CN117751129A

CN117751129A - Compositions and dosage forms for the treatment of HPV infection and HPV-induced neoplasia

Info

Publication number: CN117751129A
Application number: CN202380011993.XA
Authority: CN
Inventors: B·奥尔德斯; R·加迪拉朱; S·沃尔特; O·丹尼尔斯; G·马德里斯; A·阿加德; R·马哈林甘; 赵正乐; 秦朝阳; 蒋思懿; 李润妍
Original assignee: Antiva Biosciences Inc
Current assignee: Antiva Biosciences Inc
Priority date: 2022-07-21
Filing date: 2023-07-20
Publication date: 2024-03-22

Abstract

The present invention provides pharmaceutically acceptable salts of acyclic nucleotide phosphamide and pharmaceutical compositions, crystalline forms, and dosage forms thereof for the treatment of HPV and related conditions, including neoplasias.

Description

Compositions and dosage forms for the treatment of HPV infection and HPV-induced neoplasia

Cross Reference to Related Applications

U.S. provisional application No. 63/391,283 filed on day 21 7 of 2022; U.S. provisional application No. 63/400,661 filed on 8/24 of 2022; china patent application No. 202211206517.7 filed on 9 and 30 of 2022; united states provisional application No. 63/412,143 filed on 9 and 30 of 2022. These applications are incorporated herein by reference for all purposes.

Technical Field

The present invention provides compositions, advantageous salts, prodrugs, stereoisomers, crystalline forms, dosage forms and uses thereof for treating Human Papillomavirus (HPV) infections or related disorders, such as HPV induced neoplasias, in a host in need thereof.

Background

According to the statement of the U.S. center for disease control, there is no direct cure for human papillomaviruses. In 2018, the number of infected subjects exceeded 4300 ten thousand, and the number of newly added infected subjects exceeded 1300 ten thousand.

Current treatment options for HPV infection are only adjuvant and limited. They all have significant drawbacks. Common drug therapies include salicylic acid, trichloroacetic acid, imiquimod and pradafil. Both trichloroacetic acid and salicylic acid chemically burn wart tissue as a means of removing virus, which often causes skin irritation, ulcers and pain. In addition, salicylic acid is not used to treat HPV infections of the anogenital area. Imiquimod (Aldara) ^TM ，Zyclara ^TM ) Stimulation of the immune system by toll-like receptor signaling clears the infection and causes redness and swelling. Pradeloy (Condylox) ^TM ) Disruption of tissue by microtubule destabilization prevents host cell replication.

More problematic is that HPV infection has caused transformation of cells in human patients, which have not progressed to cancer, but have reached the neoplastic stage. HPV induced neoplasias include cervical intraepithelial neoplasia ("CIN"), anal intraepithelial neoplasia ("AIN"), perianal intraepithelial neoplasia ("PAIN"), vulval 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.

Identification and treatment of HPV-induced neoplasia is critical before it progresses to a potentially untreated cancer. Almost all cervical cancer cases are caused by sexually transmitted HPV oncogenic type infections. The primary goal of early screening (e.g., pap test (pap smear test)) is to identify abnormal cervical cells with severe cellular changes in order to remove or destroy them.

Cervical intraepithelial neoplasia is most often treated by observation (waiting and sightseeing method) or by excision or ablation of the cervical transformation zone. Techniques include cryotherapy, laser therapy, ring electro-ablation (LEEP), and cone biopsy. All of these surgical procedures damage the affected area and may lead to scarring. The most common intervention LEEP is effective in 60% -90% of cases, however it is associated with a significantly increased risk of miscarriage, ectopic pregnancy and negative psychological consequences. Despite extensive research, no drug has been approved to replace or be used in conjunction with these surgical procedures.

Papillomaviruses are a group of non-enveloped DNA viruses that infect keratinocytes of the skin and mucosa, including the anogenital area, in humans. They are known to cause cutaneous warts, genital warts, respiratory papillomatosis and cancer. Several species of the genus α -papillomavirus contain high-risk types of HPV, which are more likely to cause neoplasia, then cancer, in humans. Most oncogenic HPV types are from the alpha-7 and alpha-9 categories, including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 types. The most common oncogenic HPV types are 16 and 18.HPV-16 and-18 are the etiology of most cervical cancers. Most warts are caused by low-risk HPV types 6 and 11. Vaccines have been developed against HPV 6, 11, 16 and 18, which may be effective if administered prior to the first sexual act. However, HPV vaccines may provide little benefit to sexually active females who have been infected with HPV.

Specific prophylactic vaccines that may be used include gardsil (HPV 9 vaccine; HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58), gardsil 4 (tetravalent) and Cervarix (bivalent). These are useful if the person is vaccinated before virus exposure (which usually means before sexual activity). Prophylactic vaccines aim to produce neutralizing antibodies that clear the virus before it infects the cells. In contrast, therapeutic vaccines are vaccines designed to generate cd4+ and/or cd8+ T cell-based responses to the elimination of HPV infected cells. Exemplary antigens for therapeutic vaccines include E6 and E7. There is currently no approved therapeutic vaccine. 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 suffer from CIN (Rozendaal, L. 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 (Bethesda) scale. When women are diagnosed with a class 1 CIN, a "wait and look" approach is typically taken. Treatment is recommended only when CIN is grade 2-3 due to adverse side effects of the surgical procedure.

Cervical epithelium is composed of several layers of tissue and is called stratified squamous epithelium. These layers are the superficial cell layer, the intermediate cell layer, the sub-basal cell layer and the basal cell layer. Topical drugs for the treatment of cervical intraepithelial neoplasias must be able to penetrate these tissue multilayers to adequately reach and treat the transformed cells. This is a difficult task because the cells are tightly bound and there are no blood vessels.

In 1996, the national cancer institute consensus group determined that non-surgical intervention on cervical intraepithelial neoplasias was required (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 release of this guideline, a number of different approaches to the treatment of HPV and CIN have been explored, including immunomodulators, antiproliferative drugs, antiviral drugs and hormones. However, there is still no FDA approved therapeutic regimen that has been demonstrated to be effective in clinical trials against 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 and Karl hometler et al, as the designated inventors, have filed a series of patents for a variety of acyclic nucleotide derivatives to treat papilloma infection, including (i) U.S. patent No. 8,835,603;9,629,860;9,156,867;10,449,207;10,195,222;10,076,533;10,076,532;9,775,852;9,387,217, priority date is 15 days 3 and 3 in 2013; (ii) U.S. patent No. 10,702,532;10,213,430;9,493,493; and 9,801,884, priority date is 2014, 9, 15; and (iii) U.S. patent nos. 11,014,950 and 10,377,782, priority date 2015, 9, 15. Some of these patents have been issued to An Diwa bioscience (Antiva Biosciences) corporation, which is developing new therapies to treat pre-cancerous lesions caused by HPV.

An Diwa the bioscience company used phosphonate ABI-1968 for human clinical trials to assess its ability to penetrate sufficiently into the cervical epithelium and release the antiviral agent PMEG ((9- [ 2-phosphonomethoxy) ethyl) guanine). The PMEG is then phosphorylated to the active compound PMEGpp (PMEG polyphosphate). It was determined that when doses up to 3% were used, ABI-1968 did not reach a tissue concentration of 15ng/mg of ABI-1968 (see columns F and G in fig. 116) and was therefore unsuitable as a topical drug for the treatment of cervical intraepithelial neoplasia. Local administration of HPV-infected epithelial layered tissue in an effective manner to destroy neoplastic cells in multiple epithelial layers is a difficult challenge. The drug must be sufficiently lipophilic to pass through the tissue layers and metabolize to a sufficient concentration of active agent to kill the pathogenic cells when needed.

The publication has discussed various topical drug delivery strategies including semi-solid dosage forms, gels, tablets, films, and vaginal suppositories. See, e.g., keshari Sahoo, C.et al, "Intra vaginal Drug Delivery System: an review", 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 evaluation of a novel bioadhesive vaginal drug delivery system for clotrimazole" Journal of Controlled Release,2002,81 (3) 347-354; acarturk, F. "Mucoadhesive vaginal drug delivery systems", recent Pat Drug Deliv formula, 2009,3 (3) 193-205; and Signal, G.et al, "Exploring Novel Approaches to Vaginal Drug Delivery", recent Patents on Drug Delivery and Formulation,2011,5 (2) 82-94.

It is an object of the present invention to provide effective pharmaceutical compositions and treatments for HPV infection and related disorders such as HPV-induced neoplasia including, but not limited to, cervical Intraepithelial Neoplasia (CIN), anal Intraepithelial Neoplasia (AIN), vulva Intraepithelial Neoplasia (VIN), penile Intraepithelial Neoplasia (PIN), perianal intraepithelial neoplasia (PAIN), and vaginal intraepithelial neoplasia (VAIN) in a host in need thereof.

Disclosure of Invention

It has been found that effective compositions for the treatment of HPV infections and related diseases, including HPV-induced neoplasias, such as cervical intraepithelial neoplasia, anal intraepithelial neoplasia, perianal intraepithelial neoplasia, penile intraepithelial neoplasia, vulva intraepithelial neoplasia and vaginal intraepithelial neoplasia, need to be combined with a number of options to achieve the desired result. It is necessary to select suitable compounds having favorable lipophilic and tissue penetrating properties in combination with selected pharmaceutically acceptable salts, optionally in favorable crystalline forms, to achieve the long sought ability to penetrate (pendate) epithelial layered tissue in effective amounts to deliver the active agent. Many failures have been experienced and years of research have been required to solve this problem, thereby benefiting patients worldwide with possible cancerous intraepithelial neoplasias.

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

compound I is (ethyl (((2- (2-amino-6-methoxy-9H-purin-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 the university of california, generally claim compound I and pharmaceutically acceptable salts and methods of using the compounds and salts for treating papillomavirus infections. Compound I is an acyclic nucleotide phosphonate which can be metabolized to a known potent antiviral compound (PMEG; (9- [ 2-phosphonomethoxy) ethyl) guanine)), but PMEG has poor cell permeability and limited systemic toxicity for use. The assignee has found how to improve the locally delivered pro-drugs so that they are rapidly absorbed by the epithelial cells, a challenging task to date, and ABI-1968 has failed.

Compound I (ethyl (- ((2- (2-amino-6-methoxy-9H-purin-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 may be in the R or S stereoconfiguration. Thus, compound I is represented by four stereoisomers or two diastereomeric pairs: (R) _P ,S _C )/(S _P ,S _C ) And (R) _P ,R _C )/(S _P ,R _C ) Is present in the form of (c). Although U.S. patent nos. 9,801,884 and 11,344,555 generally describe compound I, these patents do not relate to the potential stereochemistry of phosphorus atoms. As discussed further herein, stereoisomers of compound I having R-stereochemistry at phosphorus and S-stereochemistry at the amino acid carbon have been found to have superior properties to the other three stereoisomers.

In a non-limiting example, an advantageous salt of compound I (e.g. fumarate salt) is used as a mixture of (R, S) and (S, S) diastereomers, wherein the first R/S designates 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). The (R, S) diastereomers are most prominent, although any proportion of the diastereomers that provides the desired result may be used. In other embodiments, the ratio of R to S enantiomer at the phosphorus atom is about 1:1. In some aspects, the compounds are enriched in R chiral enantiomer at the phosphorus atom, wherein the amount of R is, for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or greater by weight.

The S-configuration on the chiral carbon is advantageous in the present invention, corresponding to the natural amino acid configuration. In certain aspects, the amount of S is, for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or greater by weight. In alternative embodiments, the compounds are used with an R-stereoconfiguration at a 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 greater.

Enantiomerically pure (R) of Compound I _p ,S _c Or simply "R, S") form is the primary embodiment. Unless otherwise indicated, enantiomerically pure compound II is at least 90% free of the opposite enantiomer. Surprisingly, the compound is an oil and not a solid and therefore is not selected as an active ingredient for a topical formulation. This is especially true because the racemic mixture or enantiomerically enriched R, S and S, S are solids as the free base. Further, as shown in fig. 120, the S, S isomer has a moderate degree of crystallinity. However, when fumarate salts are formed, the R, S enantiomerically pure compound I becomes a highly crystalline material and is most advantageous for intraepithelial topical application. Thus, the monofumarate salt of compound I has unexpected stability and processability and thus has therapeutic advantages compared to the free base compound I.

The monofumarate salt of the R, S isomer can be readily crystallized from isopropanol and heptane. This crystalline form is an anhydrous compound having a melting point of about 140 ℃ (example 15). This crystalline form morphology was reproduced not only in the milligram scale but also in the multiple gram scale.

Although the S, S isomer is easier to handle as the free base, the S, S isomer monofumarate has a polymorphism with a lower melting point, about 105 ℃. Four crystalline forms of the S, S monofumarate salt were identified (example 15). In some experiments, dissociation of S, S monofumarate to hemi-fumarate was observed. This mode of synthesis is not reproducible when performed on a larger scale.

It has surprisingly been found that certain pharmaceutical composition dosage forms prepared from compound I monofumarate salt and crystalline form mode 1 thereof have advantageous properties. Tablets prepared from compound I free base degrade significantly within one month at 40 ℃ and 75% RH, but in contrast, tablets prepared from compound I monofumarate degrade to a much lower extent (example 25), significantly extending shelf life.

Compound II mentioned herein and described hereinafter is an enantiomerically enriched or pure example having mainly R-stereochemistry at the phosphorus atom and S-stereochemistry at the amino acid carbon atom. In enantiomerically pure form, compound II exhibits stability superior 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 critical to the success of topical application to the cervix, vagina, vulva, perianal area, anus or penis.

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

Compound II has been found to have high tissue penetration and is unexpectedly stable, crystalline and non-hygroscopic. Compound II and its advantageous crystalline form pattern 1 are useful in the treatment of HPV infection or diseases associated with HPV infection, such as intraepithelial neoplasia, including but not limited to cervical intraepithelial neoplasia, anal intraepithelial neoplasia, vulva intraepithelial neoplasia, penile intraepithelial neoplasia, perianal intraepithelial neoplasia, and vaginal intraepithelial neoplasia, to prevent transition to cancer.

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

In particular, it has surprisingly been found that the monofumarate salt of ethyl ((R) - ((2- (2-amino-6-methoxy-9H-purin-9-yl) ethoxy) methyl) (benzyloxy) phosphoryl) -L-alanine (compound II) has a very high tissue penetration when topically applied to the target tissue. Topical administration avoids the toxicity associated with systemic administration of drugs. Since HPV-infected pre-cancerous and/or cancerous cells have several layers in the epithelium, the compound must have high penetration to reach and treat these affected cells.

Compound I monofumarate has better tissue penetration and penetration in porcine and human vaginal tissue than ABI-1968, although ABI-1968 is also a phosphate ester of acyclopurine nucleoside, it failed in clinical trials. For a dose of 0.1%, the compound I monofumarate reached a concentration of 40-85ng/mL in vaginal tissues. ABI-1968, even at a dose of 3%, was used at concentrations of even less than 15ng/mL (see FIG. 116). Significant improvement in tissue penetration, especially in view of dose reduction, is not anticipated in advance.

Corresponding S to _P Compound II was surprisingly stable compared to the isomer (compound III). As shown in example 7, table 9, the melting point of compound II is about 140 ℃ +10 ℃, e.g. at 141.5 ℃, whereas the melting point of compound III is about 100 ℃ +10 ℃, e.g. 106.4 ℃. From the XRPD data comparing the monofumarate salts of the two compounds, it can be seen that compound II is also more crystalline than compound III (see example 13, table 37 and fig. 71, comparative example 15, table 40 and fig. 77).

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

It has been found that compound I mono fumarate, compound II or compound III can be prepared in solid dosage forms for topical administration. In some embodiments, the tablet formulation provides similar tissue penetration as a gel formulation (gel 55-85ng/mg, tablet 44-79ng/mg, fig. 121).

The high crystallinity may facilitate the isolation and processing of the pharmaceutical compounds. Compound II shows surprisingly low hygroscopicity compared to compound III. Compound II retained about 0.25% of the water content when exposed to cycles of 40% -0-95% -0-45% relative humidity, and XRPD pattern was unchanged (example 21, table 46). Compound III retained about 10% of the water content, a 40-fold increase, upon exposure to the same conditions. These conditions also lead to loss of one of the peaks in XRPD pattern, indicating that compound III changes morphology with changes in humidity. The hygroscopicity and stability advantages of compound II over compound III are surprising and unexpected in advance.

For example, compound II mode 1 can be prepared by recrystallizing compound II (example 13, table 37) and equilibrating in a suitable solvent (example 22). In certain embodiments, compound II can be dissolved in an alcohol solvent (e.g., isopropanol) and crystallized into mode 1 by the addition of an aliphatic solvent (e.g., heptane). In certain embodiments, compound II may be dissolved in an alcohol solvent (e.g., ethanol) and crystallized into mode 1 by the addition of an aliphatic solvent (e.g., heptane). Compound II, mode I, can also be prepared by equilibration in isopropanol, heptane, water, acetone, isopropanol: heptane (3:10), isopropanol: MTBE (1:3) and ethyl acetate: toluene (1:3).

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

Compound I (i.e., a mixture of R and S enantiomers at the phosphorus atom and the S stereoisomer at the amino acid carbon) monofumarate salt pattern 1 can be prepared by recrystallising compound I monofumarate salt (example 12, table 31), equilibrating compound I monofumarate salt in a suitable solvent, or by slow evaporation solvent crystallization (example 12, table 32). In certain embodiments, compound I mono fumarate can be dissolved in an alcohol solvent (e.g., isopropanol) and crystallized to mode 1 by the addition of an aliphatic solvent (e.g., heptane). In certain embodiments, compound I mono fumarate can be dissolved in an alcohol solvent (e.g., isopropanol) and crystallized to mode 1 by the addition of an ether solvent (e.g., methyl t-butyl ether). In certain embodiments, compound I monofumarate pattern 1 can be produced by equilibration in a mixture of an ether solvent (e.g., tetrahydrofuran) and an aliphatic solvent (e.g., heptane). In certain embodiments, compound I monofumarate salt mode 1 can be produced by crystallization by slow evaporation of the solvent at room temperature. Suitable solvents for slow evaporative crystallization of compound I monofumarate salt form 1 include, but are not limited to, acetone, methyl ethyl ketone, ethyl acetate, methanol, ethanol, isopropanol, and tetrahydrofuran. In certain embodiments, compound I monofumarate salt form 1 is characterized by an XRPD pattern comprising at least 32Θ values selected from: 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 mode 2, mode 3 and mode 4. However, these crystalline forms are sometimes unstable and form hemi-fumarate salts (a mixture of compound IV and compound V) even when prepared from mono-fumarate salts.

Compound I monofumarate in methyl ethyl ketone; acetone; acetone and heptane; methyl ethyl ketone and heptane; and recrystallization in ethanol and methyl tert-butyl ether both produced hemi-fumarate pattern 2. By passing through ¹ The ratio of compound I as free base to fumarate salt as measured by H NMR is about 1:0.5, e.g. 1:0.52. In one embodiment, mode 2 is characterized by an XRPD pattern comprising at least 3 2θ values selected from the group consisting of: 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 mono fumarate in acetonitrile or acetonitrile and water provides hemi-fumarate pattern 3. Filtration and separation are carried out and then pass through ¹ The ratio of compound I as free base to fumarate salt measured by H NMR was about 1:0.95, but after washing with water, the ratio was reduced to about 1:0.76. In one embodiment, mode 3 is characterized by an XRPD pattern comprising at least 3 2θ values selected from the group consisting of: 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 mono fumarate in acetone and toluene provided hemi-fumarate pattern 4. By passing through ¹ The ratio of compound I as free base to fumarate salt as measured by H NMR is about 1:0.7, e.g. 1:0.69. In one embodiment, mode 4 is characterized by an XRPD pattern comprising at least 3 2θ values selected from the group consisting of: 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 properties of the monofumarate salt over the hemi-fumarate salt, compound I monofumarate salt mode 1 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 an active compound or combination of active compounds as described herein in a topical formulation sufficient to treat neoplasia.

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

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

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

The specific dose is 0.05mg, 0.1mg, 0.2mg or 0.3mg of compound I monofumarate, compound II or compound III. In certain embodiments, a dose of 0.05mg, 0.1mg, 0.2mg, or 0.3mg of compound I monofumarate, compound II, or compound III is administered once, twice, or three times a week as desired. In certain embodiments, a dose of 0.05mg of compound I mono fumarate, compound II or compound III is administered for a time specified by the healthcare provider, including daily dosing.

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

In certain embodiments, a compound may be administered during one or more treatment cycles, including a treatment period and a withdrawal period, wherein the treatment period includes administration of a compound as described herein, followed by a withdrawal period (including a no-treatment period), and then the next treatment cycle. In certain embodiments, the withdrawal period is from about one day to about six months. In certain embodiments, the withdrawal period is one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, or longer before the next treatment period. In certain embodiments, multiple treatment cycles are administered, for example one, two, three, four, five, or six treatment cycles.

Dosage forms that do not adhere well to the target site may fall off, thereby interfering with treatment. Dosage forms have been found that adhere to the target site and dissolve rapidly in low fluid volumes. Adhesion to the target site may also prevent exposure to non-target tissues that may limit toxicity, create undesirable systemic exposure, and side effects. Dosage forms that soften, disintegrate and/or disintegrate rapidly in low fluid volumes facilitate rapid release of the active compound to the target tissue. Dosage forms that disintegrate in, for example, 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 1mL, or less than about 2mL of fluid facilitate penetration of the drug into the target site.

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

The physical size of the dosage form can affect the effectiveness of the dosage form. Thinner tablets provide a larger surface area to volume ratio and can degrade faster and better cover the target area. In certain embodiments, the minimum dimension of the dosage form is a thickness of less than 3 millimeters.

The formulation of the dosage form is important for adequate application of the active agent to the intraepithelial tissue. For example, the formulations may be formulated for use as tablets, reconstituted powders, dry powders, semi-solid dosage forms, films or pessaries (i.e. pessaries).

The tablet formulation should exhibit mucoadhesive properties and affinity (subtotantity) and include excipients with solubilizing, eroding (for disintegration), porous (for water absorption) and tackifying (to maintain the drug at the target site) properties.

Examples of excipients that cause the solid dosage form to rapidly disintegrate to cover the cervical, anal, penile, perianal, vulvar, or vaginal area include, but are not limited to, mannitol, microcrystalline cellulose, lactose, sucrose, calcium phosphate, sodium bicarbonate, citric acid, maleic acid, adipic acid, or fumaric acid. Examples of excipients that may enhance disintegration and cover the affected area include, but are not limited to, sodium starch glycolate, pregelatinized starch, crospovidone, and croscarmellose sodium. Mucoadhesive excipients useful in the present invention include, but are not limited to, microcrystalline cellulose, polycarbophil, hydroxymethyl cellulose, hypromellose, hydroxypropyl cellulose, and PVP.

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

Alternative formulations are reconstituted powders or dry powders. These formulations may include the excipients described above, and in certain embodiments xanthan gum may be added. As non-limiting examples, 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, mucoadhesive polymers, solubility/penetration enhancers, lipophilic solubilizing agents, and penetration enhancers. The mucoadhesive polymer may be, for example, but not limited to, carbomer, polyethylene glycol, crospovidone, hypromellose, polycarbophil, and/or hydroxyethyl cellulose. The solubility/penetration enhancer may be, for example, but is not limited to, polyethylene glycol 6 stearate type I, a mixture of ethylene glycol stearate and polyethylene glycol 32 stearate type I, cetyl alcohol, stearyl alcohol, polysorbate 80, sodium lauryl sulfate, mono-and diglycerides, sorbitan monostearate, glycerol isostearate, polyethylene glycol 15 hydroxystearate, poly 15 hydroxystearate, polyethylene glycol 40 hydrogenated castor oil, octyldodecanol, and/or soy lecithin. Lipophilic solubilizing agents include, but are not limited to, light mineral oils, white waxes, and silicone fluids (silicone fluids). Penetration enhancers include, but are not limited to, propylene glycol, diethylene glycol monoethyl ether (transcutol), oleic acid, isopropyl myristate, propylene glycol glycerol monooleate, propylene glycol monocaprylate, PEG-8 beeswax, cetyl alcohol, stearic acid, cetyl palmitate and/or cetostearyl alcohol.

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

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

Vaginal suppositories (pessaries) can be formulated with, for example, but not limited to, sclerosant lipids (e.g., ovucire, witepsol, supposi-Base), polyethylene glycol (polyethylene glycol or macrogol), cocoa butter and glycerol. Non-limiting examples of vaginal suppositories can be prepared from Witepsol H15 or Ovucire WL 3264.

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

(i) Compound I monofumarate;

(ii) A compound II;

(iii) A compound III;

(iv) A compound IV;

(v) A compound V;

(vi) The compound of (i), (ii), (iii), (iv) or (v), in enantiomerically enriched or enantiomerically pure form;

(vii) The compound of (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 greater;

(viii) The compound of (vii), wherein the amount of 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 greater;

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

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

(xi) Enantiomerically pure compound II of R, S in highly crystalline form;

(xii) Compound II mode 1;

(xiii) A crystalline form as described in more detail in section III.

