrogol), cocoa butter, and glycerin. Non-limiting examples of vaginal suppositories can be prepared from Wiptsol H 15 or Ovucire WL 3264.

Therefore, the present invention includes at least the following features:

(i) Compound I monofumarate;

(ii) Compound II;

(iii) Compound III;

(iv) Compound IV;

(v) Compound V;

(vi) The compounds described in (i), (ii), (iii), (iv) or (v) are in enantiomer-enriched or enantiomer-pure form;

(vii) The compound as described in (ii), wherein the amount of R by weight is, for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or higher;

(viii) The compound as described in (vii) wherein the amount of the S-stereoconfiguration at the chiral carbon is greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or higher;

(ix) Enantiomerically pure (R,S) compound I;

(x) Enantiomerically pure (S,S) compound I;

(xi)R,S enantiomerically pure compound II is in a highly crystalline form;

(xii) Compound II, Mode 1;

(xiii) Crystal form described in more detail in Part III.

(xiv) A topical pharmaceutical composition comprising an effective amount of the active compound as described herein, or its crystalline form and a pharmaceutically acceptable carrier thereof;

(xv) A topical formulation as described in (xiv), wherein the formulation is in tablet form;

(xvi) The tablet dosage form as described in (xv), the dosage form comprising compound I monofumarate, mannitol, polycrystalline cellulose and magnesium stearate;

(xvii) The tablet dosage form as described in (xv), the dosage form comprising compound II, mannitol, polycrystalline cellulose and magnesium stearate;

(xviii) A topical formulation as described in (xiv), wherein the formulation is in the form of a semi-solid dosage form;

(xix) The semi-solid dosage form as described in (xviii) comprises compound I monofumarate, light mineral oil, propylparaben, 63, water, EDTA, methylparaben and 974P.

(xx) The semi-solid dosage form as described in (xviii) comprises compound I monofumarate, water, EDTA, methyl benzoate, 974P, propylene glycol and optionally sorbic acid;

(xxi) The semi-solid dosage form as described in (xviii) comprises compound II, light mineral oil, propylparaben, 63, water, EDTA, methylparaben, and 974P.

(xxii) The semi-solid dosage form as described in (xviii), the semi-solid dosage form comprising compound II, water, EDTA, methyl benzoate, 974P, propylene glycol and optionally sorbic acid;

(xxiii) The topical formulation as described in (xiv), wherein the formulation is in the form of a reconstructed powder;

(xxiv) The topical formulation as described in (xiv), wherein the formulation is in the form of a dry powder;

(xxv) A topical formulation as described in (xiv), wherein the formulation is in the form of a film;

(xxvi) The topical preparation as described in (xiv), wherein the preparation is in the form of a vaginal suppository;

(xxvii) as described in (xv)-(xxvi) for delivery to the cervix, vagina, vulva, penis, perianal area and/or anus;

(xxviii) A method for treating HPV-induced infections or associated conditions, including but not limited to intraepithelial neoplasia, such as cervical intraepithelial neoplasia, vaginal intraepithelial neoplasia, vulvar intraepithelial neoplasia, perianal intraepithelial neoplasia, anal intraepithelial neoplasia, or penile intraepithelial neoplasia, said method comprising administering to a host in need an effective amount of a crystalline form of a compound or administering to a host in need a pharmaceutical composition as described in any of the above embodiments;

(xxix) Use of any of the embodiments described above in the preparation of a medicament for treating HPV infection or associated conditions in a host in need, including but not limited to intraepithelial neoplasia, such as cervical intraepithelial neoplasia, penile intraepithelial neoplasia, vulvar intraepithelial neoplasia, perianal intraepithelial neoplasia, anal intraepithelial neoplasia or vaginal intraepithelial neoplasia.

Examples (i)-(xxvii) are used to treat HPV infection or associated conditions in a host in need, including but not limited to intraepithelial neoplasia, such as cervical intraepithelial neoplasia, penile intraepithelial neoplasia, vulvar intraepithelial neoplasia, perianal intraepithelial neoplasia, anal intraepithelial neoplasia or vaginal intraepithelial neoplasia.

(xxxi) Any of the above embodiments, wherein the host is a human;

(xxxii) Any of the above-described topical formulations for the treatment of intraepithelial neoplasia are provided in dosage forms containing the following: from 0.005 mg to 50 mg, from 0.05 mg to 40 mg, from 0.1 mg to 30 mg, from 0.05 mg to 0.3 mg, from 0.5 mg to 20 mg, from 1 mg to 20 mg, from 1 mg to 15 mg, from 1 mg to 10 mg of the compound as described in Examples (i)-(v) and in some examples about or at least 0.005, 0.01, 0.03, 0.05, 0.1 mg, 0.3 mg, 0.5 mg, 0.7 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg or 50 mg.

(xxxiii) Any of the above-described topical formulations for treating intraepithelial neoplasia provides a compound comprising, in dosage forms, from about 0.001 to about 20 mg, from about 0.005 to about 10 mg, from about 0.01 mg to about 5 mg, from about 0.03 mg to about 1 mg, from about 0.05 mg to about 0.3 mg, from about 0.03 mg to about 0.07 mg, from about 0.05 to about 0.15 mg, or from about 0.15 mg to about 0.45 mg, the compound as described in Examples (i)-(v).

(xxxiv) The topical formulation as described in Examples (xiv) to (xxvii) may be applied twice daily, once daily, or several days weekly (e.g., 2 or 3 days weekly), as long as necessary to achieve the desired results;

(xxxv) The topical formulation as described in Examples (xiv) to (xxvii) is applied on a weekly schedule for one, two, three, four, five, six or more weeks.

(xxxvi) The method as described in (xxviii) further includes applying a lubricating method to the dosage form before inserting the dosage form into the affected area;

(xxxvii) The method as described in (xxxvi), wherein the lubricant is selected from water, glycerin-based lubricants, and hydroxyethyl cellulose-based lubricants; and

(xxxviii) A method for preparing a topical formulation as described in Examples (xiv) to (xxviii);

(xxxix) A method for treating HPV-induced infection or associated conditions, including but not limited to intraepithelial neoplasia, such as cervical intraepithelial neoplasia, penile intraepithelial neoplasia, vulvar intraepithelial neoplasia, perianal intraepithelial neoplasia, anal intraepithelial neoplasia, or vaginal intraepithelial neoplasia, the method comprising administering an effective amount of the compound as described in Examples (i)-(xxii) to a host in need, in combination with surgical treatment of the target tissue before, during, or after the administration of the compound;

(xl) The method as described in (xxxix) includes surgical treatment of the target tissue followed by administration of an effective amount of the compound as described in Examples (i)-(xxii) to a host in need;

(xli) The method as described in (xxxiv) includes administering an effective amount of the compound as described in Examples (i)-(xxii) to a host in need, followed by surgical treatment of the target tissue;

(xlii) The method as described in (xxxiv) includes administering an effective amount of the compound as described in Examples (i)-(xxii) to the host in need at approximately the same time as the time of surgical treatment of the target tissue.

(xliii) The method as described in Examples (xxxiv)-(xxxvii), wherein the surgical treatment of the target tissue is resection;

(xliv) The method as described in Examples (xxxiv)-(xxxvii), wherein the surgical treatment of the target tissue is ablation;

(xlv) The method described in (xliii) is Circular Electrosurgical Excision Procedure (LEEP);

(xlvi) The method described in (xliii) is a large loop resection of the transformation zone (LLETZ);

(xlvii) The method as described in (xliii), wherein the resection is a scalpel biopsy;

(xlviii) The method as described in (xliii), wherein the resection is laser conization;

(xlix) The method described by (xliv), wherein the ablation is laser ablation; and

(l) The method as described in (xliv), wherein the ablation is cryoablation.

(li) a method for preparing compound II as described herein; and

(lii) is a method for preparing the crystal form as described herein.

Attached Figure Description

Figure 1 is a comparison of the XRPD diffraction patterns of Compound I Mode 1 obtained in the equilibrium experiment in heptane (Example 2, Table 2, Experiment EQ10), the antisolvent precipitation in the acetone/methyl tert-butyl ether (MTBE) system (Example 2, Table 3, Experiment AS3), and the expanded preparation of Compound I Mode 1 as described in Example 3.

Figure 2 is the DSC thermogram of compound I mode 1 obtained in the equilibrium experiment in heptane (Example 2, Table 2, Experiment EQ10).

Figure 3 is the TGA thermogram of compound I mode 1 obtained in the equilibrium experiment in heptane (Example 2, Table 2, Experiment EQ10).

Figure 4 is the XRPD diffraction pattern of compound I, mode 1, prepared in Example 3.

Figure 5 is the DSC thermogram of compound I mode 1 prepared in Example 3.

Figure 6 is the TGA thermogram of compound I mode 1 prepared in Example 3.

Figure 7 is a comparison of the XRPD diffraction patterns of fumaric acid mode, compound IV mode 1 (obtained in Example 7), a mixture of compound IV mode 1 and fumaric acid mode (sample RC2-EA, obtained in Example 5, experimental RC2), and compound IV mode 1 (Example 5, sample RC3-EA).

Figure 8 is a comparison of the XRPD diffraction patterns of compound I monosuccinate mode 1 (Example 5, sample RC7-IPA) and succinic acid mode.

Figure 9 is a comparison of the XRPD diffraction patterns of compound I monosuccinate mode 1 of sample RC7-EA (Example 5, Table 5) and compound I monosuccinate mode 1 of sample AS7-B (Example 5, Table 6).

Figure 10 is a comparison of the XRPD diffraction patterns of fumarate mode, compound I monofumarate mode 1 (Example 5, Table 6, sample AS2-B) and compound IV mode 1 (Example 5, sample AS3-B).

Figure 11 is a comparison of the XRPD diffraction patterns of compound I monosuccinate mode 1 (Example 5, sample AS7-B) and free succinate mode.

Figure 12 is the XRPD diffraction pattern of compound I hemifumarate mode 1 (Example 6, sample RC13).

Figure 13 is the DSC thermogram of compound I hemifumarate mode 1 (Example 6, sample RC13).

Figure 14 is the TGA thermogram of compound I hemifumarate mode 1 (Example 6, sample RC13).

Figure 15 is the XRPD diffraction pattern of compound I monosuccinate mode 1 (Example 6, sample RC14).

Figure 16 is the DSC thermogram of compound I monosuccinate mode 1 (Example 6, sample RC14).

Figure 17 is the TGA thermogram of compound I monosuccinate mode 1 (Example 6, sample RC14).

Figure 18 is a comparison of the XRPD diffraction patterns of compound I monofumarate mode 1 (Example 6, sample RC16) and compound I hemifumarate mode 1 (Example 6, sample RC-13).

Figure 19 is a comparison of the XRPD diffraction patterns of compound I hemisuccinate mode 1 (Example 6, sample RC17) and compound I monosuccinate mode 1 (Example 6, sample RC-14).

Figure 20 is the DSC thermogram of compound I hemisuccinate mode 1 (Example 6, sample RC17).

Figure 21 is the TGA thermogram of compound I hemisuccinate mode 1 (Example 6, sample RC17).

Figure 22 is a comparison of the XRPD diffraction patterns of compound I monofumarate mode 1 (Example 6, sample RC18) and compound I monofumarate mode 1 (Example 6, sample RC-16).

Figure 23 is a comparison of the XRPD diffraction patterns of compound I hemifumarate mode 1 and compound I monofumarate mode 1 (small-scale preparation) obtained in Example 7.

Figure 24 is a DSC thermogram of compound I hemifumarate mode 1 obtained in Example 7.

Figure 25 is the TGA thermogram of compound I hemifumarate mode 1 obtained in Example 7.

Figure 26A is a DSC thermogram of compound I monofumarate mode 1 (small-scale preparation, Example 7), recorded by a recorder at a heating rate of 10 °C/min.

Figure 26B is a DSC thermogram of compound I monofumarate mode 1 (small-scale preparation, Example 7), recorded by a recorder at a heating rate of 2 °C/min.

Figure 26C shows the DSC cycle (DSC cycle, 0-150°C, 150-0°C, 0-250°C, 10°C/min) of compound I monofumarate mode 1 for small-scale sample preparation (Example 7).

Figure 27 is a TGA thermogram of compound I monofumarate mode 1 used for small-scale sample preparation (Example 7).

Figure 28 is a comparison of the XRPD diffraction patterns of the original sample of compound I monofumarate mode 1 obtained in stability test experiments as described in Example 8 at 25°C/84%RH (2 days, open container), 25°C/92%RH (1 week, open container), 40°C/75%RH (1 week, open container), and 60°C (1 week, sealed container).

Figure 29 shows the dynamic gas-phase adsorption (DVS) plot and mass plot of compound I, mode 1 (Example 10), showing the DVS changes.

Figure 30 is a comparison of the XRPD diffraction patterns of compound I mode 1 (obtained in Example 3) before and after the DVS study (Example 10).

Figure 31 shows the DVS changes in the dynamic gas-phase adsorption (DVS) and mass plots for compound I hemifumarate mode 1 (Example 10).

Figure 32 is a comparison of the XRPD diffraction patterns of compound I hemifumarate mode 1 (obtained in Example 7) before and after the DVS study (Example 10).

Figure 33 shows the DVS changes in the dynamic gas-phase adsorption (DVS) and mass plots for compound I monofumarate mode 1 (Example 10).

Figure 34 is a comparison of the XRPD diffraction patterns of compound I monofumarate mode 1 (obtained in Example 7) before and after the DVS study (Example 10).

Figure 35 is a comparison of the XRPD diffraction patterns of compound I monofumarate mode 1 obtained in Example 11 (large scale) before and after heating to 106°C.

Figure 36A is a DSC thermogram of compound I monofumarate mode 1 (Example 11), recorded at a heating rate of 10 °C/min.

Figure 36B is a DSC thermogram of compound I monofumarate mode 1 (large-scale preparation, Example 11), recorded at a heating rate of 2 °C/min.

Figure 36C is the DSC thermogram of compound I monofumarate mode 1 (obtained in Example 11).

Figure 37 is a TGA thermogram of compound I monofumarate mode 1 (obtained in Example 11).

Figure 38 is a comparison of the XRPD diffraction patterns (obtained in Example 12) of compound I monofumarate mode 1, hemifumarate mode 2, hemifumarate mode 3, mode 4 obtained from an equilibrium experiment in water at 25°C for 2 weeks, and fumarate mode.

Figure 39 is a comparison of the XRPD diffraction patterns of compound I mode 4 (obtained in Example 12) obtained from equilibrium experiments in water at 25°C for 2 and 3 weeks.

Figure 40 is the DSC thermogram of compound I mode 4 obtained in an equilibrium experiment in water at 25°C for 2 weeks (obtained in Example 12).

Figure 41 is the DSC thermogram of compound I mode 4 obtained in an equilibrium experiment in water at 25°C for 3 weeks (obtained in Example 12).

Figure 42 is the TGA thermogram of compound I mode 4 obtained in an equilibrium experiment in water at 25°C for 2 weeks (obtained in Example 12).

Figure 43 is the TGA thermogram of compound I mode 4 obtained in an equilibrium experiment in water at 25°C for 3 weeks (obtained in Example 12).

Figure 44 is a comparison of the XRPD diffraction patterns of fumarate mode and compound I hemifumarate mode C obtained in equilibration experiments EQ2 (in acetonitrile) and EQ15 (in 2.9:97.1 v/v water/acetonitrile) for 3 weeks at 25°C.

Figure 45 is the DSC thermogram of compound I hemifumarate mode C obtained in an equilibrium experiment at 25°C for 2 weeks in acetonitrile (obtained in Example 12).

Figure 46 is the TGA thermogram of compound I hemifumarate mode C obtained in an equilibrium experiment at 25°C for 2 weeks (obtained in Example 12).

Figure 47 is a comparison of the XRPD diffraction patterns (obtained in Example 12) of compound I hemifumarate mode 2 obtained in equilibration experiments EQ3 (in methyl ethyl ketone), EQ4 (in acetone), and EQ7 (in 1:1 v/v acetone/heptane) at 25°C for 3 weeks.

Figure 48 is the DSC thermogram of compound I hemifumarate mode 2 obtained in an equilibrium experiment at 25°C for 2 weeks (obtained in Example 12).

Figure 49 is the TGA thermogram of compound I hemifumarate mode 3 obtained in an equilibrium experiment at 25°C for 2 weeks (obtained in Example 12).

Figure 50 is a comparison of the XRPD diffraction patterns (obtained in Example 12) of fumaric acid and compound I monofumarate mode 1 with unknown modes obtained from equilibration experiments EQ5 (in isopropanol), EQ8 (in 1:1 v/v isopropanol/heptane), EQ9 (in 1:1 v/v isopropanol/toluene), EQ10 (in 1:3 v/v isopropanol/methyl tert-butyl ether), and EQ12 (in 1:3 v/v ethanol/heptane) conducted at 25°C for 3 weeks.

Figure 51 is a DSC thermogram of a mixture of compound I monofumarate mode 1 and an unknown mode obtained in EQ5, an equilibrium experiment conducted for 2 weeks at 25°C in isopropanol (obtained in Example 12).

Figure 52 is a TGA thermogram of a mixture of compound I monofumarate mode 1 and an unknown mode obtained in EQ5, an equilibrium experiment conducted for 2 weeks at 25°C in isopropanol (obtained in Example 12).

Figure 53 is a comparison of the XRPD diffraction patterns (obtained in Example 12) of compound I hemifumarate mode 5 obtained in equilibration experiments EQ6 (in 1:1 v/v acetone/toluene) at 25°C for 2 and 3 weeks.

Figure 54 is a DSC thermogram of compound I hemifumarate mode 5 obtained in Example 12, during an equilibrium experiment EQ6 (in 1:1 v/v acetone/toluene) at 25°C for 2 weeks.

Figure 55 is a DSC thermogram of compound I hemifumarate mode 5 obtained in Example 12, during an equilibrium experiment EQ6 (in 1:1 v/v acetone/toluene) at 25°C for 3 weeks.

Figure 56 is a TGA thermogram of compound I hemifumarate mode 5 obtained in Example 12, obtained in equilibration experiment EQ6 (in 1:1 v/v acetone/toluene) for 2 weeks at 25°C.

Figure 57 is a TGA thermogram of compound I hemifumarate mode 5 obtained in Example 12, obtained in equilibration experiment EQ6 (in 1:1 v/v acetone/toluene) for 3 weeks at 25°C.

Figure 58 is a comparison of the XRPD diffraction patterns of compound I monofumarate pattern 1 and compound I monofumarate pattern 1 (material obtained in Example 7) obtained in equilibration experiments (procedure in Example 12) for 2 weeks at 25°C (in EQ11 (in 1:3 v/v tetrahydrofuran/heptane), EQ13 (in 1:3 v/v ethyl acetate/toluene), and EQ14 (in 1:3 v/v ethanol/toluene).

Figure 59 is a comparison of the XRPD diffraction patterns (obtained in Example 12) of compound I monofumarate mode 1 obtained in equilibration experiments EQ11 (in 1:3 v/v tetrahydrofuran/heptane), EQ13 (in 1:3 v/v EA/toluene), and EQ14 (in 1:3 v/v ethanol/toluene) for 3 weeks at 25 °C.

Figure 60 is a comparison of the XRPD diffraction patterns of the unknown pattern obtained from the equilibrium experiment of compound I monofumarate mode 1 and EQ16 (in 1:4 v/v isopropanol/heptane), EQ5 (in isopropanol) at 25°C for 2 weeks, and the mixture of compound I monofumarate mode 1 obtained in Example 7 (obtained in Example 12).

Figure 61 is a comparison of the XRPD diffraction patterns of the reference fumaric acid pattern, compound I monofumarate pattern 1 (Example 7), compound I monofumarate pattern 2 (obtained by precipitation from acetone solution with heptane antisolvent in Experiment AS1, Example 12), compound I monofumarate pattern 2 (obtained by precipitation from methyl ethyl ketone solution with heptane antisolvent in Experiment AS4, Example 12), and the fumaric acid pattern obtained in Experiment AS2, Example 12.

Figure 62 is a comparison of the XRPD diffraction patterns of compound I monofumarate pattern 1 (Example 7), compound I monofumarate pattern 2 (obtained from ethanol solution by precipitation with heptane antisolvent in Experiment AS6, Example 12), and compound I monofumarate pattern 2 (obtained from tetrahydrofuran solution by precipitation with heptane antisolvent in Experiment AS7, Example 12).

Figure 63 is a comparison of the XRPD diffraction pattern of compound I monofumarate mode 1 (Example 7) with that of compound I monofumarate mode 1 obtained by slow evaporation from acetone, methyl ethyl ketone and ethyl acetate by crystallization at room temperature, as described in Table 32 of Example 12.

Figure 64 is a comparison of the XRPD diffraction pattern of compound I monofumarate mode 1 (Example 7) with that of compound I monofumarate mode 1 obtained by slow evaporation from methanol, ethanol, isopropanol and tetrahydrofuran, as described in Example 12, Table 32, by crystallization at room temperature.

Figure 65 is a comparison of the XRPD diffraction patterns of the following compounds: reference fumaric acid pattern; compound I monofumarate pattern 1 (Example 7); compound I monofumarate pattern 2 obtained by combining with a hot saturated solution of methyl ethyl ketone and then slowly cooling; compound I monofumarate pattern 2 obtained by crystallizing from a hot saturated solution of acetone and then slowly cooling; and compound I monofumarate pattern 3 obtained by crystallizing from a hot saturated solution of acetonitrile and then slowly cooling.

Figure 66 is a comparison of the XRPD diffraction patterns of compound I monofumarate pattern 1 (Example 7); compound I monofumarate pattern 1 obtained by crystallization from a hot water-saturated solution followed by slow cooling; and compound I monofumarate pattern 1 obtained by crystallization from a hot water-saturated solution in ethanol/toluene (1/1 v/v) followed by slow cooling (Example 12, Table 33).

Figure 67 is a comparison of the XRPD diffraction patterns of compound I monofumarate pattern 1 (Example 7); compound I monofumarate pattern 2 obtained by rapid cooling crystallization from a hot saturated solution of acetone; and compound I monofumarate pattern 2 obtained by rapid cooling crystallization from a hot saturated solution of methyl ethyl ketone (Example 12, Table 34).

Figure 68 is a comparison of the XRPD diffraction patterns of the following compounds: reference fumaric acid pattern; compound I monofumarate pattern 1 (Example 7); compound I monofumarate pattern 1 obtained by rapid cooling crystallization from a hot water-saturated solution; compound I monofumarate pattern 3 obtained by rapid cooling crystallization from a hot saturated solution in acetonitrile; and compound I monofumarate pattern 1 obtained by rapid cooling crystallization from a hot saturated solution in ethanol/toluene (1/1 v/v) (Example 12).

Figure 69 is a heating-cooling-heating DSC thermogram of compound I monofumarate mode 1 (heated to 106°C) (Example 12, Table 35).

Figure 70 is a heating-cooling-heating DSC thermogram of compound I monofumarate mode 1 (heated to 130°C) (Example 12, Table 35).

Figure 71 is the XRPD diffraction pattern of compound II in mode 1 (Example 13).

Figure 72 is the DSC thermogram of compound II, mode 1 (Example 13).

Figure 73 is the TGA thermogram of compound II, mode 1 (Example 13).

Figure 74 is the XRPD diffraction pattern of compound IV, mode 1 (Example 14).

Figure 75 is the DSC thermogram of compound IV mode 1 (Example 14).

Figure 76 is the TGA thermogram of compound IV mode 1 (Example 14).

Figure 77 is the XRPD diffraction pattern of compound III in mode 1 (Example 15).

Figure 78 is the DSC thermogram of compound III in mode 1 (Example 15).

Figure 79 is the TGA thermogram of compound III, mode 1 (Example 15).

Figure 80 is the XRPD diffraction pattern of compound III in mode 2 (Example 16).

Figure 81 is the DSC thermogram of compound III mode 2 (Example 16).

Figure 82 is the TGA thermogram of compound III mode 2 (Example 16).

Figure 83 is the XRPD diffraction pattern of compound V mode 1 (Example 17).

Figure 84 is the DSC thermogram of compound V mode 1 (Example 17).

Figure 85 is the TGA thermogram of compound V mode 1 (Example 17).

Figure 86 is the XRPD diffraction pattern of compound V mode 2 (Example 18).

Figure 87 is the DSC thermogram of compound V mode 2 (Example 18).

Figure 88 is the TGA thermogram of compound V mode 2 (Example 18).

Figure 89 is a comparison of the XRPD diffraction patterns of compound II mode 1 obtained from the bulk stability study (Example 19).

Figure 90 is a comparison of the XRPD diffraction patterns of compound III mode 2 obtained from volumetric stability studies (Example 19).

Figure 91 shows the DVS changes in the dynamic gas-phase adsorption (DVS) and mass plots for compound II, mode 1 (Example 21).

Figure 92 is a comparison of the XRPD diffraction patterns of compound II mode 1 before and after the DVS study (Example 21).

Figure 93 shows the DVS changes in the dynamic gas-phase adsorption (DVS) plot and mass plot for compound III, mode 2 (Example 21).

Figure 94 is a comparison of the XRPD diffraction patterns of compound III mode 2 before and after the DVS study (Example 21).

Figure 95 is the XRPD diffraction pattern of compound IV mode 1 obtained from compound II mode 1 in Example 22, Experiment PS4.

Figure 96 is the DSC thermogram of compound IV mode 1 obtained from compound II mode 1 in Example 22, Experiment PS4.

Figure 97 is the TGA thermogram of compound IV mode 1 obtained from compound II mode 1 in Example 22, Experiment PS4.

Figure 98 is the XRPD diffraction pattern of compound IV mode 2 obtained from compound II mode 1 in Example 22, Experiment PS5.

Figure 99 is the DSC thermogram of compound IV mode 2 obtained from compound II mode 1 in Example 22, Experiment PS5.

Figure 100 is the TGA thermogram of compound IV mode 2 obtained from compound II mode 1 in Example 22, Experiment PS5.

Figure 101 is the XRPD diffraction pattern of compound III mode 3 obtained from compound III mode 2 in Example 22, Experiment PS3.

Figure 102 is the DSC thermogram of compound III mode 3 obtained from compound III mode 2 in Example 22, Experiment PS3.

Figure 103 is the TGA thermogram of compound III mode 3 obtained from compound III mode 2 in Example 22, Experiment PS3.

Figure 104 is the XRPD diffraction pattern of compound III mode 4 obtained from compound III mode 2 in Example 22, Experiment PS4.

Figure 105 is the DSC thermogram of compound III mode 4 obtained from compound III mode 2 in Example 22, Experiment PS4.

Figure 106 is the TGA thermogram of compound III mode 4 obtained from compound III mode 2 in Example 22, Experiment PS4.

Figure 107 is the XRPD diffraction pattern of compound III mode 5 obtained from compound III mode 2 in Example 22, Experiment PS5.

Figure 108 is the DSC thermogram of compound III mode 5 obtained from compound III mode 2 in Example 22, Experiment PS5.

Figure 109 is the TGA thermogram of compound III mode 5 obtained from compound III mode 2 in Example 22, Experiment PS5.

Figure 110 is the XRPD diffraction pattern of compound III mode 6 obtained from compound III mode 2 in Example 22, Experiment PS9.

Figure 111 is the DSC thermogram of compound III mode 6 obtained from compound III mode 2 in Example 22, Experiment PS9.

Figure 112 is the XRPD diffraction pattern of compound IV mode 1 obtained from compound II mode 1 in Example 22, Experiment PS8.

Figure 113 is a comparison of the XRPD diffraction patterns of compound V mode 1 obtained from compound III mode 2 in Example 22, Experiment PS1, and compound V mode 1 obtained in Example 18.

Figure 114 shows the molecular structure of compound II mode 1 as determined by single-crystal X-ray diffraction analysis in Example 25.

Figure 115 shows the molecular structure of compound II in pattern 1 as determined by single-crystal X-ray diffraction analysis in Example 25. In the single-crystal form of pattern 1, there is an intermolecular interaction between the protonated N5- atom of the free base and the O7- atom of the fumarate anion (N(5)–H(5)···O(7)).

Figure 116 shows an in vitro permeation assay of vaginal tissue, comparing compound II with ABI-1968. Column A shows the tissue penetration of 0.1% compound II gel in porcine vaginal tissue. Columns B and C show the tissue penetration of 0.1% compound II gel in human cervical tissue. Column D shows the tissue penetration of 1% ABI-1968 formulation in 6% NMP in porcine vaginal tissue. Column E shows the tissue penetration of 1% ABI-1968 nanosuspension in porcine vaginal tissue. Column F shows the tissue penetration of 3% ABI-1968 formulation in 6% NMP in porcine vaginal tissue. Column G shows the tissue penetration of 3% ABI-1968 formulation in 20% NMP in porcine vaginal tissue. ABI-1968 penetrates tissue much less, which hinders the compound's ability to reach HPV-infected cells. This may be a contributing factor to the performance of ABI-1968 in clinical studies. Surprisingly, compound II exhibited high tissue penetration in both pig and human tissues. This high tissue penetration may lead to high anti-HPV activity, as described in Example 41.

Figure 117 shows a flowchart of the process for preparing the topical cream formulation described in Example 29.

Figure 118 shows a flowchart of the process for preparing the topical gel formulation described in Example 29.

Figure 119 shows a flowchart of the process for preparing the tablet formulation described in Example 30.

Figure 120 is an XRPD diffraction pattern of (S,S)-compound I with moderate crystallinity as described in Example 12.

Figure 121 is a bar chart comparing the tissue penetration of the topical gel and the topical tablet formulation as described in Example 41. The tissue penetration of the tablet formulation was similar to that of the topical gel, with an average compound concentration of 58 ng/mg in the tissue.

Figure 122 depicts the structures of compound I monofumarate, compound II, and compound III. The synthesis of these compounds is described in Examples 26-28.

Detailed Implementation

Effective compositions for treating HPV infection or HPV-related diseases or conditions (such as HPV-induced intraepithelial neoplasia, including but not limited to cervical intraepithelial neoplasia, perianal intraepithelial neoplasia, penile intraepithelial neoplasia, vulvar intraepithelial neoplasia, anal intraepithelial neoplasia, and vaginal intraepithelial neoplasia) require a combination of multiple factors working together to achieve the desired results. It is necessary to select suitable compounds with favorable lipophilicity and tissue penetration properties, combined with a selected pharmaceutically acceptable salt (optionally in a favorable crystalline form), to achieve the long-sought ability to penetrate epithelial layers of tissue to deliver the active agent in an effective amount. After numerous failures, years of research are needed to address this issue to benefit patients worldwide suffering from potentially carcinogenic intraepithelial neoplasia.

In particular, the key compounds for delivering the active agent were found to be the following specific salts:

Compound I is (ethyl(((2-(2-amino-6-methoxy-9H-purine-9-yl)ethoxy)methyl)(benzyloxy)-phosphoryl)-L-alanine ester). U.S. Patent Nos. 9,801,884 and 11,344,555, assigned to the Board of Trustees of the University of California, generally claim protection for Compound I and pharmaceutically acceptable salts, as well as methods of using said compound and salts for the treatment of human papillomavirus infection. Compound I is an acyclic nucleotide phosphonate that can be metabolized to a known potent antiviral compound (PMEG; ((9-[2-phosphonomethoxy)ethyl)guanine])), but with poor cellular penetration and systemic toxicity limiting its use. The assignee discovered how to improve the local delivery of the prodrug to enable its rapid entry into epithelial cells, a task that has been challenging to date and was a failure of ABI-1968.

Compound I (ethyl(-((2-(2-amino-6-methoxy-9H-purine-9-yl)ethoxy)methyl)-(benzyloxy)phosphoryl)-L-alanine ester) has two chiral centers, one at the phosphorus atom and one at the amino acid moiety, either of which can be either R or S stereochemistry. Therefore, compound I has four stereoisomers. While U.S. Patent Nos. 9,801,884 and 11,344,555 generally describe compound I, these patents do not address the potential stereochemistry of the phosphorus atom. As discussed further herein, stereoisomers of compound I having R-stereochemistry at the phosphorus atom and S-stereochemistry at the amino acid carbon have been found to possess properties superior to the other three stereoisomers.