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

(xv) The topical formulation of (xiv), which is in the form of a tablet;

(xvi) The tablet dosage form of (xv) comprising compound I mono fumarate, mannitol, multicrystalline cellulose and magnesium stearate;

(xvii) The tablet dosage form of (xv) comprising compound II, mannitol, polycrystalline cellulose, and magnesium stearate;

(xviii) The topical formulation of (xiv), in the form of a semi-solid dosage form;

(xix) The semi-solid dosage form according to (xviii) comprising compound I monofumarate, light mineral oil, propyl parahydroxybenzoate,63. Water, EDTA, methylparaben and +.>974P；

(xx) The semi-solid dosage form according to (xviii) comprising compound I monofumarate, water, EDTA, methyl benzoate,974P, propylene glycol, and optionally sorbic acid;

(xxi) The semi-solid dosage form of (xviii) comprising compound II, light mineral oil, propyl parahydroxybenzoate,63. Water, EDTA, methylparaben and +.>974P；

(xxii) A semisolid dosage form as described in (xviii) comprising compound II, water, EDTA, methyl benzoate,974P, propylene glycol, and optionally sorbic acid;

(xxiii) The topical formulation of (xiv), which is in the form of a reconstituted powder;

(xxiv) The topical formulation of (xiv), which is in the form of a dry powder dosage form;

(xxv) The topical formulation of (xiv), which is in the form of a film;

(xxvi) The topical formulation of (xiv), which is in the form of a vaginal suppository;

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

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

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

(xxx) Embodiments (i) - (xxvii) are used for treating HPV infection or associated disorders in a host in need thereof, including but not limited to intraepithelial neoplasia, such as cervical intraepithelial neoplasia, penile intraepithelial neoplasia, vulva 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 topical formulations described above for treating intraepithelial neoplasia provide a dosage form comprising: from 0.005mg to 50mg, from 0.05mg to 40mg, from 0.1mg to 30mg, from 0.05mg to 0.3mg, from 0.5mg to 20mg, from 1mg to 15mg, from 1mg to 10mg of a compound as described in examples (i) - (v), and in some embodiments about or at least 0.005, 0.01, 0.03, 0.05, 0.1mg, 0.3mg.0.5mg, 0.7mg, 1mg, 1.5mg, 2mg, 2.5mg, 3mg, 4mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, or 50mg.

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

(xxxiv) The topical formulations of examples (xiv) to (xxvii) are administered twice daily, once daily or for several days per week (e.g. 2 or 3 days per week) as long as necessary to achieve the desired result;

(xxxv) The topical formulation of examples (xiv) to (xxvii) is administered on a weekly schedule for one, two, three, four, five, six or more weeks.

(xxxvi) The method of (xxviii), further comprising applying lubrication to the dosage form prior to inserting the dosage form into the affected area;

(xxxvii) The method of (xxxvi), wherein the lubricant is selected from the group consisting of water, glycerol-based lubricants, and hydroxyethylcellulose-based lubricants; and

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

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

(xl) The method of (xxxix) comprising surgically treating the target tissue followed by administering to a host in need thereof an effective amount of a compound as described in examples (i) - (xxii);

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

(xlii) The method of (xxxiv), comprising administering to a host in need thereof an effective amount of a compound as described in examples (i) - (xxii) at about the same time as or near the time of surgical treatment of the target tissue;

(xliii) The method of embodiments (xxxiv) - (xxxvii), wherein the surgical treatment of the target tissue is resection;

(xliv) The method of embodiments (xxxiv) - (xxxvii), wherein the surgical treatment of the target tissue is ablation;

(xlv) The method of (xliii), wherein the excision is a loop electro-excision (LEEP);

(xlvi) The method of (xliii), wherein the excision is a transformation zone macrocyclic excision (LLETZ);

(xlvii) The method of (xliii), wherein the excision is a knife cone biopsy;

(xlviii) The method of (xliii), wherein the ablation is a laser cone ablation;

(xlix) The method of (xliv), wherein the ablating is laser ablating; and

(l) The method of (xliv), wherein the ablating is cryoablating.

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

(lii) a process for preparing a crystalline form as described herein.

Drawings

Fig. 1 is a comparison of XRPD diffractograms of compound I mode 1 obtained in an equilibration experiment in heptane (example 2, table 2, experiment EQ 10), anti-solvent precipitation in acetone/methyl tert-butyl ether (MTBE) system (example 2, table 3, experiment AS 3), and an extended preparation of compound I mode 1 described in example 3.

Fig. 2 is a DSC thermogram of compound I pattern 1 obtained in an equilibration experiment in heptane (example 2, table 2, experiment EQ 10).

Fig. 3 is a TGA thermogram of compound I mode 1 obtained in an equilibration experiment in heptane (example 2, table 2, experiment EQ 10).

Fig. 4 is an XRPD diffractogram of compound I mode 1 prepared in example 3.

FIG. 5 is a DSC thermogram of compound I mode 1 prepared in example 3.

FIG. 6 is a TGA thermogram of compound I mode 1 prepared in example 3.

FIG. 7 is a comparison of XRPD diffraction patterns for fumaric acid pattern, compound IV pattern 1 (obtained in example 7), a mixture of compound IV pattern 1 and fumaric acid pattern (sample RC2-EA, obtained in example 5, experiment RC 2), and compound IV pattern 1 (example 5, sample RC 3-EA).

FIG. 8 is a comparison of XRPD diffraction patterns for compound I monosuccinate mode 1 (example 5, sample RC 7-IPA) and succinic acid mode.

FIG. 9 is a comparison of XRPD diffraction patterns for compound I monosuccinate pattern 1 of sample RC7-EA (example 5, table 5) and compound I monosuccinate pattern 1 of sample AS7-B (example 5, table 6).

FIG. 10 is a comparison of XRPD diffraction patterns for fumaric acid mode, compound I monofumarate mode 1 (example 5, table 6, sample AS 2-B) and Compound IV mode 1 (example 5, sample AS 3-B).

FIG. 11 is a comparison of XRPD diffraction patterns for compound I monosuccinate mode 1 (example 5, sample AS 7-B) and the free succinic acid mode.

Fig. 12 is an XRPD diffractogram of compound I hemi-fumarate salt pattern 1 (example 6, sample RC 13).

Fig. 13 is a DSC thermogram of compound I hemi-fumarate salt form 1 (example 6, sample RC 13).

Fig. 14 is a TGA thermogram of compound I hemi-fumarate salt form 1 (example 6, sample RC 13).

Fig. 15 is an XRPD diffractogram of compound I monosuccinate mode 1 (example 6, sample RC 14).

FIG. 16 is a DSC thermogram of compound I monosuccinate mode 1 (example 6, sample RC 14).

FIG. 17 is a TGA thermogram of compound I monosuccinate mode 1 (example 6, sample RC 14).

FIG. 18 is a comparison of XRPD diffraction patterns for compound I monofumarate pattern 1 (example 6, sample RC 16) and compound I hemi-fumarate pattern 1 (example 6, sample RC-13).

FIG. 19 is a comparison of XRPD diffraction patterns for compound I hemisuccinate mode 1 (example 6, sample RC 17) and compound I monosuccinate mode 1 (example 6, sample RC-14).

FIG. 20 is a DSC thermogram of compound I hemisuccinate mode 1 (example 6, sample RC 17).

FIG. 21 is a TGA thermogram of compound I hemisuccinate mode 1 (example 6, sample RC 17).

FIG. 22 is a comparison of XRPD diffraction patterns for compound I monofumarate pattern 1 (example 6, sample RC 18) and compound I monofumarate pattern 1 (example 6, sample RC-16).

Fig. 23 is a comparison of XRPD diffractograms of compound I hemi-fumarate pattern 1 and compound I mono-fumarate pattern 1 obtained in example 7 (small scale preparation).

Fig. 24 is a DSC thermogram of compound I hemi-fumarate salt form 1 obtained in example 7.

Fig. 25 is a TGA thermogram of compound I hemi-fumarate salt form 1 obtained in example 7.

Fig. 26A is a DSC thermogram of compound I monofumarate salt form 1 (small scale preparation, example 7), recorded at a heating rate of 10 ℃/min.

Fig. 26B is a DSC thermogram of compound I monofumarate salt pattern 1 (small scale preparation, example 7), with a recorder running at a heating rate of 2 ℃/min.

FIG. 26C is a DSC cycle (DSC cycle, 0-150deg.C, 150-0deg.C, 0-250deg.C, 10deg.C/min) of Compound I monofumarate salt form 1 for small-scale preparation of samples (example 7).

Fig. 27 is a TGA thermogram of compound I monofumarate salt form 1 used in small scale preparation of samples (example 7).

Fig. 28 is a comparison of XRPD diffractograms of compound I monofumarate pattern 1 obtained in stability test experiments with compound I monofumarate pattern 1 prior to testing, at 25 ℃/84% rh (2 days, open vessel), 25 ℃/92% rh (1 week, open vessel), 40 ℃/75% rh (1 week, open vessel) and 60 ℃ (1 week, sealed vessel) as described in example 8.

Fig. 29 is a dynamic vapor phase adsorption (DVS) diagram and mass plot (mass plot) for compound I mode 1 (example 10) for DVS changes.

Fig. 30 is a comparison of XRPD diffractograms of compound I mode 1 (obtained in example 3) before and after DVS study (example 10).

Fig. 31 is a dynamic vapor phase adsorption (DVS) profile and DVS variation in mass profile for compound I hemi-fumarate salt pattern 1 (example 10).

Fig. 32 is a comparison of XRPD diffractograms of compound I hemi-fumarate salt pattern 1 (obtained in example 7) before and after DVS study (example 10).

Fig. 33 is a dynamic vapor phase adsorption (DVS) profile and DVS variation in mass profile for compound I monofumarate salt mode 1 (example 10).

Fig. 34 is a comparison of XRPD diffractograms of compound I monofumarate salt form 1 (obtained in example 7) before and after DVS study (example 10).

Fig. 35 is a comparison of XRPD diffractograms of compound I monofumarate salt form 1 obtained in example 11 (large scale) before and after heating to 106 ℃.

FIG. 36A is a DSC thermogram of compound I monofumarate salt form 1 (example 11) recorded at a heating rate of 10 ℃/min.

FIG. 36B is a DSC thermogram of Compound I monofumarate salt form 1 (prepared on a large scale, example 11) recorded at a heating rate of 2 ℃/min.

Fig. 36C is a DSC thermogram of compound I monofumarate salt form 1 (obtained in example 11).

Fig. 37 is a TGA thermogram of compound I monofumarate salt form 1 (obtained in example 11).

Fig. 38 is a comparison of XRPD diffractograms (obtained in example 12) for compound I mono-fumarate pattern 1, hemi-fumarate pattern 2, hemi-fumarate pattern 3, pattern 4 obtained from an equilibration experiment in water at 25 ℃ for 2 weeks.

Fig. 39 is a comparison of XRPD diffractograms of compound I pattern 4 (obtained in example 12) obtained from equilibration experiments in water at 25 ℃ for 2 and 3 weeks.

Figure 40 is a DSC thermogram of compound I pattern 4 obtained in an equilibration experiment in water at 25 ℃ for 2 weeks (obtained in example 12).

FIG. 41 is a DSC thermogram of compound I mode 4 (obtained in example 12) obtained in an equilibration experiment in water at 25℃for 3 weeks.

FIG. 42 is a TGA thermogram of compound I mode 4 (obtained in example 12) obtained in an equilibration experiment in water at 25℃for 2 weeks.

FIG. 43 is a TGA thermogram of compound I mode 4 (obtained in example 12) obtained in an equilibration experiment in water at 25℃for 3 weeks.

FIG. 44 is a comparison of XRPD diffraction patterns (obtained in example 12) for fumaric acid pattern and compound I hemi-fumarate pattern C obtained in equilibration experiments EQ2 (in acetonitrile) and EQ15 (in 2.9:97.1v/v water/acetonitrile) at 25℃for 3 weeks.

Fig. 45 is a DSC thermogram of compound I hemi-fumarate salt form C obtained in an equilibration experiment in acetonitrile at 25 ℃ for 2 weeks (obtained in example 12).

Fig. 46 is a TGA thermogram of compound I hemifumarate salt form C obtained in an equilibration experiment in acetonitrile at 25 ℃ for 2 weeks (obtained in example 12).

Fig. 47 is a comparison of XRPD diffractograms (obtained in example 12) of compound I hemifumarate pattern 2 obtained in equilibration experiments EQ3 (in methyl ethyl ketone), EQ4 (in acetone) and EQ7 (in 1:1v/v acetone/heptane) at 25 ℃ for 3 weeks.

Fig. 48 is a DSC thermogram of compound I hemi-fumarate salt form 2 obtained in an equilibration experiment in methyl ethyl ketone at 25 ℃ for 2 weeks (obtained in example 12).

Fig. 49 is a TGA thermogram of compound I hemi-fumarate salt form 3 obtained in an equilibration experiment in methyl ethyl ketone at 25 ℃ for 2 weeks (obtained in example 12).

FIG. 50 is a comparison of XRPD diffraction patterns (obtained in example 12) for a mixture of fumaric acid and compound I monofumarate pattern 1 with unknown patterns obtained in equilibrium experiments EQ5 (in isopropanol), EQ8 (in 1:1v/v isopropanol/heptane), EQ9 (in 1:1v/v isopropanol/toluene), EQ10 (in 1:3v/v isopropanol/methyl tert-butyl ether) and EQ12 (in 1:3v/v ethanol/heptane) at 25℃for 3 weeks.

Fig. 51 is a DSC thermogram of a mixture of compound I monofumarate salt pattern 1 with unknown pattern obtained in equilibration experiment EQ5 in isopropanol at 25 ℃ for 2 weeks (obtained in example 12).

Fig. 52 is a TGA thermogram of a mixture of compound I monofumarate salt pattern 1 and unknown pattern obtained in equilibration experiment EQ5 in isopropanol at 25 ℃ for 2 weeks (obtained in example 12).

FIG. 53 is a comparison of XRPD diffraction patterns (obtained in example 12) for compound I hemifumarate pattern 5 obtained in equilibration experiment EQ6 (in 1:1v/v acetone/toluene) at 25℃for 2 and 3 weeks.

FIG. 54 is a DSC thermogram of compound I hemi-fumarate salt form 5 (obtained in example 12) obtained in equilibration experiment EQ6 (in 1:1v/v acetone/toluene) at 25℃for 2 weeks.

FIG. 55 is a DSC thermogram of compound I hemi-fumarate salt form 5 (obtained in example 12) obtained in equilibration experiment EQ6 (in 1:1v/v acetone/toluene) at 25℃for 3 weeks.

FIG. 56 is a TGA thermogram of compound I hemi-fumarate salt form 5 (obtained in example 12) obtained in equilibration experiment EQ6 (in 1:1v/v acetone/toluene) at 25℃for 2 weeks.

FIG. 57 is a TGA thermogram of compound I hemi-fumarate salt form 5 (obtained in example 12) obtained in equilibration experiment EQ6 (in 1:1v/v acetone/toluene) at 25℃for 3 weeks.

FIG. 58 is a comparison of XRPD diffraction patterns for Compound I monofumarate pattern 1 and Compound I monofumarate pattern 1 (material obtained in example 7) obtained in an equilibration experiment (procedure in example 12) at 25℃for 2 weeks, EQ11 (in 1:3v/v tetrahydrofuran/heptane), EQ13 (in 1:3v/v ethyl acetate/toluene), EQ14 (in 1:3v/v ethanol/toluene).

FIG. 59 is a comparison of XRPD diffraction patterns (obtained in example 12) for Compound I monofumarate pattern 1 obtained in equilibration experiments sEQ 11 (in 1:3v/v tetrahydrofuran/heptane), EQ13 (in 1:3v/v EA/toluene), EQ14 (in 1:3v/v ethanol/toluene) at 25℃for 3 weeks.

Fig. 60 is a comparison of the XRPD diffractograms (obtained in example 12) of compound I mono fumarate pattern 1 and EQ16 (in 1:4v/v isopropanol/heptane), the unknown pattern obtained in the equilibration experiment of EQ5 (in isopropanol) at 25 ℃ for 2 weeks, and the mixture of compound I mono fumarate pattern 1 obtained in example 7.

Fig. 61 is a comparison of XRPD diffractograms for reference fumaric acid pattern, compound I mono fumarate pattern 1 (example 7), compound I mono fumarate pattern 2 (obtained by precipitation from acetone solution with heptane anti-solvent in experiment AS1, example 12), compound I mono fumarate pattern 2 (obtained by precipitation from methyl ethyl ketone solution with heptane anti-solvent in experiment AS4, example 12), and fumaric acid pattern obtained in experiment AS2, example 12.

Fig. 62 is a comparison of XRPD diffractograms of compound I mono fumarate pattern 1 (example 7), compound I mono fumarate pattern 2 (obtained by precipitation from ethanol solution with heptane anti-solvent in experiment AS6, example 12) and compound I mono fumarate pattern 2 (obtained by precipitation from tetrahydrofuran solution with heptane anti-solvent in experiment AS7, example 12).

Fig. 63 is a comparison of the XRPD diffractograms of compound I monofumarate salt form 1 (example 7) and compound I monofumarate salt form 1 obtained by slow evaporation from acetone, methyl ethyl ketone and ethyl acetate by crystallization at room temperature as described in example 12, table 32.

Fig. 64 is a comparison of the XRPD diffractograms of compound I mono fumarate pattern 1 (example 7) with compound I mono fumarate pattern 1 obtained by slow evaporation from methanol, ethanol, isopropanol and tetrahydrofuran by crystallization at room temperature as described in example 12, table 32.

FIG. 65 is a reference fumaric acid pattern; compound I monofumarate pattern 1 (example 7); and compound I monofumarate salt form 2 obtained by slow cooling by combining from a hot saturated solution of methyl ethyl ketone; compound I monofumarate salt form 2 obtained by slow cooling by crystallization from a solution hot saturated with acetone; and XRPD diffractograms of compound I monofumarate salt form 3 obtained by slow cooling by crystallization from a solution of acetonitrile heat saturated.

FIG. 66 is compound I monofumarate salt pattern 1 (example 7); compound I monofumarate salt form 1 obtained by slow cooling by crystallization from a hot water saturated solution; and a comparison of XRPD diffractograms of compound I monofumarate salt form 1 obtained by slow cooling by crystallization from a solution of hot saturated in ethanol/toluene (1/1 v/v) (example 12, table 33).

FIG. 67 is compound I monofumarate salt pattern 1 (example 7); compound I monofumarate salt form 2 obtained by crystallization from a hot acetone saturated solution by rapid cooling; and a comparison of XRPD diffractograms of compound I monofumarate salt form 2 obtained by rapid cooling crystallization from a solution of hot saturated in methyl ethyl ketone (example 12, table 34).

FIG. 68 is a reference fumaric acid pattern; compound I monofumarate pattern 1 (example 7); compound I monofumarate salt form 1 obtained by crystallization from a hot aqueous saturated solution by rapid cooling; compound I monofumarate salt form 3 obtained by crystallization from a solution saturated thermally in acetonitrile by rapid cooling; and a comparison of XRPD diffractograms of compound I monofumarate salt form 1 obtained by crystallization from a hot saturated solution in ethanol/toluene (1/1 v/v) by rapid cooling (example 12).

FIG. 69 is a heat-cool-heat DSC thermogram of compound I monofumarate salt form 1 (heated to 106 ℃ C.) (example 12, table 35).

Fig. 70 is a heat-cool-heat DSC thermogram of compound I monofumarate salt form 1 (heated to 130 ℃) (example 12, table 35).

Fig. 71 is an XRPD diffractogram of compound II mode 1 (example 13).

FIG. 72 is a DSC thermogram of compound II mode 1 (example 13).

FIG. 73 is a TGA thermogram of compound II mode 1 (example 13).

Fig. 74 is an XRPD diffractogram of compound IV pattern 1 (example 14).

Fig. 75 is a DSC thermogram of compound IV mode 1 (example 14).

FIG. 76 is a TGA thermogram of compound IV mode 1 (example 14).

FIG. 77 is an XRPD diffraction pattern for compound III pattern 1 (example 15).

FIG. 78 is a DSC thermogram of compound III mode 1 (example 15).

FIG. 79 is a TGA thermogram of compound III, mode 1 (example 15).

FIG. 80 is an XRPD diffraction pattern for compound III pattern 2 (example 16).

FIG. 81 is a DSC thermogram of compound III mode 2 (example 16).

FIG. 82 is a TGA thermogram of compound III, mode 2 (example 16).

FIG. 83 is an XRPD diffraction pattern for compound V pattern 1 (example 17).

FIG. 84 is a DSC thermogram of compound V mode 1 (example 17).

FIG. 85 is a TGA thermogram of compound V, mode 1 (example 17).

FIG. 86 is an XRPD diffraction pattern for compound V pattern 2 (example 18).

FIG. 87 is a DSC thermogram of Compound V pattern 2 (example 18).

Fig. 88 is a TGA thermogram of compound V mode 2 (example 18).

Fig. 89 is a comparison of XRPD diffractograms of compound II mode 1 (example 19) obtained from the volume stability study (bulk stability study).

Figure 90 is a comparison of XRPD patterns of compound III mode 2 (example 19) obtained from a volume stability study.

Fig. 91 is a dynamic vapor phase adsorption (DVS) profile for compound II mode 1 and DVS variation in the mass profile (example 21).

Fig. 92 is a comparison of XRPD diffractograms of compound II mode 1 before and after DVS study (example 21).

Fig. 93 is a dynamic vapor phase adsorption (DVS) profile for compound III mode 2 and DVS variation in the mass profile (example 21).

Fig. 94 is a comparison of XRPD diffractograms of compound III pattern 2 before and after DVS study (example 21).

Fig. 95 is an XRPD diffractogram of compound IV mode 1 obtained from compound II mode 1 in example 22, experiment PS 4.

Fig. 96 is a DSC thermogram of compound IV mode 1 obtained from compound II mode 1 in example 22, experiment PS 4.

FIG. 97 is a TGA thermogram of compound IV pattern 1 obtained from compound II pattern 1 in example 22, experiment PS 4.

Fig. 98 is an XRPD diffractogram of compound IV mode 2 obtained from compound II mode 1 in example 22, experiment PS 5.

FIG. 99 is a DSC thermogram of compound IV pattern 2 obtained from compound II pattern 1 in experiment PS5, example 22.

FIG. 100 is a TGA thermogram of compound IV mode 2 obtained from compound II mode 1 in example 22, experiment PS 5.

Fig. 101 is an XRPD diffractogram of compound III pattern 3 obtained from compound III pattern 2 in example 22, experiment PS 3.

FIG. 102 is a DSC thermogram of compound III pattern 3 obtained from compound III pattern 2 in experiment PS3, example 22.

FIG. 103 is a TGA thermogram of compound III mode 3 obtained from compound III mode 2 in experiment PS3, example 22.

FIG. 104 is an XRPD diffraction pattern for compound III pattern 4 obtained from compound III pattern 2 in example 22, experiment PS 4.

Fig. 105 is a DSC thermogram of compound III pattern 4 obtained from compound III pattern 2 in example 22, experiment PS 4.

FIG. 106 is a TGA thermogram of compound III mode 4 obtained from compound III mode 2 in experiment PS4, example 22.

FIG. 107 is an XRPD diffraction pattern for compound III pattern 5 obtained from compound III pattern 2 in example 22, experiment PS 5.

Fig. 108 is a DSC thermogram of compound III pattern 5 obtained from compound III pattern 2 in example 22, experiment PS 5.

FIG. 109 is a TGA thermogram of compound III mode 5 obtained from compound III mode 2 in experiment PS5, example 22.

Fig. 110 is an XRPD diffractogram of compound III pattern 6 obtained from compound III pattern 2 in example 22, experiment PS 9.

Fig. 111 is a DSC thermogram of compound III pattern 6 obtained from compound III pattern 2 in example 22, experiment PS 9.

Fig. 112 is an XRPD diffractogram of compound IV mode 1 obtained from compound II mode 1 in example 22, experiment PS 8.

FIG. 113 is a comparison of XRPD diffraction patterns of compound V pattern 1 obtained from compound III pattern 2 in example 22, experiment PS1, and compound V pattern 1 obtained in example 18.

FIG. 114 is a molecular structure of Compound II mode 1 as determined by single crystal X-ray diffraction analysis of example 25.

FIG. 115 is a molecular structure of Compound II mode 1 as determined by single crystal X-ray diffraction analysis of example 25. In the single crystal form of mode 1, there is an intermolecular interaction between the protonated N5-atom of the free base and the O7-atom of the fumaric acid anion (N (5) -H (5). Cndot.O (7)).

FIG. 116 is a vaginal tissue in vitro permeation (permeation) test 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 tissue penetration of 0.1% compound II gel in human cervical tissue. Column D shows tissue penetration of 1% ABI-1968 formulation in 6% NMP in pig vaginal tissue. Column E shows 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 the tissue to a much lesser extent, which hinders the ability of the compound to reach cells infected with HPV. This may be a contributor to the performance of ABI-1968 in clinical studies. Surprisingly, compound II showed high tissue penetration in both porcine and human tissues. High tissue penetration may lead to high activity against HPV. This is described in example 41.

Fig. 117 shows a flow chart of a process for preparing the topical cream formulation described in example 29.

Fig. 118 shows a flow chart of a process for preparing the topical gel formulation described in example 29.

Fig. 119 shows a flow chart of a process for preparing the tablet formulation described in example 30.

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

Fig. 121 is a bar graph comparing tissue penetration of topical gel and topical tablet dosage forms as described in example 41. The tablet formulation produced a tissue penetration similar to that of the topical gel, with an average of 58ng/mg of compound in the tissue.

FIG. 122 depicts the structures of Compound I monofumarate, compound II, and Compound III. See examples 26-28 for synthesis of these compounds.

Detailed Description

It has been found that effective compositions for treating HPV infection or diseases or conditions associated with HPV infection (e.g., HPV induced neoplasia including, but not limited to, cervical intraepithelial neoplasia, perianal intraepithelial neoplasia, penile intraepithelial neoplasia, vulva intraepithelial neoplasia, anal intraepithelial neoplasia, and vaginal intraepithelial neoplasia) require combinations that work in concert with selected aspects to achieve the desired result. It is necessary to select suitable compounds having favorable lipophilic and tissue penetrating properties in combination with selected pharmaceutically acceptable salts (optionally in favorable crystalline forms) to achieve the long sought ability to penetrate epithelial layered tissue in effective amounts to deliver the active agent. Many years of research have been required to solve this problem after many failures to benefit patients worldwide with possible cancerous intraepithelial neoplasias.

In particular, the key compounds found to deliver active agents are specific salts of:

compound I is (ethyl (((2- (2-amino-6-methoxy-9H-purin-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 the university of california, generally claim compound I and pharmaceutically acceptable salts and methods of using the compounds and salts for treating papillomavirus infections. Compound I is an acyclic nucleotide phosphonate which can be metabolized to the known potent antiviral compound (PMEG; (9- [ 2-phosphonomethoxy) ethyl) guanine) but has poor cell permeability and limited systemic toxicity for use. The assignee has discovered how to improve the local delivery of prodrugs into epithelial cells rapidly, a so far challenging task, also the failure of ABI-1968.

Compound I (ethyl (- ((2- (2-amino-6-methoxy-9H-purin-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 may be in the R or S stereoconfiguration. Thus, compound I has four stereoisomers. Although U.S. patent nos. 9,801,884 and 11,344,555 generally describe compound I, these patents do not relate to the potential stereochemistry of phosphorus atoms. As discussed further herein, stereoisomers of compound I having R-stereochemistry at phosphorus and S-stereochemistry at the amino acid carbon have been found to have superior properties to the other three stereoisomers.

In a non-limiting example, an advantageous salt of compound I (e.g. fumarate salt) is used as a mixture of (R, S) and (S, S) diastereomers, wherein the first R/S designates the stereochemistry at the phosphorus atom and the second S is the stereochemistry of the carbon in the amino acid moiety (corresponding to the L-alanine residue having the S-configuration). The (R, S) diastereomers are most prominent, although any proportion of the diastereomers that provides the desired result may be used. In other embodiments, the ratio of R to S enantiomer at the phosphorus atom is about 1:1. In certain aspects, the compounds are enantiomerically enriched for R at the phosphorus atom, wherein the amount of R is, for example, greater than about 50%, or equal to or greater than about 60%, 70%, 75%, 80%, or even 85% or greater by weight.

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

Thus, in a major aspect, the invention comprises administration optionally in a pharmaceutically acceptable carrierAn effective amount of (R _P ,S _C ) Fumarate salt of ethyl (((2- (2-amino-6-methoxy-9H-purin-9-yl) ethoxy) methyl) (benzyloxy) -phosphoryl) -L-alanine (compound II). The present invention provides an acyclic nucleotide drug salt, method, composition and dosage form for treating diseases associated with Human Papillomavirus (HPV).

The compounds, compositions and dosage forms are also useful for treating conditions associated with or arising from HPV viral exposure or infection. For example, the active compounds are useful for treating pre-cervical lesions, cervical intraepithelial neoplasias, vaginal cancers, vulvar cancers, penile cancers, perianal cancers, and anal intraepithelial neoplasias, cervical cancers, rectal cancers, penile cancers, vaginal cancers, and oropharyngeal cancers.

The active compounds and compositions are also useful for treating infections caused by a range of HPV types. Most oncogenic HPV types are from the alpha-7 and alpha-9 categories, including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 types. The most common oncogenic HPV types are 16 and 18.HPV-16 and HPV-18 are reported to be responsible for 50% of cervical cancers; and 90% of the warts are caused by HPV-6 and HPV-11 (World Health Organization, "Cervcal Cancer" https:// www.who.int/news-room/face-pieces/detail/Cervcal-Cancer). Infection with one genotype does not exclude infection with a different genotype after that.

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 mono fumarate, 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, a compound, formulation or solid dosage form comprising the compound may also be used prophylactically to prevent or delay the progression of clinical disease in an HPV-positive or HPV-exposed individual.

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

Compound II had R-stereochemistry at the phosphorus atom, which has been confirmed by X-ray crystallography (example 25, see fig. 114 and 121). In alternative embodiments, compound II may be used in the form of the phosphorus R-and S-enantiomers in any desired ratio, including up to the pure enantiomer. In some embodiments, compound II is used in a form that is at least 90% free of the opposite enantiomer, and may be at least 98%, 99% or even 100% free of the opposite enantiomer. Unless otherwise indicated, enantiomerically pure compound II is at least 90% free of the opposite enantiomer. In certain embodiments, compound II is used as a racemic mixture of isomers. Furthermore, in an alternative embodiment, the amino acid of the phosphoimidic acid salt may be in the D-or L-configuration, or mixtures thereof, including racemic mixtures.

If the phosphoramide has chirality, R or S chiral phosphorus derivatives or mixtures thereof, including enantiomerically enriched forms (including racemic mixtures), may be provided. All combinations of these configurations 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 certain embodiments, compound I may be:

or a pharmaceutically acceptable salt thereof. In certain embodiments, compound II may be +.>

In certain embodiments, compound III may be

I.(R _P ,S _C ) Fumarate salt of ethyl (((2- (2-amino-6-methoxy-9H-purin-9-yl) ethoxy) methyl) (benzyloxy) phosphoryl) -L-alanine ester (compound II)

In certain embodiments, the active compound of the present invention is compound II, which may be provided in a pharmaceutically acceptable composition or 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 invention also includes a non-limiting illustrative method for preparing a fumarate salt of compound I (e.g., compound II) comprising

(i) Compound I R _P ,S _C A first step of dissolving the isomer in a flask or vessel containing an organic solvent (e.g., acetone, methanol, ethanol, isopropanol, methylene chloride, tetrahydrofuran, or acetonitrile, etc.);

(ii) Adding fumaric acid to R of step (i) at a specific molar ratio (e.g. 0.5:1.0, 1.0:1.0 or 1.5:1) at ambient temperature or at a slightly elevated or reduced temperature (e.g. 23 ℃ -55 ℃) _P ,S _C A solution of compound I;

(iii) Stirring the reaction of step (ii) at ambient or slightly elevated or reduced temperature;

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

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

(vi) Optionally stirring the resulting solution at ambient temperature or slightly elevated or reduced temperature;

(vii) Cooling the resulting solution to a reduced temperature, for example, about 0 ℃ to 10 ℃, and then stirring the solution at that temperature;

(viii) Filtering the resulting solid; and

(ix) The solid is optionally dried under reduced pressure and elevated temperature, for example at or at least at 30 ℃, 35 ℃, 40 ℃, 45 ℃ or 50 ℃.

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

In one embodiment, compound I R _P ,S _C The isomer is dissolved in ethanol of step (i). In one embodiment, compound I R _P ,S _C The isomer is dissolved in the methanol of step (i). In one embodiment, compound I R _P ,S _C The isomer is dissolved in acetonitrile of step (i). In a further embodiment, compound I R _P ,S _C The isomer is dissolved in tetrahydrofuran in 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 tertiary butyl ether. In one embodiment, the second organic solvent in step (v) is water.

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

(x) Compound I S _P ,S _C A first step of dissolving the isomer in a flask or vessel containing an organic solvent (e.g., acetone, methanol, ethanol, isopropanol, methylene chloride, tetrahydrofuran, or acetonitrile, etc.);

(xi) Adding fumaric acid to the solution of compound I of step (I) at a specific molar ratio (e.g. 0.5:1.0, 1.0:1.0 or 1.5:1) at ambient temperature or at a slightly elevated or reduced temperature (e.g. about 23 ℃ -55 ℃);

(xii) Stirring the reaction of step (ii) at ambient or slightly elevated or reduced temperature;

(xiii) Optionally seeding the solution of step (iii) with crystals of compound II;

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

(xv) Optionally stirring the resulting solution at ambient temperature or slightly elevated or reduced temperature;

(xvi) The resulting solution is cooled to a reduced temperature, e.g., about 0-10 ℃, and then the solution is stirred at that temperature

(xvii) Filtering the resulting solid; and

(xviii) The solid is optionally dried under reduced pressure and elevated temperature, for example at or at least at 30 ℃, 35 ℃, 40 ℃, 45 ℃ or 50 ℃.

In one embodiment, compound I S _P ,S _C The isomer is dissolved in ethanol of step (i). In one embodiment, compound I S _P ,S _C The isomer is dissolved in the methanol of step (i). In one embodiment, compound I S _P ,S _C The isomer is dissolved in acetonitrile of step (i). In a further embodiment, compound I S _P ,S _C The isomer is dissolved in tetrahydrofuran in step (i).

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

II salts of Compound I

In certain embodiments, the present invention provides compound I, R as a monofumarate salt _P Compounds I and S _P A compound I. In certain embodiments, the present invention provides compound I, R as a hemi-fumarate salt _P Compounds I and S _P A compound I. In certain embodiments, the invention provides compound I, R as a sesquifumarate salt _P Compounds I and S _P A compound I. In certain embodiments, the present invention provides R as a sulfate _P Compounds I and S _P A compound I. In certain embodiments, the present invention provides R as the hydrochloride salt _P Compounds I and S _P A compound I. In certain embodiments, the present invention provides compound I, R as a benzenesulfonate salt _P Compounds I and S _P A compound I. In certain embodiments, the invention provides R as tosylate _P Compounds I and S _P A compound I. In certain embodiments, the invention provides R as a succinate salt _P Compounds I and S _P A compound I.

/>

_P R salts of the compounds I

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_P S salts of Compound I

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The hemi-fumarate salt and the monosuccinate salt form a solid having advantageous properties as a solid dosage form for use in the treatment of a host (e.g. a human) suffering from an HPV infection or HPV related disease (e.g. cervical intraepithelial neoplasia, anal intraepithelial neoplasia, penile intraepithelial neoplasia, vulva intraepithelial neoplasia, perianal intraepithelial neoplasia or vaginal intraepithelial neoplasia). However, monofumarate shows superior performance to hemi-fumarate and monosuccinate. Thus, the monofumarate salt is still the desired salt form of compound I.

Further embodiments of the invention

In certain embodiments, the invention comprises at least:

1. a compound having the formula:

2. a compound having the formula:

or a pharmaceutically acceptable salt thereof.

3. A compound having the formula:

Or a pharmaceutically acceptable salt thereof.

4. The compound of example 2, having the formula:

5. the compound of example 3, having the formula:

6. an isolated crystalline form of a compound having the formula:

wherein the isolated crystalline form is characterized by an XRPD pattern comprising peaks 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. An isolated crystalline form of a compound having the formula:

wherein the isolated crystalline form is characterized by an XRPD pattern comprising peaks 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. An isolated crystalline form of a compound having the formula:

wherein the isolated crystalline form is characterized by an XRPD pattern comprising 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 °, 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. An isolated crystalline form of a compound having the formula:

10. A pharmaceutical composition comprising a compound of any one of embodiments 1-5 and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition comprising the crystalline form of any one of claims 6-9 in a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of examples 10-11, which is in a solid dosage form.

13. The pharmaceutical composition of examples 10-11, which is a semi-solid dosage form.

14. The pharmaceutical composition of examples 10-11, in the form of a reconstituted powder.

15. The pharmaceutical composition of examples 10-11, which is in the form of a dry powder dosage form.

16. The pharmaceutical composition of examples 10-11, which is in the form of a film.

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

18. The pharmaceutical composition of example 12, which is in the form of a tablet.

19. The pharmaceutical composition of example 13, which is in the form of a cream.

20. The pharmaceutical composition of embodiment 13, which is in the form of a gel.

21. The pharmaceutical composition of any one of embodiments 10-20, formulated for topical administration.

22. The pharmaceutical composition of any one of embodiments 10-21, for delivery to the cervix.

23. The pharmaceutical composition of any one of embodiments 10-21 for delivery to the vagina.

24. The pharmaceutical composition of any one of embodiments 10-21 for delivery to the vulva.

25. The pharmaceutical composition of any one of embodiments 10-21, for delivery to the perianal region.

26. The pharmaceutical composition of any one of embodiments 10-21 for delivery to the anus.

27. The pharmaceutical composition of any one of embodiments 10-21 for delivery to the penis.

28. The pharmaceutical composition of embodiment 18, wherein the tablet is a bilayer tablet.

29. The pharmaceutical composition of embodiment 18, wherein the tablet disintegrates in less than about 250 μl of fluid.

30. The pharmaceutical composition of embodiment 18, wherein the tablet disintegrates in less than about 150 μl of fluid.

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

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

33. The pharmaceutical composition of embodiment 32 comprising from about 0.05mg to about 40mg of the compound.

34. The pharmaceutical composition of embodiment 32, comprising from about 0.1mg to about 30mg of the compound.

35. The pharmaceutical composition of embodiment 32 comprising at least about 0.1mg of the compound.

36. The pharmaceutical composition of embodiment 32 comprising at least about 0.3mg of the compound.

37. The pharmaceutical composition of embodiment 32 comprising at least about 1mg of the compound.

38. The pharmaceutical composition of embodiment 32, comprising at least 1.5mg of the compound.

39. The pharmaceutical composition of embodiment 32 comprising at least about 2mg of the compound.

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

41. The pharmaceutical composition of embodiment 40 comprising from about 0.01% to 0.5% of the compound.

42. The pharmaceutical composition of embodiment 40 comprising from about 0.1% to 5% of the compound.

43. The pharmaceutical composition of any one of embodiments 10-42, comprising a mucoadhesive polymer.

44. The pharmaceutical composition of embodiment 43 comprising from about 5% to about 20% mucoadhesive polymer.

45. The pharmaceutical composition of embodiment 43 comprising from about 10% to about 50% mucoadhesive polymer.

46. The pharmaceutical composition of embodiment 43 comprising from about 50% to about 90% mucoadhesive polymer.

47. The pharmaceutical composition of any one of embodiments 10-42, comprising a disintegration-enhancing excipient.

48. The pharmaceutical composition of any one of embodiments 10-42, comprising a penetration enhancing excipient.

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

50. The pharmaceutical composition of embodiment 19, wherein the pharmaceutically acceptable carrier consists of: light mineral oil, propyl parahydroxybenzoate, tefose 63, water, EDTA, methyl parahydroxybenzoate, and Carbopol 974P.

51. The pharmaceutical composition of embodiment 20, wherein the pharmaceutically acceptable carrier consists of: water, EDTA, methyl benzoate, carbopol 974P, propylene glycol and sorbic acid.

52. The pharmaceutical composition 18 of the embodiment, wherein the tablet consists of: mannitol, polycrystalline cellulose, and magnesium stearate.

53. A method of treating a human papillomavirus infection comprising administering to a host in need thereof an effective amount of a compound of any one of embodiments 1-5, optionally in a pharmaceutically acceptable carrier.

54. A method of treating a disorder caused by human papillomavirus infection, the method comprising administering to a host in need thereof an effective amount of a compound of any one of embodiments 1-5, optionally in a pharmaceutically acceptable carrier.

55. The method of embodiment 54, wherein the disorder caused by human papillomavirus infection is intraepithelial neoplasia.

56. The method of embodiment 55, wherein the disorder caused by human papillomavirus is atypical squamous cell of unknown significance (ASC-US).

57. The method of embodiment 55, wherein the condition caused by human papillomavirus is atypical Adenovirus (AGC).

58. The method of embodiment 55, wherein the disorder caused by human papillomavirus is low grade squamous intraepithelial lesions (LSIL).

59. The method of embodiment 55, wherein the disorder caused by human papillomavirus is atypical squamous cells, and high-grade squamous intraepithelial lesions (ASC-H) cannot be excluded.

60. The method of embodiment 55, wherein the disorder caused by human papillomavirus is High Squamous Intraepithelial Lesions (HSIL).

61. The method of embodiment 54, wherein the disorder caused by human papillomavirus is in situ Adenocarcinoma (AIS).

62. The method of embodiment 55, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia.

63. The method of embodiment 62, wherein the cervical intraepithelial neoplasia is grade 1 cervical intraepithelial neoplasia.

64. The method of embodiment 62, wherein the cervical intraepithelial neoplasia is a grade 2 cervical intraepithelial neoplasia.

65. The method of embodiment 62, wherein the cervical intraepithelial neoplasia is grade 3 cervical intraepithelial neoplasia.

66. The method of embodiment 55, wherein the intraepithelial neoplasia is vaginal intraepithelial neoplasia.

67. The method of embodiment 55, wherein the intraepithelial neoplasia is intraepithelial neoplasia of the vulva.

68. The method of embodiment 55, wherein the intraepithelial neoplasia is an intraanal neoplasia.

69. The method of embodiment 55, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia.

70. The method of embodiment 55, wherein the intraepithelial neoplasia is intrapenile intraepithelial neoplasia.

71. The method of any one of embodiments 53-70, wherein the host is a human.

72. The method of any one of embodiments 53-71, wherein the compound is administered topically.

73. The method of any one of embodiments 52-71, wherein from about 0.05mg to about 40 mg of the compound is administered.

74. The method of any one of embodiments 52-71, wherein from about 0.1 mg to about 30 mg is administered.

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

76. The method of embodiment 75, wherein from about 0.05mg to about 0.3mg of the compound is administered.

77. The method of any of embodiments 52-76, further comprising applying lubrication to the epithelial tissue prior to inserting the dosage form into the affected area.

78. The method of any of embodiments 52-76, further comprising applying lubrication to the dosage form prior to inserting the dosage form into the affected area.

79. The method of embodiment 77 or 78, wherein the lubricating means is selected from the group consisting of water, glycerol-based lubricants, and hydroxyethylcellulose-based lubricants.

80. The method of any one of embodiments 52-79, wherein the compound is administered once daily.

81. The method of any one of embodiments 52-79, administered twice daily.

82. The method of any one of embodiments 52-79, administered twice weekly.

83. The method of any one of embodiments 52-79, administered three or more times per week.

84. The method of any one of embodiments 52-83, administered for about one week.

85. The method of any one of embodiments 52-83, administered for about two weeks.

86. The method of any one of embodiments 52-83, administered for about three weeks.

87. The method of any one of embodiments 52-83, administered for about four weeks.

88. The method of any one of embodiments 52-83, administered for about five weeks.

89. The method of any one of embodiments 52-83, administered for about six weeks.

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

a. a treatment period comprising administration of the compound, and

b. a withdrawal period comprising a period of no treatment.

91. The method of embodiment 90, wherein the withdrawal period is about one week.

92. The method of embodiment 90, wherein the withdrawal period is about two weeks.

93. The method of embodiment 90, wherein the withdrawal period is about three weeks.

94. The method of any one of embodiments 52-79, wherein the compound is administered daily.

95. The method of example 94, wherein from about 0.01mg to about 0.5mg is administered.

96. The method of example 95, wherein from about 0.05mg to about 0.3mg is administered.

97. The method of any one of embodiments 90-93, wherein two treatment cycles are administered.

98. The method of any one of embodiments 90-93, wherein three treatment cycles are administered.

99. The method of any one of embodiments 90-93, wherein four treatment cycles are administered.

100. The method of any one of embodiments 90-93, wherein five treatment cycles are administered.

101. The method of any one of embodiments 90-93, wherein six treatment cycles are administered.

102. The method of any one of embodiments 52-101, wherein the human papillomavirus is HPV-16.

103. The method of any one of embodiments 52-101, wherein the human papillomavirus is HPV-18.

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

105. The method of embodiment 104 wherein the antiviral compound is selected from the group consisting of: protease inhibitors, other DNA polymerase inhibitors, inhibitors of E6 or E6AP, inhibitors of E7, inhibitors of E1, inhibitors of E2, inhibitors of E1-E2 protein interactions, inhibitors of L2 lipopeptides, inhibitors of L1, inhibitors of L2, degradants of L1, and degradants of L2.

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

107. The method of embodiment 106, wherein the anti-cancer compound is selected from the group consisting of: HDAC inhibitors, degradation agents of tetra-transmembrane proteins (tetraspin), immune checkpoint inhibitors, T cell therapies and antiproliferative agents.

108. The method of any one of embodiments 52-103, wherein the compound is administered in combination with surgery.

109. The method of embodiment 108, wherein the compound is administered prior to the surgical procedure.

110. The method of embodiment 108, wherein the compound is administered after the surgical procedure.

111. The method of embodiment 108, wherein the surgical procedure is performed during administration of the compound.

112. The method of any of embodiments 108-111, wherein the surgical procedure is excision of diseased tissue.

113. The method of embodiment 112, wherein the resection is loop electro-resection (LEEP).

114. The method of embodiment 112, wherein the excision is a transformation zone macrocyclic excision (LLETZ).

115. The method of embodiment 112, wherein the resection is a knife and cone resection.

116. The method of embodiment 112, wherein the ablation is a laser cone ablation.

117. The method of any of embodiments 108-111, wherein the surgical procedure is ablation of the diseased tissue.

118. The method of embodiment 117 wherein the ablating is laser ablating.

119. The method of embodiment 117, wherein the ablating is cryoablating.

120. Use of a compound as described in examples 1-5, optionally in a pharmaceutically acceptable carrier, for the manufacture of a medicament for treating a human papillomavirus infection in a host in need thereof.

121. Use of a compound as described in examples 1-5, optionally in a pharmaceutically acceptable carrier, for the manufacture of a medicament for treating a condition caused by human papillomavirus infection in a host in need thereof.

122. The use of embodiment 121, wherein the disorder caused by human papillomavirus infection is intraepithelial neoplasia.

123. The use of embodiment 122, wherein the intraepithelial neoplasia is a vaginal intraepithelial neoplasia.

124. The use of embodiment 122, wherein the intraepithelial neoplasia is intraepithelial neoplasia of the vulva.

125. The use of embodiment 122, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia.

126. The use of embodiment 122, wherein the intraepithelial neoplasia is an intraanal neoplasia.

127. The use of embodiment 122, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia.

128. The use of embodiment 122, wherein the intraepithelial neoplasia is intrapenile intraepithelial neoplasia.

129. The use of embodiments 120-128, wherein the host is a human.

130. The use of embodiments 120-129, for topical administration.

131. The compound of any one of embodiments 1-5, optionally in a pharmaceutically acceptable carrier, for use in treating human papillomavirus infection in a host in need thereof.

132. The compound of any one of embodiments 1-5, optionally in a pharmaceutically acceptable carrier, for use in treating a disorder caused by human papillomavirus infection in a host in need thereof.

133. The compound for use of embodiment 132, wherein the disorder caused by human papillomavirus infection is intraepithelial neoplasia.

134. The compound for use of embodiment 133, wherein the intraepithelial neoplasia is intravaginal intraepithelial neoplasia.

135. The compound for use of embodiment 133, wherein the intraepithelial neoplasia is intraepithelial neoplasia of the vulva.

136. The compound for use of embodiment 133, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia.

137. The compound for use of embodiment 133, wherein the intraepithelial neoplasia is anal intraepithelial neoplasia.

138. The compound for use of embodiment 133, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia.

139. The compound for use of embodiment 133, wherein the intraepithelial neoplasia is intrapenile intraepithelial neoplasia.

140. The compound for use of embodiments 131-139 wherein the host is a human.

141. The compound for use of embodiments 131-140, wherein the compound is administered topically.

142. A process for preparing the crystalline form of example 4, the process comprising:

a. r is R _P Dissolving the compound I in an alcohol solvent;

b. stirring at a temperature of from about 20 ℃ to about 70 ℃;

c. 1.0 equivalent of fumaric acid was added;

d. adding an aliphatic solvent;

e. cooling the mixture;

f. stirring the cooled solution; and is also provided with

g. The solid was isolated and dried.

143. The method of embodiment 142, wherein the alcoholic solvent of step (a) is ethanol or isopropanol.

144. The method of embodiments 142-143 wherein the alcoholic solvent of step (a) is isopropanol.

145. The method of embodiment 142, wherein the solution of step (b) is stirred at about 45 ℃ to about 55 ℃.

146. The method of embodiment 142, wherein the aliphatic solvent is hexane or heptane.

147. The method of embodiments 142 and 146, wherein the aliphatic solvent is heptane.

148. The method of embodiment 142, wherein the mixture is cooled to less than about 20 ℃.

149. The method of embodiment 142, wherein the mixture is cooled to less than about 10 ℃.

150. The method of embodiment 142, wherein the mixture is cooled to less than about 5 ℃.

151. The method of embodiment 142, wherein the mixture is cooled to about 5 ℃ to 0 ℃.

Form III

The invention provides an isolated crystalline form of compound I monofumarate, mode 1. In certain embodiments, compound I monofumarate salt form 1 is characterized by an XRPD pattern in figure 23, or an XRPD pattern substantially similar thereto.

1. In one embodiment, compound I monofumarate salt form 1 is characterized by an XRPD pattern comprising at least about 3, 4, or 5 2Θ values selected from: 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, compound I monofumarate salt form 1 is characterized by an XRPD pattern comprising at least 12 2Θ values selected from: 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, compound I monofumarate salt form 1 is characterized by an XRPD pattern comprising at least 11 2Θ values selected from: 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, compound I monofumarate salt form 1 is characterized by an XRPD pattern comprising at least 10 2Θ values selected from: 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, compound I monofumarate salt form 1 is characterized by an XRPD pattern comprising at least 9 2θ values selected from the group consisting of: 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, compound I monofumarate salt form 1 is characterized by comprising at least 8 XRPD patterns of 2θ values selected from 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, compound I monofumarate salt form 1 is characterized by comprising at least 7 XRPD patterns of 2θ values selected from 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, compound I monofumarate salt form 1 is characterized by comprising at least 6 XRPD patterns of 2θ values selected from 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, compound I monofumarate salt form 1 is characterized by comprising at least 5 XRPD patterns of 2θ values selected from 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, compound I monofumarate salt form 1 is characterized by an XRPD pattern comprising at least 4 2θ values selected from the group consisting of: 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, compound I monofumarate salt form 1 is characterized by an XRPD pattern comprising at least 3 2θ values selected from the group consisting of: 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 salt form 1 can be produced, for example, by crystallization from isopropanol and heptane, as described in example 7. The compound I free base and about 1.0 equivalent of fumaric acid may be dissolved in isopropanol, for example, at a concentration of about 25% to about 40% w/v and stirred at an elevated temperature, for example, about 45 ℃, about 50 ℃, about 55 ℃. The solution is stirred at this temperature until some solids are formed, then optionally seeded with compound I monofumarate pattern 1 crystalline solids. The mixture is stirred and cooled to a lower temperature to promote crystallization, for example, less than about 40 ℃, less than about 30 ℃, less than about 25 ℃, less than about 20 ℃, or less than about 15 ℃. The mixture is then stirred while heptane is added in an amount from about 1mL/mL of isopropanol to about 5mL/mL of isopropanol (e.g., about 4mL/mL of isopropanol). The resulting suspension is stirred while the product crystallizes, for example, at 25 ℃ for at least about 24 hours. Next, the suspension is cooled to further promote crystallization. The solution may be cooled to less than about 10 ℃, less than about 5 ℃, less than about 0 ℃, or less than about-5 ℃. The solution is stirred at a reduced temperature for a period of time, such as at least about one day, to allow additional product to crystallize, and then the solid is collected by filtration. The collected solids are dried under reduced pressure and optionally at elevated temperature to provide compound I monofumarate salt form 1.