In non-limiting embodiments, a favorable salt of compound I (e.g., fumarate) is used as a mixture of (R,S) and (S,S) diastereomers, wherein the first R/S specifies the stereochemistry at the phosphorus atom, and the second S is the stereochemistry of the carbon in the amino acid moiety (corresponding to an L-alanine residue having the S-configuration). While any ratio of diastereomers providing the desired results can be used, the (R,S) diastereomer is most prominent. In other embodiments, the ratio of the R to S enantiomers at the phosphorus atom is about 1:1. In some aspects, the compound is enriched in the R enantiomer at the phosphorus atom, wherein the amount of R by weight is, for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or higher.

The S-stereoconfiguration on the chiral carbon corresponding to the natural amino acid configuration is advantageous in this invention. In other aspects, the amount of S by weight is, for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or higher. In alternative embodiments, the R-stereoconfiguration of the chiral carbon is dominant and is greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or higher.

Therefore, in its primary aspect, the present invention comprises optionally administering, in a pharmaceutically acceptable carrier, an effective amount of ( _RP , _SC )ethyl(((2-(2-amino-6-methoxy-9H-purine-9-yl)ethoxy)methyl)(benzyloxy)phosphoryl)-L-alanine fumarate (compound II) as described herein. The present invention provides an acyclic nucleotide pharmaceutical salt, method, composition, and dosage form for treating diseases associated with human papillomavirus (HPV).

The compounds, compositions, and dosage forms described herein may also be used to treat conditions associated with or caused by HPV exposure or infection. For example, the active compounds may be used to treat cervical precancerous lesions, cervical intraepithelial neoplasia, vaginal cancer, vulvar cancer, penile cancer, perianal cancer, and anal intraepithelial neoplasia, cervical cancer, rectal cancer, penile cancer, vaginal cancer, and oropharyngeal cancer.

The active compounds and compositions described herein can also be used to treat infections caused by a range of HPV types. Most carcinogenic HPV types originate from alpha-7 and alpha-9, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82. The most common carcinogenic HPV types are 16 and 18. HPV-16 and HPV-18 have been reported to be the cause of 50% of cervical cancers; and 90% of genital warts are caused by HPV-6 and HPV-11 (World Health Organization, “Cervical Cancer” https://www.who.int/news-room/fact-sheets/detail/cervical-cancer). Infection with one genotype does not preclude future infection with different genotypes.

In one embodiment, compound I monofumarate, compound II, or compound III is used to treat HPV-16. In one embodiment, compound I monofumarate, compound II, or compound III is used to treat HPV-18. In one embodiment, compound I monofumarate, compound II, or compound III is used to treat high-risk HPV infection. In one embodiment, compound I monofumarate, compound II, or compound III is used to treat HPV types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, or 82.

In one embodiment, compounds, formulations, or solid dosage forms containing the compound may also be used preventively to prevent or delay the clinical progression of disease in individuals who are HPV positive or have been exposed to HPV.

In particular, compound II has been found to have superior drug-like and pharmacological properties.

Compound II has R-stereochemistry at the phosphorus atom, which has been confirmed by X-ray crystallography (Example 25, see Figures 114 and 121). In alternative embodiments, Compound II can be used in the form of phosphorus R- and S-enantiomers in any desired ratio, including up to a purity of enantiomers. In some embodiments, Compound II is used in a form free of at least 90% of the opposite enantiomers, and can be at least 98%, 99%, or even 100% free of the opposite enantiomers. Unless otherwise stated, enantiomerically pure Compound II is at least 90% free of the opposite enantiomers. In some embodiments, Compound II is used as a racemic mixture of isomers. Furthermore, in an alternative embodiment, the amino acid of the phosphorimide salt can be D- or L-configured, or a mixture thereof, including a racemic mixture.

If the phosphoramide is chiral, R or S chiral phosphorus derivatives or mixtures thereof can be provided, including enantiomer-enriched forms (including racemic mixtures). All combinations of these stereoconfigurations are alternative embodiments of the invention described herein. In another embodiment, at least one hydrogen atom of compound I, compound II, or compound III may be replaced by deuterium.

In some embodiments, compound I may be:

Or a pharmaceutically acceptable salt thereof. In some embodiments, compound II may be...

In some embodiments, compound III may be

I. Fumarate of ( _RP , _SC ) ethyl(((2-(2-amino-6-methoxy-9H-purin-9-yl)ethoxy)methyl)(benzyloxy)phosphoryl)-L-alanine ester (compound II)

In some embodiments, the active compound of the present invention is compound II, which may be provided as a pharmaceutically acceptable composition or in a solid dosage form thereof. In one embodiment, compound II is an amorphous solid. In another embodiment, compound II is a crystalline solid.

Synthesis of Compound II

The present invention also includes a non-limiting illustrative method for preparing a fumarate (e.g., compound II) of compound I, comprising:

(i) The first step of dissolving the _IRP , _SC isomer of compound in a flask or container containing an organic solvent (e.g., acetone, methanol, ethanol, isopropanol, dichloromethane, tetrahydrofuran or acetonitrile, etc.);

(ii) At ambient temperature or at a temperature that is slightly higher or lower (e.g., 23°C-55°C), fumaric acid is added to the solution of _RP , _SC compound I in step (i) at a specific molar ratio (e.g., 0.5:1.0, 1.0:1.0 or 1.5:1).

(iii) Stir the reaction of step (ii) at ambient temperature or at a temperature that is slightly higher or lower;

(iv) Optionally, seed the solution of step (iii) with crystals of compound II;

(v) Add a second organic solvent to induce crystallization, such as pentane, n-hexane, heptane, petroleum ether, methyl tert-butyl ether, diethyl ether, or water;

(vi) Optionally, the resulting solution is stirred at ambient temperature or at a temperature that is slightly higher or lower than ambient temperature;

(vii) Cool the resulting solution to a lower temperature, for example, about 0°C-10°C, and then stir the solution at that temperature;

(viii) The solid obtained from filtration; and

(ix) Optionally, the solid is dried under reduced pressure and increased temperature, for example at or at least 30°C, 35°C, 40°C, 45°C or 50°C.

In some embodiments, step (i) above is carried out in isopropanol. Furthermore, the second organic solvent in step (v) may be, for example, heptane.

In one embodiment, the _IRP , _SC isomer is dissolved in the ethanol of step (i). In one embodiment, the _IRP , _SC isomer is dissolved in the methanol of step (i). In one embodiment, the _IRP , _SC isomer is dissolved in the acetonitrile of step (i). In another embodiment, the _IRP , _SC isomer is dissolved in the tetrahydrofuran of step (i).

In one embodiment, the second organic solvent in step (v) is pentane. In one embodiment, the second organic solvent in step (v) is hexane. In one embodiment, the second organic solvent in step (v) is methyl tert-butyl ether. In one embodiment, the second organic solvent in step (v) is water.

The present invention further includes a non-limiting illustrative method for preparing compound III, comprising:

(x) The first step of dissolving the I _SP , _SC isomer of compound in a flask or container containing an organic solvent (e.g., acetone, methanol, ethanol, isopropanol, dichloromethane, tetrahydrofuran or acetonitrile).

(xi) At ambient temperature or at a temperature that is slightly higher or lower (e.g., about 23°C to 55°C), fumaric acid is added to the solution of compound I in step (i) at a specific molar ratio (e.g., 0.5:1.0, 1.0:1.0 or 1.5:1).

(xii) Stir the reaction in step (ii) at ambient temperature or at a temperature that is slightly raised or lowered;

(xiii) Optionally, the solution of step (iii) is seeded with crystals of compound II;

(xiv) Add a second organic solvent to induce crystallization, such as pentane, n-hexane, heptane, petroleum ether, methyl tert-butyl ether, diethyl ether, or water;

(xv) The resulting solution is optionally stirred at ambient temperature or at a temperature that is slightly higher or lower;

(xvi) Cool the resulting solution to a reduced temperature, for example, about 0°C-10°C, and then stir the solution at that temperature.

(xvii) the solid obtained from filtration; and

(xviii) Optionally, the solid is dried under reduced pressure and increased temperature, for example at or at least 30°C, 35°C, 40°C, 45°C or 50°C.

In some embodiments, step (i) above is carried out in isopropanol. Furthermore, the second organic solvent in step (v) may be, for example, heptane.

In one embodiment, the _ISP , _SC isomer is dissolved in the ethanol of step (i). In one embodiment, the _ISP , _SC isomer is dissolved in the methanol of step (i). In one embodiment, the _ISP , _SC isomer is dissolved in the acetonitrile of step (i). In another embodiment, the _ISP , _SC isomer is dissolved in the tetrahydrofuran of step (i).

In one embodiment, the second organic solvent in step (v) is pentane. In one embodiment, the second organic solvent in step (v) is hexane. In one embodiment, the second organic solvent in step (v) is methyl tert-butyl ether. In one embodiment, the second organic solvent in step (v) is water.

In some embodiments, monofumarate is synthesized from sesquifumarate (1.5 equivalents of fumaric acid). Sesquifumarate can be washed with a solvent (e.g., methyl tert-butyl ether) to remove excess fumaric acid, thereby providing monofumarate.

II. Salts of Compound I

In some embodiments, the present invention provides compound I, _RP compound I, and _SP compound I as monofumarate. In some embodiments, the present invention provides compound I, _RP compound I, and _SP compound I as hemifumarate. In some embodiments, the present invention provides compound I, _RP compound I, and _SP compound I as sesquifumarate. In some embodiments, the present invention provides _RP compound I and _SP compound I as sulfate. In some embodiments, the present invention provides _RP compound I and _SP compound I as hydrochloride. In some embodiments, the present invention provides compound I, _RP compound I, and _SP compound I as benzenesulfonate. In some embodiments, the present invention provides _RP compound I and _SP compound I as toluenesulfonate. In some embodiments, the present invention provides _RP compound I and _SP compound I as succinate.

_RP salt of compound I

S _P salt of compound I

Hemifumarate and monosuccinate form solids with advantageous properties as solid dosage forms for treating hosts (such as humans) with HPV infection or HPV-related diseases (e.g., cervical intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, vulvar intraepithelial neoplasia, perianal intraepithelial neoplasia, or vaginal intraepithelial neoplasia). However, monofumarate exhibits superior performance compared to hemifumarate and monosuccinate. Therefore, monofumarate remains the preferred salt form of compound I.

Another embodiment of the present invention

In some embodiments, the present invention includes at least:

1. A compound having the following formula:

2. A compound having the following formula:

Or its pharmaceutically acceptable salt.

3. A compound having the following formula:

Or its pharmaceutically acceptable salt.

4. The compound as described in Example 2 has the following formula:

5. The compound as described in Example 3 has the following formula:

6. A separable crystalline form of a compound having the following formula:

The isolated crystalline form is characterized by an XRPD spectrum containing the following peaks, which are independently selected from at least 3, 4, 5, or 6 of the following 2θ values: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°.

7. A separable crystalline form of a compound having the following formula:

The isolated crystalline form is characterized by an XRPD spectrum containing the following peaks, which are independently selected from at least 3, 4, 5, 6, 7, 8, or 9 of the following 2θ values: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

8. A separated crystalline form of a compound having the following formula:

The separated crystalline forms are characterized by XRPD spectra containing peaks independently selected from at least 3, 4, 5, 6, 7, 8, or 9 of the following 2θ values: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, and 17.85±0.2°. 18.15±0.2°, 19.90±0.2°, 20.38±0.2°, 22.94±0.2°, 25.09±0.2°, 26.54±0.2°, 26.90±0.2°, 27.38±0.2°, 28.28±0.2°, 28.95±0.2°, 29.64±0.2° and 38.07±0.2°.

9. A separable crystalline form of a compound having the following formula:

The isolated crystalline form is characterized by an XRPD spectrum containing the following peaks, which are independently selected from at least 3, 4, 5, or 6 of the following 2θ values: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°.

10. A pharmaceutical composition comprising a compound as described in any one of Examples 1-5 and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising a crystalline form as described in any one of Examples 6-9 in a pharmaceutically acceptable carrier.

12. The pharmaceutical composition as described in Examples 10-11 is in solid dosage form.

13. The pharmaceutical composition as described in Examples 10-11 is a semi-solid dosage form.

14. The pharmaceutical composition as described in Examples 10-11 is in reconstituted powder form.

15. The pharmaceutical composition as described in Examples 10-11 is in the form of a dry powder.

16. The pharmaceutical composition as described in Examples 10-11 is in film form.

17. The pharmaceutical composition as described in Examples 10-11, which is in the form of a vaginal suppository.

18. The pharmaceutical composition as described in Example 12, which is in tablet form.

19. The pharmaceutical composition as described in Example 13, which is in the form of an ointment.

20. The pharmaceutical composition as described in Example 13, which is in gel form.

21. The pharmaceutical composition as described in any one of Examples 10-20 is formulated for topical application.

22. The pharmaceutical composition as described in any one of Examples 10-21, for delivery to the cervix.

23. The pharmaceutical composition as described in any one of Examples 10-21, for delivery into the vagina.

24. The pharmaceutical composition as described in any one of Examples 10-21, for delivery to the vulva.

25. The pharmaceutical composition as described in any one of Examples 10-21, for delivery to the perianal area.

26. The pharmaceutical composition as described in any one of Examples 10-21, for delivery to the anus.

27. The pharmaceutical composition as described in any one of Examples 10-21, for delivery to the penis.

28. The pharmaceutical composition as described in Example 18, wherein the tablet is a bilayer tablet.

29. The pharmaceutical composition as described in Example 18, wherein the tablet disintegrates in less than about 250 μL of fluid.

30. The pharmaceutical composition as described in Example 18, wherein the tablet disintegrates in less than about 150 μL of fluid.

31. The pharmaceutical composition of any one of Examples 10-30, comprising a compound in amounts from about 0.001 to about 20 mg, from about 0.005 to about 10 mg, from about 0.01 mg to about 5 mg, from about 0.03 mg to about 1 mg, or from about 0.05 mg to about 0.3 mg.

32. The pharmaceutical composition as described in any one of Examples 10-30, comprising from about 0.005 mg to about 50 mg of the compound.

33. The pharmaceutical composition as described in Example 32, comprising from about 0.05 mg to about 40 mg of the compound.

34. The pharmaceutical composition as described in Example 32, comprising from about 0.1 mg to about 30 mg of the compound.

35. The pharmaceutical composition as described in Example 32, comprising at least about 0.1 mg of the compound.

36. The pharmaceutical composition as described in Example 32, comprising at least about 0.3 mg of the compound.

37. The pharmaceutical composition as described in Example 32, comprising at least about 1 mg of the compound.

38. The pharmaceutical composition as described in Example 32, comprising at least 1.5 mg of the compound.

39. The pharmaceutical composition as described in Example 32, comprising at least about 2 mg of the compound.

40. The pharmaceutical composition of any one of Examples 19-29, comprising from about 0.001% to about 10% of the compound.

41. The pharmaceutical composition as described in Example 40, comprising from about 0.01% to 0.5% of the compound.

42. The pharmaceutical composition as described in Example 40, comprising from about 0.1% to 5% of the compound.

43. The pharmaceutical composition as described in any one of Examples 10-42, comprising a mucosal adhesion polymer.

44. The pharmaceutical composition as described in Example 43, comprising from about 5% to about 20% of a mucosal adhesion polymer.

45. The pharmaceutical composition as described in Example 43, comprising from about 10% to about 50% of a mucosal adhesion polymer.

46. The pharmaceutical composition as described in Example 43, comprising from about 50% to about 90% of a mucosal adhesion polymer.

47. The pharmaceutical composition as described in any one of Examples 10-42, comprising a disintegration-enhancing excipient.

48. The pharmaceutical composition as described in any one of Examples 10-42, comprising a penetration-enhancing excipient.

49. The pharmaceutical composition of any one of Examples 10-42, comprising an excipient that allows for the controlled release of the active compound.

50. The pharmaceutical composition as described in Example 19, wherein the pharmaceutically acceptable carrier comprises: light mineral oil, propylparaben, Tefose 63, water, EDTA, methylparaben, and Carbopol 974P.

51. The pharmaceutical composition as described in Example 20, wherein the pharmaceutically acceptable carrier comprises water, EDTA, methyl benzoate, Carbopol 974P, propylene glycol, and sorbic acid.

52. The pharmaceutical composition 18 as described in the examples, wherein the tablet comprises mannitol, polycrystalline cellulose and magnesium stearate.

53. A method for treating human papillomavirus infection, the method comprising administering to a host in need an effective amount of a compound as described in any one of Examples 1-5, said compound optionally in a pharmaceutically acceptable carrier.

54. A method for treating a condition caused by human papillomavirus infection, the method comprising administering to a host in need an effective amount of a compound as described in any one of Examples 1-5, said compound optionally in a pharmaceutically acceptable carrier.

55. The method as described in Example 54, wherein the condition caused by human papillomavirus infection is intraepithelial neoplasia.

56. The method as described in Example 55, wherein the condition caused by human papillomavirus is atypical squamous cells of undetermined significance (ASC-US).

57. The method as described in Example 55, wherein the condition caused by human papillomavirus is atypical glandular cells (AGC).

58. The method as described in Example 55, wherein the condition caused by human papillomavirus is low-grade squamous intraepithelial lesion (LSIL).

59. The method as described in Example 55, wherein the symptom caused by human papillomavirus is atypical squamous cell disease, and high squamous intraepithelial lesion (ASC-H) cannot be ruled out.

60. The method as described in Example 55, wherein the condition caused by human papillomavirus is high-grade squamous intraepithelial lesion (HSIL).

61. The method as described in Example 54, wherein the condition caused by human papillomavirus is adenocarcinoma in situ (AIS).

62. The method as described in Example 55, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia.

63. The method as described in Example 62, wherein the cervical intraepithelial neoplasia is grade 1 cervical intraepithelial neoplasia.

64. The method as described in Example 62, wherein the cervical intraepithelial neoplasia is grade 2 cervical intraepithelial neoplasia.

65. The method as described in Example 62, wherein the cervical intraepithelial neoplasia is grade 3 cervical intraepithelial neoplasia.

66. The method as described in Example 55, wherein the intraepithelial neoplasia is vaginal intraepithelial neoplasia.

67. The method as described in Example 55, wherein the intraepithelial neoplasia is vulvar intraepithelial neoplasia.

68. The method as described in Example 55, wherein the intraepithelial neoplasia is anal intraepithelial neoplasia.

69. The method as described in Example 55, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia.

70. The method as described in Example 55, wherein the intraepithelial neoplasia is penile intraepithelial neoplasia.

71. The method as described in any one of Examples 53-70, wherein the host is a human being.

72. The method as described in any one of Examples 53-71, wherein the compound is applied topically.

73. The method as described in any one of Examples 52-71, wherein the compound is applied in doses from about 0.05 mg to about 40 mg.

74. The method as described in any one of Examples 52-71, wherein the dosage is from about 0.1 mg to about 30 mg.

75. The method of any one of Examples 52-71, wherein the compound is administered in amounts from about 0.001 to about 20 mg, from about 0.005 to about 10 mg, from about 0.01 mg to about 5 mg, from about 0.03 mg to about 1 mg, from about 0.05 mg to about 0.3 mg, from about 0.03 mg to about 0.07 mg, from about 0.05 mg to about 0.15 mg, or from about 0.15 mg to about 0.45 mg.

76. The method as described in Example 75, wherein the compound is administered in doses ranging from about 0.05 mg to about 0.3 mg.

77. The method as described in any one of Examples 52-76, further comprising applying a lubricating method to the epithelial tissue before inserting the dosage form into the affected area.

78. The method of any one of Examples 52-76, further comprising applying a lubricating method to the dosage form before inserting the dosage form into the affected area.

79. The method as described in Example 77 or 78, wherein the lubrication method is selected from water, glycerin-based lubricants, and hydroxyethyl cellulose-based lubricants.

80. The method as described in any one of Examples 52-79, wherein the compound is administered once daily.

81. The method as described in any one of Examples 52-79, applied twice daily.

82. The method as described in any one of Examples 52-79 is applied twice a week.

83. The method as described in any one of Examples 52-79 is applied three or more times per week.

84. The method as described in any one of Examples 52-83 is applied for about one week.

85. The method as described in any one of Examples 52-83 is applied for approximately two weeks.

86. The method as described in any one of Examples 52-83 is applied for approximately three weeks.

87. The method as described in any one of Examples 52-83 is applied for approximately four weeks.

88. The method as described in any one of Examples 52-83 is applied for approximately five weeks.

89. The method as described in any one of Examples 52-83 is applied for approximately six weeks.

90. The method of any one of Examples 52-89, wherein the compound is administered over a treatment cycle, the treatment cycle comprising:

a. The treatment period, which includes the administration of the compound, and

b. The withdrawal period, which includes the period of no treatment.

91. The method as described in Example 90, wherein the withdrawal period is approximately one week.

92. The method as described in Example 90, wherein the withdrawal period is approximately two weeks.

93. The method as described in Example 90, wherein the withdrawal period is approximately three weeks.

94. The method as described in any one of Examples 52-79, wherein the compound is applied daily.

95. The method as described in Example 94, wherein the dosage is from about 0.01 mg to about 0.5 mg.

96. The method as described in Example 95, wherein the dosage is from about 0.05 mg to about 0.3 mg.

97. The method as described in any one of Examples 90-93, wherein two treatment cycles are administered.

98. The method as described in any one of Examples 90-93, wherein three treatment cycles are administered.

99. The method as described in any one of Examples 90-93, wherein four treatment cycles are administered.

100. The method as described in any one of Examples 90-93, wherein five treatment cycles are administered.

101. The method as described in any one of Examples 90-93, wherein six treatment cycles are administered.

102. The method as described in any one of Examples 52-101, wherein the human papillomavirus is HPV-16.

103. The method as described in any one of Examples 52-101, wherein the human papillomavirus is HPV-18.

104. The method of any one of Examples 52-103, wherein the compound is administered in combination with another antiviral compound.

105. The method of Example 104, wherein the antiviral compound is selected from the group consisting of: protease inhibitors, another DNA polymerase inhibitor, E6 or E6AP inhibitors, E7 inhibitors, E1 inhibitors, E2 inhibitors, E1-E2 protein interaction inhibitors, L2 lipopeptides, L1 inhibitors, L2 inhibitors, L1 degraders, and L2 degraders.

106. The method of any one of Examples 52-103, wherein the compound is administered in combination with an anticancer compound.

107. The method of Example 106, wherein the anticancer compound is selected from the group consisting of: HDAC inhibitors, tetraspanin degraders, immune checkpoint inhibitors, T-cell therapies, and antiproliferative drugs.

108. The method as described in any one of Examples 52-103, wherein the compound is administered in combination with a surgical procedure.

109. The method as described in Example 108, wherein the compound is administered prior to the surgical procedure.

110. The method as described in Example 108, wherein the compound is administered after the surgical procedure.

111. The method as described in Example 108, wherein the surgical procedure is performed during the administration of the compound.

112. The method as described in any one of Examples 108-111, wherein the surgical procedure is the removal of diseased tissue.

113. The method as described in Example 112, wherein the resection is Circular Electrosurgical Excision Procedure (LEEP).

114. The method as described in Example 112, wherein the resection is a large loop resection of the transformation zone (LLETZ).

115. The method as described in Example 112, wherein the resection is a scalpel resection.

116. The method as described in Example 112, wherein the resection is a laser conization resection.

117. The method as described in any one of Examples 108-111, wherein the surgical procedure is an ablation of the diseased tissue.

118. The method as described in Example 117, wherein the ablation is laser ablation.

119. The method as described in Example 117, wherein the ablation is cryoablation.

120. Use of the compounds described in Examples 1-5 in the preparation of a medicament for treating human papillomavirus infection in a host in need, wherein the compounds are optionally in a pharmaceutically acceptable carrier.

121. Use of the compounds described in Examples 1-5 in the preparation of a medicament for treating symptoms caused by human papillomavirus infection in a host in need, wherein the compounds are optionally in a pharmaceutically acceptable carrier.

122. The use as described in Example 121, wherein the condition caused by human papillomavirus infection is intraepithelial neoplasia.

123. The use as described in Example 122, wherein the intraepithelial neoplasia is vaginal intraepithelial neoplasia.

124. The use as described in Example 122, wherein the intraepithelial neoplasia is vulvar intraepithelial neoplasia.

125. The use as described in Example 122, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia.

126. The use as described in Example 122, wherein the intraepithelial neoplasia is anal intraepithelial neoplasia.

127. The use as described in Example 122, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia.

128. The use as described in Example 122, wherein the intraepithelial neoplasia is penile intraepithelial neoplasia.

129. Use as described in Examples 120-128, wherein the host is a human being.

130. For use as described in Examples 120-129, for topical application.

131. The compound of any one of Examples 1-5, for treating human papillomavirus infection in a host in need, wherein the compound is optionally in a pharmaceutically acceptable carrier.

132. The compound of any one of Examples 1-5, for treating symptoms caused by human papillomavirus infection in a host in need, wherein the compound is optionally in a pharmaceutically acceptable carrier.

133. The compound used as described in Example 132, wherein the condition caused by human papillomavirus infection is intraepithelial neoplasia.

134. The compound used as described in Example 133, wherein the intraepithelial neoplasia is vaginal intraepithelial neoplasia.

135. The compound used as described in Example 133, wherein the intraepithelial neoplasia is vulvar intraepithelial neoplasia.

136. The compound used as described in Example 133, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia.

137. The compound used as described in Example 133, wherein the intraepithelial neoplasia is anal intraepithelial neoplasia.

138. The compound used as described in Example 133, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia.

139. The compound used as described in Example 133, wherein the intraepithelial neoplasia is penile intraepithelial neoplasia.

140. The compound used as described in Examples 131-139, wherein the host is human.

141. The compound used as described in Examples 131-140, wherein the compound is applied topically.

142. A method for preparing the crystalline form as described in Example 4, the method comprising:

a. Dissolve _RP compound I in an alcohol solvent;

b. Stir at a temperature ranging from about 20°C to about 70°C;

c. Add 1.0 equivalent of fumaric acid;

d. Add aliphatic solvents;

e. Cool the mixture;

f. Stir the cooled solution; and

g. Separating and drying solids.

143. The method as described in Example 142, wherein the alcohol solvent in step (a) is ethanol or isopropanol.

144. The method as described in Examples 142-143, wherein the alcohol solvent in step (a) is isopropanol.

145. The method as described in Example 142, wherein the solution of step (b) is stirred at about 45°C to about 55°C.

146. The method as described in Example 142, wherein the aliphatic solvent is hexane or heptane.

147. The method as described in Examples 142 and 146, wherein the aliphatic solvent is heptane.

148. The method as described in Example 142, wherein the mixture is cooled to below about 20°C.

149. The method as described in Example 142, wherein the mixture is cooled to below about 10°C.

150. The method as described in Example 142, wherein the mixture is cooled to below about 5°C.

151. The method as described in Example 142, wherein the mixture is cooled to about 5°C to 0°C.

III. Crystal Form

This invention provides a model 1 of the isolated crystalline form of compound I monofumarate. In some embodiments, model 1 of compound I monofumarate is characterized by the XRPD pattern shown in Figure 23 or an XRPD pattern substantially similar thereto.

1. In one embodiment, the compound I monofumarate pattern 1 is characterized by comprising at least about 3, 4 or 5 XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2° and 28.0±0.2°.

2. In one embodiment, the compound I monofumarate pattern 1 is characterized by comprising at least 12 XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

3. In one embodiment, the monofumarate pattern 1 of compound I is characterized by comprising at least 11 XRPD spectra selected from the following 2θ values: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

4. In one embodiment, the compound I monofumarate pattern 1 is characterized by comprising at least 10 XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

5. In one embodiment, the compound I monofumarate pattern 1 is characterized by comprising at least nine XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

6. In one embodiment, the compound I monofumarate pattern 1 is characterized by comprising at least eight XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

7. In one embodiment, the compound I monofumarate mode 1 is characterized by comprising at least seven XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

8. In one embodiment, the compound I monofumarate mode 1 is characterized by comprising at least six XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

9. In one embodiment, the compound I monofumarate pattern 1 is characterized by comprising at least five XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

10. In one embodiment, the monofumarate pattern 1 of compound I is characterized by comprising at least four XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

11. In one embodiment, the compound I monofumarate pattern 1 is characterized by comprising at least three XRPD spectra with 2θ values selected from the following: 6.0±0.2°, 8.9±0.2°, 9.3±0.2°, 9.7±0.2°, 11.9±0.2°, 14.8±0.2°, 18.0±0.2°, 20.0±0.2°, 23.4±0.2°, 25.2±0.2°, 25.9±0.2°, 26.8±0.2°, and 28.0±0.2°.

Compound I monofumarate form 1 can be produced, for example, by crystallization from isopropanol and heptane, as described in Example 7. The free base of Compound I and about 1.0 equivalent of fumaric acid can be dissolved in isopropanol, for example, at a concentration of about 25% to about 40% w/v, and stirred at elevated temperatures, for example, about 45°C, about 50°C, or about 55°C. The solution is stirred at this temperature until some solids form, and then optionally inoculated with the crystalline solids of Compound I monofumarate form 1. The mixture is stirred and cooled to a lower temperature to promote crystallization, for example, below about 40°C, below about 30°C, below about 25°C, below about 20°C, or below about 15°C. The mixture is then stirred while heptane is added in an amount of about 1 mL/mL isopropanol to about 5 mL/mL isopropanol (e.g., about 4 mL/mL isopropanol). The resulting suspension is stirred while the product crystallizes, for example, at 25°C for at least about 24 hours. The suspension is then cooled to further promote crystallization. The solution can be cooled to below about 10°C, below about 5°C, below about 0°C, or below about -5°C. The solution is stirred at the reduced temperature to allow additional time for product crystallization, such as at least about one day, and then the solid is collected by filtration. The collected solid is dried under reduced pressure and optionally at an elevated temperature to provide compound I monofumarate form 1.

This invention provides a separated crystalline form, Mode 1, of compound II. In one embodiment, Mode 1 is characterized by the XRPD pattern shown in FIG71 or an XRPD pattern substantially similar thereto.

1. In one embodiment, compound II mode 1 is characterized by comprising at least 3, 4 or 5 XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2° and 28.82±0.2°.

2. Compound II, Mode 1 as described in Example 1, characterized in that it comprises at least seven XRPD spectra selected from the following 2θ values: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°.

3. Compound II, Mode 1 as described in Example 1, characterized in that it comprises at least six XRPD spectra selected from the following 2θ values: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°.

4. Compound II, Mode 1 as described in Example 1, characterized in that it comprises at least five XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°.

5. Compound II, Mode 1 as described in Example 1, characterized in that it comprises at least four XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°.

6. Compound II, Mode 1 as described in Example 1, characterized in that it comprises at least three XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°.

7. Compound II, Mode 1 as described in Example 1, characterized in that it comprises at least two XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°.

8. Compound II, Mode 1 as described in Example 1, characterized in that it comprises at least one XRPD spectrum with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°.

9. In one embodiment, compound II, mode 1, is characterized by comprising at least 3, 4, or 5 XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 17.45±0.2°, 18.13±0.2°, 19.78±0.2°, 20.14±0.2°, 22.91±0.2°, 23.34±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.99±0.2°, and 28.82±0.2°.

10. Compound II, Mode 1 as described in Example 9, characterized in that it comprises at least 15 XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 17.45±0.2°, 18.13±0.2°, 19.78±0.2°, 20.14±0.2°, 22.91±0.2°, 23.34±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.99±0.2°, and 28.82±0.2°.

11. Compound II, Mode 1 as described in Example 9, characterized in that it comprises at least 14 XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 17.45±0.2°, 18.13±0.2°, 19.78±0.2°, 20.14±0.2°, 22.91±0.2°, 23.34±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.99±0.2°, and 28.82±0.2°.

12. Compound II, Mode 1, as described in Example 9, characterized in that it comprises at least 13 XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 17.45±0.2°, 18.13±0.2°, 19.78±0.2°, 20.14±0.2°, 22.91±0.2°, 23.34±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.99±0.2°, and 28.82±0.2°.

13. Compound II, Mode 1 as described in Example 9, characterized in that it comprises at least 12 XRPD spectra selected from the following 2θ values: 3.08±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 17.45±0.2°, 18.13±0.2°, 19.78±0.2°, 20.14±0.2°, 22.91±0.2°, 23.34±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.99±0.2°, and 28.82±0.2°.

14. Compound II, Mode 1 as described in Example 9, characterized in that it comprises at least 11 XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 17.45±0.2°, 18.13±0.2°, 19.78±0.2°, 20.14±0.2°, 22.91±0.2°, 23.34±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.99±0.2°, and 28.82±0.2°.

15. Compound II, Mode 1, as described in Example 9, characterized in that it comprises at least 10 XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 17.45±0.2°, 18.13±0.2°, 19.78±0.2°, 20.14±0.2°, 22.91±0.2°, 23.34±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.99±0.2°, and 28.82±0.2°.

16. Compound II, Mode 1 as described in Example 9, characterized in that it comprises at least nine XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 17.45±0.2°, 18.13±0.2°, 19.78±0.2°, 20.14±0.2°, 22.91±0.2°, 23.34±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.99±0.2°, and 28.82±0.2°.

17. Compound II, Mode 1, as described in Example 9, characterized in that it comprises at least eight XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 17.45±0.2°, 18.13±0.2°, 19.78±0.2°, 20.14±0.2°, 22.91±0.2°, 23.34±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.99±0.2°, and 28.82±0.2°.

18. In some embodiments, compound II, mode 1, is characterized by comprising at least 3, 4, or 5 XRPD spectra with 2θ values selected from the following: 3.08±0.2°, 6.07±0.2°, 8.80±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 12.60±0.2°, 14.92±0.2°, 15.10±0.2°, 1 7.45±0.2°, 17.838±0.2°, 18.13±0.2°, 18.63±0.2°, 18.89±0.2°, 19.78±0.2°, 20.14±0.2°, 20.81±0.2°, 21.24±0.2°, 21.59±0.2°, 21.89±0.2°, 22.91±0.2°, 23.34±0.2°, 24.2 3±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.13±0.2°, 27.59±0.2°, 27.99±0.2°, 28.82±0.2°, 29.23±0.2°, 29.45±0.2°, 30.39±0.2°, 31.18±0.2°, 31.66±0 0.2°, 32.27±0.2°, 32.69±0.2°, 33.43±0.2°, 34.06±0.2°, 34.34±0.2°, 34.69±0.2°, 35.58±0.2°, 36.19±0.2°, 36.62±0.2°, 37.41±0.2°, 38.30±0.2°, 38.77±0.2° and 39.24±0.2°.

19. Compound II, Mode 1, as described in Example 18, characterized in that it comprises at least 45 XRPD spectra selected from the following 2θ values: 3.08±0.2°, 6.07±0.2°, 8.80±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 12.60±0.2°, 14.92±0.2°, 15.10±0.2°, 17 0.45±0.2°, 17.838±0.2°, 18.13±0.2°, 18.63±0.2°, 18.89±0.2°, 19.78±0.2°, 20.14±0.2°, 20.81±0.2°, 21.24±0.2°, 21.59±0.2°, 21.89±0.2°, 22.91±0.2°, 23.34±0.2°, 24.2 3±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.13±0.2°, 27.59±0.2°, 27.99±0.2°, 28.82±0.2°, 29.23±0.2°, 29.45±0.2°, 30.39±0.2°, 31.18±0.2°, 31.66±0 0.2°, 32.27±0.2°, 32.69±0.2°, 33.43±0.2°, 34.06±0.2°, 34.34±0.2°, 34.69±0.2°, 35.58±0.2°, 36.19±0.2°, 36.62±0.2°, 37.41±0.2°, 38.30±0.2°, 38.77±0.2° and 39.24±0.2°.

20. Compound II, Mode 1, as described in Example 18, characterized in that it comprises at least 40 XRPD spectra selected from the following 2θ values: 3.08±0.2°, 6.07±0.2°, 8.80±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 12.60±0.2°, 14.92±0.2°, 15.10±0.2°, 17 0.45±0.2°, 17.838±0.2°, 18.13±0.2°, 18.63±0.2°, 18.89±0.2°, 19.78±0.2°, 20.14±0.2°, 20.81±0.2°, 21.24±0.2°, 21.59±0.2°, 21.89±0.2°, 22.91±0.2°, 23.34±0.2°, 24.2 3±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.13±0.2°, 27.59±0.2°, 27.99±0.2°, 28.82±0.2°, 29.23±0.2°, 29.45±0.2°, 30.39±0.2°, 31.18±0.2°, 31.66±0 0.2°, 32.27±0.2°, 32.69±0.2°, 33.43±0.2°, 34.06±0.2°, 34.34±0.2°, 34.69±0.2°, 35.58±0.2°, 36.19±0.2°, 36.62±0.2°, 37.41±0.2°, 38.30±0.2°, 38.77±0.2° and 39.24±0.2°.

21. Compound II, Mode 1, as described in Example 18, characterized in that it comprises at least 35 XRPD spectra selected from the following 2θ values: 3.08±0.2°, 6.07±0.2°, 8.80±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 12.60±0.2°, 14.92±0.2°, 15.10±0.2°, 17 0.45±0.2°, 17.838±0.2°, 18.13±0.2°, 18.63±0.2°, 18.89±0.2°, 19.78±0.2°, 20.14±0.2°, 20.81±0.2°, 21.24±0.2°, 21.59±0.2°, 21.89±0.2°, 22.91±0.2°, 23.34±0.2°, 24.2 3±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.13±0.2°, 27.59±0.2°, 27.99±0.2°, 28.82±0.2°, 29.23±0.2°, 29.45±0.2°, 30.39±0.2°, 31.18±0.2°, 31.66±0 0.2°, 32.27±0.2°, 32.69±0.2°, 33.43±0.2°, 34.06±0.2°, 34.34±0.2°, 34.69±0.2°, 35.58±0.2°, 36.19±0.2°, 36.62±0.2°, 37.41±0.2°, 38.30±0.2°, 38.77±0.2° and 39.24±0.2°.

22. Compound II, Mode 1, as described in Example 18, characterized in that it comprises at least 30 XRPD spectra selected from the following 2θ values: 3.08±0.2°, 6.07±0.2°, 8.80±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 12.60±0.2°, 14.92±0.2°, 15.10±0.2°, 17 0.45±0.2°, 17.838±0.2°, 18.13±0.2°, 18.63±0.2°, 18.89±0.2°, 19.78±0.2°, 20.14±0.2°, 20.81±0.2°, 21.24±0.2°, 21.59±0.2°, 21.89±0.2°, 22.91±0.2°, 23.34±0.2°, 24.2 3±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.13±0.2°, 27.59±0.2°, 27.99±0.2°, 28.82±0.2°, 29.23±0.2°, 29.45±0.2°, 30.39±0.2°, 31.18±0.2°, 31.66±0 0.2°, 32.27±0.2°, 32.69±0.2°, 33.43±0.2°, 34.06±0.2°, 34.34±0.2°, 34.69±0.2°, 35.58±0.2°, 36.19±0.2°, 36.62±0.2°, 37.41±0.2°, 38.30±0.2°, 38.77±0.2° and 39.24±0.2°.

23. Compound II, Mode 1, as described in Example 18, characterized in that it comprises at least 25 XRPD spectra selected from the following 2θ values: 3.08±0.2°, 6.07±0.2°, 8.80±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 12.60±0.2°, 14.92±0.2°, 15.10±0.2°, 17 0.45±0.2°, 17.838±0.2°, 18.13±0.2°, 18.63±0.2°, 18.89±0.2°, 19.78±0.2°, 20.14±0.2°, 20.81±0.2°, 21.24±0.2°, 21.59±0.2°, 21.89±0.2°, 22.91±0.2°, 23.34±0.2°, 24.2 3±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.13±0.2°, 27.59±0.2°, 27.99±0.2°, 28.82±0.2°, 29.23±0.2°, 29.45±0.2°, 30.39±0.2°, 31.18±0.2°, 31.66±0 0.2°, 32.27±0.2°, 32.69±0.2°, 33.43±0.2°, 34.06±0.2°, 34.34±0.2°, 34.69±0.2°, 35.58±0.2°, 36.19±0.2°, 36.62±0.2°, 37.41±0.2°, 38.30±0.2°, 38.77±0.2° and 39.24±0.2°.

24. Compound II, Mode 1, as described in Example 18, characterized in that it comprises at least 20 XRPD spectra selected from the following 2θ values: 3.08±0.2°, 6.07±0.2°, 8.80±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 12.60±0.2°, 14.92±0.2°, 15.10±0.2°, 17 0.45±0.2°, 17.838±0.2°, 18.13±0.2°, 18.63±0.2°, 18.89±0.2°, 19.78±0.2°, 20.14±0.2°, 20.81±0.2°, 21.24±0.2°, 21.59±0.2°, 21.89±0.2°, 22.91±0.2°, 23.34±0.2°, 24.2 3±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.13±0.2°, 27.59±0.2°, 27.99±0.2°, 28.82±0.2°, 29.23±0.2°, 29.45±0.2°, 30.39±0.2°, 31.18±0.2°, 31.66±0 0.2°, 32.27±0.2°, 32.69±0.2°, 33.43±0.2°, 34.06±0.2°, 34.34±0.2°, 34.69±0.2°, 35.58±0.2°, 36.19±0.2°, 36.62±0.2°, 37.41±0.2°, 38.30±0.2°, 38.77±0.2° and 39.24±0.2°.

25. Compound II, Mode 1, as described in Example 18, characterized in that it comprises at least 15 XRPD spectra selected from the following 2θ values: 3.08±0.2°, 6.07±0.2°, 8.80±0.2°, 9.30±0.2°, 10.66±0.2°, 12.08±0.2°, 12.60±0.2°, 14.92±0.2°, 15.10±0.2°, 17 0.45±0.2°, 17.838±0.2°, 18.13±0.2°, 18.63±0.2°, 18.89±0.2°, 19.78±0.2°, 20.14±0.2°, 20.81±0.2°, 21.24±0.2°, 21.59±0.2°, 21.89±0.2°, 22.91±0.2°, 23.34±0.2°, 24.2 3±0.2°, 25.14±0.2°, 25.33±0.2°, 25.86±0.2°, 26.78±0.2°, 27.13±0.2°, 27.59±0.2°, 27.99±0.2°, 28.82±0.2°, 29.23±0.2°, 29.45±0.2°, 30.39±0.2°, 31.18±0.2°, 31.66±0 0.2°, 32.27±0.2°, 32.69±0.2°, 33.43±0.2°, 34.06±0.2°, 34.34±0.2°, 34.69±0.2°, 35.58±0.2°, 36.19±0.2°, 36.62±0.2°, 37.41±0.2°, 38.30±0.2°, 38.77±0.2° and 39.24±0.2°.

26. Compound II, Mode 1, as described in any one of Examples 1-25, wherein the ratio of compound II to fumaric acid is approximately 1:1 by ^1H NMR.

Compound II, Mode 1, can be prepared, for example, by recrystallization of compound II (Example 12, Table 31), by equilibration of compound II in a suitable solvent, or by crystallization by slow evaporation of the solvent (Example 12, Table 32). Therefore, the present invention includes at least the following features:

1. In some embodiments, compound II mode 1 is prepared by recrystallization of compound II.

2. In some embodiments, compound II mode 1 is prepared by equilibrating compound II in a solvent.

3. In some embodiments, compound II (mode 1) is prepared by slowly evaporating the solvent from a solution of compound II.

4. The method as described in Example 1, wherein compound II is dissolved in an alcohol solvent and crystallized into pattern 1 by adding an ether solvent.

5. The method as described in Example 1, wherein compound II is dissolved in an alcohol solvent and crystallized into pattern 1 by adding an aliphatic solvent.

6. The method as described in Example 1, wherein compound II is dissolved in an ether solvent and crystallized into pattern 1 by adding an aliphatic solvent.

7. The method as described in Example 4 or 5, wherein the alcohol solvent is or comprises methanol.

8. The method as described in Example 4 or 5, wherein the alcohol solvent is or comprises ethanol.

9. The method as described in Example 4 or 5, wherein the alcohol solvent is or comprises isopropanol.

10. The method as described in Example 4 or 5, wherein the alcohol solvent is or comprises n-propanol.

11. The method as described in Example 4 or 5, wherein the alcohol solvent is or comprises n-butanol.

12. The method as described in Example 4 or 5, wherein the alcohol solvent is or comprises isoamyl alcohol.

13. The method as described in Example 4 or 5, wherein the alcohol solvent is or comprises cyclohexanol.

14. The method as described in Example 4 or 6, wherein the ether solvent is or comprises diethyl ether.

15. The method as described in Example 4 or 6, wherein the ether solvent is or comprises dibutyl ether.

16. The method as described in Example 4 or 6, wherein the ether solvent is or comprises methyl tert-butyl ether.

17. The method as described in Example 4 or 6, wherein the ether solvent is or comprises cyclopropyl methyl ether.

18. The method as described in Example 4 or 6, wherein the ether solvent is or comprises glycol dimethyl ether.

19. The method as described in Example 4 or 6, wherein the ether solvent is or contains tetrahydrofuran.

20. The method as described in Example 4 or 6, wherein the ether solvent is 2-methyltetrahydrofuran.

21. The method as described in Example 4 or 6, wherein the ether solvent is or comprises dioxane.

22. The method of any one of Examples 5-21, wherein the aliphatic solvent is or comprises pentane.

23. The method of any one of Examples 5-21, wherein the aliphatic solvent is or comprises n-hexane.

24. The method of any one of Examples 5-21, wherein the aliphatic solvent is or comprises heptane.

25. The method of any one of Examples 5-21, wherein the aliphatic solvent is or comprises petroleum ether.

26. The method of any one of Examples 5-21, wherein the aliphatic solvent is or comprises octane.

27. The method of any one of Examples 5-21, wherein the aliphatic solvent is or comprises cyclohexane.

28. The method of any one of Examples 5-21, wherein the aliphatic solvent is or a mixture comprising hexane isomers.

29. The method of any one of Examples 4-28, wherein the solids are collected by filtration.

30. The method as described in any one of Examples 4-28, wherein the solution is cooled after the addition of the aliphatic solvent.

31. The method as described in Example 30, wherein the solution is cooled to below about 10°C.

32. The method as described in Example 30, wherein the solution is cooled to about or less than about 5°C.

33. The method of any one of Examples 4-32, wherein the solution, suspension or slurry is stirred until crystallization occurs.

34. The method as described in Example 33, wherein the solution, suspension or slurry is stirred for at least about one day.

35. The method as described in Example 33, wherein the solution, suspension or slurry is stirred for at least about one week.

36. The method as described in Example 33, wherein the solution, suspension or slurry is stirred for at least about two weeks.

37. The method as described in Example 33, wherein the solution, suspension or slurry is stirred for at least about three weeks.

38. The method as described in Example 2, wherein compound II is dissolved in an equilibrium solvent at 25°C and stirred for a period of time from about 2 weeks to about 3 weeks.

39. The method as described in Example 2, wherein the equilibrium solvent is a binary mixture of solvents.

40. The method as described in Example 2, wherein the equilibrium solvent is isopropanol.

41. The method as described in Example 39, wherein the equilibrium solvent is about a portion of tetrahydrofuran to about a third portion of heptane.

42. The method as described in Example 39, wherein the equilibrium solvent is about a portion of ethanol to about three portions of heptane.

43. The method as described in Example 39, wherein the equilibrium solvent is about a portion of ethyl acetate to about three portions of toluene.

44. The method as described in Example 39, wherein the equilibrium solvent is about a portion of isopropanol to about a portion of heptane.

45. The method as described in Example 39, wherein the equilibrium solvent is about a portion of isopropanol to about a portion of toluene.

46. The method as described in Example 39, wherein the equilibrium solvent is about a portion of isopropanol to about three portions of methyl tert-butyl ether.

47. The method as described in Example 39, wherein the equilibrium solvent is about a portion of isopropanol to about a quarter of heptane.

48. The method as described in Example 39, wherein the equilibrium solvent is about a portion of ethanol to about a portion of toluene.

49. The method as described in Example 3, wherein compound II is dissolved in a suitable solvent, filtered through a 0.45 μM filter, and then maintained at 23°C and one atmosphere until the solvent evaporates.

50. The method as described in Example 49, wherein the solvent is or comprises acetone.

51. The method as described in Example 49, wherein the solvent is or comprises methyl ethyl ketone.

52. The method as described in Example 49, wherein the solvent is or comprises ethyl acetate.

53. The method as described in Example 49, wherein the solvent is or comprises methanol.

54. The method as described in Example 49, wherein the solvent is or comprises ethanol.

55. The method as described in Example 49, wherein the solvent is or comprises isopropanol.

56. The method as described in Example 49, wherein the solvent is or comprises tetrahydrofuran.

This invention provides isolated crystalline forms, Mode 1 and Mode 2, of Compound III. In one embodiment, Mode 1 is characterized by the XRPD pattern shown in Figure 77 or an XRPD pattern substantially similar thereto.

1. In one embodiment, compound III, mode 1, is characterized by independently comprising at least 2, 3, 4, 5, or 6 XRPD spectra with 2θ values selected from the following: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

2. Compound III, Mode 1, as described in Example 1, is characterized by comprising at least 12 XRPD spectra with 2θ values selected from the following: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

3. Compound III, Mode 1 as described in Example 1, characterized in that it comprises at least 11 XRPD spectra selected from the following 2θ values: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

4. Compound III, Mode 1, as described in Example 1, characterized in that it comprises at least 10 XRPD spectra with 2θ values selected from the following: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

5. Compound III, Mode 1 as described in Example 1, characterized in that it comprises at least nine XRPD spectra with 2θ values selected from the following: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

6. Compound III, Mode 1 as described in Example 1, characterized in that it comprises at least eight XRPD spectra with 2θ values selected from the following: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

7. Compound III, Mode 1, as described in Example 1, characterized in that it comprises at least seven XRPD spectra with 2θ values selected from the following: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

8. Compound III Mode 1 as described in Example 1, characterized in that it comprises at least six XRPD spectra selected from the following 2θ values: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

9. Compound III, Mode 1 as described in Example 1, characterized in that it comprises at least five XRPD spectra with 2θ values selected from the following: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°.

10. In one embodiment, compound III mode 2 is characterized by independently comprising at least 3, 4, 5 or 6 XRPD spectra with 2θ values selected from the following: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 20.38±0.2°, 22.94±0.2°, 25.09±0.2°, 26.54±0.2°, 26.90±0.2°, 27.38±0.2°, 28.28±0.2°, 28.95±0.2°, 29.64±0.2° and 38.07±0.2°.

11. Compound III Mode 2 as described in Example 10, characterized in that it comprises at least 12 XRPD spectra selected from the following 2θ values: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 20.38±0.2°, 22.94±0.2°, 25.09±0.2°, 26.54±0.2°, 26.90±0.2°, 27.38±0.2°, 28.28±0.2°, 28.95±0.2°, 29.64±0.2°, and 38.07±0.2°.

12. Compound III Mode 2 as described in Example 10, characterized in that it independently comprises at least 3, 4, 5 or 6 XRPD spectra with 2θ values selected from the following: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 25.09±0.2°, 29.64±0.2° and 38.07±0.2°.

13. Compound III Mode 2 as described in Example 10, characterized in that it comprises at least 11 XRPD spectra selected from the following 2θ values: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 25.09±0.2°, 29.64±0.2°, and 38.07±0.2°.

14. Compound III Mode 2 as described in Example 10, characterized in that it comprises at least 10 XRPD spectra with 2θ values selected from the following: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 25.09±0.2°, 29.64±0.2°, and 38.07±0.2°.

15. Compound III Mode 2 as described in Example 10, characterized in that it comprises at least nine XRPD spectra selected from the following 2θ values: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 25.09±0.2°, 29.64±0.2°, and 38.07±0.2°.

16. Compound III Mode 2 as described in Example 10, characterized in that it comprises at least eight XRPD spectra with 2θ values selected from the following: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 25.09±0.2°, 29.64±0.2°, and 38.07±0.2°.

17. Compound III Mode 2 as described in Example 10, characterized in that it comprises at least seven XRPD spectra with 2θ values selected from the following: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 25.09±0.2°, 29.64±0.2°, and 38.07±0.2°.

18. Compound III Mode 2 as described in Example 10, characterized in that it comprises at least six XRPD spectra selected from the following 2θ values: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 25.09±0.2°, 29.64±0.2°, and 38.07±0.2°.

19. Compound III Mode 2 as described in Example 10, characterized in that it comprises at least five XRPD spectra selected from the following 2θ values: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 25.09±0.2°, 29.64±0.2°, and 38.07±0.2°.

For example, Compound III Mode 1 can be produced by precipitation of isopropanol and heptane (Example 15, Table 40). In some non-limiting embodiments, Compound III Mode 1 is prepared by dissolving the free base of _SP -compound I in isopropanol (e.g., about 100 mg of _SP -compound I dissolved in about 0.25 mL to about 0.5 mL of isopropanol). About 1.0 eq of fumaric acid is added to the solution, and the mixture is stirred at ambient temperature or an elevated temperature (e.g., about 25 °C to about 60 °C). Next, about two to five times the amount of heptane is added. The resulting mixture is stirred at ambient temperature or an elevated temperature (e.g., about 25 °C to about 60 °C), then gradually cooled (e.g., about 0.01 °C/min to 1 °C/min), the separated solids are filtered, and then dried under ambient pressure or reduced pressure.

IV. Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, applications, and published applications cited herein are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions in this section shall prevail unless otherwise stated.

As used herein, unless otherwise stated, any abbreviations for protecting groups, amino acids and other compounds are in accordance with their common usage, accepted abbreviations or the IUPAC-IUB Biochemical Nomenclature Committee (see Biochem. 11:942-944 (1972)).

As used herein, the term "alcohol solvent" refers to a solvent having a free hydroxyl group that is a liquid at room temperature or at the operating temperature. Non-limiting examples of alcohol solvents include methanol, ethanol, ethylene glycol, isopropanol, n-propanol, glycerol, n-butanol, isobutanol, tert-butanol, 2-butanol, n-pentanol (n-pentanol), isopentanol (isopentanol), neopentanol, hexanol, cyclohexanol, cyclohexanediol, phenol, benzyl alcohol, propynyl alcohol, diethylene glycol, 1,2-propanediol, heptanol, octanol, nonanol, and decanol.

As used herein, the term "aliphatic solvent" refers to a hydrocarbon solvent that is a liquid at room temperature or at its operating temperature. Non-limiting examples of aliphatic solvents include pentane, isopentane, cyclopentane, n-hexane, hexane (a mixture of isomers), cyclohexane, 2-hexene, 3-hexene, methylcyclohexane, heptane, octane, isooctane, petroleum ether, naphtha, mineral oil, nonane, decane, undecane, and dodecane.

As used herein, the term "ethereal solvent" refers to a solvent containing at least one ether bond and being a liquid at room temperature or the operating temperature. Examples of ethereal solvents include, but are not limited to, diethyl ether, diisopropyl ether, methyl tert-butyl ether, dibutyl ether, tert-amylethyl ether, cyclopentylmethyl ether, di-tert-butyl ether, ethyl tert-butyl ether, propylene glycol methyl ether, ethylene glycol ethyl ether, glycol dimethyl ether, diethylene glycol dimethyl ether, ethylene glycol ether, dioxane, tetrahydrofuran, 2-methyltetrahydrofuran, 4-methyltetrahydrofuran, and tetrahydropyran.

When a range of values is provided, it is understood that the upper and lower limits of the range, as well as each intermediate value between the upper and lower limits, are included in the embodiments.

As used herein, “about” refers to a range that includes values less than the value by a maximum of 10% and greater than the value by a maximum of 10%. For example, “about 100 mg” includes all values from 90 mg to 110 mg.

The terms "therapeutic effective amount" and "effective amount" are used to indicate the amount of an active compound, drug, or its metabolite that produces the intended therapeutic effect. For example, an effective amount of a compound may be the amount required to prevent, reduce, or improve disease symptoms or prolong the survival of a subject (e.g., a human). This response may occur in tissues, systems, animals, or humans, including the relief of symptoms or signs of the disease being treated.

Isotope substitution

This invention includes, but is not limited to, the use of the compounds, pharmaceutical compositions, and any active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, whose atoms have a desired isotopic substitution amount higher than the natural abundance of the isotope, i.e., enrichment. An isotope is an atom having the same atomic number but different mass numbers, i.e., atoms with the same number of protons but different numbers of neutrons. As a general example and without limitation, hydrogen isotopes, such as deuterium ( ^2H ) and tritium ( ^3H ), can be used at any position in the described structure. Alternatively, carbon isotopes, such as ^13C and ^14C , can also be used. Preferred isotopic substitution is replacing hydrogen with deuterium at one or more positions on the molecule to improve the properties of the drug. During metabolism, deuterium can bind at the bond-breaking site (α-deuterium kinetic isotope effect) or near the bond-breaking site (β-deuterium kinetic isotope effect). Achillion Pharmaceuticals, Inc. (WO/2014/169278 and WO/2014/169280) describes the deuteration of nucleotides to improve their pharmacokinetics or pharmacodynamics, including the 5-position of the molecule.

Substitution with an isotope (e.g., deuterium) can provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirements. Replacing hydrogen with deuterium at a metabolic breakdown site can reduce or eliminate the metabolic rate of that bond. Hydrogen atoms can be present at any position in a compound, and these hydrogen atoms can be any isotope of hydrogen, including proton ( ^¹H ), deuterium ( ^²H ), and tritium ( ^³H ). Therefore, unless the context clearly specifies otherwise, the compounds mentioned herein encompass all potential isotopic forms.

The term "isotope-labeled" analogue refers to a "deuterated analogue," a " ^13C -labeled analogue," or a "deuterated/ ^13C -labeled analogue." The term "deuterated analogue" refers to the compounds described herein in which a hydrogen isotope, i.e., hydrogen/proton ( ^¹H ), is substituted with a hydrogen isotope, such as deuterium ( ^²H ). Deuteration substitution can be partial or complete. Partial deuteration substitution means that at least one hydrogen atom is substituted with at least one deuterium atom. In some embodiments, the isotope is an isotope enriched at 80%, 85%, 90%, 95%, or 99% or more at any site of interest. In some embodiments, deuterium is enriched at 90%, 95%, or 99% at the desired site. Unless otherwise stated, the deuteration rate at the selected site is at least 80%. Deuteration of nucleosides can occur on any substitute hydrogen atom that provides the desired result.

Treatment or prevention of HPV-induced intraepithelial neoplasia

In an exemplary non-limiting embodiment, a method for treating HPV infection or HPV-induced intraepithelial neoplasia is provided, the method comprising administering an effective amount of one or a combination of active compounds as described herein, in a topical formulation, sufficient to treat the neoplasia or produce the effects further described herein. Types of HPV-induced intraepithelial neoplasia include, but are not limited to, cervical intraepithelial neoplasia, vaginal intraepithelial neoplasia, vulvar intraepithelial neoplasia, penile intraepithelial neoplasia, perianal intraepithelial neoplasia, and anal intraepithelial neoplasia.

In exemplary embodiments, the formulation for treating intraepithelial neoplasia is a dosage form containing from about 0.005 mg to about 50 mg, from about 0.05 mg to about 40 mg, from about 0.1 mg to about 30 mg, from about 0.5 mg to about 20 mg, from about 1 mg to about 20 mg, from about 1 mg to about 15 mg, or from about 1 mg to about 10 mg of any active compound described herein (including, but not limited to, compound I monofumarate, compound II, or compound III). In some embodiments, the formulation for treating intraepithelial neoplasia is a dosage form containing from about 0.01 mg to about 10 mg, from about 0.05 mg to about 5 mg, from about 0.05 mg to about 0.15 mg, from about 0.15 mg to about 0.45 mg, or from about 0.5 mg to about 1.5 mg of any active compound described herein (including, but not limited to, compound I monofumarate, compound II, or compound III). In some embodiments, the formulation for treating intraepithelial neoplasia is a dosage form comprising about or at least 0.005, 0.01, 0.03, 0.05, 0.1 mg, 0.3 mg, 0.5 mg, 0.7 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg of compound I monofumarate, compound II, or compound III.

In some embodiments, the formulation for treating intraepithelial neoplasia is a dosage form containing from about 0.001 to about 20 mg, from about 0.005 to about 10 mg, from about 0.01 mg to about 5 mg, from about 0.03 mg to about 1 mg, or from about 0.05 mg to about 0.3 mg of compound I monofumarate, compound II, or compound III.

In some embodiments, the topical formulation is applied twice daily, once daily, or several days a week (e.g., 2 or 3 days a week), as long as necessary to achieve the desired result. In some embodiments, the topical formulation is applied on a weekly schedule for one, two, three, four, five, six, or more weeks. In some aspects, the topical formulation is applied on a schedule of three doses per week for two, three, four, five, or six weeks.

In some embodiments, the compound may be administered over one or more treatment cycles, each cycle comprising a treatment period and a withdrawal period, wherein the treatment period comprises administration of the compound as described herein, followed by a withdrawal period (including a period of no treatment), and then the next treatment cycle. In some embodiments, the withdrawal period is from about one day to about six months. In some embodiments, the withdrawal period is one, two, three, four, five, six, seven, eight weeks, or longer before the next treatment cycle. In some embodiments, multiple treatment cycles are administered, such as one, two, three, four, five, or six treatment cycles.

Dosage forms that do not adhere well to the target site may detach, interfering with treatment. Dosage forms that adhere to the target site and dissolve rapidly in low fluid volumes have been found. Adhesion to the target site also prevents exposure to healthy tissue, which may limit toxicity and side effects. Dosage forms that rapidly soften, decompose, and/or disintegrate in low fluid volumes facilitate rapid release of the active compound to the target tissue. Dosage forms that disintegrate, for example, in fluids of less than about 50 μL, less than about 100 μL, less than about 125 μL, less than about 150 μL, less than about 175 μL, less than about 200 μL, or less than about 250 μL are particularly helpful for drug penetration into the target site.

In some embodiments, the dosage form is a gel. In some embodiments, the dosage form is a cream. In some embodiments, the dosage form is a tablet. In some embodiments, the dosage form disintegrates in about one to about ten seconds. In some embodiments, the dosage form disintegrates in about ten seconds to one minute. In some embodiments, the dosage form disintegrates in about one minute to about one hour. In some embodiments, the dosage form disintegrates in about one to six hours.

The physical dimensions of a dosage form can affect its effectiveness. Thinner tablets offer a larger surface area to volume ratio and can degrade more quickly and cover the target area better. In some embodiments, the minimum size of the dosage form is a thickness of less than about 6, 5, 4, 3, or 2 millimeters.

The formulation is important for ensuring adequate delivery of the active agent to the intraepithelial tissue. For example, formulations can be prepared as tablets, reconstituted powders, dry powders, semi-solid dosage forms, films, or vaginal suppositories (i.e., vaginal suppositories).

Some embodiments disclosed herein include the use of effective amounts of compound I monofumarate, compound II, or compound III in the preparation of a medicament for improving or treating human papillomavirus (HPV) infection, wherein the infection can be improved or treated by inhibiting viral replication through inhibition of viral DNA synthesis. Other embodiments disclosed herein include the use of effective amounts of any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) for improving or treating HPV infection, wherein the HPV infection can be improved or treated by inhibiting viral replication through inhibition of viral DNA synthesis.

Some non-limiting embodiments disclosed herein include methods for improving or treating human papillomavirus (HPV) infection, the methods comprising contacting HPV-infected cells in a subject with an effective amount of any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III), wherein the infection is improved or treated by inhibiting viral DNA synthesis. Other embodiments disclosed herein include methods for improving or treating HPV infection, the methods comprising administering an effective amount of compound I monofumarate, compound II, or compound III to a subject infected with HPV, wherein the HPV infection can be improved or treated by inhibiting viral replication through inhibition of viral DNA synthesis. Some embodiments disclosed herein relate to compound I monofumarate, compound II, or compound III for use in improving or treating HPV infection, wherein the HPV infection can be improved or treated by inhibiting viral replication through inhibition of viral DNA synthesis.