The present invention provides isolated crystalline form mode 1 of compound II. In one embodiment, mode 1 is characterized by an XRPD pattern in figure 71 or an XRPD pattern substantially similar thereto.

1. In one embodiment, compound II mode 1 is characterized by an XRPD pattern comprising at least 3, 4, or 5 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 7 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 6 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 5 XRPD patterns of 2Θ values selected from 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 claimed in example 1, characterized by comprising at least 4 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 3 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 2 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 1 XRPD pattern of 2Θ values selected from: 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 an XRPD pattern comprising at least 3, 4, or 5 2Θ values selected from: 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 claimed in example 9, characterized by comprising at least 15 XRPD patterns of 2Θ values selected from: 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 claimed in example 9, characterized by an XRPD pattern comprising at least 14 2Θ values selected from: 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 claimed in example 9, characterized by an XRPD pattern comprising at least 13 2Θ values selected from: 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 claimed in example 9, characterized by an XRPD pattern comprising at least 12 2Θ values selected from: 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 claimed in example 9, characterized by comprising at least 11 XRPD patterns of 2Θ values selected from: 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 claimed in example 9, characterized by comprising at least 10 XRPD patterns of 2Θ values selected from: 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 claimed in example 9, characterized by comprising at least 9 XRPD patterns of 2Θ values selected from: 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 claimed in example 9, characterized by comprising at least 8 XRPD patterns of 2Θ values selected from: 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 certain embodiments, compound II mode 1 is characterized by an XRPD pattern comprising at least 3, 4, or 5 2Θ values selected from: 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 °, 17.45.+ -. 0.2 °, 17.838.+ -. 0.2 °, 18.13.+ -. 0.2 °, 18.63.+ -. 0.2 °, 18.89..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 °, 25.14.+ -. 0.2 °, 25.33.+ -. 0.2 ° and 3.2 ° and the like 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..2 °, 32.27.+ -. 0.2 °, 32.69..2 °, 33.43.+ -. 0.2 °, 34.06.+ -. 0.2 °, 34.34..2 °, 34.69.+ -. 0.2 °, 35.58.+ -. 0.2 °, 36.19.+ -. 0.2 °, 36.62.+ -. 0.2 °, 37.41..2 °, 38.30.+ -. 0.2 °, 38.77.+ -. 0.2 ° and 39.24..0.2 °.

19. Compound II mode 1 of example 18, characterized by an XRPD pattern comprising at least 45 2Θ values selected from: 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 °, 17.45.+ -. 0.2 °, 17.838.+ -. 0.2 °, 18.13.+ -. 0.2 °, 18.63.+ -. 0.2 °, 18.89..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 °, 25.14.+ -. 0.2 °, 25.33.+ -. 0.2 ° and 3.2 ° and the like 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..2 °, 32.27.+ -. 0.2 °, 32.69..2 °, 33.43.+ -. 0.2 °, 34.06.+ -. 0.2 °, 34.34..2 °, 34.69.+ -. 0.2 °, 35.58.+ -. 0.2 °, 36.19.+ -. 0.2 °, 36.62.+ -. 0.2 °, 37.41..2 °, 38.30.+ -. 0.2 °, 38.77.+ -. 0.2 ° and 39.24..0.2 °.

20. Compound II mode 1 of example 18, characterized by comprising at least 40 XRPD patterns of 2Θ values selected from: 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 °, 17.45.+ -. 0.2 °, 17.838.+ -. 0.2 °, 18.13.+ -. 0.2 °, 18.63.+ -. 0.2 °, 18.89..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 °, 25.14.+ -. 0.2 °, 25.33.+ -. 0.2 ° and 3.2 ° and the like 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..2 °, 32.27.+ -. 0.2 °, 32.69..2 °, 33.43.+ -. 0.2 °, 34.06.+ -. 0.2 °, 34.34..2 °, 34.69.+ -. 0.2 °, 35.58.+ -. 0.2 °, 36.19.+ -. 0.2 °, 36.62.+ -. 0.2 °, 37.41..2 °, 38.30.+ -. 0.2 °, 38.77.+ -. 0.2 ° and 39.24..0.2 °.

21. Compound II mode 1 of example 18, characterized by comprising at least 35 XRPD patterns of 2Θ values selected from: 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 °, 17.45.+ -. 0.2 °, 17.838.+ -. 0.2 °, 18.13.+ -. 0.2 °, 18.63.+ -. 0.2 °, 18.89..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 °, 25.14.+ -. 0.2 °, 25.33.+ -. 0.2 ° and 3.2 ° and the like 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..2 °, 32.27.+ -. 0.2 °, 32.69..2 °, 33.43.+ -. 0.2 °, 34.06.+ -. 0.2 °, 34.34..2 °, 34.69.+ -. 0.2 °, 35.58.+ -. 0.2 °, 36.19.+ -. 0.2 °, 36.62.+ -. 0.2 °, 37.41..2 °, 38.30.+ -. 0.2 °, 38.77.+ -. 0.2 ° and 39.24..0.2 °.

22. Compound II mode 1 of example 18, characterized by an XRPD pattern comprising at least 30 2Θ values selected from: 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 °, 17.45.+ -. 0.2 °, 17.838.+ -. 0.2 °, 18.13.+ -. 0.2 °, 18.63.+ -. 0.2 °, 18.89..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 °, 25.14.+ -. 0.2 °, 25.33.+ -. 0.2 ° and 3.2 ° and the like 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..2 °, 32.27.+ -. 0.2 °, 32.69..2 °, 33.43.+ -. 0.2 °, 34.06.+ -. 0.2 °, 34.34..2 °, 34.69.+ -. 0.2 °, 35.58.+ -. 0.2 °, 36.19.+ -. 0.2 °, 36.62.+ -. 0.2 °, 37.41..2 °, 38.30.+ -. 0.2 °, 38.77.+ -. 0.2 ° and 39.24..0.2 °.

23. Compound II mode 1 as claimed in example 18, characterized by comprising at least 25 XRPD patterns of 2Θ values selected from: 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 °, 17.45.+ -. 0.2 °, 17.838.+ -. 0.2 °, 18.13.+ -. 0.2 °, 18.63.+ -. 0.2 °, 18.89..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 °, 25.14.+ -. 0.2 °, 25.33.+ -. 0.2 ° and 3.2 ° and the like 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..2 °, 32.27.+ -. 0.2 °, 32.69..2 °, 33.43.+ -. 0.2 °, 34.06.+ -. 0.2 °, 34.34..2 °, 34.69.+ -. 0.2 °, 35.58.+ -. 0.2 °, 36.19.+ -. 0.2 °, 36.62.+ -. 0.2 °, 37.41..2 °, 38.30.+ -. 0.2 °, 38.77.+ -. 0.2 ° and 39.24..0.2 °.

24. Compound II mode 1 of example 18, characterized by comprising at least 20 XRPD patterns of 2Θ values selected from: 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 °, 17.45.+ -. 0.2 °, 17.838.+ -. 0.2 °, 18.13.+ -. 0.2 °, 18.63.+ -. 0.2 °, 18.89..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 °, 25.14.+ -. 0.2 °, 25.33.+ -. 0.2 ° and 3.2 ° and the like 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..2 °, 32.27.+ -. 0.2 °, 32.69..2 °, 33.43.+ -. 0.2 °, 34.06.+ -. 0.2 °, 34.34..2 °, 34.69.+ -. 0.2 °, 35.58.+ -. 0.2 °, 36.19.+ -. 0.2 °, 36.62.+ -. 0.2 °, 37.41..2 °, 38.30.+ -. 0.2 °, 38.77.+ -. 0.2 ° and 39.24..0.2 °.

25. Compound II mode 1 of example 18, characterized by comprising at least 15 XRPD patterns of 2Θ values selected from: 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 °, 17.45.+ -. 0.2 °, 17.838.+ -. 0.2 °, 18.13.+ -. 0.2 °, 18.63.+ -. 0.2 °, 18.89..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 °, 25.14.+ -. 0.2 °, 25.33.+ -. 0.2 ° and 3.2 ° and the like 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..2 °, 32.27.+ -. 0.2 °, 32.69..2 °, 33.43.+ -. 0.2 °, 34.06.+ -. 0.2 °, 34.34..2 °, 34.69.+ -. 0.2 °, 35.58.+ -. 0.2 °, 36.19.+ -. 0.2 °, 36.62.+ -. 0.2 °, 37.41..2 °, 38.30.+ -. 0.2 °, 38.77.+ -. 0.2 ° and 39.24..0.2 °.

26. The compound II mode 1 of any one of embodiments 1-25 wherein the compound is prepared by ¹ H NMR, the ratio of compound II to fumaric acid was about 1:1.

Compound II mode 1 can be prepared, for example, by recrystallising compound II (example 12, table 31), equilibrating compound II in a suitable solvent, or by crystallization by slow evaporation of the solvent (example 12, table 32). The invention thus includes at least the following features:

1. In certain embodiments, compound II mode 1 is prepared by recrystallizing compound II.

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

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

4. The process of example 1, wherein compound II is dissolved in an alcohol solvent and crystallized as mode 1 by the addition of an ether solvent.

5. The process of example 1, wherein compound II is dissolved in an alcohol solvent and crystallized by addition of an aliphatic solvent to form mode 1.

6. The process of example 1, wherein compound II is dissolved in an ether solvent and crystallized by addition of an aliphatic solvent to form mode 1.

7. The method of embodiment 4 or 5, wherein the alcoholic solvent is or comprises methanol.

8. The method of embodiment 4 or 5, wherein the alcoholic solvent is or comprises ethanol.

9. The method of embodiment 4 or 5, wherein the alcohol solvent is or comprises isopropanol.

10. The method of embodiment 4 or 5, wherein the alcohol solvent is or comprises n-propanol.

11. The method of embodiment 4 or 5, wherein the alcohol solvent is or comprises n-butanol.

12. The method of embodiment 4 or 5, wherein the alcohol solvent is or comprises isoamyl alcohol.

13. The method of embodiment 4 or 5, wherein the alcohol solvent is or comprises cyclohexanol.

14. The method of embodiment 4 or 6, wherein the ether solvent is or comprises diethyl ether.

15. The method of embodiment 4 or 6, wherein the ethereal solvent is or comprises dibutyl ether.

16. The method of embodiment 4 or 6, wherein the ethereal solvent is or comprises methyl tertiary butyl ether.

17. The method of embodiment 4 or 6, wherein the ethereal solvent is or comprises cyclopropyl methyl ether.

18. The method of embodiment 4 or 6, wherein the ethereal solvent is or comprises glyme.

19. The method of embodiment 4 or 6, wherein the ethereal solvent is or comprises tetrahydrofuran.

20. The method of embodiment 4 or 6, wherein the ethereal solvent is 2-methyltetrahydrofuran.

21. The method of embodiment 4 or 6, wherein the ether solvent is or comprises dioxane.

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

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

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

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

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

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

28. The method of any of embodiments 5-21 wherein the aliphatic solvent is or comprises a mixture of hexane isomers.

29. The method of any one of embodiments 4-28, wherein the solid is collected by filtration.

30. The method of any of embodiments 4-28, wherein the solution is cooled after adding the aliphatic solvent.

31. The method of embodiment 30, wherein the solution is cooled to less than about 10 ℃.

32. The method of embodiment 30, wherein the solution is cooled to about or less than about 5 ℃.

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

34. The method of embodiment 33, wherein the solution, suspension, or slurry is stirred for at least about one day.

35. The method of embodiment 33, wherein the solution, suspension, or slurry is stirred for at least about one week.

36. The method of embodiment 33, wherein the solution, suspension, or slurry is stirred for at least about two weeks.

37. The method of embodiment 33, wherein the solution, suspension, or slurry is stirred for at least about three weeks.

38. The process of example 2, wherein compound II is dissolved in an equilibration solvent at 25 ℃ and stirred for a period of time from about 2 weeks to about 3 weeks.

39. The method of embodiment 2, wherein the equilibration solvent is a binary mixture of solvents.

40. The method of embodiment 2, wherein the equilibration solvent is isopropanol.

41. The method of example 39, wherein the equilibration solvent is from about a portion of tetrahydrofuran to about three portions of heptane.

42. The method of example 39, wherein the equilibration solvent is from about a portion of ethanol to about three portions of heptane.

43. The process of example 39 wherein the equilibration solvent is from about a portion of ethyl acetate to about three portions of toluene.

44. The method of embodiment 39, wherein the equilibration solvent is from about a portion of isopropyl alcohol to about a portion of heptane.

45. The method of embodiment 39, wherein the equilibration solvent is from about a portion of isopropanol to about a portion of toluene.

46. The method of example 39, wherein the equilibration solvent is from about a portion of isopropanol to about three portions of methyl tertiary butyl ether.

47. The method of example 39, wherein the equilibration solvent is from about a portion of isopropyl alcohol to about four portions of heptane.

48. The method of embodiment 39, wherein the equilibration solvent is from about a portion of ethanol to about a portion of toluene.

49. The process of example 3, wherein compound II is dissolved in a suitable solvent, filtered through a 0.45 μm filter, and then maintained at 23 ℃ and one atmosphere until the solvent evaporates.

50. The method of embodiment 49, wherein the solvent is or comprises acetone.

51. The method of embodiment 49, wherein the solvent is or comprises methyl ethyl ketone.

52. The method of embodiment 49, wherein the solvent is or comprises ethyl acetate.

53. The method of embodiment 49, wherein the solvent is or comprises methanol.

54. The method of embodiment 49, wherein the solvent is or comprises ethanol.

55. The method of embodiment 49, wherein the solvent is or comprises isopropyl alcohol.

56. The method of embodiment 49, wherein the solvent is or comprises tetrahydrofuran.

Isolated crystalline forms of compound III, mode 1 and mode 2, are provided in the present invention. In one embodiment, mode 1 is characterized by an XRPD pattern in figure 77 or an XRPD pattern substantially similar thereto.

1. In one embodiment, compound III mode 1 is characterized by an XRPD pattern comprising independently at least 2, 3, 4, 5, or 6 2Θ values selected from: 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 claimed in example 1, characterized by an XRPD pattern comprising at least 12 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 11 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 10 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 9 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 8 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 7 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 6 XRPD patterns of 2Θ values selected from: 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 claimed in example 1, characterized by comprising at least 5 XRPD patterns of 2Θ values selected from: 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 an XRPD pattern comprising at least 3, 4, 5, or 6 2Θ values independently selected from: 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 claimed in example 10, characterized by an XRPD pattern comprising at least 12 2Θ values selected from: 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 claimed in example 10, characterized by an XRPD pattern comprising independently at least 3, 4, 5 or 6 2Θ values selected from: 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 claimed in example 10, characterized by comprising at least 11 XRPD patterns of 2Θ values selected from: 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 claimed in example 10, characterized by comprising at least 10 XRPD patterns of 2Θ values selected from: 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 claimed in example 10, characterized by comprising at least 9 XRPD patterns of 2Θ values selected from: 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 claimed in example 10, characterized by comprising at least 8 XRPD patterns of 2Θ values selected from: 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 claimed in example 10, characterized by comprising at least 7 XRPD patterns of 2Θ values selected from: 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 claimed in example 10, characterized by comprising at least 6 XRPD patterns of 2Θ values selected from: 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 claimed in example 10, characterized by comprising at least 5 XRPD patterns of 2Θ values selected from: 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 (example 15, table 40) can be produced by precipitation of isopropanol and heptane. In certain non-limiting embodiments, compound III mode 1 is prepared by reacting S _P Compound I free base is soluble in isopropanol (e.g., about 100mg S _p -compound I is dissolved in about 0.25mL to about 0.5mL isopropyl alcohol). About 1.0eq fumaric acid is added to the solution and the mixture is stirred at ambient or elevated temperature (e.g., about 25 ℃ to about 60 ℃).Next, heptane was added approximately two to five times that of isopropanol. The resulting mixture is stirred at ambient or elevated temperature (e.g., about 25 ℃ to about 60 ℃) and then gradually cooled (e.g., about 0.01 ℃/min to 1 ℃/min), the solids separated by filtration and then dried at ambient or reduced pressure.

Definition of IV

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications and published applications cited herein are incorporated by reference in their entirety unless otherwise indicated. If there are multiple definitions of terms herein, the definitions in this section control unless otherwise indicated.

As used herein, unless otherwise indicated, any protecting group, amino acid, and other compound abbreviation is in accordance with its common usage, accepted abbreviation, or IUPAC-IUB biochemical nomenclature committee (see biochem.11:942-944 (1972)).

The term "alcoholic solvent" as used herein refers to a solvent having free hydroxyl groups, which is liquid at room or use temperature. Non-limiting examples of alcohol solvents include methanol, ethanol, ethylene glycol, isopropanol, n-propanol, glycerol, n-butanol, isobutanol, t-butanol, 2-butanol, n-pentanol (n-pentanol), isopentanol (isopentanol), neopentyl alcohol, hexanol, cyclohexanol, cyclohexane diol, phenol, benzyl alcohol, propynyl alcohol, diethylene glycol, 1, 2-propanediol, heptanol, octanol, nonanol, and decanol.

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

The term "ether solvent" as used herein refers to a solvent containing at least one ether bond, and is liquid at room temperature or at the temperature of use. Examples of the ether-based solvents include, but are not limited to, diethyl ether, diisopropyl ether, methyl t-butyl ether, dibutyl ether, t-amyl ethyl ether, cyclopentyl methyl ether, di-t-butyl ether, ethyl t-butyl ether, propylene glycol methyl ether, ethylene glycol diethyl ether, glyme, diglyme, glycol ethers, dioxane, tetrahydrofuran, 2-methyltetrahydrofuran, 4-methyltetrahydrofuran, and tetrahydropyran.

Where a range of values is provided, it is understood that the upper and lower limits of the range, and each intermediate value between the upper and lower limits of the range, are encompassed within the embodiments.

As used herein, "about" refers to a range including up to 10% less than the recited value and up to 10% greater than the recited value. For example, "about 100 mg" includes all values from 90 mg to 110 mg.

The terms "therapeutically effective amount" and "effective amount" are used to denote the amount of active compound, agent, or metabolite thereof that produces the desired therapeutic effect. For example, an effective amount of a compound may be an amount required to prevent, reduce or ameliorate symptoms of a disease or to prolong survival of a subject (e.g., a human). Such a response may occur in a tissue, system, animal or human, including alleviation of the symptoms or signs of the disease being treated.

Isotopic substitution

The present 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, with the desired isotopic substitution of the atoms above the natural abundance of the isotopes, i.e., enriched. Isotopes are atoms having the same atomic number but different mass numbers, i.e., atoms of the same proton number but different neutron numbers. As a general example and without limitation, hydrogen isotopes, e.g., deuterium ² H) And tritium% ³ H) Can be used anywhere in the structure. Alternatively, carbon isotopes, for example ¹³ C and C ¹⁴ C. The preferred isotopic substitution is substitution of deuterium for hydrogen at one or more positions on the molecule to enhance the performance of the drug. Deuterium can bind to bond-breaking sites during metabolismPlaced (alpha-deuterium kinetic isotope effect) or near the bond breaking site (beta-deuterium kinetic isotope effect). Achillion Pharmaceuticals, inc. (WO/2014/169278 and WO/2014/169280) describe deuteration of nucleotides to improve their pharmacokinetics or pharmacodynamics, including the 5-position of the molecule.

Substitution with isotopes (e.g., deuterium) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. Substitution of deuterium at the metabolic decomposition site for hydrogen can reduce or eliminate the metabolic rate of the bond. Any position in the compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including protons ¹ H) Deuterium ² H) And tritium% ³ H) A. The invention relates to a method for producing a fibre-reinforced plastic composite Thus, unless the context clearly dictates otherwise, the compounds mentioned herein include all potential isotopic forms.

The term "isotopically labeled" mimetic refers to "deuterated mimetic", "and" deuterated mimetic "and" isotopically labeled mimetic "refers to" deuterated mimetic "and" deuterated mimetic "or" deuterated mimetic "are" ¹³ C-labeled mimetic "or" deuterated- ¹³ C labeling the mimetic. The term "deuterated analog" refers to a compound described herein wherein the hydrogen isotope, i.e., hydrogen/proton @, is ¹ H) By hydrogen isotopes, e.g. deuterium ² H) And (3) substitution. Deuterium substitution may be partial or complete. Partial deuterium substitution means that at least one hydrogen is substituted by at least one deuterium. In certain embodiments, the isotope is one that is 80%, 85%, 90%, 95%, or 99% or more enriched at any location of interest. In some embodiments, deuterium is enriched by 90%, 95% or 99% at the desired position. Unless otherwise indicated, the deuteration at the selected position is at least 80%. Deuteration of nucleosides can occur on any alternative hydrogen 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 an active compound or combination of active compounds as described herein in a topical formulation sufficient to treat neoplasia or produce the effects further described herein. Types of HPV-induced intraepithelial neoplasias include, but are not limited to, cervical intraepithelial neoplasias, vaginal intraepithelial neoplasias, penile intraepithelial neoplasias, perianal intraepithelial neoplasias, and anal intraepithelial neoplasias.

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

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

Dosage forms that do not adhere well to the target site may fall off, thereby interfering with treatment. Dosage forms have been found that adhere to the target site and dissolve rapidly in low fluid volumes. Adhesion to the target site may also prevent exposure to healthy tissue, which may limit toxicity and produce side effects. Dosage forms that soften, disintegrate and/or disintegrate rapidly in low fluid volumes facilitate rapid release of the active compound to the target tissue. Dosage forms that disintegrate in, for example, 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 of fluid are particularly useful for penetration of a drug into a target site.

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

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

The formulation of the dosage form is important for adequate application of the active agent to the intraepithelial tissue. For example, the formulation may be prepared as a tablet, reconstituted powder, dry powder, semi-solid dosage form, film or vaginal suppository (i.e. pessary).

Some embodiments disclosed herein include the use of an effective amount of compound I monofumarate, compound II, or compound III in the manufacture of a medicament for ameliorating or treating an infection by a human papillomavirus, wherein the infection can be ameliorated or treated by inhibiting viral replication by inhibiting synthesis of viral DNA. Other embodiments disclosed herein include the use of an effective amount of any of the active compounds described herein (including but not limited to compound I monofumarate, compound II, or compound III) for ameliorating or treating a human papillomavirus infection, wherein the human papillomavirus infection can be ameliorated or treated by inhibiting viral replication by inhibiting viral DNA synthesis.

Certain non-limiting embodiments disclosed herein include methods for ameliorating or treating a human papillomavirus infection, which methods may include contacting a cell in a subject infected with a human papillomavirus 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 ameliorated or treated by inhibiting synthesis of viral DNA. Still other embodiments disclosed herein include methods for ameliorating or treating a human papillomavirus infection, which methods may include administering to a subject infected with a human papillomavirus an effective amount of compound I monofumarate, compound II, or compound III, wherein the human papillomavirus infection may be ameliorated or treated by inhibiting viral replication by inhibiting viral DNA synthesis. Some embodiments disclosed herein relate to compound I monofumarate, compound II, or compound III for use in ameliorating or treating a human papillomavirus infection, wherein the human papillomavirus infection may be ameliorated or treated by inhibiting viral replication by inhibiting synthesis of viral DNA.

In some embodiments, the human papillomavirus may be a high-risk human papillomavirus, such as those described herein. For example, the high-risk human papillomavirus may be selected from the group consisting of 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 human papillomavirus may be HPV-16. In some embodiments, the human papillomavirus may be HPV-11. In some embodiments, the human papillomavirus may be HPV-18. In some embodiments, the human papillomavirus 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 may be detected using a pap test (cervical smear test) and/or a DNA probe test (e.g., HPV DNA probe test for one or more high risk HPV types). Thus, in some embodiments, an effective amount of compound I monofumarate, compound II, or compound III may be provided to a subject diagnosed with an HPV infection, e.g., 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 identified by a pap test as diagnosed as having an HPV infection or a disease associated with an HPV infection.

In certain embodiments, an effective amount of compound I mono fumarate, compound II, or compound III may be provided to the subject, wherein the pap test results do not indicate that the disease has progressed to cervical cancer. The Besselda system is a standardized scoring system for reporting pap smear test results and assigns the results to a scale of 1-3 according to severity. Grade 1 CIN (CIN 1) indicates mild dysplasia. The 2-and 3-stage CINs (CIN 2, CIN 3) are more severe and typically require intervention. In certain embodiments, compound I monofumarate, compound II, or compound III is used to treat CIN 1 (grade 1 cervical intraepithelial neoplasia). In certain embodiments, compound I monofumarate, compound II, or compound III is used to treat CIN2 (grade 2 cervical intraepithelial neoplasia). In certain embodiments, compound I monofumarate, compound II, or compound III is used to treat CIN3 (grade 3 cervical intraepithelial neoplasia).

In certain embodiments, the pharmaceutical composition comprising compound I mono fumarate, compound II or compound III is used in the manufacture of a medicament for treating CIN 1 (cervical intraepithelial neoplasia grade 1). In certain embodiments, the pharmaceutical composition comprising compound I mono fumarate, compound II or compound III is used in the manufacture of a medicament for treating CIN2 (cervical intraepithelial neoplasia grade 2). In certain embodiments, the pharmaceutical composition comprising compound I mono fumarate, compound II or compound III is used in the manufacture of a medicament for treating CIN3 (grade 3 cervical intraepithelial neoplasia).