In some embodiments, the human papillomavirus (HPV) may be a high-risk HPV, such as those described herein. For example, a high-risk HPV may be selected from HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-73, and HPV-82. In some embodiments, the HPV may be HPV-16. In some embodiments, the HPV may be HPV-11. In some embodiments, the HPV may be HPV-18. In some embodiments, the human papillomavirus (HPV) may be one or more of the following high-risk types: HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68, HPV-73, and HPV-82. As described herein, the presence of HPV infection can be detected using a Pap smear (cervical smear) and/or a DNA probe test (e.g., an HPV DNA probe test targeting one or more high-risk HPV types). Therefore, in some embodiments, an effective amount of compound I monofumarate, compound II, or compound III may be provided to a subject diagnosed with HPV infection, such as a high-risk HPV infection, by a DNA test. In some embodiments, an effective amount of compound I monofumarate, compound II, or compound III may be provided to a subject diagnosed with HPV infection or an HPV-related condition identified by a Pap smear test.

In some embodiments, subjects may be given an effective amount of compound I monofumarate, compound II, or compound III, wherein the Pap smear result does not indicate that the disease has progressed to cervical cancer. The Bethesda system is a standardized scoring system for reporting Pap smear test results, assigning results to grades 1–3 according to severity. Grade 1 CIN (CIN 1) indicates mild dysplasia. Grades 2 and 3 CIN (CIN 2, CIN 3) are more severe and usually require intervention. In some embodiments, compound I monofumarate, compound II, or compound III is used to treat CIN 1 (grade 1 cervical intraepithelial neoplasia). In some embodiments, compound I monofumarate, compound II, or compound III is used to treat CIN 2 (grade 2 cervical intraepithelial neoplasia). In some embodiments, compound I monofumarate, compound II, or compound III is used to treat CIN 3 (grade 3 cervical intraepithelial neoplasia).

In some embodiments, a pharmaceutical composition comprising compound I monofumarate, compound II, or compound III is used in the preparation of a medicament for treating CIN 1 (grade 1 cervical intraepithelial neoplasia). In some embodiments, a pharmaceutical composition comprising compound I monofumarate, compound II, or compound III is used in the preparation of a medicament for treating CIN 2 (grade 2 cervical intraepithelial neoplasia). In some embodiments, a pharmaceutical composition comprising compound I monofumarate, compound II, or compound III is used in the preparation of a medicament for treating CIN 3 (grade 3 cervical intraepithelial neoplasia).

In some embodiments, compound I monofumarate, compound II, or compound III, optionally in a pharmaceutically acceptable carrier, is used to treat conditions selected from the group consisting of: atypical squamous cells of undetermined significance (ASC-US), atypical glandular cells (AGC), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cells (with high-grade squamous intraepithelial lesion not excluded) (ASC-H), high-grade squamous intraepithelial lesion (HSIL), adenocarcinoma in situ (AIS), and cervical cancer (e.g., squamous cell carcinoma or adenocarcinoma).

In some embodiments, an effective amount of compound II may be provided to the subject, wherein the Pap test result does not indicate that the disease has progressed to cervical cancer. In some embodiments, compound II is used to treat CIN 1 (grade 1 cervical intraepithelial neoplasia). In some embodiments, compound II is used to treat CIN 2 (grade 2 cervical intraepithelial neoplasia). In some embodiments, compound II is used to treat CIN 3 (grade 3 cervical intraepithelial neoplasia).

In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating CIN 1 (grade 1 cervical intraepithelial neoplasia). In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating CIN 2 (grade 2 cervical intraepithelial neoplasia). In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating CIN 3 (grade 3 cervical intraepithelial neoplasia).

In some embodiments, compound II, optionally in a pharmaceutically acceptable carrier, is used to treat conditions selected from the group consisting of: atypical squamous cells of undetermined significance (ASC-US), atypical glandular cells (AGC), low-grade squamous intraepithelial lesion (LSIL), atypical squamous cells (with high-grade squamous intraepithelial lesion not excluded) (ASC-H), high-grade squamous intraepithelial lesion (HSIL), adenocarcinoma in situ (AIS), and cervical cancer (e.g., squamous cell carcinoma or adenocarcinoma).

In some embodiments, compound I (monofumatate), compound II, or compound III is used in the preparation of a medicament for treating anal intraepithelial neoplasia. In some embodiments, compound I (monofumatate), compound II, or compound III is used in the preparation of a medicament for treating perianal intraepithelial neoplasia. In some embodiments, compound I (monofumatate), compound II, or compound III is used in the preparation of a medicament for treating vulvar intraepithelial neoplasia. In some embodiments, compound I (monofumatate), compound II, or compound III is used in the preparation of a medicament for treating penile intraepithelial neoplasia. In some embodiments, compound I (monofumatate), compound II, or compound III is used in the preparation of a medicament for treating vaginal intraepithelial neoplasia.

In some embodiments, compound I (monofumatate), compound II, or compound III is used to treat anal intraepithelial neoplasia. In some embodiments, compound I (monofumatate), compound II, or compound III is used to treat perianal intraepithelial neoplasia. In some embodiments, compound I (monofumatate), compound II, or compound III is used to treat vulvar intraepithelial neoplasia. In some embodiments, compound I (monofumatate), compound II, or compound III is used to treat penile intraepithelial neoplasia. In some embodiments, compound I (monofumatate), compound II, or compound III is used to treat vaginal intraepithelial neoplasia.

In some embodiments, the human papillomavirus (HPV) may be a low-risk HPV type, including those described herein. In some embodiments, the HPV may be HPV-6. In some embodiments, the HPV may be HPV-11.

Compound I (monofumatate), Compound II, or Compound III can be used to improve and/or treat infections caused by one or more types of human papillomavirus (HPV). For example, Compound I (monofumatate), Compound II, or Compound III can be used to improve and/or treat HPV-16 and/or HPV-18 infections. In some embodiments, Compound I (monofumatate), Compound II, or Compound III can be used to treat high-risk HPV infections. In some embodiments, Compound I (monofumatate), Compound II, or Compound III can be used to treat associated diseases or conditions that occur as a result of high-risk HPV infection. In some embodiments, Compound I (monofumatate), Compound II, or Compound III can be used to improve and/or treat infections including both high-risk and low-risk HPV types.

Compound I (monofumatate), Compound II, or Compound III may be used in the manufacture of medicaments for improving and/or treating infections caused by one or more types of human papillomavirus. For example, Compound I (monofumatate), Compound II, or Compound III may be used in the preparation of medicaments for improving and/or treating HPV-16 and/or HPV-18 infections. In some embodiments, Compound I (monofumatate), Compound II, or Compound III may be used in the preparation of medicaments for treating high-risk HPV infections. In some embodiments, Compound I (monofumatate), Compound II, or Compound III may be used in the preparation of medicaments for treating related diseases or conditions that occur as a result of high-risk HPV infection. In some embodiments, Compound I (monofumatate), Compound II, or Compound III may be used in the preparation of medicaments for improving and/or treating infections including both high-risk and low-risk HPV types.

It will be apparent to those skilled in the art that the useful in vivo dose and specific administration method will vary depending on age, weight, severity of illness, the specific compound used, and the specific purpose for which the compound is used. The determination of the effective dose level, i.e., the dose level necessary to achieve the desired results, can be accomplished by those skilled in the art using conventional methods, such as human clinical trials and in vitro studies.

In some embodiments, the pharmaceutical composition comprising compound II is used to treat conditions associated with or resulting from HPV exposure or infection. In some embodiments, the pharmaceutical composition comprising compound II is used to treat cervical precancerous lesions. In some embodiments, the pharmaceutical composition comprising compound II is used to treat cervical intraepithelial neoplasia. In some embodiments, the pharmaceutical composition comprising compound II is used to treat vaginal and anal intraepithelial neoplasia. In some embodiments, the pharmaceutical composition comprising compound II is used to treat cervical cancer. In some embodiments, the pharmaceutical composition comprising compound II is used to treat rectal cancer. In some embodiments, the pharmaceutical composition comprising compound II is used to treat penile cancer. In some embodiments, the pharmaceutical composition comprising compound II is used to treat vaginal cancer. In some embodiments, the pharmaceutical composition comprising compound II is used to treat oropharyngeal cancer.

In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating conditions associated with or resulting from HPV exposure or infection. In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating precancerous cervical lesions. In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating cervical intraepithelial neoplasia. In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating vaginal and anal intraepithelial neoplasia. In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating cervical cancer. In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating rectal cancer. In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating penile cancer. In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating vaginal cancer. In some embodiments, the pharmaceutical composition comprising compound II is used in the preparation of a medicament for treating oropharyngeal cancer.

Advantageously, the dosage form is easy to apply to the target site. Direct application to the target site prevents systemic exposure and toxicity. To place the dosage form at the target site, it can be applied using a drug applicator. In some embodiments, the dosage form is applied using a vaginal applicator. In some embodiments, the dosage form is applied without a drug applicator. In some embodiments, an additional liquid (e.g., a lubricant) is delivered or applied to the dosage form, or to the target site or surrounding tissue, along with the dosage form.

In some embodiments, the lubricant is applied in combination with the dosage form to enhance coverage of the cervix, vagina, vulva, anus, perianal area, or penis. In some embodiments, water is used as the liquid applied with the dosage form. In some embodiments, a water-soluble lubricant-type liquid based on glycerin or hydroxyethyl cellulose is applied with the dosage form. In some embodiments, the dosage form can be applied without additional liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 5 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 4 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 3 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 2 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 1 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.75 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.5 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.25 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.2 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.15 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.125 ml of liquid. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in less than about 0.1 ml of liquid.

In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in a liquid ranging from about 10 μL to about 100 μL. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in a liquid ranging from about 75 μL to about 250 μL. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in a liquid ranging from about 200 μL to about 500 μL. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in a liquid ranging from about 400 μL to about 750 μL. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in a liquid ranging from about 700 μL to about 1,000 μL. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in a liquid ranging from about 1 mL to about 2 mL. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in a liquid ranging from about 2 mL to about 3 mL. In some embodiments, the dosage form will soften, disintegrate, and/or dissolve in a liquid ranging from about 3 mL to about 4 mL. In some embodiments, the dosage form will be softened, disintegrated, and/or dissolved in a liquid ranging from about 4 ml to about 5 ml.

In some embodiments, compound II is administered over consecutive or discontinuous days of at least 1, 2, 3, 4, 5, or 6 days.

In some embodiments, compound II is administered once weekly. In some embodiments, compound II is administered once weekly for up to 12 weeks. In some embodiments, compound II is administered once weekly for up to 10 weeks. In some embodiments, compound II is administered once weekly for up to 8 weeks. In some embodiments, compound II is administered once weekly for up to 6 weeks. In some embodiments, compound II is administered once weekly for up to 4 weeks. In some embodiments, compound II is administered once weekly for up to 2 weeks. In some embodiments, compound II is administered once weekly for up to 1 week.

In some embodiments, compound II is administered twice weekly. In some embodiments, compound II is administered twice weekly for up to 12 weeks. In some embodiments, compound II is administered twice weekly for up to 10 weeks. In some embodiments, compound II is administered twice weekly for up to 8 weeks. In some embodiments, compound II is administered twice weekly for up to 6 weeks. In some embodiments, compound II is administered twice weekly for up to 4 weeks. In some embodiments, compound II is administered twice weekly for up to 2 weeks. In some embodiments, compound II is administered twice weekly for up to 1 week.

In some embodiments, compound II is administered three times a week. In some embodiments, compound II is administered three times a week for up to 12 weeks. In some embodiments, compound II is administered three times a week for up to 10 weeks. In some embodiments, compound II is administered three times a week for up to 8 weeks. In some embodiments, compound II is administered three times a week for up to 6 weeks. In some embodiments, compound II is administered three times a week for up to 4 weeks. In some embodiments, compound II is administered three times a week for up to 2 weeks. In some embodiments, compound II is administered three times a week for up to 1 week.

In some embodiments, compound II is administered daily. In some embodiments, compound II is administered daily for up to 12 weeks or for a period specified by a healthcare provider. In some embodiments, compound II is administered daily for up to 10 weeks. In some embodiments, compound II is administered daily for up to 8 weeks. In some embodiments, compound II is administered daily for up to 6 weeks. In some embodiments, compound II is administered daily for up to 4 weeks. In some embodiments, compound II is administered daily for up to 2 weeks. In some embodiments, compound II is administered daily for up to 1 week. In some embodiments, compound II is administered daily at a dose from about 0.05 mg to about 0.3 mg for one, two, three, four, five, six or more weeks, as specified by a healthcare provider.

In some embodiments, compound I monofumarate is administered three times a week. In some embodiments, compound I monofumarate is administered three times a week for up to 12 weeks. In some embodiments, compound I monofumarate is administered three times a week for up to 10 weeks. In some embodiments, compound I monofumarate is administered three times a week for up to 8 weeks. In some embodiments, compound I monofumarate is administered three times a week for up to 6 weeks. In some embodiments, compound I monofumarate is administered three times a week for up to 4 weeks. In some embodiments, compound I monofumarate is administered three times a week for up to 2 weeks. In some embodiments, compound I monofumarate is administered three times a week for up to 1 week.

In some embodiments, compound I monofumarate is administered daily. In some embodiments, compound I monofumarate is administered daily for up to 12 weeks or for a period specified by a healthcare provider. In some embodiments, compound I monofumarate is administered daily for up to 10 weeks. In some embodiments, compound I monofumarate is administered daily for up to 8 weeks. In some embodiments, compound I monofumarate is administered daily for up to 6 weeks. In some embodiments, compound I monofumarate is administered daily for up to 4 weeks. In some embodiments, compound I monofumarate is administered daily for up to 2 weeks. In some embodiments, compound I monofumarate is administered daily for up to 1 week.

In some embodiments, compound I monofumarate, compound II, or compound III may be administered three, four, five, or six times per week. In some embodiments, compound I monofumarate, compound II, or compound III may be administered once daily. In some embodiments, compound I monofumarate, compound II, or compound III may be administered twice daily. In some embodiments, compound I monofumarate, compound II, or compound III may be administered three, four, or more times daily. In some embodiments, compound I monofumarate, compound II, or compound III is administered daily.

In some embodiments, the compound may be administered over one or more treatment cycles, each cycle comprising a treatment period and a withdrawal period, wherein the treatment period comprises administration of the compound as described herein, followed by a withdrawal period (including a period of no treatment), and then the next treatment cycle. In some embodiments, the withdrawal period is from about one day to about six months. In some embodiments, the withdrawal period is one, two, three, four, five, six, seven, eight weeks, or longer before the next treatment cycle. In some embodiments, multiple treatment cycles are administered, such as one, two, three, four, five, or six treatment cycles.

As mentioned above, many compounds have been investigated for the treatment of HPV-induced cervical lesions, but none have been approved. For non-limiting examples of the methods investigated, see: Ahn W.S. et al. Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions. Eur. J. Cancer Prev. 2003; 12:383-390; Ashrafian L et al. Double-blind randomized placebo-controlled multicenter clinical trial (phase IIa) on diindolymethane’s efficacy and safety in the treatment of CIN:impl ications for cervical cancer prevention.EPMA J.2015;6:doi:10.1186/s13167-13015-10048-13169; Bossens M., et al.Safety and tolerance of cidofovir as a 2% gel for loc alapplication in high-grade cervical intraepithelial neoplasia:A phase 1investigation.Int.J.Clin.Pharmacol.2018;56:134-141; Chen F.P.Efficacy of imiquimod5%c ream for persistent human papillomavirus in genital intraepithelialneoplasm.Taiwanese J.Obstetrics Gynecol.2013;52(4):475-478;Choo Y., et al.Intravaginal applic ation of leukocyte interferon gel in the treatment ofcervical intraepithelial neoplasia(CIN)Arch Gynecol.1985;237:51-54; de WitteC.J et al.Imiquimod in cervical, vaginal and vulvar intraepithelial neoplasia:areview.Gynecol.Oncol.2015;139:377-384; Desravines N, et al. Low dose 5-fluorouracil intravaginal therapy for the trea Treatment of cervical intraepithelial neoplasia 2/3:A case series.J.Gynecol.Surg.2020;36; DiSilvestro P.A., et al. Treatment of cervical intraepithelial neoplasia levels 2and 3with adapalene, a retinoid-related molecule.J.Low Genit Tract.Dis.2001;5:33-37;Graham V., et al.PhaseIItrial of beta-all-transretinoic acid forcervical int raepithelial neoplasia via a collagen sponge and cervicalcap.West.J.Med.1986;145:192-195; Grimm C., et al.Treatment of cervicalintraepithelial neoplasia with top ical imiquimod: arandomized controlledtrial.Obstet.Gynecol.2012;120(1):152-159; Hampson L., et al. A single-arm, proof-of-concept trial of lopimune(lopinavir/ritonav ir)as treatment for HPV-relatedpre-invasive cervical disease.PLoS ONE.2016;11; Helm C.W. et al. Retinoids forpreventing the progression of cervical intra-epithelial neoplasia.CochraneSystematic Review.2013; Hubert P., et al.Local applications of GM-CSF induce therecruitment of immune cells in cervical low-grade squamous int raepithelial lesions.Am.J.Reprod.Immunol.2010;64:126-136;Koeneman MM, et al. Topical Imiquimodtreatment of high-grade Cervical intraepithelial neoplasia (TOPIC tri al): studyprotocol for a randomized controlled trial.BMC Cancer.2016:doi:10.1186/s12885-12016-12187-12883; Krause S., et al.Interferon and cervical dysplasia:CINII I treated with local interferon application.Colposcopy Gynecologic LaserSurgery.1987;3:195-198;Krebs H.B., et alChronic ulcerations following topicaltherapy with 5-fluorouracil for vaginal human papillomavirus-associatedlesions.Obstet.Gynecol.1991;78(2):205-208; Laccetta G. et al. Effect of the treatment with beta-glucan in women with cervical cytologic report ofatypical squamous cells of undetermined significance(ASCUS)and low-gradeintraepithelial lesions(L-SIL)Minerva Ginecol .2015;67:113-120; Meyskens F.L., et al. A phaseItrial of beta-all-transretinoic acid delivered via a collagensponge and a cervical cap for mild or moderate intraep ithelial cervicalneoplasia.J.Natl Cancer Inst.1983;71:921-925;Niwa K., et alTopical vidarabineof 5-fluoruracil treatment against persistent HPV in genital(pre)ca ncerouslesions.Oncol Reports.2003;10:1437-1441;Pachman DR, et al. Randomized clinical trial of imiquimod: an adjunct to treating cervical dysplasia.Am.J.Obstet.Gyne col.2012;206(1):42e41-47;Rahangdale L et alTopical 5-fluorouracil fortreatment of Cervical Intraepithelial Neoplasia 2:a randomized controlled trial.Am.J.Obstet. Gynecol.2014;210:e1-e8; Schneider A., et al Efficacy trial oftopically administered Interferon gamma-1beta gel in comparison to lasertreatment in cervical intraep ithelial neoplasia.Arch.Gynecol Obste.1995;256:75-83;Silman F.H., et al5-fluorouracil/chemosurgery for intraepithelialneoplasia of the lower genital tract.Obstet .Gynecol.1981;58:356-360;SnoeckR.,Noel J.C.,Muller C.,Clercq De,Bossens M.Cidofovir, a new approach for the treatment of cervix intraepithelial neoplasiaIII(C IN III) J. Med. Virol. 2000; 60: 205–209; Stentella P., Biamonti A., Carraro C. Efficacy of carboxymethyl beta-glucan in cervical intraepithelial neoplasia: a retrospe ctive,case-controlstudy.Minerva Ginecol.2017;69:425-430;Suh-Burgmann E.,Sivret J.,Duska L.R.,Del Carmen M.,Seiden M.V.Long-term administration of intravagin aldehydroepiandrosterone on regression of low-grade cervical dysplasia-a pilotstudy.Gynecol.Obstet.Invest.2003;55:25-31;Valencia M.H.,Pacheco A.C.,QuijanoT.H .,Giron A.V.,Lopez C.V.Clinical response to glycyrrhizinic acid in genitalinfection due to human papillomavirus and low-grade squamous intraepitheliallesion .Clin.Pract.2011 1(e93);van de Sande A.,Koeneman M.,Gerestein C.,KruseA.,van Kemenade F.,van Beekhuizen H.Topical Imiquimod treatment of residualor recurren t cervical intraepithelial neoplasia (TOPIC-2 trial): a studyprotocol for a randomized controlled trial. BMC Cancer. 2018; 18:4510-4517; and Van Pachterbeke C., Buce lla D., Rozenberg S. Topical treatment of CIN 2+ bycidofovir: Results of a phase II, double-blind, prospective, placebo-controlled study. Gynecol Onc. 2009; 115:69-74.

VI. Pharmaceutical Compositions and Dosage Forms

In one aspect of the invention, the pharmaceutical composition according to the invention comprises an anti-HPV effective amount of any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, optionally in combination with a pharmaceutically acceptable carrier, additive, or excipient, and/or in combination with or alternating with at least one other active compound. In one embodiment, the invention comprises a solid dosage form of compound II in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is applied directly to the cervix, vagina, vulva, perianal area, anus, or penis. In some embodiments, the dosage form adheres to the cervix, vagina, vulva, perianal area, anus, or penis.

In one aspect of the invention, the pharmaceutical composition according to the invention comprises an effective amount of anti-HPV compound II as described herein, optionally combined with a pharmaceutically acceptable carrier, additive, or excipient, and further optionally combined with at least one other antitumor or antiviral agent (e.g., an anti-HPV agent). In some embodiments, the pharmaceutical composition comprises compound II in combination with a second antiviral drug. In some embodiments, the pharmaceutical composition comprises compound II in combination with an anticancer drug.

This invention includes pharmaceutical compositions comprising an effective amount for treating HPV infection in a pharmaceutically acceptable carrier or excipient of any of the active compounds described herein (including, but not limited to, Compound I monofumarate, Compound II, or Compound III of this invention). In an alternative embodiment, this invention includes pharmaceutical compositions comprising an effective amount for preventing HPV infection in a pharmaceutically acceptable carrier or excipient of Compound I monofumarate or Compound II or a prodrug of this invention.

Those skilled in the art will recognize that the effective therapeutic dose will vary depending on the infection or condition to be treated, its severity, the proposed treatment regimen, the pharmacokinetics of the drug used, and the patient or subject (animal or human) to be treated, and that the therapeutic dose can be determined by the attending physician or expert.

The compound I monofumarate, compound II, or compound III, or any active compound described herein according to the invention, may be formulated in a mixture having a pharmaceutically acceptable carrier. For the treatment of HPV infection, the pharmaceutical composition is preferably applied directly to the cervix, vagina, vulva, perianal area, anus, or penis.

In certain pharmaceutical dosage forms, prodrug forms of compounds, particularly including acylated (acetylated or otherwise) and ether (alkyl and related) derivatives, phosphate esters, thiophosphorimide esters, phosphorimide esters, and various salts of the compounds of the present invention, can be used to achieve the desired effects. Those skilled in the art will recognize how readily the compounds of the present invention can be modified into prodrug forms to facilitate the delivery of the active compound to a target site within the host organism or patient. Those skilled in the art will also utilize the favorable pharmacokinetic parameters of the prodrug forms to, where applicable, deliver the compounds of the present invention to target sites within the host organism or patient to maximize the intended effects of the compound.

The amount of any active compound described herein, including but not limited to compound I monofumarate, compound II, or compound III, contained in the therapeutically active formulation according to the invention is an effective amount to achieve the desired results according to the invention, for example, for treating HPV infection, reducing the likelihood of HCV infection, or inhibiting, reducing, and/or eliminating HPV or its side effects (including disease states, symptoms, and/or complications following HPV infection).

Typically, for the treatment, prevention, or delay of the occurrence of these infections and/or reduction of the likelihood of HPV infection, or secondary disease states, conditions, or complications of HPV, dosage forms containing any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, are administered in amounts from about 0.001 mg to about 100 mg. In some embodiments, solid dosage forms contain amounts from about 0.001 mg to about 0.005 mg, from about 0.005 mg to about 0.01 mg, from about 0.01 mg to about 0.03 mg, from about 0.03 mg to about 0.25 mg, from about 0.20 mg to about 0.5 mg, from about 0.4 mg to about 1 mg, from about 0.75 mg to about 3 mg, from about 1 mg to about 10 mg, or from about 5 mg to about 20 mg. In some embodiments, the solid dosage form comprises at least about 0.001, 0.003, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 10, 20, 30, 40, or 50 mg or more of any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III.

In some embodiments, to treat or delay the occurrence of these infections and/or reduce the likelihood of HPV infection, or secondary HPV-related conditions, symptoms, or complications, dosage forms containing any of the active compounds described herein (including, but not limited to, Compound I monofumarate, Compound II, or Compound III) are administered in amounts ranging from about 0.001 to about 20 mg, from about 0.005 to about 10 mg, from about 0.01 mg to about 5 mg, from about 0.03 mg to about 1 mg, or from about 0.05 mg to about 0.3 mg of Compound I monofumarate, Compound II, or Compound III. Typically, dosage forms of Compound I monofumarate, Compound II, or Compound III are administered 1, 2, 3, or more times per week, up to daily, in doses from 0.05 mg to 0.3 mg.

In some embodiments, for the treatment of infection with high-risk HPV strains, dosage forms containing any of the active compounds described herein (including, but not limited to, Compound I monofumarate, Compound II, or Compound III) are administered in amounts ranging from about 0.001 to about 20 mg, from about 0.005 to about 10 mg, from about 0.01 mg to about 5 mg, from about 0.03 mg to about 1 mg, or from about 0.05 mg to about 0.3 mg of Compound I monofumarate, Compound II, or Compound III. Typically, dosage forms for treating high-risk HPV strains containing 0.05 mg to 0.3 mg of Compound I monofumarate, Compound II, or Compound III may be administered 1, 2, 3, or more times per week, up to daily.

In some embodiments, compound I monofumarate, compound II, or compound III may be applied as a gel. In some embodiments, the gel contains from about 0.001% to about 10%, from about 0.01% to about 10%, from about 0.05% to about 5%, from about 0.1% to about 3%, or from about 0.1% to about 2% (by weight). In some embodiments, the gel contains from about 0.001% to about 0.05% of compound I monofumarate, compound II, or compound III. In some embodiments, the gel contains from about 0.01% to about 0.5% of compound I monofumarate, compound II, or compound III. In some embodiments, the gel contains from about 0.1% to about 5% of compound I monofumarate, compound II, or compound III.

In some non-limiting embodiments, any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, may be administered topically. More generally, compound I monofumarate, compound II, or compound III may be administered in the form of tablets, capsules, suspensions, liquids, emulsions, implants, particles, spheres, creams, ointments, suppositories, transdermal forms, gels, mucosal agents, etc. Dosage forms may also be bilayer tablets, wherein the full dose of the active compound is released in one direction (e.g., toward the target tissue).

In some embodiments, the dosage form may soften, disintegrate, and/or release in a low liquid volume. In some embodiments, the dosage form softens and begins immediate drug release. In some embodiments, the dosage form softens and begins gradual drug release. In some embodiments, the dosage form softens and begins drug release within one hour. In some embodiments, the dosage form softens and begins drug release within two hours. The dosage form may be prepared to maximize surface area and promote disintegration. In some embodiments, the dosage form is a spherical tablet. In some embodiments, the dosage form is an oval tablet. In some embodiments, the dosage form is a capsule tablet. The tablet width has a maximum dimension, and the tablet thickness has a minimum dimension. In some embodiments, the width of the dosage form is twice the thickness. In some embodiments, the width of the dosage form is three times the thickness. In some embodiments, the width of the dosage form is four times or more the thickness. In some embodiments, the dosage form is about 0.1 mm thick to about 5 mm thick. In some embodiments, the dosage form is about 1 mm thick to about 2 mm thick. In some embodiments, the dosage form is about 2 mm thick to about 3 mm thick. In some embodiments, the dosage form is about 3 mm thick to about 4 mm thick. In some embodiments, the dosage form is about 4 mm to about 5 mm thick. In some embodiments, the tablet is about 5 mm to about 15 mm thick. In some embodiments, the dosage form is less than 5 grams. In some embodiments, the dosage form is about 0.05 grams to about 0.15 grams. In some embodiments, the dosage form is about 0.1 to about 1 gram. In some embodiments, the dosage form is about 0.75 grams to about 2 grams. In some embodiments, the dosage form is from about 1 gram to about 5 grams.

In some embodiments, the dosage form is not easily removed, removed, or moved from the target site. These desired properties can be achieved by including a mucosal adhesion polymer in the pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a mucosal adhesion polymer or a mucosal adhesion excipient. Non-limiting examples of mucosal adhesion polymers and excipients include: hydroxypropyl methylcellulose, lectins, thiolated polymers (e.g., chitosan-iminothiacyclopentane, poly(acrylic acid)-cysteine, poly(acrylic acid)-homocysteine, chitosan-mercaptoglycolic acid, chitosan-mercaptoethylamidine, alginate-cysteine, poly(methacrylic acid)-cysteine, and sodium carboxymethyl cellulose-cysteine), polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylate polyhydroxyethyl methacrylate, chitosan, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, Methylcellulose, sodium carboxymethylcellulose, aminated corn starch, cellulose derivatives, poly(acrylic acid) polymers, poly(hydroxyethyl methacrylate), poly(ethylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), astragalus gum, sodium alginate, carrageenan gum, guar gum, xanthan gum, soluble starch, gelatin, pectin, chitosan, methylcellulose, hyaluronic acid, hydroxypropyl methylcellulose, hydroxypropyl cellulose, gellan gum, carrageenan, cationic hydroxyethyl cellulose, hydrogel, dihydroxyphenylalanine, and alginate-polyethylene glycol acrylate. In some embodiments, the pharmaceutical composition comprises from about 0% to about 10% of a mucosal adhesion polymer excipient selected from the group consisting of: carbomer, polyethylene glycol, cropovidone, polycarbofil, hydroxypropyl methylcellulose, and hydroxyethyl cellulose.

In some embodiments, the pharmaceutical composition comprises from at least about 0.1% to about 90%, about 92%, about 93%, about 95%, about 98%, about 97%, about 98%, or about 99% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises from about 0.1% to about 1% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises from about 0.5% to about 5% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises from about 1% to about 10% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises from about 5% to about 20% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises from about 10% to about 50% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises from about 20% to about 75% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises from about 50% to about 90% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises from about 75% to about 99% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises at least about 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises no more than about 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of a mucosal adhesion polymer. In some embodiments, the pharmaceutical composition comprises 0% of a mucosal adhesion polymer. In this case, adhesion to the target site is achieved by using other pharmaceutically acceptable excipients.

To prepare the pharmaceutical compositions of the present invention, a therapeutically effective amount of any active compound described herein (including, but not limited to, Compound I monofumarate, Compound II, or Compound III of the present invention) is typically mixed with a pharmaceutically acceptable carrier to produce a dose, according to conventional pharmaceutical compounding techniques. The carrier can take various forms depending on the desired formulation for application, such as topical, oral, or parenteral administration. Any commonly used pharmaceutical medium can be used when preparing topical dosage forms of pharmaceutical compositions. Therefore, suitable carriers and additives, including water, glycols, oils, alcohols, preservatives, etc., can be used for liquid or semi-solid topical formulations such as gels, creams, ointments, suspensions, elixirs, and solutions. In some embodiments, the pharmaceutical composition comprises propylene glycol. In some embodiments, the pharmaceutical composition comprises carboxylated polymethylene. In some embodiments, the pharmaceutical composition comprises ethylenediaminetetraacetic acid (EDTA). In some embodiments, the pharmaceutical composition comprises sorbic acid. In some embodiments, the pharmaceutical composition comprises carbomer. In some embodiments, the pharmaceutical composition comprises hydroxyethyl cellulose. In some embodiments, the pharmaceutical composition comprises polyethylene glycol.

For solid topical dosage forms such as powders, tablets, and capsules, and for solid dosage forms such as suppositories, suitable carriers and additives can be used, including starch, sugar carriers (such as dextrose, mannitol, lactose, and related carriers), diluents, granulating agents, lubricants, binders, mucosal adhesion polymers, disintegrants, etc. If desired, tablets or capsules can be coated or sustained-released using standard techniques. The use of these dosage forms can significantly enhance the bioavailability of the compound in patients. In some embodiments, the pharmaceutical composition comprises mannitol. In some embodiments, the pharmaceutical composition comprises magnesium stearate. In some embodiments, the pharmaceutical composition comprises microcrystalline cellulose. In some embodiments, the pharmaceutical composition comprises polycarboxymethyl cellulose. In some embodiments, the pharmaceutical composition comprises polyethylene oxide. In some embodiments, the pharmaceutical composition comprises colloidal silica. In some embodiments, the pharmaceutical composition comprises povidone. In some embodiments, the pharmaceutical composition comprises isopropyl alcohol. In some embodiments, the pharmaceutical composition comprises sodium carboxymethyl starch. In some embodiments, the pharmaceutical composition comprises croscarmellose sodium. In some embodiments, the pharmaceutical composition comprises croscarmellose. In some embodiments, the pharmaceutical composition comprises hydroxypropyl methylcellulose. In some embodiments, the pharmaceutical composition comprises lactose. In some embodiments, the powder pharmaceutical composition comprises one or more excipients selected from the group consisting of: xanthan gum, microcrystalline cellulose, polyethylene oxide, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, povidone, mannitol, colloidal silica, sodium benzoate, sodium carboxymethyl starch, sodium lauryl sulfate, poloxamer 407, polyoxypropylene-polyoxyethylene copolymer, etc.

In some embodiments, pharmaceutical compositions comprising effective amounts of any of the active compounds described herein, including but not limited to compound I, further comprising pharmaceutically acceptable excipients selected from the following list, which consists of: gum arabic, agar, alginic acid, ascorbyl palmitate, bentonite, benzoic acid, butylated hydroxyanisole, butylated hydroxytoluene, butanediol, calcium acetate, calcium hydroxide, rapeseed oil, carob gum, carrageenan, castor oil, cellulose, corn starch, disodium edetate, isoascorbic acid, ethyl lactate, ethylcellulose, glycerin, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, hydroxyethyl methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose Cellulose, lactic acid, lauric acid, lecithin, linoleic acid, medium-chain triglycerides, methyl benzoate, methylcellulose, microcrystalline cellulose, microcrystalline wax, myristic acid, oleic acid, palmitic acid, peanut oil, pectin, phosphoric acid, polycarboxymethyl cellulose, potassium alginate, propionic acid, propyl gallate, propylparaben, propylene glycol, propylene glycol alginate, silicon dioxide, dimethyl silicone oil, sodium alginate, sodium benzoate, sodium bicarbonate, sodium carboxymethyl cellulose, sodium chloride, sodium citrate, sodium lactate, sodium lauryl sulfate, sodium metabisulfite, sodium phosphate, sodium sulfite, sodium thiosulfate, sorbic acid, stearic acid, talc, tapioca starch, tartaric acid, thymol, urea, vitamin E polyvinyl succinate, beeswax, xanthan gum, and zinc acetate.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient for use as a vaginal suppository. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, Compound I monofumarate, Compound II, or Compound III) further comprises up to 99.9% of a vaginal suppository excipient selected from the group consisting of: cleaved lipids, PEG, polyethylene glycols (macrogols), cocoa butter, and glycerol. Non-limiting examples of cleaved lipids include (monoglycerides, diglycerides, and triglycerides of fatty acids ( _C10 to _C18 ), primarily the triglyceride portion and ethoxylated fatty alcohols), (glycerides of saturated plant fatty acids, such as lauric acid), and Supposi-base ^™ (a mixture of saturated polyethylene glycol-modified glycerides).

In some embodiments, a pharmaceutical composition comprising an effective amount of any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) further comprises pharmaceutically acceptable excipients that enhance penetration, disintegration, film formation, and/or control the release properties of the composition.

In one embodiment, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) further comprises a penetration-enhancing excipient. In some embodiments, the penetration-enhancing excipient is selected from the group consisting of: oleic acid; eucalyptol; caprylyl alcohol; Labrafil; Labrasol; lauryl alcohol; diethylene glycol monomethyl ether; propylene glycol; sodium lauryl sulfate; cetyltrimethylammonium bromide; poloxamer (231, 182, 184); Tween 20, 40, 60, 80; fatty acids and fatty acid esters; isostearic acid; glycerol; and chitosan. In some embodiments, the pharmaceutical composition comprising the fumarate of compound I contains from 0% to about 20% a penetration-enhancing excipient selected from the group consisting of: cetyl alcohol, propylene glycol, transcutol P, oleic acid, isopropyl myristate, propylene glycol dicaprylyl, glyceryl monooleate, propylene glycol monocaprylyl, PEG-8 beeswax, cetyl alcohol, stearic acid, cetyl palmitate, and cetyl sterol. In some embodiments, the pharmaceutical composition contains about 0% to about 25% a penetration-enhancing excipient selected from the list consisting of: stearyl alcohol, polysorbate 80, sodium lauryl sulfate, monoglycerides and diglycerides, sorbitan monostearate, glyceryl isostearate, polyethylene glycol 15-hydroxystearate, polyethylene glycol 40 hydrogenated castor oil, octyl dodecyl alcohol, and soybean lecithin.

In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) further comprises a film-forming excipient. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) contains from 0% to about 99% of a film-forming excipient selected from the group consisting of: hydroxypropyl methylcellulose, polyethylene glycol, polymethyl methacrylate, microcrystalline cellulose, guar gum, xanthan gum, and polyvinylpyrrolidone.

In some embodiments, pharmaceutical compositions comprising any of the active compounds described herein (including, but not limited to, Compound I monofumarate, Compound II, or Compound III) further comprise excipients that allow for the controlled release of the active compound. In some embodiments, the controlled-release pharmaceutical compositions comprise ethyl cellulose, hydroxypropyl methylcellulose, microcrystalline wax, polycarboxymethyl ether, or beeswax.

Unless otherwise stated, the percentage range of excipients and other components in a pharmaceutical composition is expressed as a weight percentage.

In some embodiments, pharmaceutical compositions comprising any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, further comprise disintegration-enhancing excipients. In some embodiments, the disintegration-enhancing excipients are selected from the group consisting of: cellulose, guar gum, crospovidone, crospovidone, soybean polysaccharide, calcium silicate, gelatin, cation exchange resin, bentonite, citrus pulp, alginate, calcium alginate, methylcellulose, microcrystalline cellulose, sodium carboxymethyl cellulose, crospovidone, filter residue, corn starch, sodium carboxymethyl starch (Explotab, Primojel), glycine, hydroxypropyl starch, and starch 1500. In some embodiments, the pharmaceutical composition comprises up to about 99% of disintegration-enhancing excipients, such as mannitol and/or microcrystalline cellulose. In some embodiments, the pharmaceutical composition comprises from about 0% to about 70% of disintegration-enhancing excipients selected from the list consisting of: lactose, sucrose, and calcium phosphate. In some embodiments, the pharmaceutical composition comprises from about 0% to about 50% of a disintegration-enhancing excipient selected from the list below, which includes sodium bicarbonate, citric acid, maleic acid, adipic acid, and fumaric acid. In some embodiments, the pharmaceutical composition comprises from about 0% to about 20% of a disintegration-enhancing excipient selected from the list below, which includes sodium glycolate starch, pregelatinized starch, crospovidone, and croscarmellose sodium.

In some embodiments, pharmaceutical compositions comprising any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, further comprise from 0% to about 70% mannitol, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In some embodiments, pharmaceutical compositions comprising the fumarate of compound I further comprise from 0% to about 70% lactose, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) further comprises from about 0% to about 70% sucrose, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including but not limited to, compound I monofumarate, compound II, or compound III) further comprises from about 0% to about 70% microcrystalline cellulose, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) further comprises from 0% to about 20% sodium carboxymethyl starch, including but not limited to any amount to achieve the desired binding, such as up to about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 12%, about 15%, or about 20%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including but not limited to, compound I monofumarate, compound II, or compound III) further comprises from about 0% to about 20% pregelatinized starch, including but not limited to any amount to achieve the desired binding, such as up to about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 12%, about 15%, or about 20%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, Compound I monofumarate, Compound II, or Compound III) further comprises from about 0% to about 20% crospovidone, including but not limited to any amount to achieve the desired binding, such as up to about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 12%, about 15%, or about 20%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including but not limited to, Compound I monofumarate, Compound II, or Compound III) further comprises from about 0% to about 20% crospovidone sodium carboxymethyl cellulose, including but not limited to any amount to achieve the desired binding, such as up to about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 12%, about 15%, or about 20%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) further comprises from 0% to about 50% xanthan gum, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including but not limited to, compound I monofumarate, compound II, or compound III) further comprises from 0% to about 50% polycarboflavone, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, Compound I monofumarate, Compound II, or Compound III) further comprises from 0% to about 50% polyethylene oxide, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including but not limited to, Compound I monofumarate, Compound II, or Compound III) further comprises from 0% to about 50% hydroxyethyl methylcellulose, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) further comprises from 0% to about 50% hydroxyethyl cellulose, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including but not limited to, compound I monofumarate, compound II, or compound III) further comprises from 0% to about 50% hydroxypropyl methylcellulose, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) further comprises from 0% to about 50% hydroxypropyl cellulose, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including but not limited to, compound I monofumarate, compound II, or compound III) further comprises from 0% to about 50% PVP, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%. In some embodiments, a pharmaceutical composition comprising any of the active compounds described herein (including, but not limited to, compound I monofumarate, compound II, or compound III) further comprises from 0% to about 50% microcrystalline cellulose, including but not limited to any amount to achieve the desired binding, such as up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45%.

In typical embodiments of the invention, any active compound described herein, including but not limited to compound I monofumarate, compound II or compound III, and the composition is used to treat, prevent or delay HPV infection or secondary disease states, conditions or complications of HPV.

In some embodiments, tablets for treating, preventing, or delaying HPV infection or secondary disease states, conditions, or complications of HPV contain any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, and further contain about 250 mg of microcrystalline cellulose, about 20 mg of cropovidone, about 5 mg of magnesium stearate, about 5 mg of silica, about 5 mg of polyethylene oxide, and about 100 mg of mannitol. In some embodiments, tablets for treating, preventing, or delaying HPV infection or secondary disease states, conditions, or complications of HPV further contain about 155 mg of microcrystalline cellulose, about 1.75 mg of magnesium stearate, and about 17.5 mg of mannitol.

In some embodiments, a semi-solid formulation for treating, preventing, or delaying HPV infection or secondary disease states, conditions, or complications of HPV comprises any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, and further comprises about 15 mg carbomer, about 50 mg propylene glycol, about 10 mg sorbic acid, about 5 mg EDTA, and about 920 mg water. In some embodiments, a semi-solid formulation for treating, preventing, or delaying HPV infection or secondary disease states, conditions, or complications of HPV comprises any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, and further comprises about 20 mg carbomer; about 70 mg mineral oil; about 80 mg a mixture of polyethylene glycol 6 stearate type I, polyethylene glycol stearate, and polyethylene glycol 32 stearate type I; about 5 mg parabens; about 60 mg propylene glycol; about 5 mg EDTA; and about 760 mg water.

In some embodiments, the reconstitutional dry powder for treating, preventing, or delaying HPV infection or secondary disease states, conditions, or complications of HPV comprises any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, and further comprises about 15.5 mg xanthan gum, about 19.8 mg mannitol, about 5 mg silica, and about 0.5 mg sodium benzoate.

VII. Combination and Alternating Therapies

The treatments for cervical intraepithelial neoplasia described herein can be combined with conventional methods (e.g., but not limited to, resection or ablation of the transformation zone). Techniques include cryotherapy, laser therapy, loop electrosurgical excision procedure (LEEP), and cone biopsy. All of these surgical procedures can damage the affected area and may result in scarring. LEEP, the most common intervention for cervical intraepithelial neoplasia, is effective in 60%–90% of cases; however, it is associated with a significantly increased risk of miscarriage, ectopic pregnancy, and negative psychological outcomes. In some embodiments, the treatments described herein are used to reduce, improve, or replace the use of these conventional procedures.

In some embodiments, the treatments described herein may be used in combination with surgical techniques. In some embodiments, patients in need may undergo surgery before, during, and/or after administration of an effective amount of the compounds described herein. In some embodiments, surgery may be the removal of target and/or diseased tissue, including but not limited to loop electrosurgical excision (LEEP), wide loop excision of the transformation zone (LLETZ), scalpel conization, cold scalpel conization, scalpel biopsy, or laser conization. In some embodiments, surgery may be ablation, including but not limited to laser ablation or cryoablation.

The efficacy of drugs against HPV infection can be prolonged, enhanced, or restored by combining or alternating one, or even two or three, other antiviral compounds that induce mutations different from the primary drug or act through different pathways. Alternatively, the pharmacokinetics, biodistribution, half-life, or other parameters of a drug can be altered through such combination therapy (which may include alternating therapy if synergy is considered). Furthermore, since HPV is associated with several types of cancer, combination therapy administered with anticancer drugs can provide better outcomes for patients. Because the disclosed compound II is a DNA polymerase inhibitor, it may be useful to administer the compound in combination with, for example, drugs such as:

a) Protease inhibitors;

b) Another DNA polymerase inhibitor;

c) Inhibitors of E6 or E6AP, such as MEDI0457, luteolin, CAF-24 or gossypol;

d) E7 inhibitors;

e) E1 or E2 inhibitors, including inhibitors of E1-E2 protein interactions;

f) L2 lipopeptide;

g) L1 or L2 inhibitors or degrading agents;

h) HDAC inhibitors (such as vorinostat);

i) Degraders of tetraspan membrane proteins, such as CD9, CD63, or CD151;

j) Immunotherapy, such as T-cell therapy (including adoptive T-cell therapy) and checkpoint inhibitors;

k) Antiproliferative drugs;

l) Therapeutic vaccines;

m) Preventive vaccines;

n) Trichloroacetic acid;

o) Salicylic acid;

p) Imiquimod;

q) Pudafilo;

r)9；

s)4;

t)Cervarix;

u)VGX-3100;

v)GGX-188E; and/or

w)ADXS11-001.

Example

General annotations

The following instrumental analysis methods were used to characterize the crystal form of the present invention.

X-ray powder diffraction (XRPD)

Differential scanning calorimetry (DSC)

Thermogravimetric analysis (TGA)

Dynamic gas phase adsorption (DVS)

Karl Fischer water test

Instrument: Mettler Toledo Coulometric KF Titrator C30

Method: Coulomb method

Polarized light microscopy (PLM)

Nuclear magnetic resonance (NMR)

instrument Bruker Avance-AV 400M frequency 400MHz probe 5mm PABBO BB-1H/D Number of scans 8 temperature 297.6K Relaxation delay 1 second

Fourier transform infrared spectroscopy (FT-IR)

High-performance liquid chromatography (HPLC)

1. Stability Study

Gradient procedure:

Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 0.01 95 5 9.0 5 95 13.0 5 95 13.1 95 5 17.0 95 5

2. Chiral purity

Gradient procedure:

Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 35.0 60 40 45.0 55 45 50.0 10 90 55.0 10 90 55.5 95 5 60.0 95 5

Abbreviations:

DMF: N,N-dimethylformamide;

DCM: Dichloromethane, methylene dichloro;

MeOH: Methanol, methyl alcohol;

ACN: Acetonitrile;

EtOH: Ethanol; Ethyl alcohol;

IPAc: Isopropyl acetate;

IPA: Isopropyl alcohol;

THF: Tetrahydrofuran;

MEK: Methyl ethyl ketone;

DIAD: Diisopropyl azodicarbonate;

DEAD: Diethyl azodicarbonate;

MTBE: Methyl tert-butyl ether;

DMSO-d6: Deuterated dimethyl sulfoxide;

_Cs₂CO₃ : Cesium carbonate _;

TMSBr: Trimethylsilyl bromide;

NaOMe: Sodium methoxy;

TEA: Triethylamine;

Ph ₃ P: Triphenylphosphine;

_Na₂SO₄ : _Sodium sulfate;

NaOH: Sodium hydroxide;

HCl: hydrochloric acid;

H ₂ SO ₄ : sulfuric acid;

BsOH: Benzenesulfonic acid;

p-TsOH: p-Toluenesulfonic acid;

MsOH: Methanesulfonic acid;

^1H NMR: Proton nuclear magnetic resonance;

LCMS: Liquid Chromatography-Mass Spectrophotometer;

HPLC: High-performance liquid chromatography;

^1H NMR section: s = singlet; bs = broad singlet; d = doublet; dd = double doublet; t = triplet; m = multiplet; J = spin coupling constant.

Example 1: Approximate solubility of free base of compound I at 25°C

Approximately 2 mg of the free form of compound I was weighed into a 2 mL glass vial, and 20 μL of each solvent (Table 1) of the aliquot was added to determine the solubility at 25 °C. In the second set of experiments, approximately 10 mg of the free form of compound I was added to a 2 mL glass vial, and 20 μL of each solvent (aliquot) of the aliquot was added to determine the solubility at 50 °C. The maximum volume of each solvent added was 1 mL. Approximate solubility was determined by visual observation. The results are presented in Table 1.

Table 1. Approximate solubility of compound I in its free form at 25°C and 50°C

Example 2: Crystallization screening of compound I in its free base form

Equilibrate with solvent at 25°C

As shown in Table 2, approximately 30 mg of the free base of compound I was stirred in a suitable amount of solvent on a stirring pan at 25 °C for one week to reach equilibrium. On the eighth day of the study, no precipitated solid was observed, and all ten samples were stirred at 5 °C for approximately 3 days. In Experiment 10, the suspension in heptane was filtered. The solid material (wet cake) obtained in Experiment 10 was studied by XRPD, DSC, TGA, and NMR.

Table 2. Equilibration with solvent at 25°C for 1 week

According to XRPD measurements (Figure 1), the solid material obtained in Experiment 10 forms compound I in free form, mode 1. This mode is characterized by DSC (Figure 2) showing a melting onset temperature of 40.3 °C (with a transformation enthalpy of 43 J/g); and by TGA (Figure 3) showing a weight loss of 0.1% at 40 °C. According to ^1H NMR, the material contains 0.2% residual heptane.

By adding antisolvent precipitation

Approximately 30 mg of the free base of compound I was dissolved in the solvents listed below (Table 3). The antisolvent was slowly added to the resulting solution. The precipitate from Experiment 3 was collected by filtration and analyzed by XRPD.

Table 3. Results of adding antisolvent precipitation

Based on XRPD data (Figure 1), the solid materials formed are as shown in Experiment 3, and Table 3 shows the free form of compound I, pattern 1.

A crystalline free form, compound I free form mode 1, was obtained from equilibrium experiments in heptane and antisolvent experiments in acetone/MTBE.

Example 3: Synthesis of the salt of compound I

Compound I sulfate

Two methods are used to synthesize the sulfate of compound I.

Method A

At 0–10 °C, sulfuric acid solution (1 N in THF) was slowly added to a solution of compound I (0.049 g, 0.1 mmol) in dry THF (1 mL). During the addition of sulfuric acid, the pink, clear THF solution of the free base of compound I turned into a grayish-white semi-solid. The reaction mixture was allowed to reach room temperature over 20–30 minutes and shaken. After allowing the solid material to settle, the supernatant was carefully decanted. The resulting semi-solid was washed with an additional amount of dry THF (2 x 2 mL) and the resulting solid was dried under high vacuum to yield 0.053 g of compound I sulfate-1 as a grayish-white solid.

Method B

Sulfuric acid solution (1N, in EtOAc) was slowly added to a solution of compound I (0.024 g, 0.05 mmol) in dry ethyl acetate (EtOAc, 0.5 mL) at 0–10 °C. During the addition of sulfuric acid, the pink, clear EtOAc solution of the free base of compound I turned into a grayish-white solid. The reaction mixture was allowed to reach room temperature over 20–30 min and shaken thoroughly. After allowing the solid material to settle, the supernatant was carefully decanted. The grayish-white solid was washed with an additional amount of EtOAc (2 x 2 mL). The resulting grayish-white solid was dried under high vacuum to yield 0.023 g of compound I sulfate-2.

Compound I methanesulfonate

Two methods were used to synthesize the methanesulfonate of compound I.

Method A

At 0–10 °C, purified methanesulfonic acid (MSA; MW = 96.11; d = 1.47; 0.007 mL; 0.11 mmol) was added dropwise to a solution of compound I (0.049 g, 0.1 mmol) in dried EtOAc (1 mL). During the addition of MSA, the pink, clear EtOAc solution of the free base of compound I turned into a grayish-white semi-solid (glue-like). The heterogeneous mixture was allowed to reach room temperature over 20–30 min and shaken thoroughly. After the semi-solid material settled, the supernatant was carefully decanted. The resulting semi-solid (glue-like) was washed with methyl tert-butyl ether (MTBE; 2 x 2 mL) and dried under high vacuum to yield 0.054 g of compound I methanesulfonate-1.

Method B

At 0–10 °C, a solution of methanesulfonic acid (1 N, in THF; 0.11 mmol; 0.110 mL) was added dropwise to a solution of compound I (0.049 g, 0.1 mmol) in dry IPA (1 mL). During the addition of the MSA solution, the pink EtOAc clear solution of the free base of compound I turned into a grayish-white solid. The heterogeneous mixture was allowed to reach room temperature over 20–30 min and shaken thoroughly. After allowing the solid material to settle, the supernatant was carefully decanted. The resulting solid was washed with methyl tert-butyl ether (MTBE; 2 x 2 mL) and dried under high vacuum to yield 0.049 g of compound I methanesulfonate-2.

Compound I hydrochloride

At 0–10 °C, HCl solution (4 N, in dioxane; 0.11 mmol; 0.027 mL) was added dropwise to a solution of compound I (MW = 492; 0.049 g, 0.1 mmol) in dried EtOAc (1 mL). During the addition of HCl solution, the pink, clear EtOAc solution of the free base of compound I turned into a grayish-white solid. The heterogeneous mixture was allowed to reach room temperature over 20–30 min and shaken thoroughly. After allowing the solid material to settle, the supernatant was carefully decanted. The resulting solid was washed with methyl tert-butyl ether (MTBE; 2 x 2 mL) and dried under high vacuum to yield 0.045 g of HCl salt of compound I.

Compound I monofumarate

Fumaric acid (MW = 116; 12.3 mg; 0.106 mmol; 1.5 equivalences) was added to a solution of compound I (0.035 g, 0.071 mmol) in dry isopropanol (0.1 mL) at 0–10 °C. The reaction mixture was brought to room temperature, then heated at 60 °C for 30 min and stirred at room temperature for 12 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure at 40 °C. The mixture was diluted with MTBE (2 mL), shaken thoroughly, and the MTBE was carefully decanted. The colorless solid was washed with an additional amount of MTBE (2 mL) and dried under high vacuum to give 0.022 g of compound I sesquifumarate. Compound I monofumarate can be synthesized by washing compound I sesquifumarate with additional MTBE and drying under high vacuum.

Compound I benzenesulfonate

At 0–10 °C, a benzenesulfonic acid solution (BsOH, MW = 158; 0.020 g; 0.127 mmol in 0.2 mL EtOAc) was added dropwise to a solution of compound I (0.057 g, 0.115 mmol) in 1.0 mL of dried EtOAc. During the addition of the BsOH solution, a pink, clear solution of the free base of compound I precipitated as a colorless solid in EtOAc. The reaction mixture was allowed to reach room temperature over 20–30 min and shaken thoroughly. After allowing the solid material to settle, the supernatant was carefully decanted. The colorless solid was washed with an additional amount of MTBE (2 x 2 mL). The resulting solid was dried under high vacuum to yield 0.064 g of compound I benzenesulfonate.

Compound I toluenesulfonate

At 0–10 °C, a p-toluenesulfonic acid solution (p-TsOH, 0.055 mL; 0.055 mmol; 1 N, in EtOAc) was added dropwise to a solution of compound I (0.024 g, 0.05 mmol) in dry EtOAc (0.5 mL). During the addition of the p-TsOH solution, a pink, clear solution of the free base of compound I in EtOAc precipitated as a colorless solid. The reaction mixture was allowed to reach room temperature over 20–30 min and shaken thoroughly. After allowing the solid material to settle, the supernatant was carefully decanted. The colorless solid was washed with an additional amount of MTBE (2 x 2 mL). The resulting solid was dried under high vacuum to yield 0.027 g of compound I toluenesulfonate.

The melting point of the salt of compound I obtained in this example was determined by differential scanning calorimetry. The results are presented in Table 4. The monofumarate obtained by washing the sesquifumarate had the highest melting point among the tested salts.

Table 4. Melting points of salts of compound I

Example 5: Salt screening of the free base of compound I

Eight acids (fumaric acid, citric acid, L-malic acid, L-tartaric acid, succinic acid, benzenesulfonic acid, oxalic acid, and maleic acid) and two co-formations (L-proline and nicotinamide) were selected to screen for potential salt and/or co-crystal formations. Compound I was used as a mixture of two diastereomers ( ^1H NMR purity approximately 98%–99%). Isopropanol (IPA), ethanol, and ethyl acetate (EA) were selected as screening solvents. Slurry equilibrium, antisolvent addition, and slow evaporation were applied as screening methods.

Salt screening by slurry equilibrium

A mixture of the free base of compound I as diastereomers (approximately 30 mg) was added to a suitable solvent, followed by the addition of 1 or 0.5 molar equivalents of acid (as shown in Table 5 below) or co-formations (experiments RC2 to RC10) at 50 °C with stirring. The mixture was stirred at 50 °C for 2 hours, and then at 25 °C for at least 12 hours. For those experiments in which a clear solution was formed, half the volume was evaporated in a fume hood, and the remainder was treated by adding an antisolvent. The resulting suspension was removed and centrifuged. The solids obtained in experiments RC2 (sample RC2-EA), RC3 (sample RC3-EA), RC7 (samples RC7-IPA and RC7-EA), and RC11 (sample RC11-EA) were analyzed by XRPD. The results are presented in Table 5.

Table 5. Screening of salts (eutectic) by slurry equilibrium

In experiment RC2, the solid material obtained in EA (sample RC2-EA) showed a mixture of fumaric acid and, according to XRPD (Figs. 7 and 10), resembled the phase of hemifumarate mode 1 (sample AS3-B in Table 6). In experiment RC3, the solid material obtained in EA (sample RC3-EA) showed a phase similar to hemifumarate mode 1 according to XRPD data (Fig. 7). In experiment RC11, the solid material obtained in EA showed a mixture of L-proline co-formation and free form mode 1. In experiment RC7, the solid materials obtained from isopropanol (sample RC7-IPA, Fig. 8) and ethyl acetate (sample RC7-EA, Fig. 9) were characterized by XRPD resembling the phase of monosuccinate mode 1 (see below, Table 6, experiment AS7, sample AS7-B).

Salt screening results by antisolvent addition

Antisolvents (methyl tert-butyl ether (MTBE) or heptane) were slowly added to the clear solutions obtained in the experiments in Table 5 (Experiments AS1 to AS12 in Table 6). In experiments AS2, AS3, and AS7, heptane antisolvent was added to the solution in EtOH to form precipitates of solid materials AS2-B, AS3-B, and AS7-B, which were characterized by XRPD.

Next, at 25°C, with stirring, 0.5, 1, or 1.5 moles of acid or co-formation were added to the clear solutions (from experiments AS1, AS3, and AS12) and sample AS5-A containing a small amount of solid material obtained after the addition of antisolvent, and the mixture was stirred at this temperature for at least 12 hours. No solid phase was formed in experiments AS13 to AS17. The results are summarized in Table 6.

Table 6. Results of salt screening by antisolvent addition

Adding heptane as an antisolvent to a clear solution obtained from experiment RC2 (ethanol as solvent, 1 molar equivalent of fumaric acid) leads to the formation of monofumarate mode 1, characterized by XRPD (sample AS2-B, Fig. 10). The composition of this material as monofumarate was established by ^1H NMR (free base form to fumaric acid ratio = 1:0.98); according to the XRPD, no free fumaric acid phase was found in this sample. Adding heptane as an antisolvent to a clear solution obtained from experiment RC3 (ethanol as solvent, 0.5 molar equivalent of fumaric acid) leads to the formation of hemifumarate mode 1, characterized by XRPD (sample AS3-B, Fig. 10). The composition of this material as hemifumarate was established by ^1H NMR (free base form to fumaric acid ratio = 1:0.54); according to the XRPD, no free fumaric acid phase was found in this sample. Adding heptane as an antisolvent to a clear solution obtained from entry RC7 (ethanol as solvent, 1 molar equivalent of succinic acid) results in the formation of monosuccinate mode 1, characterized by XRPD (sample AS7-B, Fig. 11). The composition of this material as monosuccinate was established by ^1H NMR (free base form to succinic acid ratio = 1:1.06); according to XRPD, no free succinic acid phase was found in this sample.

Reequilibrium Experiment

The solvent was evaporated from the gel-like or oily samples obtained by salt screening with antisolvent addition as described above. The residual mixture was then liquefied in MTBE or heptane at 25°C, as shown in Table 7. The experimental results are given in Table 7.

Table 7. Results of Rebalancing

Citric acid, L-malic acid, L-tartaric acid, oxalic acid, maleic acid, and nicotinamide do not form the crystalline phase of compound I during the reequilibrium experiment.

Example 6: Formation of Compound I salt by slurry crystallization

In experiment RC13, approximately 50 mg of the free base of compound I was added to EtOH, and 0.5 moles of fumaric acid were added over 2 hours at 50 °C with stirring, followed by stirring at 25 °C for at least 12 hours. Seed crystals of sample AS3-B were added, and heptane (0.4 mL) was added as an antisolvent. The resulting mixture was stirred at 5 °C for approximately 3 days. The suspension was then removed and centrifuged. The resulting solid was dried in an oven under vacuum at 50 °C for approximately 1 hour and analyzed by XRPD (Figure 12), NMR, DSC (Figure 13), and TGA (Figure 14).

In experiment RC14, approximately 50 mg of compound I free base was added to EtOH, and an equimolar amount of succinic acid was added at 50 °C with stirring for 2 hours, followed by stirring at 25 °C for at least 12 hours. Seed crystals of sample AS7-B were added, along with heptane (0.2 mL) as an antisolvent. The resulting suspension was removed and centrifuged. The resulting solid was dried in an oven under vacuum at 50 °C for approximately 1 hour and analyzed by XRPD (Figure 15), NMR, DSC (Figure 16), and TGA (Figure 17).

In experiment RC15, approximately 50 mg of compound I free base was added to 0.1 mL of EtOH. An equimolar amount of benzenesulfonic acid was dissolved in 0.2 mL of EtOH. The benzenesulfonic acid solution was then added dropwise to the free base solution at 25 °C. MTBE (0.8 mL) was added as an antisolvent, and the mixture was stirred at 5 °C, during which some solids formed.

In experiment RC16, approximately 50 mg of compound I free base was added to 0.1 mL of IPA. 1.5 moles of fumaric acid were added to the mixture, and the mixture was stirred at 25 °C. After approximately 3 hours, the sample became too viscous, so 0.1 mL of additional IPA was added, and stirring was continued at 25 °C for approximately 22 hours. A viscous sample was obtained and dried in a vacuum oven at 50 °C for approximately 2 hours, and then re-liquefied in 1.0 mL of MTBE at 25 °C for approximately 5 days. The resulting suspension was removed and centrifuged. The obtained solid was dried in an oven at 40 °C under vacuum for approximately 1 hour and analyzed by XRPD (Figure 18) and NMR. The residual solid was added to RC18 as seed crystals.

In experiment RC17, approximately 30 mg of compound I free base was added to 0.1 mL of EtOH. At 50 °C, with stirring, 0.5 molar amount of succinic acid was added to the mixture for 2 hours, resulting in a clear solution. Heptane (0.3 mL) was added as an antisolvent. An oil was obtained, and the mixture was stirred at 25 °C for approximately 5 days. The resulting suspension was removed and centrifuged. The obtained solid was dried in an oven under vacuum at 40 °C for approximately 1 hour and analyzed by XRPD (Figure 19), NMR, DSC (Figure 20), and TGA (Figure 21).

In experiment RC18, approximately 30 mg of compound I free base was added to 0.1 mL IPA. An equimolar amount of fumaric acid was added to the mixture at 50 °C with stirring for 2 hours, yielding a clear yellow solution. 0.3 mL of heptane was added as an antisolvent. Some seed crystals from RC16 were added. A suspension was obtained and stirred at 25 °C for at least 12 hours. The resulting suspension was removed, centrifuged, and analyzed by XRPD (Figure 22). Monofumarate pattern 1 was obtained and added as seed crystals to the expanded sample.