In certain embodiments, compound I mono fumarate, compound II or compound III, optionally in a pharmaceutically acceptable carrier, is used to treat a disorder selected from the group consisting of: atypical squamous cell of unknown significance (ASC-US), atypical Glandular Cell (AGC), low-grade squamous intraepithelial lesions (LSIL), atypical squamous cell (high-grade squamous intraepithelial lesions cannot be excluded) (ASC-H), high-grade squamous intraepithelial lesions (HSIL), in-situ Adenocarcinoma (AIS), and cervical cancer (e.g., squamous cell carcinoma or adenocarcinoma).

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

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

In certain embodiments, compound II, optionally in a pharmaceutically acceptable carrier, is used to treat a disorder selected from the group consisting of: atypical squamous cell of unknown significance (ASC-US), atypical Glandular Cell (AGC), low-grade squamous intraepithelial lesions (LSIL), atypical squamous cell (high-grade squamous intraepithelial lesions cannot be excluded) (ASC-H), high-grade squamous intraepithelial lesions (HSIL), in-situ Adenocarcinoma (AIS), and cervical cancer (e.g., squamous cell carcinoma or adenocarcinoma).

In certain embodiments, compound I monofumarate, compound II, or compound III is used in the manufacture of a medicament for treating anal intraepithelial neoplasia. In certain embodiments, compound I monofumarate, compound II, or compound III is used in the manufacture of a medicament for treating perianal intraepithelial neoplasia. In certain embodiments, compound I monofumarate, compound II, or compound III is used in the manufacture of a medicament for treating intraepithelial neoplasia of the vulva. In certain embodiments, compound I monofumarate, compound II or compound III is used in the manufacture of a medicament for treating intraepithelial neoplasia of the penis. In certain embodiments, compound I monofumarate, compound II, or compound III is used in the manufacture of a medicament for treating a neoplasia in the vaginal epithelium.

In certain embodiments, compound I monofumarate, compound II, or compound III is used to treat anal intraepithelial neoplasia. In certain embodiments, compound I monofumarate, compound II, or compound III is used to treat perianal intraepithelial neoplasia. In certain embodiments, compound I monofumarate, compound II, or compound III is used to treat vulvar intraepithelial neoplasia. In certain embodiments, compound I monofumarate, compound II, or compound III is used to treat intraepithelial neoplasia of the penis. In certain embodiments, compound I monofumarate, compound II, or compound III is used to treat a vaginal intraepithelial neoplasia.

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

Compound I monofumarate, compound II or compound III can be used to ameliorate and/or treat infections caused by one or more types of human papillomaviruses. For example, compound I monofumarate, compound II or compound III can be used to ameliorate and/or treat HPV-16 and/or HPV-18 infection. In certain embodiments, compound I monofumarate, compound II, or compound III can be used to treat high-risk HPV infection. In certain embodiments, compound I monofumarate, compound II or compound III can be used to treat a related disease or disorder that occurs as a result of a high risk HPV infection. In some embodiments, compound I monofumarate, compound II, or compound III can be used to ameliorate and/or treat infections including high-risk and low-risk HPV types.

Compound I monofumarate, compound II or compound III can be used in the manufacture of a medicament for use in ameliorating and/or treating an infection caused by one or more types of human papillomaviruses. For example, compound I monofumarate, compound II or compound III may be used in the manufacture of a medicament for ameliorating and/or treating an infection of HPV-16 and/or HPV-18. In certain embodiments, compound I monofumarate, compound II or compound III may be used in the manufacture of a medicament for the treatment of high risk HPV infection. In certain embodiments, compound I monofumarate, compound II or compound III may be used in the manufacture of a medicament for the treatment of a related disease or disorder that occurs as a result of a high risk HPV infection. In some embodiments, compound I monofumarate, compound II or compound III can be used in the manufacture of a medicament for ameliorating and/or treating infections including high-risk and low-risk HPV types.

It will be apparent to those skilled in the art that the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, severity of the affliction, the particular compound used and the particular use for which the compound is used. Determination of an effective dosage level, i.e., the dosage level necessary to achieve the desired result, can be accomplished by one of ordinary skill in the art using routine methods, such as human clinical trials and in vitro studies.

In certain embodiments, the pharmaceutical composition comprising compound II is used to treat a disorder associated with or occurring as a result of exposure or infection of HPV. In certain embodiments, the pharmaceutical composition comprising compound II is used to treat a pre-cervical lesion. In certain embodiments, the pharmaceutical composition comprising compound II is used to treat cervical intraepithelial neoplasia. In certain embodiments, the pharmaceutical compositions comprising compound II are used to treat intraepithelial neoplasia of the vagina and anus. In certain embodiments, the pharmaceutical composition comprising compound II is used to treat cervical cancer. In certain embodiments, the pharmaceutical composition comprising compound II is for use in treating colorectal cancer. In certain embodiments, the pharmaceutical composition comprising compound II is for treating penile cancer. In certain embodiments, the pharmaceutical composition comprising compound II is used to treat vaginal cancer. In certain embodiments, the pharmaceutical composition comprising compound II is used to treat oropharyngeal cancer.

In certain embodiments, the pharmaceutical composition comprising compound II is used in the manufacture of a medicament for treating a condition associated with or resulting from exposure to HPV or infection. In certain embodiments, the pharmaceutical composition comprising compound II is used in the manufacture of a medicament for treating a pre-cervical cancer lesion. In certain embodiments, the pharmaceutical composition comprising compound II is used in the manufacture of a medicament for the treatment of cervical intraepithelial neoplasia. In certain embodiments, the pharmaceutical composition comprising compound II is used in the manufacture of a medicament for the treatment of intraepithelial neoplasia of the vagina and anus. In certain embodiments, the pharmaceutical composition comprising compound II is used in the manufacture of a medicament for the treatment of cervical cancer. In certain embodiments, the pharmaceutical composition comprising compound II is used in the manufacture of a medicament for the treatment of rectal cancer. In certain embodiments, the pharmaceutical composition comprising compound II is used in the manufacture of a medicament for treating penile cancer. In certain embodiments, the pharmaceutical composition comprising compound II is used in the manufacture of a medicament for treating vaginal cancer. In certain embodiments, the pharmaceutical composition comprising compound II is used in the manufacture of a medicament for the treatment of oropharyngeal cancer.

Advantageously, the dosage form is easy to apply to the target site. Direct application to the target site may prevent systemic exposure and toxicity. To place the dosage form at the target site, the dosage form may be administered with an applicator. In certain embodiments, the dosage form is administered with a vaginal applicator. In certain embodiments, the dosage form is administered without an applicator. In certain embodiments, an additional liquid (e.g., lubricant) is delivered with the dosage form, applied to the dosage form, or applied to the target site or surrounding tissue.

In certain embodiments, the lubricating fluid is administered in combination with a dosage form to enhance coverage of the cervix, vagina, vulva, anus, perianal area, or penis. In certain embodiments, water is used as the liquid to be administered with the dosage form. In certain embodiments, a lubricious glycerol or hydroxyethylcellulose-based water-soluble liquid is administered with the dosage form. In certain embodiments, the dosage form is administered without additional liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 5 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 4 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 3 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 2 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 1 milliliter of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 0.75 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 0.5 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 0.25 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 0.2 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 0.15 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 0.125 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in less than about 0.1 milliliters of liquid.

In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in from about 10 microliters to about 100 microliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in from about 75 microliters to about 250 microliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in from about 200 microliters to about 500 microliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in from about 400 microliters to about 750 microliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in from about 700 microliters to about 1,000 microliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in from about 1 milliliter to about 2 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in from about 2 milliliters to about 3 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in from about 3 milliliters to about 4 milliliters of liquid. In certain embodiments, the dosage form will soften, disintegrate and/or dissolve in from about 4 milliliters to about 5 milliliters of liquid.

In certain embodiments, compound II is administered for at least 1, 2, 3, 4, 5, or 6 consecutive or non-consecutive days.

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

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

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

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

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

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

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

As described above, many compounds for treating HPV-induced neoplasias have been studied, 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 versions, eur.j.cancer prev.2003;12:383-390; ashfafan L, et al Double-blind randomized placebo-controlled multicenter clinical trial (phase IIa) on diindolymethane's efficacy and safety in the treatment of CIN: implications for cervical cancer precursor. 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 local application in high-grade cervical intraepithelial neoplasia: A phase 1 inventorization.int.J.Clin. Pharmacol.2018;56:134-141; chen f.p. efficiency of imiquimod 5%cream for persistent human papillomavirus in genital intraepithelial neoplasm.Taiwanese J.Obstetrics Gynecol.2013;52 (4) 475-478; choo y, et al Intravaginal application of leukocyte interferon gel in the treatment of Cervical Intraepithelial Neoplasia (CIN) Arch gynecol.1985;237:51-54; de Witte C.J et al Imiquimod in cervical, vaginal and vulvar intraepithelial neoplasia: a review. Gynecol. 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PLoS ONE.2016;11; helm C.W. et al Retinoids for preventing the progression of cervical intra-epi-chiral system review.2013; hubert p., et al Local applications of GM-CSF induce the recruitment of immune cells in cervical low-grade squamous intraepithelial versions.am.j. Reprod.immunol.2010;64:126-136; koeneman MM, et al Topical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC three): study protocol for a randomized controlled three. BMC cancer.2016:doi:10.1186/s12885-12016-12187-12883; krause S. et al Interferon and cervical dysplasia: CINIIItreated with local interferon application.Colposcope Gynecologic Laser surgery.1987;3:195-198; krebs H.B., et al Chronic ulcerations following topical therapy with-fluorouracil for vaginal human papillomavirus-associated versions, obstet, gynecol 1991;78 205-208; laccetta G. Et al Effect of the treatment with beta-glucan in women with cervical cytologic report of Atypical Squamous Cells of Undetermined Significance (ASCUS) and Low-grade intraepithelial lesions (L-SIL) Minerva Ginecol.2015;67:113-120; meyskes F.L., et al A phaseItrial of beta-all-transretinoic acid delivered via a collagen sponge and a cervical cap for mild or moderate intraepithelial cervical neoplasia.J.Natl Cancer Inst.1983;71:921-925; niwa K, et al Topical vidarabine of-fluoruracil treatment against persistent HPV in genital (pre) cancelius version.Oncol reports.2003;10:1437-1441; pachman DR, et al Randomized clinical trial of imiquimod: an adjunct to treating cervical dysplasia.am.J.Obstet.Gynecol.2012;206 42e41-47; rahangdale L et al Topical 5-fluorouracil for treatment of Cervical Intraepithelial Neoplasia 2:a randomized controlled trial.Am.J.Obstet.Gynecol.2014;210:e1-e8; schneider a et al Efficacy trial of topically administered Interferon gamma-1beta gel in comparison to laser treatment in cervical intraepithelial neoplasia.Arch.Gynecol Obste.1995;256:75-83; silman F.H., et al 5-fluorous cil/chemosurgery for intraepithelial neoplasia of the lower genital ct. 58:356-360; snoeck r., noel j.c., muller c., clercq De, bossens m.cidofovir, a new approach for the treatment of cervix intraepithelial neoplasiaIII (CIN III) j.med.virol.2000;60:205-209; stemella p., biamonti a., carraro c.effect of carboxymethyl beta-glucan in cervical intraepithelial neoplasia: a retroactive, case-control student.miniva ginecol.2017;69:425-430; suh-Burgmann E, sivret J, duska L.R., del Carmen M, seiden M.V. Long-term administration of intravaginal dehydroepiandrosterone on regression of low-grade cervical dysplasia-a pilot student.Gynecol. Obset. 2003;55:25-31; valencia M.H., pacheco A.C., quijano T.H., giron A.V., lopez C.V. clinical response to glycyrrhizinic acid in genital infection due to human papillomavirus and low-grade squamous intraepithelial version.Clin. Practice.2011 (e 93); van de Sande a, koeneman m, gerestein c, kruse a, van kemende f, van Beekhuizen h.topical Imiquimod treatment of residual or recurrent cervical intraepithelial neoplasia (TOPIC-2 three): a study protocol for a randomized controlled three.bmc cancer.2018;18:4510-4517; and Van Pachterbeke C., bucella D., rozenberg S.temporal treatment of CIN2+by cidofovir Results of a phase II, double-blank, prophetic, plant-controlled student.Gynecol Onc.2009;115:69-74.

VI pharmaceutical composition and dosage form

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 mono fumarate, compound II or compound III described herein, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient, and/or in combination or alternation with at least one other active compound. In one embodiment, the invention includes a solid dosage form of compound II in a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition is administered directly to the cervix, vagina, vulva, perianal region, anus, or penis. In certain embodiments, the dosage form adheres to the cervix, vagina, vulva, perianal region, anus, or penis.

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

The invention includes pharmaceutical compositions comprising an effective amount to treat HPV infection of any of the active compounds described herein, including but not limited to compound I monofumarate, compound II or compound III of the invention, in a pharmaceutically acceptable carrier or excipient. In an alternative embodiment, the invention includes a pharmaceutical composition comprising an effective amount to prevent HPV infection of the present compound I monofumarate or compound II or prodrug in a pharmaceutically acceptable carrier or excipient.

One of ordinary skill in the art will recognize that the therapeutically effective amount will vary with the infection or condition being treated, its severity, the treatment regimen to be employed, the pharmacokinetics of the drug being administered, and the patient or subject (animal or human) being treated, and that the amount of treatment can be determined by the attending physician or specialist.

Compound I monofumarate, compound II or compound III or any active compound described herein according to the invention may be formulated in a mixture with a pharmaceutically acceptable carrier. For the treatment of HPV infections, 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 the compounds, particularly various salt forms including acylated (acetylated or otherwise) and ether (alkyl and related) derivatives, phosphates, thiophosphoryl imidoesters, phosphonyl imidoesters, and compounds of the present invention, may be used to achieve the desired effect. One of ordinary skill in the art will recognize how to readily modify the compounds of the present invention into prodrug forms to facilitate delivery of the active compound to a target site within a host organism or patient. Those of ordinary skill in the art will also utilize the favorable pharmacokinetic parameters of the prodrug forms, where applicable, to deliver the compounds of the present invention to a target site within a host organism or patient to maximize the intended effect of the compound.

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

Typically, to treat, prevent or delay the onset of such infections and/or reduce the likelihood of HPV infection, or a disease state, condition or complication secondary to 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 an amount of about 0.001 mg to about 100 mg. In certain embodiments, the solid dosage form comprises 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, from about or 5 mg to about 20 mg. In certain 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 milligrams or more of any of the active compounds described herein, including, but not limited to, compound I monofumarate, compound II, or compound III.

In certain embodiments, to treat or delay the onset of these infections and/or reduce the likelihood of HPV infection, or a secondary disease state, condition, or complication of HPV, a dosage form containing any of the active compounds described herein (including but not limited to compound I monofumarate, compound II, or compound III) is administered in an amount of from about 0.001 to about 20mg, from about 0.005 to about 10mg, from about 0.01mg to about 5mg, from about 0.03mg to about 1mg, or from about 0.05mg to about 0.3mg of compound I monofumarate, compound II, or compound III. Typically, dosage forms of compound I mono fumarate, compound II, or compound III at dosages of 0.05mg to 0.3mg are administered 1, 2, 3 or more times per week until daily administration.

In certain embodiments, for the treatment of infection by a high risk strain of HPV, a dosage form containing any of the active compounds described herein (including but not limited to compound I monofumarate, compound II, or compound III) is administered in an amount of from about 0.001 to about 20mg, from about 0.005 to about 10mg, from about 0.01mg to about 5mg, from about 0.03mg to about 1mg, or from about 0.05mg to about 0.3mg of compound I monofumarate, compound II, or compound III. Typically, dosage forms of compound I monofumarate, compound II, or compound III that treat high risk strains of HPV at a dose of 0.05mg to 0.3mg can be administered 1, 2, 3 or more times per week until daily administration.

In certain embodiments, compound I monofumarate, compound II, or compound III can be administered as a gel. In certain embodiments, the gel comprises 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%, from about 0.1 to about 2% compound I monofumarate, compound II, or compound III (weight/weight). In certain embodiments, the gel comprises from about 0.001% to about 0.05% compound I monofumarate, compound II, or compound III. In certain embodiments, the gel comprises from about 0.01% to about 0.5% compound I monofumarate, compound II, or compound III. In certain embodiments, the gel comprises from about 0.1% to about 5% compound I mono fumarate, compound II or compound III.

In certain non-limiting embodiments, any of the active compounds described herein, including but not limited to compound I mono fumarate, compound II, or compound III, is topically administered. More generally, compound I monofumarate, compound II or compound III can be administered in the form of a tablet, capsule, suspension, liquid, emulsion, implant, granule, sphere, cream, ointment, suppository, transdermal form, gel, mucosal agent, or the like. The dosage form may also be a bilayer tablet wherein the full dose of the active compound is released in one direction (e.g., toward the target tissue).

In certain embodiments, the dosage form may soften, disintegrate and/or be released in a low liquid volume. In certain embodiments, the dosage form softens and begins immediate release of the drug. In certain embodiments, the dosage form softens and begins to release the drug gradually. In certain embodiments, the dosage form softens and begins to release the drug within one hour. In certain embodiments, the dosage form softens and begins to release the drug within two hours. Dosage forms may be prepared to maximize surface area, promoting disintegration. In certain embodiments, the dosage form is a circular tablet. In certain embodiments, the dosage form is an oblong tablet. In certain embodiments, the dosage form is a caplet. The tablet width has a maximum dimension and the tablet thickness has a smaller dimension. In certain embodiments, the dosage form has a width that is twice the thickness. In certain embodiments, the dosage form has a width that is three times the thickness. In certain embodiments, the width of the dosage form is four or more times the thickness. In certain embodiments, the dosage form is about 0.1mm thick to about 5mm thick. In certain embodiments, the dosage form is about 1mm to about 2mm thick. In certain embodiments, the dosage form is about 2mm to about 3mm thick. In certain embodiments, the dosage form is about 3mm to about 4mm thick. In certain embodiments, the dosage form is about 4mm to about 5mm thick. In certain embodiments, the tablet is about 5mm to about 15mm thick. In certain embodiments, the dosage form is less than 5 grams. In certain embodiments, the dosage form is from about 0.05 grams to about 0.15 grams. In certain embodiments, the dosage form is from about 0.1 to about 1 gram. In certain embodiments, the dosage form is from about 0.75 grams to about 2 grams. In certain embodiments, the dosage form is from about 1 gram to about 5 grams.

In certain embodiments, the dosage form is not easily removed, moved or mobilized from the target site. These desirable properties may be achieved by including a mucoadhesive polymer in the pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises a mucoadhesive polymer or mucoadhesive excipient. Non-limiting examples of mucoadhesive polymers and excipients include: hypromellose, lectin, thiolated polymer (e.g., chitosan-iminothiolane, poly (acrylic acid) -cysteine, poly (acrylic acid) -homocysteine, chitosan)Thioglycollic acid, chitosan-mercaptoethylamidine, alginate-cysteine, poly (methacrylic acid) -cysteine and sodium carboxymethyl cellulose-cysteine), polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acidPolyhydroxyethyl methacrylate, chitosan, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, sodium carboxymethyl cellulose, aminated corn starch, cellulose derivatives, poly (acrylic acid) polymers, poly (hydroxyethyl methacrylate), poly (ethylene oxide), poly (vinyl pyrrolidone), poly (vinyl alcohol), tragacanth, sodium alginate, karaya gum, guar gum, xanthan gum, soluble starch, gelatin, pectin, chitosan, methyl cellulose, hyaluronic acid, hydroxypropyl methylcellulose, hydroxypropyl cellulose, gellan gum (gellan gum), carrageenan, cationic hydroxyethyl cellulose, hydrogels, dihydroxyphenylalanine and alginate-polyethylene glycol acrylates. In certain embodiments, the pharmaceutical composition comprises from about 0% to about 10% of a mucoadhesive polymeric excipient selected from the group consisting of: carbomers, polyethylene glycols, crospovidone, polycarbophil, hypromellose, and hydroxyethyl cellulose.

In certain 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% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 0.1% to about 1% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 0.5% to about 5% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 1% to about 10% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 5% to about 20% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 10% to about 50% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 20% to about 75% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 50% to about 90% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises from about 75% to about 99% mucoadhesive polymer. In certain 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% mucoadhesive polymer. In certain 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% mucoadhesive polymer. In certain embodiments, the pharmaceutical composition comprises 0% mucoadhesive polymer. In this case, adhesion to the target site is achieved by using other pharmaceutically acceptable excipients.

For preparing the pharmaceutical compositions of the present invention, a therapeutically effective amount of any of the active compounds described herein (including but not limited to compound I monofumarate, compound II or compound III of the present invention) is typically admixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dose. The carrier may take a variety of forms depending on the form of formulation desired for administration, such as topical, oral or parenteral administration. In preparing the pharmaceutical composition in topical dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid or semi-solid topical formulations such as gels, creams, ointments, suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, preservatives and the like may be employed. In certain embodiments, the pharmaceutical composition comprises propylene glycol. In certain embodiments, the pharmaceutical composition comprises carboxypolymethylene. In certain embodiments, the pharmaceutical composition comprises ethylenediamine tetraacetic acid (EDTA). In certain embodiments, the pharmaceutical composition comprises sorbic acid. In certain embodiments, the pharmaceutical composition comprises carbomers. In certain embodiments, the pharmaceutical composition comprises hydroxyethylcellulose. In certain embodiments, the pharmaceutical composition comprises polyethylene glycol.

For solid topical formulations such as powders, tablets, capsules and for solid formulations such as suppositories, suitable carriers and additives may be used, including starches, sugar carriers (such as dextrose, mannitol, lactose and related carriers), diluents, granulating agents, lubricants, binders, mucoadhesive polymers, disintegrating agents and the like. If desired, the tablets or capsules may be coated or sustained release by standard techniques. The use of these dosage forms can significantly enhance the bioavailability of the compound in a patient. In certain embodiments, the pharmaceutical composition comprises mannitol. In certain embodiments, the pharmaceutical composition comprises magnesium stearate. In certain embodiments, the pharmaceutical composition comprises microcrystalline cellulose. In certain embodiments, the pharmaceutical composition comprises polycarbophil. In certain embodiments, the pharmaceutical composition comprises polyethylene oxide. In certain embodiments, the pharmaceutical composition comprises colloidal silica. In certain embodiments, the pharmaceutical composition comprises povidone. In certain embodiments, the pharmaceutical composition comprises isopropyl alcohol. In certain embodiments, the pharmaceutical composition comprises sodium carboxymethyl starch. In certain embodiments, the pharmaceutical composition comprises croscarmellose sodium. In certain embodiments, the pharmaceutical composition comprises crospovidone. In certain embodiments, the pharmaceutical composition comprises hydroxypropyl methylcellulose. In certain embodiments, the pharmaceutical composition comprises lactose. In certain embodiments, the powder pharmaceutical composition comprises one or more excipients selected from the group consisting of: xanthan gum, microcrystalline cellulose, polyethylene oxide, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, povidone, mannitol, colloidal silicon dioxide, sodium benzoate, sodium carboxymethyl starch, sodium lauryl sulfate, poloxamer 407, polyoxypropylene-polyoxyethylene copolymer, and the like.

In certain embodiments, pharmaceutical compositions comprising an effective amount of a fumarate salt of any of the active compounds described herein include, but are not limited to, compound I, further comprising a pharmaceutically acceptable excipient selected from the list consisting of: gum arabic, agar, alginic acid, ascorbyl palmitate, bentonite, benzoic acid, butylated hydroxyanisole, butylated hydroxytoluene, butylene glycol, calcium acetate, calcium hydroxide, rapeseed oil, carob bean gum, carrageenan, castor oil, cellulose, corn starch, disodium edentate, isoascorbic acid, ethyl lactate, ethylcellulose, glycerol, glyceryl behenate, glyceryl monooleate, glyceryl monostearate, hydroxyethyl methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactic acid, lauric acid, lecithin, linoleic acid, medium chain triglycerides, methyl benzoate, methylcellulose, methyl cellulose microcrystalline cellulose, microcrystalline wax, myristic acid, oleic acid, palmitic acid, peanut oil, pectin, phosphoric acid, polycarbophil, potassium alginate, propionic acid, propyl gallate, propyl parahydroxybenzoate, propylene glycol alginate, silicon dioxide, dimethicone, 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 polyethylene succinate, beeswax, xanthan gum and zinc acetate.

In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient for use as a vaginal suppository. In certain embodiments, the 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: hardened fat, PEG, polyethylene glycols (macrogols), cocoa butter and glycerol. Non-limiting examples of hardened lipids include(fatty acid (C) ₁₀ To C ₁₈ ) Mono-, di-and triglycerides, mainly triester moieties and ethoxylated fatty alcohols), -and-in>(glycerides of vegetable saturated fatty acids, such as lauric acid) and Supposi-base ^TM (mixtures of saturated polyglycolized glycerides).

In certain 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 a pharmaceutically acceptable excipient that enhances penetration, disintegration, film formation, and/or controls the release characteristics of the composition.

In a certain 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 certain embodiments, the penetration enhancing excipient is selected from the group consisting of: oleic acid; eucalyptus extract; xin Xianchun; labrafil; labrasol; lauryl alcohol; diethylene glycol monomethyl ether; propylene glycol; sodium laurate; sodium lauryl sulfate; cetyl trimethylammonium bromide; poloxamers (231, 182, 184); tween 20, 40, 60, 80; fatty acids and fatty acid esters; isostearic acid; glycerol; and chitosan. In certain embodiments, the pharmaceutical composition comprising the fumarate salt of compound I contains from 0% to about 20% of a penetration enhancing excipient selected from the group consisting of: cetyl alcohol, propylene glycol, transcutol P, oleic acid, isopropyl myristate, propylene glycol dioctanoate, glyceryl monooleate, propylene monocaprylate, PEG-8 beeswax, cetyl alcohol, stearic acid, cetyl palmitate and cetyl sterols. In certain embodiments, the pharmaceutical composition comprises from about 0% to about 25% of a penetration enhancing excipient selected from the list consisting of: stearyl alcohol, polysorbate 80, sodium lauryl sulfate, mono-and diglycerides, sorbitan monostearate, glycerol isostearate, polyethylene glycol 15 hydroxystearate, polyethylene glycol 40 hydrogenated castor oil, octyldodecanol and soybean lecithin.