Table 8. Results of salt screening by slurry crystallization

Note:

*The salt ratio is as determined by proton integration in ^1H NMR spectroscopy for compound I.

Alkali dissociation: the ratio of counterions;

**ND: Data not collected.

Based on salt screening experiments, four patterns were identified: hemifumarate pattern 1, monofumarate pattern 1, hemisuccinate pattern 1, and monosuccinate pattern 1. The four patterns exhibited comparable characteristics, such as low to moderate crystallinity relative to their low melting points.

Example 7: Preparation of fumarate

Preparation of compound I hemifumarate in mode 1

Approximately 200 mg of the free base of Compound I was added to 0.5 mL of EtOH. With stirring, 0.5 moles of fumaric acid were added at 50 °C, and the mixture was stirred for 2 hours. A clear solution was obtained. The solution was then cooled to 25 °C over 1 hour. Hemifumarate seed crystals of sample RC13 (Example 5, Table 7) were added, followed by 2.5 mL of heptane to induce precipitation. An oil was obtained and stirred continuously at 25 °C for approximately 4 days. After 4 days, the resulting suspension was cooled to 5 °C. After stirring at 5 °C for approximately 4 days, the precipitated solid was collected by filtration and dried under vacuum at 40 °C for approximately 3 hours. As a result, 116 mg of pale orange hemifumarate, Pattern 1, was obtained with a yield of 52%. XRPD is shown in Figure 23; DSC is shown in Figure 24; and TGA is shown in Figure 25.

Preparation of Compound I monofumarate, Mode 1 (Small-scale preparation)

Approximately 244 mg of free base of compound I was added to 0.8 mL of IPA. Then, 1.0 equivalent of fumaric acid was added, accompanied by stirring at 50 °C for approximately 1.5 h. The resulting clear yellow solution was cooled to 25 °C and stirred for approximately 5 min. Monofumarate seed crystals of sample RC18 (Table 7) were added to the mixture, followed by 4 mL of heptane as an antisolvent. The mixture was stirred at 25 °C for 4 days. The precipitated material was collected by filtration and dried under vacuum at 40 °C for approximately 2 h. As a result, 208 mg of monofumarate pattern 1 solid was obtained, with a yield of 69%. XRPD is shown in Figure 23. DSC performed at 10 °C/min is shown in Figure 26, and DSC performed at 2 °C/min is shown in Figure 27b. The DSC cycling results are shown in Figure 27c. TGA is shown in Figure 28.

Table 9. Characterization of Compound I in its free base mode and its monofumarate and hemifumarate modes

Compound I, mode 1, is highly crystalline. The hemifumarate and monofumarate modes are of moderate crystalline quality.

Compound I, mode 1, is an anhydrous compound and has a melting peak at a T _-start of 75.0 °C, with an enthalpy of about 64 J/g. It shows a weight loss of about 0.3% at about 70 °C. KF analysis shows that it contains about 1.7% water. Approximately 0.7% MTBE (by weight) residue was detected by ^1H NMR.

Compound I, hemifumarate, mode 1, is an anhydrous compound. Based on ^1H NMR results, the stoichiometry of free form:fumaric acid is approximately 1:0.5. It has a melting peak at a T _onset of 85.2 °C, with an enthalpy of approximately 37 J/g. It shows a weight loss of approximately 1.0% at approximately 85 °C. KF indicates that it contains approximately 1.7% water. Approximately 0.7% EtOH and 0.7% heptane (residue, by weight) were detected by ^1H NMR.

Compound I, monofumarate, mode 1, is an anhydrous compound. Based on ^1H NMR results, the stoichiometry of free form:fumaric acid is approximately 1:1.0. It exhibits melting and splitting peaks at a T _onset of 107.2 °C, with an enthalpy of approximately 78 J/g. The two thermal events cannot be resolved by heating rates of 2 K/min and 0.5 K/min, or by DSC. It shows a weight loss of approximately 0.3% at approximately 107 °C. KF indicates that it contains approximately 1.2% water. Residue of approximately 0.7% IPA and 2.2% heptane (by weight) was detected by ^1H NMR.

Example 8. Stability of Compound I in free base mode 1, Compound I in monofumarate mode 1 and Compound I in hemifumarate mode 1

Initial chemical purity: The initial purities of Compound I (free base mode 1), Compound I (monofumarate mode 1), and Compound I (hemofumarate mode 1) were 98.7%, 98.6%, and 97.8%, respectively.

Volumetric stability: Accelerated stability tests were conducted for one week in open containers at 25°C/92%RH and 40°C/75%RH, and in sealed containers at 60°C. The results are presented in Table 10. The results for compound I monofumarate mode 1 are also presented in Figure 28.

All three candidates exhibited good physical stability after exposure to the three conditions. They all showed some degradation after exposure to 40°C/75% RH for one week, with two of them becoming unstable at elevated temperatures (60°C for one week). Under both of the above conditions, compound I hemifumarate mode 1 was more prone to degradation than compound I monofumarate mode 1.

Table 10. Volumetric stability of Compound I in free base mode 1, Compound I in monofumarate mode 1 and Compound I in hemifumarate mode 1

Example 9. Solubility of Compound I in free base mode 1, Compound I in monofumarate mode 1 and Compound I in hemifumarate mode 1

Approximately 4 mg of Compound I, Mode 1, was added to 2 mL of buffer solution. Approximately 4 mg of Compound I, Hemifumarate Mode 1, and Compound I, Monofumarate Mode 1, were added to 1.8 mL and 1.6 mL of buffer solution, respectively. The pH was adjusted to simulated vaginal secretions using 0.2 N NaOH. Clear solutions of all three candidates were obtained after stirring at 37 °C for 0.5 h and 2 h.

Table 11. Solubility of Compound I in Free Base Mode 1, Compound I in Monofumarate Mode 1, and Compound I in Hemifumarate Mode 1

Solubility was tested at 37°C for 0.5 h and 2 h in five media: pH 3.0 citrate buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer, water, and simulated vaginal secretions (pH 4.2). All three candidates were highly soluble in the media (>2 mg/mL).

Example 10: Hygroscopicity of Compound I in Free Base Mode 1, Compound I in Hemifumarate Mode 1, and Compound I in Monofumarate Mode 1

The hygroscopicity was investigated using DVS at 25°C using the following method:

Method: 40-0-95-0-40% RH, dm/dt = 0.002

The results are presented in Table 12 and Figures 29 to 34.

Table 12. Hygroscopicity of Compound I in Free Base Mode 1, Compound I in Hemifumarate Mode 1, and Compound I in Monofumarate Mode 1

Note: D – desorption; S – adsorption

Hygroscopicity was investigated by DVS at 25°C. All three modes exhibited moderate hygroscopicity. Compound I, mode 1, showed approximately 4.3% water absorption up to 90% RH, but absorbed approximately 14.3% water at 95% RH. For Compound I, monofumarate mode 1 and Compound I, hemifumarate mode 1, they showed approximately 2.9% and 6.9% water absorption, respectively, up to 95% RH. No form change was observed after DVS testing.

Example 11: Large-scale preparation of monofumarate mode 1

Approximately 4.16 g of the free base of compound I was dissolved in 11 mL of IPA. Then, 1.0 equivalent of fumaric acid was added to the clear yellow solution at 50 °C with stirring. After approximately 1 hour, some solid precipitate formed. Then, 10 mg of monofumarate seed crystals (from the ‘Small-scale Preparation’ above) were added. The mixture was stirred at 50 °C for approximately 1.5 hours and cooled to 25 °C, then stirred at 25 °C for approximately 10 minutes. Then, 40 mL of heptane was added as an antisolvent. The resulting suspension was stirred at 25 °C for approximately 24 hours, then cooled to 5 °C at a rate of 0.1 °C/min and stirred at 5 °C for approximately 1 day. The solid was collected by filtration and dried in an oven under vacuum at 40 °C for approximately 2 hours. Approximately 4.1 g of a pale pink solid was obtained, with a yield of 81.2%. XRPD is shown in Figure 35; DSC at a rate of 10 °C/min is shown in Figure 36a; DSC at a rate of 2 °C/min is shown in Figure 36b; mDSC thermogram is shown in Figure 36c; TGA is shown in Figure 37.

Table 13. Characteristics of Compound I monofumarate in Mode 1

Example 12: Screening study of polymorphs of compound I fumarate

A screening study was conducted on the polymorphisms of the fumarate of compound I (a mixture of diastereomers). The polymorphic behavior was investigated through equilibrium, precipitation (by adding an antisolvent), slow cooling, rapid cooling, and slow evaporation experiments.

Approximate solubility of compound I monofumarate in mode 1 at 25°C and 50°C

Approximately 2 mg of Compound I monofumarate, Pattern 1 (Example 11), was weighed into a 2 mL glass vial, and 20 μL of each solvent (as shown in Table 14) was added to determine the solubility at 25°C. Approximately 10 mg of Compound I monofumarate, Pattern 1 (Example 11), was weighed into a 2 mL glass vial, and 20 μL of each solvent (as shown in Table 14 below) was added to determine the solubility at 50°C. The maximum volume of each solvent added was 1 mL. Approximate solubility was determined by visual observation.

Table 14. Approximate solubility of compound I monofumarate (mode 1) at 25 °C and 50 °C

Equilibrate with solvent at 25°C for 2 and 3 weeks.

Approximately 30 mg of Compound I monofumarate (obtained in Example 11) was equilibrated with a suitable amount of solvent at 25°C using a stir bar for 2 and 3 weeks. The resulting suspension was filtered. The solid fraction (wet cake) was studied by XRPD. Further studies (e.g., NMR, DSC, TGA, HPLC, and PLM) were performed when differences were observed. The results are presented in Tables 15-30 and Figures 38-60.

Table 15. Results of equilibration of compound I monofumarate in mode 1 with water at 25°C for 2 and 3 weeks.

For mode D, the XRPD diffraction patterns are shown in Figures 38 and 39; the DSC thermograms are shown in Figures 40 and 41; and the TGA thermograms are shown in Figures 42 and 43.

Table 16. Results of equilibration of compound I monofumarate in mode 1 at 25 °C for 2 and 3 weeks with acetonitrile.

Note: The salt ratio is in free base form: fumaric acid ratio.

For mode C, the XRPD diffraction pattern is shown in Figure 44; the DSC thermogram is shown in Figure 45; and the TGA thermogram is shown in Figure 46.

Table 17. Results of equilibration of compound I monofumarate in mode 1 at 25 °C for 2 and 3 weeks using MEK.

Note: The salt ratio is in free base form: fumaric acid ratio.

For mode B, the XRPD diffraction pattern is shown in Figure 47; the DSC thermogram is shown in Figure 48; and the TGA thermogram is shown in Figure 49.

Table 18. Results of equilibration of compound I monofumarate mode 1 with acetone at 25°C for 2 and 3 weeks.

The XRPD diffraction pattern is shown in Figure 47 (3 weeks).

Table 19. Equilibrium persistence of compound I monofumarate at 25 °C using isopropanol in mode 1

Results after 2 weeks and 3 weeks.

Note: The salt ratio is in free base form: fumaric acid ratio.

For mode 1, the XRPD diffraction pattern is shown in Figure 50; the DSC thermogram is shown in Figure 51; and the TGA thermogram is shown in Figure 52.

Table 20. Results of equilibration of compound I monofumarate in mode 1 at 25 °C for 2 and 3 weeks using acetone/toluene (1:1 v/v).

For mode E, the XRPD diffraction pattern is shown in Figure 53; the DSC thermogram is shown in Figures 54 and 55; and the TGA thermogram is shown in Figures 56 and 57.

Table 21. Modulation of compound I monofumarate using acetone/heptane (1:1 v/v) at 25 °C

Equation 1 shows the results after balancing for 2 weeks and 3 weeks.

Note: The salt ratio is in free base form: fumaric acid ratio.

The XRPD diffraction pattern is shown in Figure 47 (3 weeks).

Table 22. Results of equilibration of compound I monofumarate in mode 1 at 25 °C for 2 and 3 weeks using IPA/heptane (1:1 v/v).

Note: The salt ratio is in free base form: fumaric acid ratio.

The XRPD diffraction pattern is shown in Figure 50.

Table 23. Results of equilibration of compound I monofumarate in mode 1 at 25 °C for 2 and 3 weeks using IPA/toluene (1:1 v/v).

Note: The salt ratio is in free base form: fumaric acid ratio.

The XRPD diffraction pattern is shown in Figure 50.

Table 24. Results of equilibration of compound I monofumarate in mode 1 at 25 °C for 2 and 3 weeks using IPA/MTBE (1:3 v/v).

Note: The salt ratio is in free base form: fumaric acid ratio.

The XRPD diffraction pattern is shown in Figure 50.

Table 25. Results of equilibration of compound I monofumarate in mode 1 at 25 °C for 2 and 3 weeks using THF/heptane (1:3 v/v).

Note: The salt ratio is in free base form: fumaric acid ratio.

XRPD is shown in Figures 58 and 59.

Table 26. Results of equilibration of compound I monofumarate in mode 1 at 25 °C for 2 and 3 weeks using EtOH/heptane (1:3 v/v).

Note: The salt ratio is in free base form: fumaric acid ratio.

The XRPD diffraction pattern is shown in Figure 50.

Table 27. Results of equilibration of compound I monofumarate in mode 1 for 2 and 3 weeks at 25 °C using EA/toluene (1:3 v/v).

Note: The salt ratio is in free base form: fumaric acid ratio.

XRPD is shown in Figures 58 and 59.

Table 28. Results of equilibration of compound I monofumarate in mode 1 at 25 °C for 2 and 3 weeks using EtOH/toluene (1:3 v/v).

Note: The salt ratio is in free base form: fumaric acid ratio.

XRPD is shown in Figures 58 and 59.

Table 29. Results of equilibration of compound I monofumarate mode 1 for 2 and 3 weeks at 25 °C with water/ACN (2.9:97.1 v/v).

Note: The salt ratio is in free base form: fumaric acid ratio.

The XRPD diffraction pattern is shown in Figure 44.

Table 30. Modeling of compound I monofumarate at 25 °C using IPA/heptane (1:4 v/v).

Equation 1 balances the results of 2 weeks and 3 weeks.

Note: The salt ratio is in free base form: the ratio of fumaric acid.

The XRPD diffraction pattern is shown in Figure 60.

By adding antisolvent precipitation

Approximately 30 mg of compound I monofumarate, mode 1 (Example 7), was dissolved in a good solvent. An antisolvent was slowly added to the resulting solution. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD. When differences were observed, further studies (e.g., NMR, DSC, TGA) were performed. If no precipitation was obtained, the solution was cooled to 5°C for crystallization. After stirring at 5°C for approximately 23 days without obtaining a precipitate, the solution was placed in a -20°C freezer for crystallization.

The results are presented in Table 31. The XRPD diffraction patterns are shown in Figures 61 and 62.

Table 31. Precipitation of Compound I Fumarate by Adding Antisolvent

Crystallization by slow evaporation at room temperature

Combined with approximate solubility experiments, the solubility sample was filtered through a 0.45 μm nylon filter. The resulting solution was then slowly evaporated under ambient conditions. The polymorphic form of the solid residue was examined.

The results are presented in Table 32. The XRPD diffraction patterns are shown in Figures 63 and 64.

Table 32. Crystallization at room temperature by slow evaporation

Crystallization from a hot saturated solution by slow cooling

Approximately 30 mg of compound I monofumarate (Example 7) was dissolved in a minimal amount of a selected solvent at 50 °C. The resulting solution was cooled to 5 °C at a rate of 0.1 °C/min. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD. When differences were observed, further studies (e.g., NMR, DSC, TGA) were performed. If no precipitate was obtained, the solution was placed in a -20 °C freezer for crystallization. After storage in a -20 °C freezer for approximately 5 days without obtaining a precipitate, heptane was added as an antisolvent. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD.

The results are presented in Table 33. The XRPD diffraction patterns are shown in Figures 65 and 66.

Table 33. Crystallization from a hot saturated solution by slow cooling

Crystallization from a hot saturated solution by rapid cooling

At 50°C, approximately 30 mg of compound I monofumarate (Example 7) was dissolved in a minimal amount of a selected solvent. The resulting solution was placed in an ice bath and shaken. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD. When differences were observed, further studies (e.g., NMR, DSC, TGA) were performed. If no precipitate was obtained, the solution was placed in a -20°C freezer for crystallization. After storage in a -20°C freezer for approximately 7 days, if no precipitate was obtained, heptane was added as an antisolvent. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD.

The results are presented in Table 34. The XRPD diffraction patterns are shown in Figures 67 and 68.

Table 34. Crystallization from a hot saturated solution by rapid cooling

Behavior of compound I monofumarate in mode 1 under heating and cooling

The polymorphic behavior of compound I monofumarate mode was investigated by DSC under two different heating-cooling cycles.

Cycle 1: 0°C to 106°C at 10°C/min; 106°C to 0°C at 10°C/min; reheat from 0°C to 250°C at 10°C/min.

Cycle 2: Reheat at 10℃/min from 0℃ to 130℃; at 10℃/min from 130℃ to 0℃; at 10℃/min from 0℃ to 250℃.

The results are presented in Table 35 and Figures 69 and 70.

Table 35. Behavior of Compound I monofumarate in Mode 1 under heating and cooling

Compressed behavior

Approximately 10 mg of Compound I monofumarate (Example 7) was compressed for 5 minutes at 10 MPa using a hydraulic press. XRPD characterization was performed to investigate the polymorphic behavior after compression. According to XRPD, no change in form was observed.

Grinding simulation experiment

Approximately 10 mg of compound I monofumarate (Example 7) was manually ground in a mortar and pestle for 5 min. Crystallization transformation and crystallinity were assessed by XRPD. According to XRPD, no change in form was observed; crystallinity decreased slightly.

Granulation simulation experiment

Water or ethanol was added dropwise to approximately 10 mg of Compound I monofumarate, Mode 1 (Example 7), until the solid was fully wetted. The sample was manually ground and pounded in a mortar for 3 minutes. The sample was dried under ambient conditions for 10 minutes. The crystal form transformation and crystallinity were evaluated by XRPD. According to XRPD, no form change was observed in either ethanol or water.

Summary of identified monofumarate and hemifumarate polymorphs

The monofumarate used in the study, Model 1, was prepared from the free base of Compound I according to Example 7. The initial form of the monofumarate used in the polymorphic studies described below (Model 1) was an anhydrous compound of monofumarate with an HPLC purity of approximately 99.3%. The ratio of the free form to fumaric acid was approximately 1:0.96 (according to ^1H NMR). As measured by differential scanning calorimetry (DSC), it exhibited two melting peaks at a T _-start of approximately 98.5 °C (where the enthalpy is approximately 14 J/g) and at 109.6 °C (where the enthalpy is approximately 25 J/g). By thermogravimetric analysis (TGA), Model 1 showed approximately 0.5% weight loss at approximately 98 °C and approximately 0.6% weight loss from 98 °C to 140 °C. Approximately 1.0% (by weight) heptane and 0.2% (by weight) IPA residue were detected by ^1H NMR. Karl Fischer titration showed that it contained approximately 1.3% water.

During the polymorph studies, four new modes were identified. Despite using monofumarate as the initial physical form, the new modes exhibited different stoichiometry. Modes B, C, and E are hemifumarate, while mode D is a degradation product.

Pattern B is an anhydrous compound of hemifumarate with an HPLC purity of approximately 99.6%. It was obtained using MEK, acetone, acetone/heptane, MEK/heptane, and EtOH/MTBE as solvents, through equilibrium experiments, antisolvent addition experiments, slow cooling, and rapid cooling experiments. The ratio of the free base form to fumaric acid was approximately 1:0.52 (by 1H-NMR). It exhibits two thermal events at a T- _initial of approximately 77.4 °C (with an enthalpy of approximately 71 J/g) and at 88.4 °C (with an enthalpy of approximately 18 J/g). It shows approximately 0.7% weight loss at approximately 77 °C and approximately 4.2% weight loss from 77 °C to 130 °C. Approximately 4.6% (by weight) MEK residue was detected by ^1H NMR.

Using ACN and ACN/water as solvents, a mixture of pattern C and fumaric acid was obtained through equilibrium, slow cooling, and rapid cooling experiments. The ratio of the free base form of the mixture to fumaric acid was approximately 1:0.95 (according to ^1H NMR). After washing with water, this ratio decreased to approximately 1:0.76. This indicates that pattern C is not a monofumarate. It exhibits two thermal events at a T _-start of approximately 74.0 °C (with an enthalpy of approximately 89 J/g) and at a T _-start of approximately 90.6 °C (with an enthalpy of approximately 15 J/g). It shows approximately 0.4% weight loss at approximately 73 °C and approximately 2.1% weight loss from 73 °C to 144 °C. Approximately 2.0% (by weight) of ACN residue was detected by ^1H NMR.

Pattern D is the degradation product with an HPLC purity of approximately 0.2%. It was obtained in water via equilibrium experiments. It exhibits two thermal events at _{an initial} temperature (T₀) of approximately 41.4 °C (with an enthalpy of approximately 67 J/g) and at _{an initial} temperature (T₀) of approximately 72.1 °C (with an enthalpy of approximately 29 J/g). It shows a weight loss of approximately 0.6% at approximately 41 °C and approximately 8.5% from 41 °C to 178 °C.

Pattern E is an anhydrous compound of hemifumarate with an HPLC purity of approximately 98.9%. It was obtained by equilibrium experiments in acetone/toluene. The ratio of the free base form of the mixture to fumaric acid was approximately 1:0.69 (by ^1H NMR). It exhibits two thermal events at a T _-start at approximately 53.1 °C (with an enthalpy of approximately 33 J/g) and at a T _-start at approximately 96.5 °C (with an enthalpy of approximately 34 J/g). It shows approximately 1.0% weight loss at approximately 53 °C and approximately 3.6% weight loss from 53 °C to 96 °C. Approximately 0.6% (by weight) acetone residue was detected by ^1H NMR.

The results of the studies on the polymorphs of compound I monofumarate and hemifumarate are summarized in Table 36 below. The salt ratio is the ratio between the free base and the salt counterion of compound I. "AS" indicates the form prepared by adding an antisolvent using the solvent/antisolvent pairs listed in the table. "EQ" indicates the form prepared by equilibration in the listed solvents. "SC" indicates the form prepared by slowly cooling a solution of compound I monofumarate in the listed solvents. "FC" indicates the form prepared by rapidly cooling a solution of compound I monofumarate in the listed solvents.

Table 36. Summary of identified compound I fumarate patterns A, B, C, and E

Example 13: Preparation of Compound II, Mode 1

Experiment 1. Small-scale synthesis and preparation of seed crystals

100 mg of _RP compound I free base and 0.3 mL of IPA were added to a glass vial. 1.0 equivalent of fumaric acid was added, and the resulting mixture was stirred at 50 °C for 2 min, resulting in the precipitation of most of the material. 1.0 mL of heptane was added. The sample was stirred at 50 °C for 1 h, then cooled to 3 °C at a rate of 0.1 °C/min. After stirring at 3 °C for approximately 8 h, 0.4 mL of heptane was added. The obtained solid was separated by filtration and dried in a vacuum oven at 40 °C for approximately 2 h to obtain compound II, pattern 1. The characterization results are reported in Table 37. The XRPD diffraction pattern of compound II, pattern 1, is shown in Figure 71.

Table 37. Characteristics of Compound II, Mode 1 (Small-scale preparation)

Experiment 2. Large-scale preparation

3 g of _RP compound I free base and 9 mL of IPA were added to a glass vial. 1.0 equivalent of fumaric acid was added, and the resulting mixture was stirred at 50 °C for 5 min. Approximately 21 mg of compound II mode 1 seed crystals from Experiment 1 were added to the mixture. 30 mL of heptane was added to the mixture. The sample was stirred at 50 °C for 1 h, then cooled to 3 °C at a rate of 0.1 °C/min. After stirring at 3 °C for approximately 16 h, the obtained solid was separated by filtration and dried in a vacuum oven at 40 °C for approximately 4 h and then at 50 °C for approximately 3 h. This yielded 2.9 g of white solid compound II mode 1, with a yield of 78.3%. The XRPD diffraction pattern of compound II mode 1 is shown in Figure 71. The DSC thermogram of compound II mode 1 is shown in Figure 72. The TGA thermogram of compound II mode 1 is shown in Figure 73.

Table 38. Characteristics of Compound II, Mode 1 (Large-scale preparation)

Compound II, mode 1, is an anhydrous compound. Based on ^1H NMR results, the stoichiometry of free base:fumaric acid is approximately 1:1.0. It exhibits a melting peak at a T _initiation of 141.5 °C, accompanied by decomposition. TGA showed a weight loss of approximately 0.3% at approximately 130 °C. No residual solvent was detected by ^1H NMR.

Example 14: Preparation of Compound IV Mode 1

66 mg of the free base of _RP compound I and 0.2 mL of EtOH were added to a glass vial. 0.5 equivalents of fumaric acid were added, and the resulting mixture was stirred at 50 °C for 2 h, after which some solid precipitate formed. 0.4 mL of heptane was added to the suspension, and the mixture was stirred further at 25 °C for approximately 4 days. The solid was separated by filtration and dried in a vacuum oven at 50 °C for approximately 2 h. The obtained compound IV, pattern 1, was characterized as reported in Table 39. The XRPD diffraction pattern of compound IV, pattern 1 is shown in Figure 74. The DSC thermogram of compound IV, pattern 1 is shown in Figure 75. The TGA thermogram of compound IV, pattern 1 is shown in Figure 76.

Table 39. Characteristics of Compound IV, Mode 1

Example 15: Preparation of Compound III Mode 1

100 mg of the free base of _SP compound I and 0.3 mL of IPA were added to a glass vial. 1.0 equivalent of fumaric acid was added, and the mixture was stirred at 50 °C for 15 min, resulting in the precipitation of most of the material. After adding 1.0 mL of heptane, the sample was stirred at 50 °C for 1 h, then cooled to 3 °C at a rate of 0.1 °C/min. After stirring at 3 °C for approximately 8 h, 0.4 mL of heptane was added to the mixture to obtain a better suspension. The white solid was separated by filtration and dried in a vacuum oven at 40 °C for approximately 2 h to yield compound III mode 1. The characterization results are reported in Table 40. The XRPD diffraction pattern of compound III mode 1 is shown in Figure 77. The DSC thermogram of compound III mode 1 is shown in Figure 78. The TGA thermogram of compound III mode 1 is shown in Figure 79.

Table 40. Properties of Compound III, Mode 1

Example 16: Preparation of Compound III Mode 2

3 g of the free base of _SP compound I and 9 mL of IPA were added to a glass vial. Upon addition of 1.0 equivalent of fumaric acid, a large amount of solid precipitated immediately. 30 mL of heptane was added to the mixture, followed by approximately 20 mg of compound III mode 1 seed crystals. The sample was stirred at 50 °C for 1 h, then cooled to 3 °C at a rate of 0.1 °C/min. After stirring at 3 °C for approximately 20 h, the sample was heated from 3 °C to 50 °C over 20 min, and then 0.2 equivalents of fumaric acid and 1.5 mL of heptane were added to the mixture. The resulting mixture was stirred at 50 °C for approximately 2 h, then cooled to 3 °C at a rate of 0.1 °C/min. After stirring at 3 °C for approximately 13 h, the mixture was reheated to 50 °C over 20 min and stirred at 50 °C for approximately 6 h. The mixture was then cooled to 3 °C at a rate of 0.1 °C/min and stirred at 3 °C for approximately 2 days. The obtained solid was separated by filtration and dried in a vacuum oven at 50 °C for about 3 h to obtain 3.1 g of white solid, with a yield of 83.6%. The results are reported in Table 41. The XRPD diffraction pattern of compound III, mode 2 is shown in Figure 80. The DSC thermogram of compound III, mode 2 is shown in Figure 81. The TGA thermogram of compound III, mode 2 is shown in Figure 82.

Table 41. Characteristics of Compound III, Mode 2

Compound III, mode 2, is an anhydrous compound. Based on ^1H NMR results, the stoichiometry of free base:fumaric acid is approximately 1:1.2. It exhibits two melting peaks at a T _-start of 106.4 °C (with an enthalpy of approximately 47 J/g) and at a T _-start of 118.8 °C (with an enthalpy of approximately 63 J/g). It shows a weight loss of approximately 0.3% at approximately 105 °C. No residual solvent was detected by ^1H NMR.

Example 17: Preparation of compound V, mode 1

100 mg of the free base of _SP compound I and 1.0 equivalent of fumaric acid were added to a glass vial, followed by 0.8 mL of IPA. After stirring at 50 °C for about 1 h, a clear solution was obtained. Approximately 2 mg of compound III mode 1 seed crystals were added to the mixture. After observing some solid precipitation, 1 mL of heptane was added to the mixture. The mixture was stirred at 50 °C for 2 h and then cooled to 3 °C at 0.1 °C/min. It was stirred at 3 °C for about 3 days. The solid was separated by filtration and dried in a vacuum oven at 50 °C for about 2 h to produce compound V mode 1. The results are reported in Table 42. The XRPD diffraction pattern of compound V mode 1 is shown in Figure 83. The DSC thermogram of compound V mode 1 is shown in Figure 84. The TGA thermogram of compound V mode 1 is shown in Figure 85.

Table 42. Properties of Compound V, Mode 1

Example 18: Preparation of compound V mode 2

100 mg of the free base of _SP compound I and 1.0 equivalent of fumaric acid were added to a glass vial, followed by 0.8 mL of IPA. 1 mL of heptane was added to the resulting clear solution, and the mixture was stirred at 50 °C for 2 h and then cooled to 3 °C at 0.1 °C/min. It was stirred at 3 °C for approximately 3 days. The resulting solid was separated by filtration and dried in a vacuum oven at 50 °C for approximately 2 h to obtain compound V, mode 2. The results are reported in Table 43. The XRPD diffraction pattern of compound V, mode 2 is shown in Figure 86. The DSC thermogram of compound V, mode 2 is shown in Figure 87. The TGA thermogram of compound V, mode 2 is shown in Figure 88.

Table 43. Properties of Compound V, Mode 2

Example 19: Volumetric stability of compound II mode 1 and compound III mode 2

Compound II (mode 1) and Compound III (mode 2) were placed in open containers at 25°C/92% RH, open containers at 40°C/75% RH, and sealed containers at 60°C for one week. The samples were characterized by XRPD and HPLC, and color changes were examined. The results are presented in Table 44.

Table 44. Stability: Purity and Appearance

Initialization and chiral purity

The initial chemical purities of compound II (mode 1) and compound III (mode 2) were 99.7% and 98.8%, respectively. The chiral purity (%de) of compound II (mode 1) was 98.4%.

Volumetric stability

Accelerated stability tests were conducted for one week at 25°C/92% RH in an open container, at 40°C/75% RH in an open container, and at 60°C in a sealed container. Compound II (mode 1) showed good physical and chemical stability after exposure to all three conditions. Compound III (mode 2) showed good physical stability under all three conditions mentioned above. Degradation products increased by 1.6% and 1.5% after exposure to open container at 40°C/75% RH and sealed container at 60°C, respectively.

Example 20: Solubility study of compound II (mode 1) and compound III (mode 2)

12 mg of compound II (mode 1) and 12 mg of compound III (mode 2) were accurately weighed into 8 mL glass vials, and 5 mL of solubility medium was added. The amount of salt used was equivalent to 10 mg of anhydrous free base. After 0.5 and 2 h at 37 °C, all samples were clear solutions in the medium. The pH of the obtained clear solutions was analyzed using a pH meter, and the solubility was determined by observation.

Table 45. Solubility at 37℃, target concentration 10 mg/5 mL, equilibration duration 0.5 h and 2 h

Solubility was tested at 37°C for 0.5 h and 2 h in five media (pH 3.0 citrate buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer, water, and simulated vaginal secretions (pH 4.2)). Both candidates, compound II (mode 1) and compound III (mode 2), were highly soluble in the media (>2 mg/mL).