In certain embodiments, the 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 a film-forming excipient. In certain 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: hypromellose, polyethylene glycol, polymethacrylate, microcrystalline cellulose, guar gum, xanthan gum, and polyvinylpyrrolidone.

In certain embodiments, the 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 controlled release of the active compound. In certain embodiments, the controlled release pharmaceutical composition comprises ethylcellulose, hypromellose, microcrystalline wax, polycarbophil, beeswax.

Unless otherwise indicated, the percentage ranges of excipients and other components of the pharmaceutical composition are expressed in weight percent.

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

In certain embodiments, the 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 70% mannitol, including but not limited to any amount up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% to achieve the desired binding. In certain embodiments, the pharmaceutical composition comprising the fumarate salt of compound I further comprises from 0% to about 70% lactose, including but not limited to any amount to achieve the desired binding, e.g., up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In certain 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 about 0% to about 70% sucrose, including but not limited to any amount up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% to achieve the desired binding. In certain 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 about 0% to about 70% microcrystalline cellulose, including but not limited to any amount up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% to achieve the desired binding. In certain 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 20% sodium carboxymethyl starch, including but not limited to any amount up to about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 12%, about 15%, or about 20% to achieve the desired binding. In certain 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 about 0% to about 20% pregelatinized starch, including but not limited to any amount up to about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 12%, about 15%, or about 20% to achieve the desired binding. In certain 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 about 0% to about 20% crospovidone, including but not limited to any amount up to about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 12%, about 15%, or about 20% to achieve the desired binding. In certain 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 about 0% to about 20% croscarmellose sodium, including but not limited to any amount up to about 1%, about 2%, about 3%, about 5%, about 7%, about 10%, about 12%, about 15%, or about 20% to achieve the desired binding. In certain embodiments, the 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 up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% to achieve the desired binding. In certain 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 50% polycarbophil, including but not limited to any amount up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% to achieve the desired binding. In certain embodiments, the 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 50% polyethylene oxide, including but not limited to any amount up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% to achieve the desired binding. In certain 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 50% hydroxyethyl methylcellulose, including but not limited to any amount up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% to achieve the desired binding. In certain 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 50% hydroxyethylcellulose, including but not limited to any amount up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% to achieve the desired binding. In certain 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 50% hypromellose, including but not limited to any amount up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% to achieve the desired binding. In certain 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 50% hydroxypropyl cellulose, including but not limited to any amount up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% to achieve the desired binding. In certain 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 up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% to achieve the desired binding. In certain 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 50% microcrystalline cellulose, including, but not limited to, any amount up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, or about 45% to achieve the desired binding.

In typical embodiments according to the invention, any of the active compounds described herein, including but not limited to compound I monofumarate, compound II or compound III, and the compositions are useful for treating, preventing or delaying HPV infection or a secondary disease state, disorder or complications of HPV.

In certain embodiments, a tablet for treating, preventing or delaying HPV infection or a secondary disease state, disorder or complication of HPV comprises any of the active compounds described herein, including but not limited to compound I monofumarate, compound II or compound III, further comprising about 250mg microcrystalline cellulose, about 20mg crospovidone, about 5mg magnesium stearate, about 5mg silicon dioxide, about 5mg polyethylene oxide, and about 100mg mannitol. In certain embodiments, a tablet for treating, preventing or delaying HPV infection or a secondary disease state, disorder or complication of HPV further comprises about 155mg microcrystalline cellulose, about 1.75mg magnesium stearate, and about 17.5mg mannitol.

In certain embodiments, a semi-solid formulation for treating, preventing, or delaying HPV infection or a secondary disease state, disorder, or complication of HPV comprises any of the active compounds described herein, including but not limited to compound I monofumarate, compound II, or compound III, further comprising about 15mg carbomer, about 50mg propylene glycol, about 10mg sorbic acid, about 5mg EDTA, and about 920mg water. In certain embodiments, a semi-solid formulation for treating, preventing or delaying HPV infection or a secondary disease state, disorder or complication of HPV comprises any of the active compounds described herein, including but not limited to compound I monofumarate, compound II or compound III, further comprising about 20mg carbomer; about 70mg mineral oil; about 80mg of a mixture of polyethylene glycol 6 stearate form I, ethylene glycol stearate and polyethylene glycol 32 stearate form 1; about 5mg of paraben; about 60mg propylene glycol; about 5mg EDTA; and about 760mg of water.

In certain embodiments, a dry powder for reconstitution for treating, preventing or delaying HPV infection or a secondary disease state, disorder or complication of HPV comprises any of the active compounds described herein, including but not limited to compound I monofumarate, compound II or compound III, further comprising about 15.5mg xanthan gum, about 19.8mg mannitol, about 5mg silica and about 0.5mg sodium benzoate.

VII combination and alternation therapy

The treatment for intraepithelial neoplasia described herein may be combined with conventional methods (e.g., without limitation, excision or ablation of the transformation zone). Techniques include cryotherapy, laser therapy, ring electro-ablation (LEEP), and cone biopsy. All of these surgical procedures damage the affected area and may lead to scarring. The most common intervention LEEP 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 consequences. In certain embodiments, the treatments described herein are used to reduce, improve, or replace the use of these routine procedures.

In certain embodiments, the treatments described herein can be used in combination with surgical techniques. In certain embodiments, a patient in need thereof may be subjected to surgery prior to, during, and/or after administration of an effective amount of a compound described herein. In certain embodiments, the surgical procedure may be the excision of the target tissue and/or diseased tissue, including but not limited to annular electrotomy (LEEP), conversion zone macrocyclic excision (LLETZ), knife cone excision, cold knife cone excision, knife cone biopsy, or laser cone excision. In certain embodiments, the surgical procedure may be ablation, including, but not limited to, laser ablation or cryoablation.

The efficacy of a drug against HPV infection may be prolonged, enhanced or restored by the combined or alternating use of another, possibly 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 the drug may be altered by such combination therapy (which may include alternation therapy if coordination is considered). Furthermore, since HPV is associated with several types of cancer, combination therapies administered with anticancer therapeutic agents may provide better results for patients. Since the disclosed compound II is a DNA polymerase inhibitor, it may be useful to administer to a host in need thereof a compound in combination with, for example:

a) Protease inhibitors;

b) Another DNA polymerase inhibitor;

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

d) An E7 inhibitor;

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

f) An L2 lipopeptide;

g) An L1 or L2 inhibitor or degradant;

h) HDAC inhibitors (e.g., vorinostat);

i) Degradation agents for four transmembrane proteins, such as CD9, CD63 or CD151;

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

k) Antiproliferative agents;

l) therapeutic vaccine;

m) a prophylactic vaccine;

n) trichloroacetic acid;

o) salicylic acid;

p) imiquimod;

q) pridafil;

r)9；

s)4；

t)Cervarix；

u)VGX-3100；

v) GGX-188E; and/or

w)ADXS11-001。

Examples

General comments

The following instrumental analysis methods were used to characterize the crystalline forms of the invention.

X-ray powder diffraction (XRPD)

Differential Scanning Calorimetry (DSC)

Thermogravimetric analysis (TGA)

Dynamic vapor phase adsorption (DVS)

Karl Fischer water assay

Instrument: mettler Toledo Coulometric KF Titrator C30A 30

The method comprises the following steps: coulomb method

Polarized Light Microscopy (PLM)

Nuclear Magnetic Resonance (NMR)

Instrument for measuring and controlling the intensity of light	Bruker Avance-AV 400M
		Frequency of	400MHz
Probe with a probe tip	5mm PABBO BB-1H/D
		Number of scans	8
Temperature (temperature)	297.6K
		Relaxation delay	1 second

Fourier transform infrared spectrum (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: methylene chloride, methylene dichloride;

MeOH: methanol, methyl alcohol;

ACN: acetonitrile;

EtOH: ethanol; ethyl alcohol;

IPAc: isopropyl acetate;

IPA: isopropyl alcohol, isopropyl alcohol;

THF: tetrahydrofuran;

MEK, methyl ethyl ketone;

DIAD: diisopropyl azodicarboxylate;

DEAD: diethyl azodicarboxylate;

MTBE: methyl tertiary butyl ether;

DMSO-d6: deuterated dimethyl sulfoxide;

Cs ₂ CO ₃ cesium carbonate;

TMSBr: trimethylsilyl bromide;

NaOMe: sodium methoxide;

TEA: triethylamine;

Ph ₃ p: triphenylphosphine;

Na ₂ SO ₄ sodium sulfate;

NaOH: sodium hydroxide;

HCl: hydrochloric acid;

H ₂ SO ₄ : sulfuric acid;

BsOH: benzenesulfonic acid;

p-TsOH: p-toluene sulfonic acid;

MsOH: methanesulfonic acid;

¹ h NMR: proton nuclear magnetic resonance;

LCMS: a liquid chromatograph mass spectrometer;

HPLC: high pressure liquid chromatography;

¹ h NMR part: s = single peak; bs = broad unimodal; d = double peak; dd = double doublet; t = triplet; m = multiple peaks; j = spin coupling constant.

Example 1: approximate solubility of Compound I free base at 25℃

About 2mg of compound I in free form was weighed into a 2mL glass vial and an aliquot of 20 μl of each solvent was added (table 1) to determine the solubility at 25 ℃. In a second set of experiments, about 10mg of compound I in free form was added to a 2mL glass vial, and an aliquot of 20 μl of each solvent was added to determine solubility at 50 ℃. The maximum volume of each solvent added was 1mL. The approximate solubility is determined by visual observation. The results are presented in table 1.

TABLE 1 approximate solubility of the free form of Compound I at 25℃and 50 ℃C

Example 2: crystallization screening of the free base form of Compound I

Equilibrated with solvent at 25 DEG C

As shown in table 2, about 30mg of compound I free base was equilibrated in the appropriate amount of solvent on a stirring plate at 25 ℃ for 1 week. On the eighth day of the study, no precipitated solids were observed, and all ten samples were stirred at 5 ℃ for about 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℃for 1 week

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The solid material obtained in experiment 10 formed compound I free form pattern 1 according to XRPD measurement (fig. 1). This mode is characterized by DSC (FIG. 2) having a melting onset temperature of 40.3deg.C (where the enthalpy of transition is 43J/g); and is in TGA (fig. 3) which has a weight loss of 0.1% at 40 ℃. According to ¹ H NMR, the material contained 0.2% residual heptane.

Precipitation by addition of anti-solvent

About 30mg of compound I free base was dissolved in the solvents listed below (table 3). An anti-solvent was slowly added to the obtained solution. The precipitate in experiment 3 was collected by filtration and analyzed by XRPD.

TABLE 3 results of precipitation by addition of anti-solvent

According to the XRPD data (fig. 1), the solid material formed as shown in experiment 3, table 3 is compound I free form pattern 1.

A crystalline free form, compound I free form mode 1, was obtained from an equilibration experiment in heptane and an anti-solvent experiment in acetone/MTBE.

Example 3: synthesis of salts of Compound I

Compound I sulfate

Two methods were used to synthesize the sulfate salt of compound I.

Method A

To a solution of compound I (0.049 g,0.1 mmol) in dry THF (1 mL) at 0-10deg.C was slowly added a sulfuric acid solution (1N in THF). During the addition of the sulfuric acid solution, the pink clear THF solution of compound I free base turned off-white semi-solid. The reaction mixture was brought to room temperature in 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 x2 mL) and the resulting solid was dried under high vacuum to yield 0.053g of compound I sulfate-1 as an off-white solid.

Method B

To a solution of compound I (0.024 g,0.05 mmol) in dry ethyl acetate (EtOAc, 0.5 mL) was slowly added a sulfuric acid solution (1N in EtOAc) at 0-10 ℃. During the addition of the sulfuric acid solution, the pink EtOAc clear solution of compound I free base turned into an off-white colored solid. The reaction mixture was brought to room temperature in 20-30min and shaken well. After allowing the solid material to settle, the supernatant was carefully decanted. The off-white solid was washed with additional amounts of EtOAc (2×2 mL). The resulting off-white solid was dried under high vacuum to yield 0.023g of compound I sulfate-2.

Compound I mesylate

Two methods were used to synthesize the mesylate salt of compound I.

Method A

To a solution of compound I (0.049 g,0.1 mmol) in dry EtOAc (1 mL) was added dropwise pure methanesulfonic acid (MSA; mw=96.11; d=1.47; 0.0070 mL;0.11 mmol) at 0-10 ℃. During addition of the MSA solution, the pink EtOAc clear solution of compound I free base turned to an off-white semisolid (glue-like). The heterogeneous mixture was brought to room temperature within 20-30min and shaken well. After the semi-solid material subsided, the supernatant was carefully decanted. The resulting semi-solid (gum-like) was washed with methyl tert-butyl ether (MTBE; 2X2 mL) and dried under high vacuum to yield 0.054g of Compound I mesylate-1.

Method B

To a solution of compound I (0.049 g,0.1 mmol) in dry IPA (1 mL) was added dropwise methanesulfonic acid solution (1N in THF; 0.11mmol;0.110 mL) at 0-10deg.C. During addition of the MSA solution, the pink EtOAc clear solution of compound I free base turned into an off-white solid. The heterogeneous mixture was brought to room temperature within 20-30min and shaken well. After allowing the solid material to settle, the supernatant was carefully decanted. The resulting solid was washed with methyl tert-butyl ether (MTBE; 2X2 mL) and dried under high vacuum to yield 0.049g of Compound I mesylate-2.

Compound I hydrochloride

To a solution of compound I (mw=492; 0.049g,0.1 mmol) in dry EtOAc (1 mL) was added dropwise HCl solution (4N in dioxane; 0.11mmole;0.027 mL) at 0-10 ℃. During addition of the HCl solution, the pink EtOAc clear solution of compound I free base turned into an off-white solid. The heterogeneous mixture was brought to room temperature within 20-30min and shaken well. After allowing the solid material to settle, the supernatant was carefully decanted. The resulting solid was washed with methyl tert-butyl ether (MTBE; 2X2 mL) and dried under high vacuum to yield 0.045g of Compound I HCl salt.

Compound I monofumarate salt

To a solution of compound I (0.035 g,0.071 mmol) in dry isopropanol (0.1 mL) was added fumaric acid (mw=116; 12.3mg;0.106mmol;1.5 eq.) at 0-10 ℃. The reaction mixture was brought to room temperature, then heated at 60 ℃ for 30min, and stirred at room temperature for 12h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure at 40 ℃. The mixture was diluted with MTBE (2 mL), shaken well, and the MTBE was carefully decanted. The colorless solid was washed with additional amounts of MTBE (2 mL) and dried under high vacuum to yield 0.022g 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 benzenesulfonic acidSalt

To a solution of compound I (0.057 g,0.115 mmol) in dry EtOAc (1.0 mL) was added dropwise a solution of benzenesulfonic acid (BsOH, mw=158; 0.020g;0.127mmol in 0.2mL EtOAc) at 0-10 ℃. During the addition of BsOH solution, a clear solution of compound I free base in pink EtOAc precipitated as a colorless solid. The reaction mixture was allowed to reach room temperature over 20-30min and shaken well. After allowing the solid material to settle, the supernatant was carefully decanted. The colorless solid was washed with additional amounts of MTBE (2 x2 mL). The resulting solid was dried under high vacuum to yield 0.064g of compound I benzenesulfonate.

Compound I tosylate salt

To a solution of compound I (0.024 g,0.05 mmol) in dry EtOAc (0.5 mL) was added dropwise p-toluene sulfonic acid solution (p-TsOH, 0.055mL;0.055mmol;1N in EtOAc) at 0-10 ℃. During the addition of the p-TsOH solution, a clear solution of compound I free base in pink EtOAc precipitated as a colorless solid. The reaction mixture was brought to room temperature in 20-30min and shaken well. After allowing the solid material to settle, the supernatant was carefully decanted. The colorless solid was washed with additional amounts of MTBE (2 x2 ml). The resulting solid was dried under high vacuum to yield 0.027g of compound I tosylate.

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 salt produced by washing the sesquifumarate salt had the highest melting point of the tested salts.

TABLE 4 melting Point of salts of Compound I

Example 5: salt screening of Compound I free base

Selection ofEight acids (fumaric acid, citric acid, L-malic acid, L-tartaric acid, succinic acid, benzenesulfonic acid, oxalic acid and maleic acid) and two co-formers (L-proline and nicotinamide) were screened for potential salt and/or co-crystal formation. The compound I is used as a mixture of two diastereoisomers ¹ H NMR purity was about 98% -99%). Isopropyl alcohol (IPA), ethanol, and Ethyl Acetate (EA) were selected as screening solvents. Slurry equilibration, anti-solvent addition and slow evaporation were applied as screening methods.

Salt screening by slurry equilibration

Compound I free base was added as a mixture of diastereomers (about 30 mg) to a suitable solvent followed by 1 or 0.5 molar equivalents (as shown in table 5 below) of acid (experiment RC2 to RC 10) or coformer (experiment RC11 and RC 12) with stirring at 50 ℃. The mixture was stirred at 50 ℃ for 2 hours and then at 25 ℃ for at least 12 hours. For those experiments in which a clear solution was formed, half of the volume was evaporated in a fume hood and the residue was treated by addition of anti-solvent. The obtained suspension was taken out and centrifuged. The solids obtained in experiments RC2 (sample RC 2-EA), RC3 (sample RC 3-EA), RC7 (samples RC7-IPA and RC 7-EA) and RC11 (samples RC 11-EA) were analyzed by XRPD. The results are presented in table 5.

TABLE 5 screening of salts (co-crystals) by slurry equilibration

In experiment RC2, the solid material obtained in EA (samples RC 2-EA) was shown to be a mixture of fumaric acid and was similar to the phase of hemi-fumarate pattern 1 (sample AS3-B in table 6) according to XRPD (fig. 7 and 10). In experiment RC3, the solid material obtained in EA (sample RC 3-EA) was shown to be a phase similar to hemi-fumarate pattern 1 according to XRPD data (fig. 7). In experiment RC11, the solid material obtained in EA was shown to be a mixture of L-proline coform and free form mode 1. In experiment RC7, the solid material obtained from isopropanol (sample RC7-IPA, FIG. 8) and ethyl acetate (sample RC7-EA, FIG. 9) was characterized by XRPD in a phase similar to that of monosuccinate mode 1 (see below, table 6, experiment AS7, sample AS 7-B).

Salt screening results by anti-solvent addition

To the clear solutions obtained in the experiments of table 5, an antisolvent (methyl tert-butyl ether (MTBE) or heptane) was slowly added (experiments AS1 to AS12 in table 6). In the case of experiments AS2, AS3 and AS7, the addition of heptane anti-solvent to the solution in EtOH formed a precipitate of solid materials AS2-B, AS3-B and AS7-B, which were characterized by XRPD.

Next, to the clear solution (from experiments AS1, AS3 and AS 12) and sample AS5-a containing the small amount of solid material obtained after the addition of the anti-solvent, 0.5, 1 or 1.5 molar amount of acid or co-former was added with stirring at 25 ℃, and the mixture was stirred at this temperature for at least 12 hours. No solid phase was formed in experiments AS13 to AS 17. The results are summarized in table 6.

TABLE 6 salt screening results by anti-solvent addition

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The addition of heptane anti-solvent to the clear solution obtained from experiment RC2 (ethanol AS solvent, 1 molar equivalent of fumaric acid) resulted in the formation of monofumarate salt pattern 1, characterized by XRPD (sample AS2-B, fig. 10). The composition of the material being a monofumarate salt is prepared by ¹ H NMR (free base form to fumaric acid ratio = 1:0.98) was established; according to XRPD, no free fumaric acid phase was found in this sample. To the reaction mixture from experiment RC3 (ethanol as solvent, 0.5 mol DangAmount of fumaric acid) to the clear solution obtained, a heptane anti-solvent was added, resulting in the formation of the hemi-fumarate salt form 1, characterized by XRPD (sample AS3-B, fig. 10). The composition of the material being a hemi-fumarate salt is prepared by ¹ H NMR (free base form to fumaric acid ratio = 1:0.54) was established; according to XRPD, no free fumaric acid phase was found in this sample. The addition of heptane anti-solvent to the clear solution obtained from entry RC7 (ethanol AS solvent, 1 molar equivalent of succinic acid) resulted in the formation of monosuccinate mode 1, characterized by XRPD (sample AS7-B, fig. 11). The composition of the material being a monosuccinate salt is prepared by ¹ H NMR (ratio of free base form to succinic acid=1:1.06) was established; according to XRPD, no free succinic acid phase was found in this sample.

Rebalancing test

The solvent was evaporated from the gel-like or oily samples obtained in the salt screening by anti-solvent addition described above. The residual mixture was slurried in MTBE or heptane at 25 ℃ as shown in table 7. The results of the experiment are given in table 7.

TABLE 7 results of rebalancing

Citric acid, L-malic acid, L-tartaric acid, oxalic acid, maleic acid and nicotinamide did not form crystalline phases of compound I in the rebalancing experiments.

Example 6: salt formation of compound I by slurry crystallization

In experiment RC13, about 50mg of compound I free base was added to EtOH and fumaric acid was added in an amount of 0.5 molar for 2 hours under stirring at 50 ℃ and then stirring was continued for at least 12 hours at 25 ℃. Seed crystals of sample AS3-B were added, and heptane (0.4 mL) was added AS an anti-solvent. The resulting mixture was stirred at 5 ℃ for about 3 days. The suspension was then removed and centrifuged. The resulting solid was dried in the oven at 50 ℃ under vacuum for about 1 hour and analyzed by XRPD (fig. 12), NMR, DSC (fig. 13) and TGA (fig. 14).

In experiment RC14, about 50mg of compound I free base was added to EtOH and an equimolar amount of succinic acid was added with stirring at 50 ℃ for 2 hours and then stirring at 25 ℃ for at least 12 hours. Seed crystals of sample AS7-B were added, and heptane (0.2 mL) was added AS an anti-solvent. The obtained suspension was taken out and centrifuged. The resulting solid was dried in the oven at 50 ℃ under vacuum for about 1 hour and analyzed by XRPD (fig. 15), NMR, DSC (fig. 16) and TGA (fig. 17).

In experiment RC15, about 50mg of Compound I free base was added to 0.1mL EtOH. An equimolar amount of benzenesulfonic acid was dissolved in 0.2mL EtOH. The solution of benzenesulfonic acid was then added drop-wise to the free base solution at 25 ℃. MTBE (0.8 mL) was added as an anti-solvent and the mixture was stirred at 5 ℃ with some solids formed.

In experiment RC16, about 50mg of Compound I free base was added to 0.1mL of IPA. A 1.5 molar amount of fumaric acid was added to the mixture, and the mixture was stirred at 25 ℃. After about 3 hours, the sample was too viscous, 0.1mL of additional IPA was added, and stirring was maintained at 25 ℃ for about 22 hours. A viscous sample was obtained and dried in a vacuum oven at 50 ℃ for about 2 hours and reslurried in 1.0mL MTBE at 25 ℃ for about 5 days. The obtained suspension was taken out and centrifuged. The solid obtained was dried in an oven at 40 ℃ under vacuum for about 1 hour and analyzed by XRPD (fig. 18) and NMR. Residual solids were added as seed in RC 18.

In experiment RC17, about 30mg of Compound I free base was added to 0.1mL EtOH. Succinic acid was added to the mixture in an amount of 0.5 molar for 2 hours at 50 ℃ with stirring, and a clear solution was obtained. Heptane (0.3 mL) was added as an anti-solvent. An oil was obtained and the mixture was stirred at 25 ℃ for about 5 days. The obtained suspension was taken out and centrifuged. The solid obtained was dried in an oven at 40 ℃ under vacuum for about 1 hour and analyzed by XRPD (fig. 19), NMR, DSC (fig. 20) and TGA (fig. 21).

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

TABLE 8 results of salt screening by slurry crystallization

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Note that:

* The salt ratio is as in ¹ Compound I wander as determined by proton integration in H NMR spectra

Alkali separation and counter ion ratio;

* ND: data were not collected.

According to the salt screening experiments, 4 modes, hemi-fumarate mode 1, mono-fumarate mode 1, hemi-succinate mode 1 and mono-succinate mode 1 were identified. The four modes show comparable properties, such as low to medium crystallinity with respect to low melting point.

Example 7: preparation of fumarate salt

Preparation of Compound I hemi-fumarate salt form 1

About 200mg of compound I free base was added to 0.5mL EtOH. With stirring, 0.5 molar amount of fumaric acid was added at 50 ℃ and the mixture was stirred for 2 hours. A clear solution was obtained. The solution was then cooled to 25 ℃ over 1 hour. Sample RC13 (example 5, table 7) was seeded with hemi-fumarate salt followed by 2.5mL of heptane to cause precipitation. An oil was obtained and stirring was continued for about 4 days at 25 ℃. After 4 days, the resulting suspension was cooled to 5 ℃. After stirring at 5 ℃ for about 4 days, the precipitated solid was collected by filtration and dried at 40 ℃ under vacuum for about 3 hours. As a result, 116mg of pale orange hemi-fumarate pattern 1 was obtained in 52% yield. XRPD is shown in fig. 23; DSC is shown in figure 24; and TGA is shown in fig. 25.

Preparation of Compound I monofumarate salt form 1 (Small Scale preparation)

About 244mg of compound I free base was added to 0.8mL IPA. Then, 1.0 equivalent of fumaric acid was added with stirring at 50℃for about 1.5 hours. The resulting yellow clear solution was cooled to 25 ℃ and stirred for about 5 minutes. The monofumarate seeds of sample RC 18 (table 7) were added to the mixture followed by 4mL of heptane as anti-solvent. The mixture was stirred at 25 ℃ for 4 days. The precipitated material was collected by filtration and dried under vacuum at 40 ℃ for about 2 hours. As a result, 208mg of monofumarate pattern 1 solid was obtained in a yield of 69%. XRPD is shown in fig. 23. DSC at 10deg.C/min is shown in FIG. 26 and DSC at 2deg.C/min is shown in FIG. 27 b. The DSC cycle results are shown in fig. 27 c. TGA is shown in fig. 28.

TABLE 9 characterization of the free base form of Compound I and its monofumarate and hemi-fumarate forms

Compound I mode 1 is high crystallinity. The hemi-fumarate and mono-fumarate modes are medium crystallinity.

Compound I, mode 1, is anhydrous and T at 75.0 °c _Initiation The lower part has a melting peak, wherein the enthalpy is about 64J/g. It shows about 0.3% weight loss at about 70 ℃. KF display Showing that it contains about 1.7% water. By passing through ¹ H NMR detects about 0.7% MTBE (by weight) residue.

Compound I hemifumarate pattern 1 is an anhydrous compound. Based on ¹ As a result of H NMR, the stoichiometry of fumaric acid in the free form was about 1:0.5. It has a T at 85.2 DEG C _Initiation The lower part has a melting peak, wherein the enthalpy is about 37J/g. It shows about 1.0% weight loss at about 85 ℃. KF showed it to contain about 1.7% water. By passing through ¹ H NMR detected about 0.7% EtOH and 0.7% heptane (residue by weight).

Compound I monofumarate salt form 1 is an anhydrous compound. Based on ¹ As a result of H NMR, the stoichiometry of fumaric acid in the free form was about 1:1.0. It has a T at 107.2 DEG C _Initiation The lower has a melting peak and a split peak, and wherein the enthalpy is about 78J/g. The two thermal events were not resolved by DSC with heating rates of 2K/min and 0.5K/min. It shows about 0.3% weight loss at about 107 ℃. KF showed it to contain about 1.2% water. By passing through ¹ H NMR detects about 0.7% IPA and 2.2% heptane (by weight) residues.

Example 8 stability of Compound I free base Pattern 1, compound I Monofumarate Pattern 1 and Compound I hemi-fumarate Pattern 1

Initial chemical purity: the initial purities of compound I free base pattern 1, compound I monofumarate pattern 1 and compound I hemi-fumarate pattern 1 were 98.7%, 98.6% and 97.8%, respectively.

Volume stability: accelerated stability experiments were performed in an open vessel at 25 ℃/92% rh and 40 ℃/75% rh in a sealed vessel at 60 ℃ for one week. The results are presented in table 10. The results of compound I monofumarate salt pattern 1 are also presented in fig. 28.

All three candidates showed good physical stability after exposure to three conditions. They all showed some degradation after exposure to 40 ℃/75% RH for one week, both of which were unstable at elevated temperatures (60 ℃ for one week). Under both conditions, compound I hemi-fumarate pattern 1 is more prone to degradation than compound I mono-fumarate pattern 1.

TABLE 10 volume stability of Compound I free base form 1, compound I monofumarate form 1 and Compound I hemi-fumarate form 1

Example 9 solubility of Compound I free base form 1, compound I monofumarate form 1 and Compound I hemi-fumarate form 1

About 4mg of compound I mode 1 was added to 2mL of buffer solution. About 4mg of compound I hemi-fumarate form 1 and compound I mono-fumarate form 1 were added to 1.8mL and 1.6mL buffer solutions, respectively. The pH was adjusted with 0.2N NaOH in the simulated vaginal secretion. After stirring at 37 ℃ for 0.5 and 2 hours, clear solutions of all three candidates were obtained.

TABLE 11 solubility of Compound I free base form 1, compound I monofumarate form 1 and Compound I hemi-fumarate form 1

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

Example 10: hygroscopicity of compound I free base pattern 1, compound I hemi-fumarate pattern 1 and compound I mono-fumarate pattern 1

Hygroscopicity was studied by DVS at 25 ℃ using the following method:

the method comprises the following steps: 40-0-95-0-40% RH, dm/dt=0.002

The results are presented in table 12 and fig. 29 to 34.

TABLE 12 hygroscopicity of Compound I free base Pattern 1, compound I hemi-fumarate Pattern 1 and Compound I mono-fumarate Pattern 1

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Note that: d. desorption; s. adsorption

Hygroscopicity was studied by DVS at 25 ℃. All three modes were moderately hygroscopic. Compound I mode 1 shows about 4.3% water uptake to 90% RH, but about 14.3% water uptake at 95% RH. For compound I mono-fumarate pattern 1 and compound I hemi-fumarate pattern 1, they showed about 2.9% and 6.9% water absorption at up to 95% RH. There was no form change after DVS testing.

Example 11: large-scale preparation of monofumarate salt form 1

About 4.16g of compound I free base was dissolved in 11mL of IPA. Then, 1.0 equivalent of fumaric acid was added to the yellow clear solution at 50 ℃ with stirring. After about 1 hour, some solids precipitated out. Then, 10mg of monofumarate seed crystals (from the above-mentioned 'small scale preparation') were added. The mixture was stirred at 50 ℃ for about 1.5 hours and cooled to 25 ℃ and then stirred at 25 ℃ for about 10min. Then, 40mL of heptane was added as an anti-solvent. The resulting suspension was stirred at 25 ℃ for about 24 hours, then cooled to 5 ℃ at a rate of 0.1 ℃/min, and stirred at 5 ℃ for about 1 day. The solid was collected by filtration and dried in an oven at 40 ℃ under vacuum for about 2 hours. About 4.1g of a pale pink solid was obtained in a yield of 81.2%. XRPD is shown in fig. 35; DSC at a rate of 10 ℃/min is shown in FIG. 36 a; DSC at the rate of 2 ℃/min is shown in FIG. 36 b; the mDSC thermogram is shown in FIG. 36 c; TGA is shown in fig. 37.

TABLE 13 characterization of Compound I monofumarate salt form 1

Example 12: polymorph screening study of Compound I fumarate

Polymorph screening studies of the fumarate salt of compound I (mixture of diastereomers) were performed. The behavior of its polymorphs was studied by equilibration, precipitation (by addition of anti-solvent), slow cooling, rapid cooling and slow evaporation experiments.

Compound I monofumarate salt form 1 has approximate solubility at 25℃and 50 ℃

About 2mg of compound I monofumarate salt form 1 (example 11) was weighed into a 2mL glass vial and 20 μl aliquots of each solvent (as shown in table 14) were added to determine the solubility at 25 ℃. Approximately 10mg of compound I monofumarate salt form 1 (example 11) was weighed into a 2mL glass vial and an aliquot of 20 μl of each solvent was added (as shown in table 14 below) to determine the solubility at 50 ℃. The maximum volume of each solvent added was 1mL. The approximate solubility is determined by visual observation.

TABLE 14 approximate solubility of Compound I monofumarate salt form 1 at 25℃and 50 ℃

Equilibration with solvent at 25 ℃ was continued for 2 and 3 weeks

About 30mg of compound I monofumarate salt form 1 (obtained in example 11) was equilibrated with a stirring blade at 25 ℃ for 2 and 3 weeks in the appropriate amount of solvent. The suspension obtained was filtered. The solid fraction (wet cake) was studied by XRPD. When differences are observed, additional studies (e.g., NMR, DSC, TGA, HPLC and PLM) are performed. The results are presented in tables 15-30 and FIGS. 38-60.

Table 15. Results of compound I monofumarate pattern 1 equilibration with water at 25 ℃ for 2 and 3 weeks.

For mode D, XRPD diffractograms are shown in fig. 38 and 39; DSC thermograms are shown in figures 40 and 41; TGA thermograms are shown in fig. 42 and 43.

Table 16. Results of compound I monofumarate pattern 1 equilibration with acetonitrile at 25 ℃ for 2 and 3 weeks.

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Note that: the salt ratio is the ratio of free base form to fumaric acid

For mode C, the XRPD diffractogram 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 compound I monofumarate pattern 1 equilibration with MEK at 25 ℃ for 2 and 3 weeks.

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Note that: the salt ratio is the ratio of free base form to fumaric acid

For mode B, the XRPD diffractogram is shown in figure 47; DSC thermogram shown in figure 48; and the TGA thermogram is shown in figure 49.

Table 18. Results of compound I monofumarate pattern 1 equilibration with acetone at 25 ℃ for 2 and 3 weeks.

The XRPD diffractogram is shown in figure 47 (3 weeks).

TABLE 19 sustained equilibration of Compound I monofumarate form 1 with isopropanol at 25℃

Results for 2 weeks and 3 weeks.

Note that: the salt ratio is the ratio of free base form to fumaric acid

For mode 1, the xrpd diffractogram 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 for compound I monofumarate salt form 1 equilibrated with acetone/toluene (1:1 v/v) at 25℃for 2 and 3 weeks.

For mode E, the XRPD diffractogram is shown in figure 53; DSC thermograms are shown in figures 54 and 55; and TGA thermograms are shown in fig. 56 and 57.

TABLE 21 Monofumarate die of Compound I with acetone/heptane (1:1 v/v) at 25℃

The equilibrium of formula 1 persists for 2 weeks and 3 weeks of results.

Note that: the salt ratio is the ratio of free base form to fumaric acid

The XRPD diffractogram is shown in figure 47 (3 weeks).

Table 22. Results of compound I monofumarate salt form 1 equilibrated with IPA/heptane (1:1 v/v) at 25℃for 2 and 3 weeks.

Note that: the salt ratio is the ratio of free base form to fumaric acid

The XRPD diffractogram is shown in figure 50.

Table 23. Results of 2 and 3 weeks equilibration of Compound I monofumarate salt form 1 with IPA/toluene (1:1 v/v) at 25 ℃.

Note that: the salt ratio is the ratio of free base form to fumaric acid

The XRPD diffractogram is shown in figure 50.

Table 24. Results of compound I monofumarate salt form 1 equilibrated with IPA/MTBE (1:3 v/v) at 25℃for 2 and 3 weeks.

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Note that: the salt ratio is the ratio of free base form to fumaric acid

The XRPD diffractogram is shown in figure 50.

Table 25 results of compound I monofumarate salt form 1 equilibrated with THF/heptane (1:3 v/v) at 25℃for 2 and 3 weeks.

Note that: the salt ratio is the ratio of free base form to fumaric acid

XRPD is shown in fig. 58 and 59.

Table 26 results of compound I monofumarate salt form 1 equilibrated with EtOH/heptane (1:3 v/v) at 25℃for 2 and 3 weeks.

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Note that: the salt ratio is the ratio of free base form to fumaric acid

The XRPD diffractogram is shown in figure 50.

Table 27. Results for 2 and 3 weeks of compound I monofumarate salt form 1 equilibrated with EA/toluene (1:3 v/v) at 25 ℃.

Note that: the salt ratio is the ratio of free base form to fumaric acid

XRPD is shown in fig. 58 and 59

Table 28 results of compound I monofumarate salt form 1 equilibrated with EtOH/toluene (1:3 v/v) at 25℃for 2 and 3 weeks.

Note that: the salt ratio is the ratio of free base form to fumaric acid

XRPD is shown in fig. 58 and 59.

Table 29 results of compound I monofumarate salt form 1 equilibrated with water/ACN (2.9:97.1 v/v) at 25℃for 2 and 3 weeks.

Note that: the salt ratio is the ratio of free base form to fumaric acid

The XRPD diffractogram is shown in figure 44.

TABLE 30 Monofumarate die of Compound I with IPA/heptane (1:4 v/v) at 25℃

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

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Note that: the salt ratio is the ratio of free base form to fumaric acid

The XRPD diffractogram is shown in figure 60.

Precipitation by addition of anti-solvent

About 30mg of compound I monofumarate salt form 1 (example 7) was dissolved in a good solvent. An anti-solvent was slowly added to the obtained solution. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD. When a difference is observed, additional studies (e.g., NMR, DSC, TGA) are performed. If no precipitate is obtained, the solution is cooled to 5℃for crystallization. After stirring at 5℃for about 23 days, no precipitate was obtained and the solution was placed in a-20℃freezer for crystallization.

The results are presented in table 31. XRPD diffractograms are shown in fig. 61 and 62.

TABLE 31 precipitation of Compound I fumarate salt by addition of anti-solvent

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Crystallization at room temperature by slow evaporation

In combination with the approximate solubility experiment, the solubility samples were filtered through a 0.45 μm nylon filter screen. The resulting solution was slowly evaporated under ambient conditions. The solid residue was checked for polymorphic forms.

The results are presented in table 32. XRPD diffractograms are shown in fig. 63 and 64.

TABLE 32 crystallization at room temperature by slow evaporation

Crystallization from a hot saturated solution by slow cooling

About 30mg of compound I monofumarate salt form 1 (example 7) was dissolved in the minimum amount of the selected solvent at 50 ℃. The resulting solution was cooled to 5℃at a rate of 0.1℃per minute. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD. When a difference is observed, additional studies (e.g., NMR, DSC, TGA) are performed. If no precipitate is obtained, the solution is placed in a freezer at-20℃for crystallization. After storage in a-20 ℃ freezer for about 5 days, no precipitate was obtained and heptane was added as an anti-solvent. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD.

The results are presented in table 33. XRPD diffractograms are shown in fig. 65 and 66.

TABLE 33 crystallization from a hot saturated solution by slow cooling

From thermal saturation by rapid coolingSolution crystallization

About 30mg of compound I monofumarate salt form 1 (example 7) was dissolved in the minimum amount of the selected solvent at 50 ℃. The obtained solution was put into an ice bath and shaken. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD. When a difference is observed, additional studies (e.g., NMR, DSC, TGA) are performed. If no precipitate is obtained, the solution is placed in a freezer at-20℃for crystallization. After storage in a-20 ℃ freezer for about 7 days, no precipitate was obtained and heptane was added as an anti-solvent. The precipitate was collected by filtration. The solid fraction (wet cake) was studied by XRPD.

The results are presented in table 34. XRPD diffractograms are shown in fig. 67 and 68.

TABLE 34 crystallization from hot saturated solution by rapid cooling

Behavior of Compound I monofumarate salt form 1 under heating and Cooling

The polymorphic behavior of the compound I monofumarate salt pattern was investigated by DSC for two different heating-cooling cycles.

Cycle 1: at 10 ℃/min,0 ℃ to 106 ℃; at 10 ℃/min,106 ℃ to 0 ℃; reheating from 0 ℃ to 250 ℃ at 10 ℃/min.

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

The results are presented in table 35 and fig. 69 and 70.

TABLE 35 behavior of Compound I monofumarate salt form 1 under heating and cooling

Post compression behavior

About 10mg of compound I monofumarate salt form 1 (example 7) was compressed at 10MPa for 5min using a hydraulic press. XRPD characterization was performed to investigate the polymorphic behavior after compression according to XRPD, no form change was observed.

Grinding simulation experiment

About 10mg of compound I monofumarate salt form 1 (example 7) was ground manually with a mortar and pestle for 5min. The crystalline transformation and crystallinity were assessed by XRPD. According to XRPD, no form change was observed; crystallinity is slightly reduced.

Granulating simulation experiment

Water or ethanol was added dropwise to about 10mg of compound I monofumarate salt form 1 (example 7) until the solid was sufficiently wet. The sample was manually ground with a mortar and triturated for 3 minutes. The samples were dried for 10min at ambient conditions. The crystalline transformation and crystallinity were assessed by XRPD. According to XRPD, no form change was observed in both ethanol and water.

Summary of identified monofumarate and hemi-fumarate polymorphs

Compound I monofumarate pattern 1 used in the study was prepared from compound I free base according to example 7. The initial form of monofumarate salt used in the polymorphic studies described below (mode 1) was the anhydrous compound of monofumarate salt with an HPLC purity of about 99.3%. The ratio of free form to fumaric acid was about 1:0.96 (according to ¹ H NMR). It was measured by Differential Scanning Calorimetry (DSC) at a T of about 98.5 DEG C _Initiation At about 14J/g enthalpy) and at 109.6C (about 25J/g enthalpy) have two melting peaks. Mode 1 shows about 0.5% weight loss at about 98 ℃ and about 0.6% weight loss at 98 ℃ to 140 ℃ by thermogravimetric analysis (TGA). By passing through ¹ HNMR detected about 1.0% (by weight) heptane and 0.2% (by weight) IPA residues. Karl Fischer titration shows that it contains about 1.3% water.

Four new modes were identified during the polymorph study. The new pattern obtained shows different stoichiometric ratios, although mono fumarate was used as the initial physical form. Pattern B, pattern C and pattern E are hemi-fumarate salts and pattern D is a degradation product.

Mode B is the anhydrous compound of the hemi-fumarate salt with an HPLC purity of about 99.6%. MEK, acetone/heptane, MEK/heptane and EtOH/MTBE were used as solvents, which can be obtained from equilibration experiments, anti-solvent addition, slow cooling and rapid cooling experiments. The ratio of free base form to fumaric acid was about 1:0.52 (by 1H-NMR). It has a T at about 77.4 DEG C _Initiation At (where enthalpy is about 71J/g), and at 88.4 ℃ (where enthalpy is about 18J/g) there are two thermal events. It shows about 0.7% weight loss at about 77 ℃ and about 4.2% weight loss at 77 ℃ to 130 ℃. By passing through ¹ H NMR detected about 4.6% (by weight) MEK residue.

Using ACN and ACN/water as solvents, a mixture of mode C and fumaric acid can be obtained by equilibrium, slow cooling and rapid cooling experiments. The ratio of the free base form of the mixture to fumaric acid was about 1:0.95 (according to ¹ H NMR). After washing with water, the ratio was reduced to about 1:0.76. This means that pattern C is not monofumarate. It has a T at about 74.0deg.C _Initiation At (wherein the enthalpy is about 89J/g) and T at about 90.6 DEG C _Initiation The lower (where enthalpy is about 15J/g) has two thermal events. It shows about 0.4% weight loss at about 73 ℃ and about 2.1% weight loss at 73 ℃ to 144 ℃. By passing through ¹ H NMR detected about 2.0% (by weight) ACN residue.

Mode D is a degradation product, wherein HPLC purity is about 0.2%. It was obtained in water by an equilibrium experiment. It has a T at about 41.4 DEG C _Initiation At (wherein enthalpy is about 67J/g) and T at about 72.1 DEG C _Initiation The lower (where enthalpy is about 29J/g) has two thermal events. It shows about 0.6% weight loss at about 41 ℃ and about 8.5% weight loss at 41 ℃ to 178 ℃.

Mode E is the anhydrous compound of the hemi-fumarate salt, wherein HPLC purity is about 98.9%. Obtained by equilibration experiments in acetone/toluene. The ratio of the free base form of the mixture to fumaric acid was about 1:0.69 (by ¹ H NMR). It has a T at about 53.1 DEG C _Initiation Lower (wherein enthalpy is about 33J/g)T at about 96.5C _Initiation The lower (where enthalpy is about 34J/g) has two thermal events. It shows about 1.0% weight loss at about 53 ℃ and about 3.6% weight loss at 53 ℃ to 96 ℃. By passing through ¹ H NMR detected about 0.6% (by weight) acetone residue.

The results of the study of the monofumarate and hemi-fumarate polymorphs of compound I are summarized in table 36 below. The ratio of salts is the ratio between the free base of compound I and the salt counter ion. "AS" means a form that can be prepared by adding an anti-solvent using the solvent/anti-solvent pairs listed in the table. "EQ" means a form that can be prepared by equilibration in the listed solvents. "SC" means a form which can be prepared by slowly cooling a solution of the monofumarate salt of Compound I in the listed solvents. "FC" means that the form may be prepared by rapid cooling of a solution of compound I monofumarate in the listed solvents.

TABLE 36 summary of identified Compound I fumarate patterns A, B, C and E

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Example 13: preparation of Compound II mode 1

Experiment 1 Small Scale Synthesis and preparation of seed crystals

100mg of R _P Compound I free base and 0.3mL IPA were added to a glass vial. To this was added 1.0 equivalent of fumaric acid and the resulting mixture was stirred at 50℃for 2min, most of the material precipitated out. To this was added 1.0mL of heptane. The sample was stirred at 50℃for 1h and then cooled to 3℃at 0.1℃per minute. After stirring at 3℃for about 8h, 0.4mL of heptane was added. The solid obtained was isolated by filtration and dried in a vacuum oven at 40 ℃ for about 2h to obtain compound II, mode 1. The characterization results are reported in table 37. XRPD derivatives of compound II mode 1 The shot is shown in fig. 71.

TABLE 37 characterization of Compound II Pattern 1 (Small Scale preparation)

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Experiment 2 Large Scale preparation

3g of R _P Compound I free base and 9mL of IPA were added to a glass vial. To this was added 1.0 equivalent of fumaric acid and the resulting mixture was stirred at 50℃for 5 minutes. Approximately 21mg of compound II mode 1 seed crystals from experiment 1 were added to the mixture. 30mL of heptane was added to the mixture. The sample was stirred at 50℃for 1h and then cooled to 3℃at a rate of 0.1℃per minute. After stirring at 3 ℃ for about 16h, the solid obtained was isolated by filtration and dried in a vacuum oven at 40 ℃ for about 4h and at 50 ℃ for about 3h. This gave 2.9g of compound II, pattern 1, as a white solid in 78.3% yield. The XRPD diffractogram 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 characterization of Compound II Pattern 1 (Large Scale preparation)

Compound II mode 1 is anhydrous. Based on ¹ As a result of H NMR, the stoichiometry of free base to fumaric acid was about 1:1.0. It has a T at 141.5 DEG C _Initiation The lower part has a melting peak accompanied by decomposition. TGA is shown at about 13 About 0.3% weight loss at 0 ℃. By passing through ¹ No residual solvent was detected by H NMR.

Example 14: preparation of Compound IV, mode 1

66mg of R _P Compound I free base and 0.2mL EtOH were added to a glass vial. To this was added 0.5 equivalent of fumaric acid and the resulting mixture was stirred at 50 ℃ for 2h, after which some solids precipitated out. To this suspension 0.4ml of heptane was added and stirred further at 25 ℃ for about 4 days. The solid was isolated by filtration and dried in a vacuum oven at 50 ℃ for about 2h. The compound IV obtained, mode 1, is characterized as reported in table 39. The XRPD diffractogram of compound IV mode 1 is shown in figure 74. The DSC thermogram of compound IV mode 1 is shown in figure 75. TGA thermogram of compound IV mode 1 is shown in figure 76.

TABLE 39 characterization of Compound IV Pattern 1

Example 15: preparation of Compound III, mode 1

100mg of S _P Compound I free base and 0.3mL IPA were added to a glass vial. To this was added 1.0 equivalent of fumaric acid and the mixture was stirred at 50℃for 15min, most of the material precipitated out. After 1.0mL of heptane was added, the sample was stirred at 50deg.C for 1h and then cooled to 3deg.C at 0.1deg.C/min. After stirring at 3 ℃ for about 8 hours, 0.4mL of heptane was added to the mixture to get a better suspension. The white solid was isolated by filtration and dried in a vacuum oven at 40 ℃ for about 2h to yield compound III, pattern 1. The characterization results are reported in table 40. The XRPD diffractogram of compound III, mode 1, is shown in figure 77. The DSC thermogram of compound III mode 1 is shown in figure 78. TGA thermogram of compound III mode 1 is shown in figure 79.

TABLE 40 characterization of Compound III Pattern 1

Example 16: preparation of Compound III, mode 2

3g of S _P Compound I free base and 9mL of IPA were added to a glass vial. After the addition of 1.0 equivalent of fumaric acid, a large amount of solids precipitated immediately. 30mL of heptane was added to the mixture, followed by about 20mg of compound III mode 1 seed crystal added to the mixture. The sample was stirred at 50℃for 1h and then cooled to 3℃at 0.1℃per minute. After stirring at 3 ℃ for about 20 hours, the sample was heated from 3 ℃ to 50 ℃ over 20min, then 0.2 equivalents of fumaric acid and 1.5ml of heptane were added to the mixture. The resulting mixture was stirred at 50 ℃ for about 2 hours and then cooled to 3 ℃ at a rate of 0.1 ℃/min. After stirring at 3 ℃ for about 13 hours, the mixture was reheated to 50 ℃ over 20 minutes and stirred at 50 ℃ for about 6 hours. Cooled to 3 ℃ at 0.1 ℃/min and stirred at 3 ℃ for about 2 days. The resulting solid was isolated by filtration and dried in a vacuum oven at 50 ℃ for about 3 hours to give 3.1g of a white solid in 83.6% yield. The results are reported in table 41. The XRPD diffractogram 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 characterization of Compound III Pattern 2

Compound III, pattern 2, is anhydrous. Based on ¹ As a result of H NMR, the stoichiometry of free base to fumaric acid was about 1:1.2. It has a T at 106.4 DEG C _Initiation At (wherein the enthalpy is about 47J/g) and T at 118.8 DEG C _Initiation The lower (where enthalpy is about 63J/g) has two melting peaks. It shows about 0.3% weight loss at about 105 ℃. By passing through ¹ No residual solvent was detected by H NMR.

Example 17: preparation of Compound V Pattern 1

100mg of S _P Compound I free base and 1.0 equivalent of fumaric acid were added to a glass vial followed by 0.8mL of IPA. At 50 DEG CAfter stirring for about 1h, a clear solution was obtained. About 2mg of compound III mode 1 seed crystals were added to the mixture. After some solid precipitation was observed, 1mL of heptane was added to the mixture. The mixture was stirred at 50 ℃ for 2h and then cooled to 3 ℃ at 0.1 ℃/min. It was stirred at 3℃for about 3 days. The solid was isolated by filtration and dried in a vacuum oven at 50 ℃ for about 2h to yield compound V, pattern 1. The results are reported in table 42. The XRPD diffractogram of compound V mode 1 is shown in figure 83. The DSC thermogram of compound V mode 1 is shown in figure 84. TGA thermogram of compound V mode 1 is shown in figure 85.