Example 21: Hygroscopicity of Compound II (Mode 1) and Compound III (Mode 2)

The hygroscopicity was investigated using DVS at 25°C using the following method:

Method: 40-0-95-0-40% RH, dm/dt = 0.002

Compound II (mode 1) and Compound III (mode 2) are slightly hygroscopic. Compound II (mode 1) showed approximately 0.2% water absorption at up to 95% RH. No change in form was observed after DSC testing. Compound III (mode 2) showed approximately 1.0% water absorption at up to 95% RH. No change in form was observed after DSC testing. The results are presented in Table 46.

Table 46.

Example 22: Screening of polymorphs of Compound II Mode 1 and Compound III Mode 2

50 mg of monofumarate was equilibrated in an appropriate amount of solvent or solvent mixture. The resulting suspension was equilibrated for 1 week. The solids were separated by centrifugation and filtration. The wet cake obtained after equilibration was analyzed by XRPD to determine the change in crystal form.

Table 47. Screening of micro polymorphs: Crystal modification after equilibration at 25°C for 1 week (the salt ratio is defined as the ratio of free base to fumaric acid).

Polymorphism assessment of Compound II Mode 1

In this study, no dissociation was observed during the equilibrium experiments, and two new potential polymorphs of the fumarate of isomer I (compound II mode 2 and compound II mode 3) were obtained from acetonitrile and MEK, respectively. These both exhibited lower melting temperatures than compound II mode 1. The two polymorphs of isomer I showed unchanged high chiral purity.

Polymorphism assessment of compound III mode 2

In this study, dissociation was observed during the equilibrium experiment, yielding the hemifumarate of isomer II (hemifumarate mode 2). Additionally, four new monofumarates of isomer II were obtained (compound mode 3, compound mode 4, compound mode 5, and compound mode 6).

Example 23: X-ray powder diffraction (XRPD)

XRPD analysis was performed on a Bruker D8 Advance diffractometer.

Table 48 below provides the results of XRPD performed on Compound II in Mode 1. The XRPD shows sharp peaks, indicating that the sample is composed of crystalline material. In the XRPD of Compound II in Mode 1, significant peaks were observed at approximately 3.1 ± 0.2°, approximately 9.3 ± 0.2°, approximately 12.1 ± 0.2°, approximately 14.9 ± 0.2°, approximately 15.1 ± 0.2°, approximately 18.1 ± 0.2°, approximately 19.8 ± 0.2°, approximately 20.1 ± 0.2°, approximately 25.1 ± 0.2°, approximately 25.9 ± 0.2°, and approximately 28.8 ± 0.2°.

Table 48. List of XRPD peaks for Compound II in Mode 1

Table 49 below provides the results of XRPD performed on compound IV in mode 1. Significant peaks were observed in the XRPD of compound IV in mode 1 at approximately 6.5 ± 0.2°, approximately 12.1 ± 0.2°, approximately 17.5 ± 0.2°, approximately 18.1 ± 0.2°, approximately 18.5 ± 0.2°, approximately 19.6 ± 0.2°, approximately 19.8 ± 0.2°, approximately 20.2 ± 0.2°, approximately 20.6 ± 0.2°, and approximately 21.3 ± 0.2°.

Table 49. List of XRPD peaks for Compound IV, Mode 1

Table 50 below provides the results of XRPD performed on compound III in mode 1. Significant peaks were observed in the XRPD of compound III in mode 1 at approximately 9.5 ± 0.2°, approximately 11.7 ± 0.2°, approximately 14.6 ± 0.2°, approximately 17.5 ± 0.2°, approximately 18.0 ± 0.2°, approximately 20.0 ± 0.2°, approximately 20.4 ± 0.2°, approximately 22.3 ± 0.2°, approximately 23.7 ± 0.2°, and approximately 25.5 ± 0.2°.

Table 50. List of XRPD peaks for Compound III, Mode 1

Table 51 below presents the results of XRPD performed on compound III, mode 2. The XRPD shows sharp peaks, indicating that the sample is composed of crystalline material. In the XRPD of compound III, mode 2, significant peaks were observed at approximately 8.9 ± 0.2°, approximately 9.9 ± 0.2°, approximately 11.7 ± 0.2°, approximately 12.1 ± 0.2°, approximately 15.1 ± 0.2°, approximately 17.9 ± 0.2°, approximately 18.2 ± 0.2°, approximately 19.9 ± 0.2°, approximately 25.1 ± 0.2°, approximately 29.6 ± 0.2°, and approximately 38.1 ± 0.2°.

Table 51. List of XRPD peaks for Compound III, Mode 2

Table 52 below provides the results of XRPD performed on compound V in mode 1. Significant peaks were observed in the XRPD of compound V in mode 1 at approximately 5.0 ± 0.2°, approximately 7.2 ± 0.2°, approximately 10.1 ± 0.2°, approximately 12.1 ± 0.2°, approximately 17.5 ± 0.2°, approximately 17.9 ± 0.2°, approximately 19.3 ± 0.2°, approximately 22.0 ± 0.2°, approximately 24.3 ± 0.2°, approximately 25.1 ± 0.2°, and approximately 26.3 ± 0.2°.

Table 52. List of XRPD peaks for Compound V in Mode 1

Table 53 below provides the results of XRPD performed on compound V, mode 2. Significant peaks were observed in the XRPD of compound V, mode 2, at approximately 5.1 ± 0.2°, approximately 6.9 ± 0.2°, approximately 7.6 ± 0.2°, approximately 10.2 ± 0.2°, approximately 11.6 ± 0.2°, approximately 12.1 ± 0.2°, approximately 15.1 ± 0.2°, approximately 17.6 ± 0.2°, approximately 18.1 ± 0.2°, approximately 18.7 ± 0.2°, approximately 19.5 ± 0.2°, approximately 19.8 ± 0.2°, and approximately 25.1 ± 0.2°.

Table 53. List of XRPD peaks for Compound V in Mode 2

Example 24: Single-crystal X-ray diffraction (SC-XRD) study of compound II in mode 1

Single crystals of compound II mode 1 suitable for SC-XRD studies were obtained in temperature cycling experiments in MeOH. X-ray diffraction data were collected at 170(2) K using a D8 Venture diffractometer equipped with a CMOS surface detector, using a Cu-Kα radiation X-ray generator with a power of 50 kV and 1.4 mA; a sample-to-detector distance of 40 mm; an exposure time of 150 s; and a resolution of 0.81. Structural refinement: on F ^2. Hydrogen site positions: mixed. H atoms were treated with a mixture of independent and constrained refinements. X-ray diffraction data and crystal data are presented in Table 54.

Table 54. Crystallographic parameters and X-ray diffraction data

In the monoclinic system, the crystalline form of compound II mode 1, crystallized in space group P2 ₁ with R _int = 5.7% and final R ₁ [I>2σ(I)] = 7.4%, is shown. The crystalline form does not contain solvent molecules. This crystalline form of compound II mode 1 is found to have a free base to fumarate ratio of 1:1 and corresponds to the monofumarate of isomer I with a Flack parameter (absolute structural parameter) of 0.16(10). As shown in Figure 115, in the single crystal structure, the protonated free base and fumarate anion are connected by N ⁺ (5)-H(5)···O(7) ionic bonds. Proton transfer is observed from the N(5)-nitrogen atom of fumarate to purine.

Example 25. Tablet stability study

Tablets were prepared using the ratios of one or more excipients shown in the first column of the table below. The tablets were then stored under the conditions shown, and samples were taken periodically for purity determination by HPLC. In a range of different formulations, tablets produced from compound I monofumarate showed less degradation than tablets produced from compound I.

Tablets made with compound I

Tablets produced using compound I monofumarate

PEO: Polyethylene oxide; PC: Polycarboxylic acid; MCC: Microcrystalline cellulose; MgS: Magnesium stearate

Example 26: Synthesis of a mixture of (R,S) and (S,S) ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (compound I)

Step 1: Preparation of diethyl-((2-(2-amino-6-chloro-9H-purin-9-yl)-ethoxy)-methyl)-phosphonate (3)

Under a _nitrogen atmosphere and at room temperature, a dry reaction vessel was charged with 50 g (0.296 mol, 1 equivalent) of 2-amino-6 _- chloropurine ₁ , 96.37 g (0.296 mol, 1 equivalent) of Cs₂CO₃, and 250 mL of DMF. Diethyl-2-chloroethoxymethylphosphonate 2 (74.85 g, 0.325 mol, 1.1 equivalent) was added dropwise with stirring at the same room temperature. The reaction was stirred at 40–50 °C for 0.5–1.5 h, heated to 60–70 °C and stirred for 0.5–1.5 h, and then stirred at 75–85 °C for 18–24 h. After the reaction temperature reached 20–30 °C, the reaction mixture was filtered, and the resulting cake was washed with DMF (100 mL x 2). At below 70°C, the combined filtrates were concentrated to half their volume, diluted with n-heptane (250 mL), and concentrated again to half their volume at below 75°C. At 20–30°C, the resulting solution was poured into DCM (1 L), stirred for 20–40 min, and then 10% aqueous _Na₂SO₄ solution (approximately ₁₀₀ mL) was added. The resulting biphasic solution was stirred for 20–40 _min , filtered through diatomaceous earth, and the wet cake was washed with DCM (approximately 100 mL). The aqueous phase was separated from the filtrate, and the organic phase was washed again with 10% aqueous _Na₂SO₄ solution (approximately 100 mL). The combined aqueous phases were washed (back-extracted) with DCM (200–300 mL), the organic phases were combined, and concentrated. The resulting crude product 3 was then purified by silica gel column chromatography (using DCM to 1% MeOH in the DCM). The fractions containing the product were combined, and the solvent was evaporated at below 40°C. Solid product 3 was treated with repeatedly diluted MTBE and concentrated (to 1/3 ^rd volume). The resulting slurry was then diluted with MTBE (400-500 mL) and stirred at 40-50 °C for 4-6 h, and then at 15-25 °C for 8-15 h. The suspension was filtered, washed with MTBE, and dried at 35-40 °C for 15-20 h to provide the desired product, diethyl-((2-(2-amino-6-chloro-9H-purin-9-yl)-ethoxy)-methyl)-phosphonate 3, in a separated yield of 43.4% (48.66 g) with a purity of 91.8% according to HPLC. ¹ H NMR (400MHz, DMSO-d6), δppm: 8.08 (s, 1H), 6.91 (s, 2H), 4.24 (d, 2H, J = 8Hz), 3.92 (m, 4H), 3.86 (q, 4H, J = 8Hz), 1.14 (t, 6H, J = 8Hz). LCMS (m/z): 364.2(MH+) and 366.2(MH+).

Step 2: Preparation of ((2-(2-amino-6-chloro-9H-purine-9-yl)-ethoxy)-methyl)-phosphonic acid (4)

Under a _nitrogen atmosphere, diethyl-((2-(2-amino-6-chloro-9H-purin-9-yl)-ethoxy)-methyl)phosphonate 3 (100 g, 0.275 mol) was loaded into a dry reaction vessel containing 1 L of DCM, followed by 2,6-rutidine (147.33 g, 1.375 mol, 5 equivalents), and the temperature was adjusted to 0–5 °C. TMSBr (167.47 g, 1.102 mol, 4.0 equivalents) was added dropwise, and the mixture was stirred at 0–5 °C for 0.5–1 h, followed by stirring at 20–25 °C for 15–20 h. After adjusting the reaction temperature to 0–5 °C, 1144 g of aqueous 1NNaOH was added dropwise. After maintaining the temperature at 20–30 °C for 1–2 h, the aqueous alkaline layer was separated and repeatedly washed with MTBE. At 15-25°C, the aqueous solution was acidified to pH 6-7 by dropwise addition of aqueous 2N HCl, and then MeOH (10 volumes) was added. At 35-45°C, the resulting methanol solution was further acidified to pH 3-4 by dropwise addition of aqueous 2N HCl. After adding the seed crystals of product 4, the methanol acid solution was stirred at 35-45°C for 3-5 hours, and further acidified to pH 1.5-2.5 by dropwise addition of aqueous 2N HCl, and stirred at 15-20°C for 11-20 hours. The obtained solid was separated by filtration, washed with MeOH (2 x 100 mL), and dried at 45–55 °C for 20–30 h to give the desired product, ((2-(2-amino-6-chloro-9H-purin-9-yl)-ethoxy)-methyl)-phosphonic acid 4, in a yield of 96.5% (84.4 g) with a purity of 99.8% according to HPLC. ^¹H NMR (400 MHz, DMSO-d6), δppm: 8.1 (s, 1H), 6.92 (bs, 2H), 4.5–5.5 (bs, 2H), 4.22 (dd = t, 2H, J = 8 Hz), 3.84 (t, 2H, J = 8 Hz), 3.58 (t, 2H, J = 8 Hz). LCMS (m/z): 308 (MH+) and 310 (MH+).

Step 3: Preparation of ((2-(2-amino-6-methoxy-9H-purine-9-yl)-ethoxy)-methyl)-phosphonic acid (5)

Add 50 g (0.162 mol) of (2-(2-amino-6-chloro-9H-purine-9-yl)-ethoxy)-methyl)phosphonic acid 4 to a flask containing 350 mL of MeOH at 20-30 °C and stir for 10-30 min. Add dropwise 30 wt% NaOMe solution (1.62 mol, 10 equivalents) in MeOH to this solution, and then stir at 50-60 °C for 15-24 h. Maintain the reaction at 20-30 °C for 20-40 min, and then filter. Acidify the filtrate at 20-30 °C to adjust the pH to 6-7 by dropwise addition of concentrated HCl, and concentrate to one-third of its volume at below 40 °C. Raise the temperature of the concentrated solution to 35-45 °C and acidify by dropwise addition of concentrated HCl to adjust the pH to 3-4. The resulting acidic solution was loaded into the seed crystals of product 5 and stirred at 35-45°C for 1.5-2.5 h. At this temperature, concentrated HCl was added dropwise to adjust the pH to 2-3, and the mixture was stirred for 3-5 h. The mixture was then cooled to -3°C to 3°C and stirred for 8-15 h. The resulting solid was filtered and washed with MeOH (approximately 100 mL) and n-heptane (approximately 100 mL). The resulting cake was dried under vacuum at 50-60°C for 16-24 h to provide the desired product, ((2-(2-amino-6-methoxy-9H-purine-9-yl)-ethoxy)-methyl)-phosphonic acid 5, in a separated yield of 89.3% (48.22 g) with a purity of 99.5% according to HPLC. ¹ H NMR (400MHz, DMSO-d6), δ, ppm: 7.88 (s, 1H), 6.47 (bs, 4H), 4.18 (t, 2H, J = 8Hz), 3.96 (t, 2H, J = 8Hz), 3.60 (d, 2H, J = 12Hz). LCMS(m/z):304.20(MH+).

Step 4a: Preparation of a mixture of (R,S)- and (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (8)

At 20-30℃, with stirring, (S)-ethyl 2-aminopropionate hydrochloride 6 (20.19 g, 0.132 mol, 1 equivalent), benzyl alcohol 7 (71.28 g, 0.66 mol, 5 equivalents) and TEA (159.98 g, 1.58 mol, 12 equivalents) were added to a solution of ((2-(2-amino-6-methoxy-9H-purine-9-yl)-ethoxy)-methyl)-phosphonic acid 5 (40 g, 0.132 mol, 1 equivalent) in DCM (560 mL), and the solution was stirred for 10-30 min. At 20-30°C, over 60 min, a solution prepared from Ph ₃ P (207.5 g, 0.792 mol, 6 equivalents) and 2,2'-dithiopyridine (Aldrithiol-2) (174.24 g, 0.792 mol, 6 equivalents) in DCM (320 mL) was added to the solution. The resulting reaction mixture was stirred at 35-45°C for 15-20 h and concentrated under vacuum at below 40°C to remove ^3/4 of the solvent. MeOH (approximately 120 mL), distilled water (approximately 400 mL), toluene (approximately 400 mL), and n-heptane (approximately 400 mL) were added to the residue, and the mixture was stirred at 20-30°C for 0.5-1 h. After allowing the reaction mixture to stand at 20–30 °C for 0.5 to 1 h, the organic phase is separated, and the aqueous phase is extracted several times with a mixture of toluene (approximately 400 mL) and n-heptane (approximately 400 mL) to remove the maximum amount of residual reagents and byproducts. The remaining aqueous phase is then extracted with DCM (2 x 400 mL), and after concentrating the DCM under vacuum at below 40 °C, the crude product is purified by silica gel column chromatography (using DCM to 2% MeOH in the DCM as the mobile phase). The elution fractions containing the product were combined, and the solvent was removed under vacuum at below 40°C to give the desired product as a mixture of diastereomers, namely (R,S) and (S,S); (±)(2S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate 8 (compound I), in a yield of 45.8% (29.74 g) with a purity of 98.8% according to HPLC. ¹ H NMR(400MHz,DMSO-d6),δ,ppm:7.85(s,1H),7.34(m,5H),6.44(s,2H),5.36(m,1H), 4.90(m,2H),4.17(m,2H),4.07(m,2H),3.95(s,3H),3.82(m,5H),1.18-1.24(m,6H). LCMS(m/z):493.3(MH+).

Step 4b: Preparation of a mixture of (R,R)- and (S,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate esters

To synthesize a mixture of (R,R) and (S,R), D-alanine ethyl ester ((R)-ethyl 2-aminopropionate hydrogen chloride) can be replaced with L-alanine ethyl ester ((S)-ethyl 2-aminopropionate hydrogen chloride) in step 4a.

Example 27: Preparation of (±)-Compound I monofumarate ((±)-(2S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate)(9)

At 45-55°C, through a filter, a solution of fumaric acid (7.66 mol, 1.1 equivalent) in IPA was added to a solution of (±)-(2S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (compound I) 8 (29.72 g, 0.06 mol, 1 equivalent) in IPA, and stirring was continued for 1-2 h. Seed crystals of compound 9 were added to the reaction mixture, and stirring was continued at 45-55°C for 1-2 h. After allowing the reaction mixture to settle at 20-30°C for 4-6 h, n-heptane (approximately 300 mL) was added dropwise, and stirring was continued at 20-30°C for 8-15 h, followed by stirring at 0-5°C for 8-15 h. The observed solid was filtered, and the wet cake was washed with a mixture of IPA/n-heptane (1/3, v/v, about 50-60 mL). The solid cake was dried under vacuum at 35-45 °C for 16-24 h to provide the desired product, (±)-(2S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate 9 (compound I monofumarate), in a yield of 87.9% (32.74 g) with a purity of 99.1% according to HPLC. ¹ H NMR (DMSO-d ₆ ), δ, ppm: 1.14 (t, 3H, J = 7.2Hz), 1.22 (d, 3H, J = 7.2Hz), 3.82 (m, 2H; dd, 1H, J = 4.0Hz; bs, 2H), 3.95 (s, 3H), 4.06 (m, 2H ), 4.17 (m, 2H), 4.87 (m, 2H), 5.38 (q, 1H, J = 4Hz), 6.44 (s, 2H), 6.64 (s, 2H), 7.33 (m, 5H), 7.82 (s, 1H), 13.18 (bs, 2H). LCMS(m/z):493.20(MH+).

Example 28: Chiral separation of the (R,S)- and (S,S)- isomers of compound I and preparation of their monofumarate, compound II, and compound III

Step 1a: Chiral separation of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate and (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate

Compound I (a mixture of diastereomers of isomers I and II) (22.50 g) was subjected to chiral chromatography under the SFC separation conditions shown below to separate and obtain 11.7 g (R,S)-isomer I (10) (with 98.6% purity according to HPLC) and 9.1 g (S,S)-isomer II (11) (with 95.6% purity according to HPLC).

SFC conditions:

Column: ChiralPak AD, 250×30mm I.D., 10μm;

Mobile phase: A: _CO2 and B: _ethanol (0.1% _NH3H2O );

Gradient: B 45% isotropic;

Flow rate: 200 mL/min;

Wavelength: 310nm;

Cycle time: Approximately 6 minutes;

Back pressure: 100 bar;

Injection dose: approximately 1g.

Characterization of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (isomer I) as a free base: Purity by HPLC: 98.6%; ^1H NMR (DMSO-d6), δ, ppm: 7.82 (s, 1H), 7.30 (m, 5H), 6.38 (s, 2H), 5.30 (t, 1H), 4.83 (d, 2H), 4.18 (t, 2H), 4.05 (m, 2H), 3.95 (s, 3H), 3.84 (m, 2H), 3.60 (m, 5H), 1.20 (d, 3H), 1.15 (t, 3H); LCMS (m/z): 493 (MH+).

Characterization of (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (isomer II) as a free base: Purity by HPLC: 95.6%; ^1H NMR (DMSO-d6): δppm 7.82 (s, 1H), 7.35 (m, 5H), 6.45 (s, 2H), 5.30 (t, 1H), 4.80 (d, 2H), 4.18 (t, 2H), 4.05 (m, 2H), 3.95 (s, 3H), 3.80 (m, 3H), 3.70 (m, 2H), 1.20 (d, 3H), 1.15 (t, 3H); LCMS (m/z): 493 (MH+).

In some non-limiting embodiments, stereoisomers are separated using HPLC or SFC with achiral or chiral stationary phases. Non-limiting examples of chiral stationary phases that can be used include Chiralpak AD, Chiralpak AS, Chiralcel OG, and Chiralcel OJ.

In alternative, non-limiting embodiments, a single isomer can be synthesized asymmetrically. For non-limiting examples of asymmetric synthesis of phosphonoamides, see Numan, A. et al., “Asymmetric Synthesis of Stereogenic Phosphorus P(V) Centers Using Chiral Nucleophilic Catalysis”, Molecules 2021, 26, 3661 and Ambrosi, A. et al., “Synthesis of Rovafovir Etalafenamide (Part III): Evolution of the Synthetic Process to the Phosphonamidate Fragment”, 2021, Org. Process Res. Dev. 25, 5, 1247-1262.

Step 1b: Chiral separation of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate and (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate

The same technique used for the mixture of (R,R) and (S,S) as described above can be used to separate (R,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purine-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate and (S,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purine-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate (synthesized in step 4b of Example 26).

Step 2a: Preparation of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate (compound II)

A solution of fumaric acid (0.765 g, 6.6 mmol, 1.1 equivalent) in IPA was added to a solution of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate 10 (3 g, 6 mmol, 1 equivalent) in IPA. The mixture was filtered at 45-55 °C and stirred continuously for 1-2 hours. Seed crystals of compound 12 were added to the reaction mixture, and stirring was continued at 45-55 °C for 1-2 hours. After allowing the reaction mixture to settle at 20-30 °C for 4-6 hours, n-heptane (approximately 30 mL) was added dropwise, and stirring was continued at 20-30 °C for 8-15 hours, followed by stirring at 0-5 °C for 8-15 hours. The observed solids were filtered, and the wet cake was washed with a mixture of IPA/n-heptane (1/3, v/v, about 5 mL). The solid cake was dried under vacuum at 35–45 °C for 16–24 h to provide the desired product, (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate 12 (isomer I monofumarate or compound II), in a yield of 85% (3.1 g) of isolated product with a purity of 98.6% according to HPLC. ^1H NMR (DMSO- _d6 ), δ, ppm: δ 7.80 (s, 1H), 7.35 (m, 5H), 6.63 (s, 2H), 6.40 (s, 2H), 5.53 (t, 1H), 4.84 (d, 2H), 4.15 (t, 2H), 4.00 (m, 2H), 3.92 (s, 3H), 3.80 (m, 3H), 3.75 (m, 2H), 1.20 (d, 3H), 1.13 (t, 3H); base (10): fumaric acid ratio: 1:1.00 (by ^1H NMR).

Step 2b: Preparation of (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate (13) (also known as compound III)

A solution of fumaric acid (0.765 g, 6.6 mmol, 1.1 equivalent) in IPA was added to a solution of (R,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate 11 (3 g, 6 mmol, 1 equivalent) in IPA. The mixture was filtered at 45-55 °C and stirred continuously for 1-2 hours. Seed crystals of compound 13 were added to the reaction mixture, and stirring was continued at 45-55 °C for 1-2 hours. After allowing the reaction mixture to settle at 20-30 °C for 4-6 hours, n-heptane (approximately 30 mL) was added dropwise, and stirring was continued at 20-30 °C for 8-15 hours, followed by stirring at 0-5 °C for 8-15 hours. The observed solids were filtered, and the wet cake was washed with a mixture of IPA/n-heptane (1/3, v/v, about 5 mL). The solid cake was dried under vacuum at 35–45 °C for 16–24 h to provide the desired product, (S,S)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate 13 (isomer II monofumarate or compound III), in a yield of 80% (2.9 g) of isolated product with a purity of 95.6% according to HPLC. ^1H NMR (DMSO- _d6 ), δ, ppm: δ 7.82 (s, 1H), 7.35 (m, 5H), 6.62 (s, 2H), 6.35 (s, 2H), 5.30 (t, 1H), 4.90 (d, 2H), 4.15 (t, 2H), 4.05 (m, 2H), 3.95 (s, 3H), 3.80 (m, 3H), 3.70 (m, 2H), 1.20 (d, 3H), 1.15 (t, 3H); base (11): fumaric acid ratio: 1:1.2 (by ^1H NMR).

Step 2c: Preparation of (R,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (S,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate

The synthesis of (R,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate and (S,R)-ethyl-2-((((2-(2-amino-6-methoxy-9H-purin-9-yl)-ethoxy)-methyl)-(benzyloxy)-phosphoryl)-amino)-propionate monofumarate can be performed as in steps 2b and 2c for the (R,S) and (S,S) stereoisomers, by replacing the starting materials in steps 2b and 2c with the products of chiral separation in step 1b.

Example 29. Non-limiting example of preparation of semi-solid dosage forms

Topical cream formulations can be prepared, for example, by emulsifying an oil phase and an aqueous phase together with the active pharmaceutical ingredient. In a non-limiting embodiment, the oil phase of the cream is prepared by mixing light mineral oil, propylparaben, and 63. Next, the aqueous phase of the cream is prepared by mixing water, EDTA, methylparaben, and 974P, and then the oil and aqueous phases are emulsified. The active pharmaceutical ingredient and propylene glycol are added to the emulsified mixture. The mixture is pH adjusted and then filled into tubes.

Topical gel formulations can be prepared, for example, by mixing an aqueous gel carrier with an active pharmaceutical ingredient. In a non-limiting embodiment, the aqueous phase of the topical gel is prepared by mixing water, EDTA, methylparaben (or sorbic acid), and 974P. The active pharmaceutical ingredient and propylene glycol are added to this solution, mixed, and the pH is adjusted before filling into tubes.

In some non-limiting embodiments, the active pharmaceutical ingredient is added from about 0.001% w/w to about 10% w/w to the semi-solid dosage form. For example, from about 0.0025% w/w to about 2.5% w/w, such as 0.003%, 0.01%, 0.03%, 0.1%, 0.3%, or 1%.

Example 30. Preparation of Compound I monofumarate tablets

The following provides a non-limiting example of the preparation of cervical tablets containing compound I monofumarate (see flowchart in Figure 119). Two or more excipients are combined, blended, and screened to prepare excipient blends. The active pharmaceutical ingredient (e.g., compound I monofumarate) is then screened and added to a portion of the excipient blend. The resulting mixture is then blended, and more excipient blends are then added. Thus, the mixture is progressively diluted with excipient blends, with thorough mixing after each addition of excipient blend. Once the excipient blend is exhausted, magnesium stearate is added, and the mixture is blended together again. The mixture is then compressed into tablets and packaged.

Table 57. Formulation composition of Compound I monofumarate vaginal tablets, batch size of 0.3 mg is 1.0 kg.

Element %w/w Quantity/batch (g) Compound I monofumarate, Compound II or Compound III 0.212 2.126 Microcrystalline cellulose (pH 102) 88.788 887.88 Mannitol 10.0 100.0 magnesium stearate 1.0 10 Total 100.0 1000.0

Single tablet weight: 175mg

Compound I monofumarate vaginal tablets, 0.3 mg

The following provides a non-limiting example of a process for preparing vaginal tablets of compound I monofumarate.

Repackaging

1. Weigh the materials according to the batch manufacturing formula and package them into individual plastic bags.

filter

1. Provides screening for all excipients.

Blending and screening of surfactants

1. The selected excipients, microcrystalline cellulose and mannitol, were blended in a diffusion blender.

2. Take 49.5 g of the excipient blend and add 2.12 g of compound I monofumarate.

3. Blend the surfactant and excipients and screen to remove large pieces.

4. Add 148.5 grams of excipient blend to the blend.

5. Blend the reactive blend and excipients and screen to remove large pieces.

6. Add 247.5 grams of excipient blend to the blend.

7. Blend the reactive blend and excipients and screen to remove large pieces.

8. Add the remaining 495 grams of excipient blend to the blend.

9. Blend the reactive blend and excipients and screen to remove large pieces.

Final blending

10. Add magnesium stearate to the diffusion mixer and mix the contents.

11. Discharge and blend the mixture.

compression

1. Compress the blend on a rotary tablet press using appropriate tools (punching and dies) to achieve the target weight. Check for friability and disintegration at the start of the compression run, and periodically check the weight, thickness, and hardness of individual tablets.

Package

1. Pack the bulk tablets into double-stitched, resealable transparent PE bags with a desiccant between the bags, and further pack them into aluminum foil bags with a desiccant and heat-seal them.

Example 31. Exemplary excipients for tablet formulations

Tablet formulations are selected to exhibit mucosal adhesion and affinity properties, and include excipients with solubilizing, erosive (for disintegration), porosity (for water absorption), and viscosity-enhancing (to maintain the drug at the target site) properties. Examples of excipients that will cause rapid disintegration to cover the cervix, anus, or vaginal area include, but are not limited to, mannitol, microcrystalline cellulose, lactose, sucrose, calcium phosphate, sodium phosphate, sodium bicarbonate, citric acid, maleic acid, adipic acid, or fumaric acid. Examples of excipients that can enhance disintegration and cover the affected area include, but are not limited to, sodium glycolate starch, pregelatinized starch, crospovidone, and croscarmellose sodium. Mucosal adhesion excipients that can be used in this invention include, but are not limited to, microcrystalline cellulose, polycarboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, and PVP.

The table below lists combinations of excipients that have the desired properties for tablet formulations. The tablet formulation contains an active pharmaceutical ingredient, microcrystalline cellulose, and may contain mannitol. In some non-limiting embodiments, the tablet formulation contains one or more excipients selected from the rapid disintegrant class (left column of Table 58). In some non-limiting embodiments, the tablet formulation contains one or more excipients selected from the disintegration enhancer class (middle column of Table 58). In some non-limiting embodiments, the tablet formulation contains one or more excipients selected from the mucosal adhesion excipient class (right column of Table 58).

Table 58. Excipients for Tablets

(Percentages are expressed as weight/weight%)

Example 32. Exemplary excipients for reconstructing powder or dry powder formulations

Reconstructing powder or dry powder formulations can improve the shelf stability of pharmaceuticals or formulations. In some non-limiting embodiments, the dry powder formulation may be mixed with brine, propylene glycol, or other aqueous carriers shortly before application to minimize degradation time. In some non-limiting embodiments, the dry powder formulation may be mixed with oil, cream, or other non-aqueous carriers shortly before application.

In some embodiments, the reconstituted powder or dry powder formulation rapidly covers infected or diseased tissue in the cervix, vulva, vagina, perianal area, penis, or anus. Excipients that enhance rapid coverage of the cervix, vulva, vagina, perianal area, penis, or anus include, but are not limited to, mannitol, lactose, sucrose, calcium phosphate, and microcrystalline cellulose. In some embodiments, mannitol is used as an excipient for rapid coverage of the cervix, vulva, vagina, perianal area, penis, or anus.

In some embodiments, the reconstituted powder or dry powder formulation provides good coverage of the cervix, vulva, vagina, perianal area, penis, or anus. Non-limiting examples of excipients that enhance coverage of the cervix, vulva, vagina, perianal area, penis, or anus include sodium glycolate starch, pregelatinized starch, crospovidone, and croscarmellose sodium.

In some embodiments, the reconstituted powder or dry powder formulation inherently possesses mucosal adhesion properties upon reconstitution. This prevents the application of the dosage form or other methods that expose healthy tissue to the active pharmaceutical ingredient. Excipients that enhance the mucosal adhesion properties of the reconstituted powder or dry powder formulation include, but are not limited to, xanthan gum, polycarboxylic acid, polyethylene glycol, hydroxyethyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, PVP, and microcrystalline cellulose. In some embodiments, xanthan gum is the excipient that improves mucosal adhesion.

The table below lists combinations of excipients having the desired properties for reconstituted powder or dry powder formulations. Dry powder or reconstituted powder formulations contain an active pharmaceutical ingredient and may contain mannitol and/or xanthan gum. In some non-limiting embodiments, the dry powder or reconstituted powder formulation contains one or more excipients selected from the rapid coverage category (left column of Table 59). In some non-limiting embodiments, the dry powder or reconstituted powder formulation contains one or more excipients selected from the coverage-enhancing category (middle column of Table 59). In some non-limiting embodiments, the dry powder or reconstituted powder formulation contains one or more excipients selected from the mucosal adhesion excipient category (right column of Table 58).

Table 59. Excipients used for reconstructing powder or dry powder formulations

(Percentages are expressed as weight/weight%)

Example 33. Exemplary excipients for semi-solid dosage forms

Semi-solid formulations are chosen to exhibit mucosal adhesion properties and facilitate drug penetration into tissues. Semi-solid formulations may contain excipients with solubilizing, lipophilic (to facilitate the dissolution of lipophilic compounds), penetrating (higher activity), and mucosal adhesion (to maintain the drug at the target site) properties.

In some embodiments, the semi-solid formulation has mucosal adhesive properties. Excipients that contribute to mucosal adhesive properties include, but are not limited to, carbomer, polyethylene glycol, cropovidone, polycarbofil, hydroxypropyl methylcellulose, and hydroxyethyl cellulose.

In some embodiments, semi-solid formulations enhance the penetration and/or solubility of the active pharmaceutical ingredient. Excipients that enhance the penetration and/or solubility of the active pharmaceutical ingredient include, but are not limited to, polyethylene glycol 6 stearate type I, polyethylene stearate, polyethylene glycol 32 stearate type I, and propylene glycol.

The table below lists combinations of excipients that possess the desired properties for semi-solid formulations. Semi-solid formulations comprise an active pharmaceutical ingredient and one or more excipients from each column of Table 60. In some non-limiting embodiments, the semi-solid formulation comprises one or more excipients selected from mucosal adhesion polymers (left column of Table 60). In some non-limiting embodiments, the tablet formulation comprises one or more excipients selected from solubility and penetration enhancers (second column of Table 60). In some non-limiting embodiments, the semi-solid formulation comprises one or more excipients selected from lipophilic solubilizers (third column of Table 60). In some non-limiting embodiments, the semi-solid formulation comprises one or more excipients selected from penetration enhancers (right column of Table 60).

Table 60. Excipients for Semi-solid Dosage Forms

(Percentages are expressed as weight/weight%)

Example 34. Exemplary excipients for semi-solid dosage forms

The vaginal suppositories and film-forming formulations are chosen to be solid at room temperature but soften at body temperature to release the active pharmaceutical ingredient. This allows for convenient handling and storage of the formulation, as well as achieving desired tissue coverage at the cervix, vulva, vagina, perianal area, penis, or anus. In non-limiting embodiments of the film-forming formulation, one or more excipients from the left column of Table 61 provide the desired properties. In non-limiting embodiments of the vaginal suppository formulation, one or more excipients from the right column of Table 61 provide the desired properties.

Table 61. Excipients for use in films and vaginal suppositories

(Percentages are expressed as weight/weight%)

Example 35. Exemplary tablet formulation

In some non-limiting embodiments, the tablet dosage form comprises the ingredients listed in Table 62. In some non-limiting embodiments, the tablet dosage form comprises the ingredients listed in Table 63. An illustrative process for combining these ingredients into a tablet dosage form can be found in Example 29.

Table 62. Examples of Tablet Formulations

Table 63. Examples of Tablet Formulations

Example 36. Exemplary semi-solid dosage form

In some non-limiting embodiments, the formulation for a cream semi-solid dosage form comprises the ingredients in Table 64. In some non-limiting embodiments, the formulation for a gel semi-solid dosage form comprises the ingredients in Table 65. Illustrative processes for combining these ingredients into a cream or gel semi-solid dosage form can be found in Example 27.

Table 64. Examples of semi-solid dosage forms (creams)

Table 65. Examples of semi-solid dosage forms (gels)

Example 37. Exemplary film-forming formulation

Film dosage forms can be prepared by solvent casting or hot melt extrusion. For example, to prepare a film dosage form, the active pharmaceutical ingredient is dissolved in a solution of excipients and water. The solution is then optionally degassed and cast into a film, which is then dried in an oven. Film dosage forms can also be prepared by hot melt extrusion. In some embodiments, the active pharmaceutical ingredient is mixed with one or more excipients in a hopper. These components are then mixed, ground, and kneaded into a homogeneous mixture. The mixture is heated until it flows and extruded through a die onto rollers, where it is cooled. In some embodiments, the components of the film dosage form can be found in Table 66.

Table 66. Examples of film formulations

Example 38. Exemplary dry powder or reconstituted powder formulation

In some non-limiting embodiments, the dry powder or reconstituted powder formulation comprises the ingredients listed in Table 67. These ingredients may be mixed in a suitable device (e.g., a V-type mixer) and then dispensed into sterile vials for reconstitution.

Table 67. Examples of formulations of dry powder or reconstituted powder

Example 39. Exemplary vaginal suppository preparation

In some non-limiting embodiments, the vaginal suppository formulation comprises the ingredients listed in Table 68 or Table 69. For example, the vaginal suppository formulation can be prepared by mixing the active pharmaceutical ingredient with an excipient. In one non-limiting embodiment, the excipient is heated and stirred in a mixing apparatus until it softens or melts, and then the active pharmaceutical ingredient is added in batches. The temperature, stirring speed, and addition rate are controlled to ensure uniform distribution of the active pharmaceutical ingredient. The mixture is then mixed until homogeneous and solidified in a vaginal suppository or suppository mold.

Table 68. Examples of formulations for vaginal suppositories

Table 69. Examples of formulations for use as vaginal suppositories

Example 40. In vitro cytotoxicity test

Compounds:

Three compounds (compound I, compound II, and compound III) were dissolved in DMSO at 40 mM and stored at -20°C. The test compounds were evaluated using a high test concentration of 50 μM. Serial semi-logarithmic dilutions were performed for in vitro cytotoxicity assays. Tamoxifen citrate was purchased from Sigma-Aldrich (St. Louis, MO). Tamoxifen citrate was dissolved in DMSO at 40 mM and used as a positive control compound at a high test concentration of 100 μM for cytotoxicity assays.

In vitro cytotoxicity assessment :

Cells listed in Table 70 were counted using the trypan blue dye exclusion method and seeded at 100 μL/well in the wells of a 96-well flat-bottom microtiter plate. Proliferating cells were incubated overnight at 37°C/5% CO2 to allow for approximately 70% confluence adhesion to the plate. Tissue culture medium was removed and replaced with 100 μL/well of fresh medium. One hundred microliters (100 μL) of each compound was transferred to 96-well plates containing triplicate sets of cells at six different concentrations. Table 70 lists the cell lines; cell types; source of cell stock; basic tissue culture medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin; and the microtiter plate seeding density.

Table 70:

Cytotoxic XTT:

After incubation at 37°C in a 5% _CO2 incubator for five days, the test plates were stained with tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide). XTT-tetrazolium is metabolized by mitochondrial enzymes of metabolically active cells to soluble formazan products. XTT solutions were prepared daily as a stock solution of 1 mg/ml in RPM11640. Phenazine methyl sulfate (PMS) solutions were prepared in PBS at 0.15 mg/ml and stored in the dark at -20°C. XTT/PMS stock solutions were prepared immediately before use by adding 40 μL of PMS/ml XTT solution. 50 μL of XTT/PMS was added to each well of the plate, and the plate was incubated at 37°C for another 4 hours. Seal the plate with adhesive plate sealant and gently shake or invert it several times to mix the soluble formazan product. Then, use a Molecular Devices Vmax plate reader to perform spectrophotometric readings of the plate at 450/650 nm.

Data analysis and evaluation:

Microsoft Excel 2010 was used to analyze and graph the raw data. CCso (50% reduction in cell viability) values were tabulated and provided. Raw cytotoxicity data with graphical representations were provided in a printed output, summarizing the combined effects on cell viability.

In vitro cytotoxicity assessment:

The cytotoxicity of compounds I, II, and III on the proliferation of Hs27, HeLa, C33A, and HEK293 cells was assessed by measuring cell viability after 5 days of culture using XTI tetrazolium dye (Table 71). The CC ₅₀ values calculated from these assays are summarized in the table below.

Tamoxifen citrate was evaluated in parallel as a control compound. The CC ₅₀ values of tamoxifen citrate in the proliferation of C33A, HeLa, Hs27, and HEK293 cells were 17.2, 19.9, 21.2, and 21.3 μM, respectively. When evaluated simultaneously, compound I and its two isomers showed similar cytotoxicity against each of the four cell lines. In C33A cells, the CC ₅₀ values of the three test compounds ranged from approximately 0.1 to 0.28 μM. In HeLa cells, the CC ₅₀ values ranged from 15.1 to 18.6 μM. In Hs27 cells, the CC ₅₀ values ranged from approximately 7.62 to 23.2 μM. In HEK293 cells, the CC ₅₀ value of the three test compounds was approximately 0.1 μM.

Table 71: In vitro cytotoxicity

Example 41. In vitro permeation and penetration of antiviral drugs in porcine vaginal tissue

Preparation of pig vaginal tissue

Freshly harvested porcine vaginal tissue was purchased from a local slaughterhouse and stored in an icebox. The vaginal tissue was incised to expose the mucosal surface and washed with a gentle stream of PBS at pH 7.4. With the aid of telemetry perforation, the porcine vaginal tissue was cut into circular sections (approximately 2 ^cm² ).

Pig vaginal tissue was fixed onto the Franz diffusion cell.

A circular portion of the tissue was sandwiched between two chambers of a Franz diffusion cell, with an active diffusion area of 1 ^cm² , and the mucosal layer exposed to the drug delivery chamber. Resistance within the porcine vaginal tissue was measured using a waveform generator to ensure the integrity of the tissue segment used for penetration studies. Porcine vaginal tissue with a resistance ≥3 kΩ· ^cm² was used for the study. The receiving chamber was filled with 8 ml of 5% solubilized PBS (pH 7.4), stirred at 600 rpm with a 3 mm magnetic stir bar, and the temperature was maintained at 37°C using a circulating water bath.

Loading the formulation into the drug supply chamber

Inject approximately 200 mg of gel into a 1 mL syringe (tare weight only) and dispense the gel into the delivery chamber. Apply the gel to the mucosal surface using a pre-weighed applicator. After loading and spreading the gel onto the mucosal surface, record the weight of the 1 mL syringe and applicator to determine the exact amount of gel loaded into the delivery chamber.

Penetration and penetration research

A time-course (2h, 4h, and 8h) porcine vaginal permeation study was conducted. After gel loading, 500 μL samples were removed from the receiving chamber at different time intervals, with an equal volume of fresh receiving medium used to replace the removed sample each time. Samples removed at each time interval were immediately stored at -20°C until analysis. At 2, 4, and 8 hours, the preparation was removed from the supply chamber with the aid of a syringe and cleaned with cotton swabs. The tissue was removed and gently washed five times with a washing solution (50% methanol in water), alternating with cotton swabs.

Table 72: Time-Process IVPT Study Design

Research Liquid sampling time point in the receiving cavity During the research period 2h IVPT 0 and 2h 2h 4h IVPT 0, 2 and 4h 4h 8h IVPT 0, 2, 4, 6 and 8h 8h

Following IVPT, the porcine vaginal tissue (active diffusion area) was minced.

Remove the 8 mm perforation from the active diffusion area of the washed porcine vaginal tissue, weigh it, and transfer it to a test tube. Immediately place the tube on dry ice for approximately 15 min. After the specified time, remove the tissue and place it on a pre-chilled plate. Use a pre-chilled scalpel blade to mince the tissue into small pieces on the plate. Transfer the minced tissue to a sample tube, rinse the plate with 1 ml of 5% solubilizer in PBS 7.4 pH, and transfer the rinsed tissue to the same tissue sample tube. Store these tubes at -70°C until analysis.

Preparation of the receiving cavity liquid for analysis

Remove the samples stored at -20°C and thaw at room temperature for 30 min. Centrifuge the drug from the receiving chamber liquid at 13000 rpm for 5 min, and add an equal volume of extraction solvent to 200 μL of supernatant. Centrifuge these samples at 13000 rpm for 5 min, and transfer the supernatant to vials for analysis.

Drugs extracted from pig vaginal tissue

Remove the shredded tissue stored at -70°C and thaw at room temperature for approximately 90 minutes. Incubate the samples in a BioShaker at room temperature with shaking for 4 hours. After 4 hours, centrifuge the samples at 13,000 rpm for 5 minutes. Add 400 μL of extraction solvent to 100 μL of the supernatant and vortex for 2 minutes. Centrifuge these samples at 13,000 rpm for 5 minutes and transfer the supernatant to vials for analysis.

All publications and patent applications referenced in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the invention as defined in the embodiments and/or claims.

Claims (184)

1. A compound having the following formula: 2. A compound having the following formula: Or its pharmaceutically acceptable salt. 3. A compound having the following formula: Or its pharmaceutically acceptable salt. 4. The compound of claim 2, having the following formula: 5. The compound of claim 3, having the following formula: 6. A separated crystalline form of a compound having the following formula: The separated crystalline form is characterized by containing at least five ZRPD spectra selected from the following 2θ values: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°. 7. A separable crystalline form of a compound having the following formula: The separated crystalline form is characterized by containing at least seven ZRPD spectra selected from the following 2θ values: 9.53±0.2°, 10.04±0.2°, 11.60±0.2°, 14.57±0.2°, 17.22±0.2°, 17.50±0.2°, 20.04±0.2°, 20.36±0.2°, 22.34±0.2°, 23.73±0.2°, 25.48±0.2°, 26.06±0.2°, 27.38±0.2°, and 32.20±0.2°. 8. A separable crystalline form of a compound having the following formula: The separated crystalline form is characterized by containing at least seven XRPD spectra selected from the following 2θ values: 8.94±0.2°, 9.89±0.2°, 9.91±0.2°, 11.66±0.2°, 12.11±0.2°, 15.13±0.2°, 17.85±0.2°, 18.15±0.2°, 19.90±0.2°, 20.38±0.2°, 22.94±0.2°, 25.09±0.2°, 26.54±0.2°, 26.90±0.2°, 27.38±0.2°, 28.28±0.2°, 28.95±0.2°, 29.64±0.2°, and 38.07±0.2°. 9. A separable crystalline form of a compound having the following formula: The separated crystalline form is characterized by containing at least five XRPD spectra selected from the following 2θ values: 3.08±0.2°, 9.30±0.2°, 12.08±0.2°, 14.92±0.2°, 15.10±0.2°, 20.14±0.2°, 25.14±0.2°, and 28.82±0.2°. 10. A pharmaceutical composition comprising a compound as described in any one of claims 1-5 and a pharmaceutically acceptable carrier. 11. A pharmaceutical composition comprising, in any one of claims 6-9, a crystalline form in a pharmaceutically acceptable carrier. 12. The pharmaceutical composition according to claims 10-11, wherein it is in solid dosage form. 13. The pharmaceutical composition according to claims 10-11, wherein it is a semi-solid dosage form. 14. The pharmaceutical composition of claims 10-11, wherein it is in reconstituted powder form. 15. The pharmaceutical composition according to claims 10-11, wherein it is in the form of a dry powder dosage form. 16. The pharmaceutical composition according to claims 10-11, wherein it is in film form. 17. The pharmaceutical composition of claims 10-11, wherein it is in the form of a vaginal suppository. 18. The pharmaceutical composition of claim 12, wherein it is in tablet form. 19. The pharmaceutical composition of claim 13, wherein it is in cream form. 20. The pharmaceutical composition of claim 13, wherein it is in gel form. 21. The pharmaceutical composition according to any one of claims 10-20, formulated for topical application. 22. The pharmaceutical composition according to any one of claims 10-21, for delivery to the cervix. 23. The pharmaceutical composition according to any one of claims 10-21, for delivery into the vagina. 24. The pharmaceutical composition according to any one of claims 10-21, for delivery to the vulva. 25. The pharmaceutical composition according to any one of claims 10-21, for delivery to the perianal area. 26. The pharmaceutical composition according to any one of claims 10-21, for delivery to the anus. 27. The pharmaceutical composition according to any one of claims 10-21, for delivery to the penis. 28. The pharmaceutical composition of claim 18, wherein the tablet is a bilayer tablet. 29. The pharmaceutical composition of claim 18, wherein the tablet disintegrates in less than about 250 μL of fluid. 30. The pharmaceutical composition of claim 18, wherein the tablet disintegrates in less than about 150 μL of fluid. 31. The pharmaceutical composition according to any one of claims 10-30, comprising about 0.01 mg to about 10 mg of the compound. 32. The pharmaceutical composition according to any one of claims 10-30, comprising about 0.01 mg to about 5 mg of the compound. 33. The pharmaceutical composition according to any one of claims 10-30, comprising about 0.03 mg to about 1 mg of the compound. 34. The pharmaceutical composition according to any one of claims 10-30, comprising about 0.03 mg to about 0.07 mg of the compound. 35. The pharmaceutical composition according to any one of claims 10-30, comprising about 0.05 mg to about 0.15 mg of the compound. 36. The pharmaceutical composition according to any one of claims 10-30, comprising about 0.15 mg to about 0.45 mg of the compound. 37. The pharmaceutical composition according to any one of claims 10-30, comprising at least about 0.05 mg of the compound. 38. The pharmaceutical composition according to any one of claims 10-30, comprising at least about 0.1 mg of the compound. 39. The pharmaceutical composition according to any one of claims 10-30, comprising about 0.001% to about 10% of the compound. 40. The pharmaceutical composition according to any one of claims 10-30, comprising about 0.01% to 0.5% of the compound. 41. The pharmaceutical composition according to any one of claims 10-30, comprising about 0.1% to 5% of the compound. 42. The pharmaceutical composition according to any one of claims 10-41, comprising a mucosal adhesion polymer. 43. The pharmaceutical composition of claim 42, comprising about 5% to about 20% of a mucosal adhesion polymer. 44. The pharmaceutical composition of claim 42, comprising about 10% to about 50% of a mucosal adhesion polymer. 45. The pharmaceutical composition of claim 42, comprising about 50% to about 90% of a mucosal adhesion polymer. 46. The pharmaceutical composition of any one of claims 10-41, comprising a disintegration-enhancing excipient. 47. The pharmaceutical composition of any one of claims 10-41, comprising a penetration-enhancing excipient. 48. The pharmaceutical composition of any one of claims 10-41, comprising an excipient that allows for the controlled release of the active compound. 49. The pharmaceutical composition of claim 19, wherein the pharmaceutically acceptable carrier comprises: light mineral oil, propylparaben, Tefose 63, water, EDTA, methylparaben, and Carbopol 974P. 50. The pharmaceutical composition of claim 20, wherein the pharmaceutically acceptable carrier comprises: water, EDTA, methyl benzoate, Carbopol 974P, propylene glycol, and sorbic acid. 51. The pharmaceutical composition of claim 18, wherein the tablet comprises: mannitol, polycrystalline cellulose, and magnesium stearate. 52. A method for treating human papillomavirus infection, the method comprising administering to a host in need an effective amount of the compound as described in any one of claims 1-5, said compound optionally in a pharmaceutically acceptable carrier. 53. A method for treating a condition caused by human papillomavirus infection, the method comprising administering to a host in need an effective amount of the compound as described in any one of claims 1-5, the compound optionally in a pharmaceutically acceptable carrier. 54. The method of claim 53, wherein the condition caused by human papillomavirus infection is intraepithelial neoplasia. 55. The method of claim 54, wherein the condition caused by human papillomavirus is atypical squamous cells of undetermined significance (ASC-US). 56. The method of claim 54, wherein the condition caused by human papillomavirus is atypical glandular cells (AGC). 57. The method of claim 54, wherein the condition caused by human papillomavirus is low-grade squamous intraepithelial lesion (LSIL). 58. The method of claim 54, wherein the symptom caused by human papillomavirus is atypical squamous cell disease, and high squamous intraepithelial lesion (ASC-H) cannot be ruled out. 59. The method of claim 54, wherein the condition caused by human papillomavirus is high-grade squamous intraepithelial lesion (HSIL). 60. The method of claim 54, wherein the condition caused by human papillomavirus is adenocarcinoma in situ (AIS). 61. The method of claim 54, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia. 62. The method of claim 61, wherein the cervical intraepithelial neoplasia is grade 1 cervical intraepithelial neoplasia. 63. The method of claim 61, wherein the cervical intraepithelial neoplasia is grade 2 cervical intraepithelial neoplasia. 64. The method of claim 61, wherein the cervical intraepithelial neoplasia is grade 3 cervical intraepithelial neoplasia. 65. The method of claim 54, wherein the intraepithelial neoplasia is vaginal intraepithelial neoplasia. 66. The method of claim 54, wherein the intraepithelial neoplasia is vulvar intraepithelial neoplasia. 67. The method of claim 54, wherein the intraepithelial neoplasia is anal intraepithelial neoplasia. 68. The method of claim 54, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia. 69. The method of claim 54, wherein the intraepithelial neoplasia is penile intraepithelial neoplasia. 70. The method of any one of claims 52-69, wherein the host is a human being. 71. The method of any one of claims 52-70, wherein the compound is applied topically. 72. The method of any one of claims 52-71, wherein about 0.01 mg to about 1 mg of the compound is administered. 73. The method of any one of claims 52-71, wherein about 0.05 mg to about 0.3 mg is administered. 74. The method of any one of claims 52-73, further comprising applying a lubricating method to the epithelial tissue before inserting the dosage form into the affected area. 75. The method of any one of claims 52-73, further comprising applying a lubricating agent to the dosage form before inserting it into the affected area. 76. The method of claim 74 or 75, wherein the lubrication method is selected from water, glycerin-based lubricants, and hydroxyethyl cellulose-based lubricants. 77. The method of any one of claims 52-76, wherein the compound is applied daily. 78. The method as described in any one of claims 52-76, applied approximately once daily. 79. The method as described in any one of claims 52-76, applied approximately twice a week. 80. The method of any one of claims 52-76, applied about three times or more per week. 81. The method as described in any one of claims 52-80, applied for about one week. 82. The method as described in any one of claims 52-80, applied for approximately two weeks. 83. The method as described in any one of claims 52-80, applied for approximately three weeks. 84. The method as described in any one of claims 52-80, applied for approximately four weeks. 85. The method as described in any one of claims 52-80, applied for approximately five weeks. 86. The method as described in any one of claims 52-80, applied for approximately six weeks. 87. The method of any one of claims 52-86, wherein the compound is administered over a treatment cycle, the treatment cycle comprising: a. The treatment period, which includes the administration of the compound, and b. The withdrawal period, which includes the period of no treatment. 88. The method of claim 87, wherein the withdrawal period is approximately one week. 89. The method of claim 87, wherein the withdrawal period is approximately two weeks. 90. The method of claim 87, wherein the withdrawal period is approximately three weeks. 91. The method of claim 87, wherein the withdrawal period is approximately four weeks. 92. The method of claim 87, wherein the withdrawal period is approximately five weeks. 93. The method of claim 87, wherein the withdrawal period is approximately six weeks. 94. The method of any one of claims 87-93, wherein at least two treatment cycles are administered. 95. The method of any one of claims 87-93, wherein at least three treatment cycles are administered. 96. The method of any one of claims 52-80, wherein about 0.05 mg to about 0.3 mg of the compound is administered daily. 97. The method of any one of claims 52-80, wherein about 0.05 mg to about 0.3 mg of the compound is administered three times a week. 98. The method of any one of claims 52-80, wherein about 0.05 mg to about 0.3 mg of the compound is administered once weekly. 99. The method of any one of claims 52-98, wherein the human papillomavirus is a high-risk strain. 100. The method of any one of claims 52-98, wherein the human papillomavirus is HPV-16 or HPV-18. 101. The method of any one of claims 52-100, wherein the compound is administered in combination with another antiviral compound. 102. The method of claim 101, wherein the antiviral compound is selected from the group consisting of: protease inhibitors, another DNA polymerase inhibitor, E6 or E6AP inhibitors, E7 inhibitors, E1 inhibitors, E2 inhibitors, E1-E2 protein interaction inhibitors, L2 lipopeptides, L1 inhibitors, L2 inhibitors, L1 degraders, and L2 degraders. 103. The method of any one of claims 52-100, wherein the compound is administered in combination with an anticancer compound. 104. The method of claim 103, wherein the anticancer compound is selected from the group consisting of: HDAC inhibitors, degraders of tetraspan membrane proteins, immune checkpoint inhibitors, T-cell therapies, and antiproliferative drugs. 105. The method of any one of claims 52-100, wherein the compound is administered in combination with a surgical procedure. 106. The method of claim 105, wherein the compound is administered prior to the surgical procedure. 107. The method of claim 105, wherein the compound is administered after the surgical procedure. 108. The method of claim 105, wherein the surgical procedure is performed during the administration of the compound. 109. The method of any one of claims 105-108, wherein the surgical procedure is the removal of diseased tissue. 110. The method of claim 109, wherein the resection is Circular Electrosurgical Excision Procedure (LEEP). 111. The method of claim 109, wherein the resection is a large loop resection of the transformation zone (LLETZ). 112. The method of claim 109, wherein the resection is a scalpel resection. 113. The method of claim 109, wherein the resection is a laser conization resection. 114. The method of any one of claims 105-108, wherein the surgical procedure is the ablation of the diseased tissue. 115. The method of claim 114, wherein the ablation is laser ablation. 116. The method of claim 114, wherein the ablation is cryoablation. 117. Use of the compounds of claims 1-5 in the preparation of a medicament for treating human papillomavirus infection in a host in need, wherein the compounds are optionally in a pharmaceutically acceptable carrier. 118. Use of the compounds of claims 1-5 in the preparation of a medicament for treating a condition caused by human papillomavirus infection in a host in need, wherein the compounds are optionally in a pharmaceutically acceptable carrier. 119. The use as claimed in claim 118, wherein the condition caused by human papillomavirus infection is intraepithelial neoplasia. 120. The use as described in claim 119, wherein the intraepithelial neoplasia is vaginal intraepithelial neoplasia. 121. The use as described in claim 119, wherein the intraepithelial neoplasia is vulvar intraepithelial neoplasia. 122. The use as described in claim 119, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia. 123. The use as described in claim 119, wherein the intraepithelial neoplasia is anal intraepithelial neoplasia. 124. The use as described in claim 119, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia. 125. The use as described in claim 119, wherein the intraepithelial neoplasia is penile intraepithelial neoplasia. 126. The use as described in claims 117-125, wherein the host is a human being. 127. The use as described in claims 117-126, for topical application. 128. The compound of any one of claims 1-5, for treating human papillomavirus infection in a host in need, wherein the compound is optionally in a pharmaceutically acceptable carrier. 129. The compound of any one of claims 1-5, for treating a condition caused by human papillomavirus infection in a host in need, wherein the compound is optionally in a pharmaceutically acceptable carrier. 130. The compound for the use of claim 129, wherein the condition caused by human papillomavirus infection is intraepithelial neoplasia. 131. The compound for the use of claim 130, wherein the intraepithelial neoplasia is vaginal intraepithelial neoplasia. 132. The compound for the use of claim 130, wherein the intraepithelial neoplasia is vulvar intraepithelial neoplasia. 133. The compound for the use of claim 130, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia. 134. The compound for the use of claim 130, wherein the intraepithelial neoplasia is anal intraepithelial neoplasia. 135. The compound for the use of claim 130, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia. 136. The compound for the use of claim 130, wherein the intraepithelial neoplasia is penile intraepithelial neoplasia. 137. The compound for the use described in claims 128-136, wherein the host is a human. 138. The compound for the use described in claims 128-137, wherein the compound is applied topically. 139. A method for preparing the crystalline form as described in claim 4, the method comprising: a. Dissolve _RP compound I in a solvent selected from methanol, ethanol and isopropanol; b. Stir at a temperature of about 20°C to about 70°C; c. Add 1.0 equivalent of fumaric acid; d. Add a solvent selected from pentane, hexane, and heptane; e. Cooling the mixture; f. Stirring and cooling the solution; and g. Separating and drying solids. 140. The method of claim 139, wherein the alcohol solvent in step (a) is ethanol or isopropanol. 141. The method of claims 139-140, wherein the alcohol solvent in step (a) is isopropanol. 142. The method of claim 139, wherein the solution of step (b) is stirred at about 45°C to about 55°C. 143. The method of claim 139, wherein the aliphatic solvent is hexane or heptane. 144. The method of claims 139 and 143, wherein the aliphatic solvent is heptane. 145. The method of claim 139, wherein the mixture is cooled to below about 20°C. 146. The method of claim 139, wherein the mixture is cooled to below about 10°C. 147. The method of claim 139, wherein the mixture is cooled to below about 5°C. 148. The method of claim 139, wherein the mixture is cooled to about 5°C to 0°C. 149. The pharmaceutical composition of claim 10 or 11, comprising microcrystalline cellulose. 150. The pharmaceutical composition of claim 10 or 11, wherein it comprises lactose. 151. The pharmaceutical composition of claim 10 or 11, comprising sucrose. 152. The pharmaceutical composition of claim 10 or 11, comprising calcium phosphate. 153. The pharmaceutical composition of claim 10 or 11, comprising sodium bicarbonate. 154. The pharmaceutical composition of claim 10 or 11, comprising citric acid. 155. The pharmaceutical composition of claim 10 or 11, comprising sodium glycolate starch. 156. The pharmaceutical composition of claim 10 or 11, comprising pregelatinized starch. 157. The pharmaceutical composition of claim 10 or 11, comprising crosspovidone. 158. The pharmaceutical composition of claim 10 or 11, comprising croscarmellose sodium. 159. The pharmaceutical composition of claim 10 or 11, comprising polycarboxyfil. 160. The pharmaceutical composition of claim 10 or 11, comprising polyethylene oxide. 161. The pharmaceutical composition of claim 10 or 11, comprising hydroxyethyl methylcellulose. 162. The pharmaceutical composition of claim 10 or 11, comprising hydroxyethyl cellulose. 163. The pharmaceutical composition of claim 10 or 11, comprising hydroxypropyl methylcellulose. 164. The pharmaceutical composition of claim 10 or 11, comprising maleic acid. 165. The pharmaceutical composition of claim 10 or 11, comprising adipic acid. 166. The pharmaceutical composition of claim 10 or 11, comprising fumaric acid. 167. The pharmaceutical composition of claim 10 or 11, comprising hydroxypropyl cellulose. 168. The pharmaceutical composition of claim 10 or 11, wherein it comprises PVP. 169. The pharmaceutical composition of claim 10 or 11, comprising xanthan gum. 170. The pharmaceutical composition of claim 10 or 11, comprising mannitol. 171. The pharmaceutical composition of claim 10 or 11, wherein the pharmaceutical composition comprises carbomer. 172. The pharmaceutical composition of claim 10 or 11, comprising polyethylene glycol. 173. The pharmaceutical composition of claim 10 or 11, comprising cetyl alcohol. 174. The pharmaceutical composition of claim 10 or 11, comprising stearyl alcohol. 175. The pharmaceutical composition of claim 10 or 11, comprising polysorbate. 176. The pharmaceutical composition of claim 10 or 11, comprising sodium lauryl sulfate. 177. The pharmaceutical composition of claim 10 or 11, comprising light mineral oil or mineral oil. 178. The pharmaceutical composition of claim 10 or 11, comprising beeswax. 179. The pharmaceutical composition of claim 10 or 11, comprising a silicon fluid. 180. The pharmaceutical composition of claim 10 or 11, comprising diethylene glycol monoethyl ether. 181. The pharmaceutical composition of claim 10 or 11, comprising oleic acid. 182. The pharmaceutical composition of claim 10 or 11, comprising isopropyl myristate. 183. The pharmaceutical composition of claim 10 or 11, comprising propylene glycol dioctanoate. 184. The pharmaceutical composition of claim 10 or 11, comprising glyceryl monooleate.