TABLE 42 characterization of Compound V Pattern 1

Example 18: preparation of Compound V Pattern 2

100mg of S _P Compound I free base and 1.0 equivalent of fumaric acid were added to a glass vial followed by 0.8mL of IPA. To the resulting clear solution was added 1ml of heptane and the mixture was stirred at 50 ℃ for 2h and then cooled to 3 ℃ at 0.1 ℃/min. It was stirred at 3℃for about 3 days. The solid formed was isolated by filtration and dried in a vacuum oven at 50 ℃ for about 2h to give compound V mode 2. The results are reported in table 43. The XRPD diffractogram of compound V mode 2 is shown in figure 86. The DSC thermogram of compound V mode 2 is shown in figure 87. TGA thermogram of compound V mode 2 is shown in figure 88.

TABLE 43 characterization of Compound V Pattern 2

Example 19: volume stability of Compound II Pattern 1 and Compound III Pattern 2

Compound ii mode 1 and compound iii mode 2 were placed in an open vessel at 25 ℃/92% RH, in an open vessel at 40 ℃/75% RH, and in a sealed vessel at 60 ℃ for 1 week. The samples were characterized by XRPD and HPLC and checked for color change. The results are presented in table 44.

Table 44. Stability: purity and appearance

Initial chemical 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%.

Volume stability

The accelerated stability test was performed at 25 ℃/92% RH in an open vessel, at 40 ℃/75% RH in an open vessel and at 60 ℃ in a sealed vessel for one week. Compound II mode 1 shows good physical and chemical stability after exposure to three conditions. Compound III mode 2 shows good physical stability under the three conditions mentioned above. Degradation products increased by 1.6% and 1.5% after exposure to 40 ℃/75% RH in an open vessel and 60 ℃ in a sealed vessel, respectively.

Example 20: solubility studies of compound II mode 1 and compound III mode 2

12mg of compound II, mode 1, 12mg of compound III, mode 2 were accurately weighed into an 8mL glass vial, and 5mL of solubility medium (solubility medium) was added thereto. The amount of salt used corresponds to 10mg of anhydrous free base. After 0.5 and 2h at 37 ℃, all samples were clear solutions in medium. The pH of the resulting clear solution was analyzed with a pH meter and the solubility was determined by observation.

TABLE 45 solubility at 37℃target concentration 10mg/5mL, equilibration duration 0.5h and 2h

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

Example 21: hygroscopicity of Compound II Pattern 1 and Compound III Pattern 2

Hygroscopicity was studied by DVS at 25 ℃ using the following method:

the method comprises the following steps: 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 about 0.2% water absorption at up to 95% RH. No form change was observed after DSC testing. Compound III mode 2 shows about 1.0% water absorption at up to 95% RH. No form change was observed after DSC testing. The results are presented in table 46.

Table 46.

Example 22: polymorph screening of Compound II Pattern 1 and Compound III Pattern 2

50mg of monofumarate is equilibrated in a suitable amount of solvent or solvent mixture. The resulting suspension was equilibrated for 1 week. The solids were separated by centrifugation. The wet cake obtained after equilibration was analyzed by XRPD to determine the crystal modification.

Table 47. Mini-polymorph screening: crystal modification after 1 week of equilibration at 25 ℃ (salt ratio is defined as free base to fumaric acid ratio)

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Polymorphism assessment of Compound II Pattern 1

In this study, no dissociation was observed during the equilibration experiments, and 2 new potential polymorphs of the fumarate salt of isomer I (compound II mode 2 and compound II mode 3) were obtained from acetonitrile and MEK, respectively. These all show lower melting temperatures than compound II mode 1. Both polymorphs of isomer 1 show unchanged high chiral purity.

Polymorphism assessment of Compound III Pattern 2

In this study, dissociation was observed during the equilibration experiment to give the hemi-fumarate salt of isomer II (hemi-fumarate salt pattern 2). In addition, 4 new monofumarate salts of isomer II (compound III pattern 3, compound III pattern 4, compound III pattern 5 and compound III pattern 6) were obtained.

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 mode 1. XRPD showed sharp peaks, indicating that the sample consisted of crystalline material. In the XRPD of compound II mode 1, significant peaks were observed at about 3.1±0.2°, about 9.3±0.2°, about 12.1±0.2°, about 14.9±0.2°, about 15.1±0.2°, about 18.1±0.2°, about 19.8±0.2°, about 20.1±0.2°, about 25.1±0.2°, about 25.9±0.2°, and about 28.8±0.2°.

TABLE 48 XRPD peak list for Compound II Pattern 1

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Table 49 below provides the results of XRPD performed on compound IV mode 1. In XRPD of compound IV pattern 1, significant peaks were observed at about 6.5±0.2°, about 12.1±0.2°, about 17.5±0.2°, about 18.1±0.2°, about 18.5±0.2°, about 19.6±0.2°, about 19.8±0.2°, about 20.2±0.2°, about 20.6±0.2° and about 21.3±0.2°.

TABLE 49 XRPD peak list for Compound IV pattern 1

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Table 50 below provides the results of XRPD performed on compound III, mode 1. In XRPD of compound III pattern 1, significant peaks were observed at about 9.5±0.2°, about 11.7±0.2°, about 14.6±0.2°, about 17.5±0.2°, about 18.0±0.2°, about 20.0±0.2°, about 20.4±0.2°, about 22.3±0.2°, about 23.7±0.2° and about 25.5±0.2°.

TABLE 50 XRPD peak list for Compound III mode 1

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Table 51 below provides the results of XRPD performed on compound III, mode 2. XRPD showed sharp peaks, indicating that the sample consisted of crystalline material. In XRPD of compound III pattern 2, significant peaks are observed at about 8.9±0.2°, about 9.9±0.2°, about 11.7±0.2°, about 12.1±0.2°, about 15.1±0.2°, about 17.9±0.2°, about 18.2±0.2°, about 19.9±0.2°, about 25.1±0.2°, about 29.6±0.2°, and about 38.1±0.2°.

TABLE 51 XRPD peak List for Compound III Pattern 2

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Table 52 below provides the results of XRPD performed on compound V mode 1. In XRPD of compound V pattern 1, significant peaks were observed at about 5.0±0.2°, about 7.2±0.2°, about 10.1±0.2°, about 12.1±0.2°, about 17.5±0.2°, about 17.9±0.2°, about 19.3±0.2°, about 22.0±0.2°, about 24.3±0.2°, about 25.1±0.2°, and about 26.3±0.2°.

TABLE 52 XRPD peak list for Compound V Pattern 1

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Table 53 below provides the results of XRPD on compound V mode 2. Significant peaks were observed at about 5.1±0.2°, about 6.9±0.2°, about 7.6±0.2°, about 10.2±0.2°, about 11.6±0.2°, about 12.1±0.2°, about 15.1±0.2°, about 17.6±0.2°, about 18.1±0.2°, about 18.7±0.2°, about 19.5±0.2°, about 19.8±0.2° and about 25.1±0.2° in the XRPD of compound V pattern 2.

TABLE 53 XRPD peak list for Compound V Pattern 2

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Example 24: single crystal X-ray diffraction (SC-XRD) study of compound II mode 1

In a temperature cycling experiment in MeOH, single crystals of compound II mode 1 suitable for SC-XRD studies were obtained. On a D8 Venture diffractometer equipped with a CMOS area detector, at 170 (2) K, cu-K alpha radiation was used X-ray generator power: 50kV,1.4mA; distance between sample and face detector: 40mm; exposure time 150 seconds; resolution ratio: 0.81, and collecting X-ray diffraction data. Refining the structure: at F ² And (3) upper part. Hydrogen site position: and (3) mixing. The H atoms are treated with an independent and constrained refining mixture. The X-ray diffraction data and crystal data are presented in table 54.

TABLE 54 crystallographic parameters and X-ray diffraction data

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In a monoclinic system, there is R _int =5.7% and final R ₁ [I>2σ(I)]P2 =7.4% ₁ A crystalline form of compound II mode 1 crystallized in the space group. The crystalline form does not contain solvent molecules. Discovery of CompoundsThis crystalline form of mode II mode 1 has a free base to fumaric acid ratio of 1:1 and corresponds to the monofumarate salt of isomer I having a fly parameter (absolute structural parameter) of 0.16 (10). As shown in fig. 115, in the single crystal structure, the protonated free base and fumaric acid anion pass through N ⁺ (5) H (5) O (7) ionic bond linkage. Proton transfer was observed from fumaric acid to the N (5) -nitrogen atom of the 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 indicated conditions and sampled periodically and checked for purity by HPLC. In a series of different formulations, the tablets produced from compound I monofumarate salt degrade less than the tablets produced from compound I.

With compound IRaw materialsRaw tablet

Tablets produced with compound I monofumarate

PEO: polyethylene oxide; PC: polycarbophil; 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) -propionic acid ester (Compound I)

Step 1: preparation of diethyl- ((2- (2-amino-6-chloro-9H-purin-9-yl) -ethoxy) -methyl) -phosphonate (3)

At N ₂ The dry reaction vessel was charged with 2-amino-6-chloropurine 1 (50 g, 0.292 mol,1 eq.) Cs under ambient temperature ₂ CO ₃ (96.37 g,0.296mol,1 eq.) and DMF (250 mL). Diethyl 2-chloroethoxymethylphosphonate 2 (74.85 g,0.325mol,1.1 eq) was added dropwise at room temperature with stirring. The reaction was stirred at 40-50 ℃ for 0.5 to 1.5 hours, heated to 60-70 ℃ and stirred for 0.5-1.5 hours, and then stirred at 75-85 ℃ for 18-24 hours. After the reaction temperature was brought to 20-30 ℃, the reaction mixture was filtered and the resulting cake was washed with DMF (100 ml x 2). The combined filtrates were concentrated to half volume at less than 70 ℃, diluted with n-heptane (250 mL), and concentrated again to half volume at less than 75 ℃. Pouring the obtained solution into DCM (1L) at 20-30deg.C, stirring for 20-40min, and adding aqueous 10% Na ₂ SO ₄ Solution (about 100 mL). The resulting biphasic solution was stirred for 20-40 min, then filtered through celite, and the wet cake was washed with DCM (about 100 mL). The aqueous phase was separated from the filtrate and the organic phase was re-used with aqueous 10% Na ₂ SO ₄ The solution (about 100 mL) was washed. 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 DCM). The fractions containing the product were combined and the solvent was evaporated at below 40 ℃. The solid product 3 was treated with repeated dilutions of MTBE and concentrated (to 1/3 ^rd Volume). The resulting slurry was then diluted with MTBE (400-500 mL) and stirred at 40-50deg.C for 4-6h and 15-25deg.C for 8-15h. The suspension was filtered and washed with MTBE and dried at 35-40 ℃ for 15-20h to provide the desired product, diethyl- ((2- (2-ammonia)base-6-chloro-9H-purin-9-yl) -ethoxy) -methyl) -phosphonate 3 in 43.4% (48.66 g) isolated yield with 91.8% purity according to HPLC. ¹ H NMR (400 mhz, dmso-d 6), δppm:8.08 (s, 1H), 6.91 (s, 2H), 4.24 (d, 2H, j=8 Hz), 3.92 (m, 4H), 3.86 (q, 4H, j=8 Hz), 1.14 (t, 6H, j=8 Hz). LCMS (m/z): 364.2 (MH+) and 366.2 (MH+).

Step 2: preparation of ((2- (2-amino-6-chloro-9H-purin-9-yl) -ethoxy) -methyl) -phosphonic acid (4)

At N ₂ A dry reaction vessel containing DCM (1L) was charged under an atmosphere with diethyl- ((2- (2-amino-6-chloro-9H-purin-9-yl) -ethoxy) -methyl) -phosphonate 3 (100 g,0.275 mol), followed by 2, 6-lutidine (147.33 g,1.375mol,5 eq.) and the temperature was adjusted to 0-5 ℃. TMSBr (167.47 g,1.102mol,4.0 eq.) was added dropwise thereto and stirred at 0-5℃for a further 0.5-1 and at 20-25℃for 15-20h. After adjusting the reaction temperature at 0-5 ℃, 1144g of aqueous 1n noh was added dropwise. After maintaining the temperature at 20-30 ℃ for 1-2h, the aqueous alkaline layer was separated and the wash repeated with MTBE. The aqueous solution was acidified to ph=6-7 with dropwise addition of aqueous 2N HCl at 15-25 ℃ and charged with MeOH (10 volumes). The resulting methanol solution was further acidified to ph=3-4 with the addition of aqueous 2N HCl dropwise at 35-45 ℃. After the seeding of the product 4, the methanolic acid solution is stirred at 35-45 ℃ for 3-5h and, in addition, acidified to ph=1.5-2.5 using dropwise addition of aqueous 2N HCl and stirred at 15-20 ℃ for 11-20h. The resulting solid was isolated by filtration, washed with MeOH (2 x100 mL) and dried at 45-55 ℃ for 20-30H to yield 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) isolation with 99.8% purity according to HPLC. ¹ H NMR(400MHz,DMSO-d6),δppm:8.1(s,1H),6.92(bs,2H),4.5-5.5(bs,2H),4.22(dd＝t,2H,J＝8Hz),3.84(t,2H,J＝8Hz),3.58(t,2H,J＝8Hz)。LCMS(m/z):308 (MH+) and 310 (MH+).

Step 3: preparation of ((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) -phosphonic acid (5)

A flask containing MeOH (350 mL) was charged with ((2- (2-amino-6-chloro-9H-purin-9-yl) -ethoxy) -methyl) -phosphonic acid 4 (50 g,0.162 mol) at 20-30℃and stirred for 10-30min. To this solution was added 30wt% NaOMe solution (1.62 mol,10 eq.) in MeOH in a dropwise manner and then stirred at 50-60℃for 15-24h. The reaction was maintained at 20-30 ℃ for 20-40min and then filtered. The filtrate was then acidified at 20-30 ℃ to adjust ph=6-7 by dropwise addition of concentrated HCl and concentrated to one third of the volume at less than 40 ℃. The temperature of the concentrated solution was raised to 35-45 ℃ and acidified by dropwise addition of concentrated HCl to adjust ph=3-4. The resulting acidic solution was seeded with product 5 and stirred at 35-45 ℃ for 1.5-2.5h. At this temperature, the ph=2-3 adjustment was completed by adding concentrated HCl at a dropwise rate, stirring for 3-5h, cooling to a range of-3 ℃ to 3 ℃, and stirring for 8-15h. The resulting solid was filtered, washed with MeOH (about 100 mL) and n-heptane (about 100 mL). The resulting cake was dried under vacuum at 50-60 ℃ for 16-24H to provide the desired product, ((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) -phosphonic acid 5, in 89.3% (48.22 g) isolated yield with 99.5% purity 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) -propionic acid ester (8)

A solution of ((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) -phosphonic acid 5 (40 g,0.132mol,1 eq.) in DCM (560 mL) was charged with (S) -ethyl 2-aminopropionate hydrochloride 6 (20.19 g,0.132mol,1 eq.), benzyl alcohol 7 (71.28 g,0.66mol,5 eq.) and TEA (159.98 g,1.58mol,12 eq.) under stirring at 20-30℃and the solution stirred for 10-30min. Adding a solution of Ph at 20-30deg.C for 60min ₃ A solution of P (207.5 g,0.792mol,6 eq.) and 2,2' -dithiopyridine (Aldrithio-2) (174.24 g,0.792mol,6 eq.) in DCM (320 mL). The resulting reaction mixture was stirred at 35-45℃for 15-20h and concentrated under vacuum at less than 40℃to remove 3/4 ^th Is a solvent of (a) and (b). To the resulting residue were added MeOH (about 120 mL), distilled water (about 400 mL), toluene (about 400 mL), and n-heptane (about 400 mL), and stirred at 20-30deg.C for 0.5-1h. After allowing the reaction mixture to stand at 20-30 ℃ for 0.5 to 1h, the organic phase was separated and the aqueous phase was extracted several times with a mixture of toluene (about 400 mL) and n-heptane (about 400 mL) to remove the maximum amount of remaining reagents and byproducts. The remaining aqueous phase was then extracted with DCM (2×400 mL) and after concentrating the DCM under vacuum at below 40 ℃, the crude product was purified by silica gel column chromatography (with DCM to 2% MeOH in DCM as mobile phase). The product-containing eluted fractions are combined and the solvent is removed under vacuum at less than 40 ℃ to give the desired product as a mixture of diastereomers, i.e., (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) was isolated in 45.8% (29.74 g) yield with 98.8% purity 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) -propionic acid ester

To synthesize a mixture of (R, R) and (S, R), the procedure of step 4a may be performed by replacing D-alanine ethyl ester ((R) -ethyl 2-aminopropionate hydrochloride) with L-alanine ethyl ester ((S) -ethyl 2-aminopropionate hydrochloride).

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)

To a solution of (±) - (2S) -ethyl-2- ((((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) - (benzyloxy) -phosphoryl) -amino) -propionic acid ester (compound I) 8 (29.72 g,0.06mol,1 eq.) in IPA was added a solution of fumaric acid (7.66 mol,1.1 eq.) in IPA through the filter at 45-55 ℃ and stirring was continued for 1-2H. Seed crystals of compound 9 were added to the reaction mixture and stirring was continued at 45-55 ℃ for 1-2h. After allowing the reaction mixture to settle at 20-30 ℃ for 4-6 hours, dropwise addition of n-heptane (about 300 mL) is performed and stirring is continued for a further 8-15 hours at 20-30 ℃ and for a further 8-15 hours at 0-5 ℃. 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 ℃ for 16-24H 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 an isolated yield of 87.9% (32.74 g), with 99.1% purity 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 Monofumarate salts thereof, 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 (diastereomeric mixture of isomers I and II) (22.50 g) was subjected to chiral chromatographic separation under SFC separation conditions as shown below to separate and obtain 11.7g (R, S) -isomer I (10) (with 98.6% purity according to HPLC) and 9.1g (S, S) -isomer II (11) (with 95.6% purity according to HPLC).

SFC conditions:

column: chiralPak AD, 250X 30mm I.D.,10 μm;

mobile phase: a: CO ₂ And B: ethanol (0.1% NH) ₃ H ₂ O)；

Gradient: b45% isocratic;

flow rate: 200mL/min;

wavelength: 310nm;

cycle time: about 6 minutes;

back pressure: 100bar;

injection amount: about 1g.

Characterization of (R, S) -ethyl-2- ((((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) - (benzyloxy) -phosphoryl) -amino) -propionic acid ester (isomer I) as the free base: purity by HPLC: 98.6%; ¹ H 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) -propionic acid ester (isomer II) as the free base: purity by HPLC: 95.6%; ¹ H 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 certain non-limiting embodiments, the stereoisomers are separated using HPLC or SFC with an achiral or chiral stationary phase. 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, individual isomers may be asymmetrically synthesized. For non-limiting examples of asymmetric syntheses of phosphonyl amides, see, for example, numan, A et al, "Asymmetric Synthesis of Stereogenic Phosphorus P (V) Centers Using Chiral Nucleophilic Catalysis", molecular 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

Isolation of (R, R) -ethyl-2- ((((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) - (benzyloxy) -phosphoryl) -amino) -propionate and (S, R) -ethyl-2- ((((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) - (benzyloxy) -phosphoryl) -amino) -propionate (synthesis was described in step 4b of example 26) can be performed using the same techniques for mixtures of (R, S) and (S, S) as described above.

Step 2a preparation of (R, S) -ethyl-2- ((((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) - (benzyloxy) -phosphoryl) -amino) -propionic acid ester monofumarate (Compound II)

To a solution of (R, S) -ethyl-2- ((((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) - (benzyloxy) -phosphoryl) -amino) -propionic acid ester 10 (3 g,6mmol,1 eq.) in IPA was added a solution of fumaric acid (0.765 g,6.6mmol,1.1 eq.) in IPA, passed through the filter at 45 ℃ to 55 ℃ and stirring was continued for 1-2. Seed crystals of compound 12 were added to the reaction mixture and stirring was continued at 45-55 ℃ for 1-2h. After allowing the reaction mixture to settle at 20-30 ℃ for 4-6 hours, dropwise addition of n-heptane (about 30 mL) is performed and stirring is continued for a further 8-15 hours at 20-30 ℃ and for a further 8-15 hours at 0-5 ℃. The observed solid was 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 ℃ for 16-24H 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 85% (3.1 g) isolated yield with 98.6% purity according to HPLC. ¹ H NMR(DMSO-d ₆ ) Delta, ppm delta 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 of 1:1.00 (by ¹ H NMR)。

Step 2b preparation of (S, S) -ethyl-2- ((((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) - (benzyloxy) -phosphoryl) -amino) -propionic acid ester monofumarate salt (13) (also referred to as Compound III)

To a solution of (R, S) -ethyl-2- ((((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) - (benzyloxy) -phosphoryl) -amino) -propionic acid ester 11 (3 g,6mmol,1 eq.) in IPA was added a solution of fumaric acid (0.765 g,6.6mmol,1.1 eq.) in IPA, passed through the filter at 45 ℃ to 55 ℃ and stirring was continued for 1-2. Seed crystals of compound 13 were added to the reaction mixture and stirring was continued at 45-55 ℃ for 1-2h. After allowing the reaction mixture to settle at 20-30 ℃ for 4-6 hours, dropwise addition of n-heptane (about 30 mL) is performed and stirring is continued for a further 8-15 hours at 20-30 ℃ and for a further 8-15 hours at 0-5 ℃. The observed solid was 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 ℃ for 16-24H to provide the desired product, (S, S) -ethyl-2- ((((2- (2-amino-6-methoxy-9H-purin-9-yl) -ethoxy) -methyl) - (benzyloxy) -phosphoryl) -amino) -propionic acid ester monofumarate 13 (isomer II monofumarate or compound III) in 80% (2.9 g) isolated yield with 95.6% purity according to HPLC. ¹ H NMR(DMSO-d ₆ ) Delta, ppm delta 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); the ratio of base (11) to fumaric acid is 1:1.2 (by ¹ H 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 carried out as in steps 2b and 2c for the (R, S) and (S, S) stereoisomers by substituting the chiral isolated product of step 1b for the starting material of steps 2b and 2 c.

EXAMPLE 29 non-limiting example of the preparation of semi-solid formulations

Topical cream formulations can be prepared, for example, by emulsifying the oil and water phases together with the active pharmaceutical ingredient. In a non-limiting example, by blending light mineral oil, propyl parahydroxybenzoate and 63 to prepare the oil phase of the cream. Next, by mixing water, EDTA, methylparaben and +.>974P were mixed to prepare the aqueous phase of the cream and then the oil phase and aqueous phase were emulsified. To the emulsified mixture are added the active pharmaceutical ingredient and propylene glycol. The mixture was pH adjusted and then filled into a tube.

Topical gel formulations can be prepared, for example, by mixing an aqueous gel carrier with the active pharmaceutical ingredient. In a non-limiting example, by combining water, EDTA, methylparaben (or sorbic acid) and974P were mixed to prepare the aqueous phase of the topical gel. The active pharmaceutical ingredient and propylene glycol are added to the solution, mixed and pH adjusted, and then filled into tubes.

In certain non-limiting embodiments, from about 0.001% w/w to about 10% w/w active pharmaceutical ingredient is added to the semi-solid formulation. 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 Monofamate tablet

Non-limiting examples of cervical tablet preparation of compound I monofumarate are provided below (see the flow chart of fig. 119). Two or more excipients are combined, blended, and screened to produce an excipient blend. The active pharmaceutical ingredient (e.g., compound I mono fumarate) is then screened and added to a portion of the excipient blend. The resulting mixture is then blended and then more excipient blend is added. Thus, the mixture is gradually diluted with the excipient blend, with thorough mixing after each addition of the excipient blend. Once the excipient blend is used up, magnesium stearate is added and the mixture is blended together again. The mixture is then compressed into tablets and packaged.

TABLE 57 formulation of Compound I monofumarate vaginal tablets, 0.3mg batch size 1.0Kg

Composition of the components	％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 (mannitol)	10.0	100.0
Magnesium stearate	1.0	10
			Together, a total of	100.0	1000.0

Weight of individual tablets 175mg

Compound I monofumarate vaginal tablets, 0.3mg

Non-limiting examples of processes for preparing vaginal tablets of compound I monofumarate are provided below.

Sub-packaging

1. The materials were weighed according to a batch manufacturing recipe and dispensed into individual plastic bags.

Screening

1. Screening is provided for all excipients.

Blending and screening of active agents

1. The screened excipients, microcrystalline cellulose and mannitol were blended in a diffusion blender.

2. 49.5 g of excipient blend was taken and 2.12 g of compound I monofumarate was added.

3. The active agent and excipients are blended and screened to remove large lumps.

4. To this blend was added 148.5 grams of excipient blend.

5. The active blend and excipients are blended and screened to remove large lumps.

6. 247.5 grams of excipient blend was added to the blend.

7. The active blend and excipients are blended and screened to remove large lumps.

8. To this blend was added the remaining 495 grams of excipient blend.

9. The active blend and excipients are blended and screened to remove large lumps.

Final blending

10. Magnesium stearate was added to the diffusion blender and the contents were mixed.

11. The blend was drained and blended.

Compression

1. The blend is compressed on a rotary tablet press using appropriate tools (punches and dies) to achieve the target weight. Friability and disintegration were checked at the beginning of the compression run and individual tablet weights, thicknesses and hardness were checked periodically.

Packaging arrangement

1. The bulk pharmaceutical tablets were packaged in double-line resealable transparent PE bags with desiccant between the bags, and further packaged in aluminum foil bags with desiccant and heat sealed.

Example 31 exemplary excipients for tablet formulations

The tablet formulation is selected to exhibit mucoadhesive and affinity properties and includes excipients that have solubility enhancing, erosion (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 cervical, anal or vaginal area include, but are not limited to, mannitol, microcrystalline cellulose, lactose, sucrose, calcium phosphate, sodium bicarbonate, citric acid, maleic acid, adipic acid or fumaric acid. Examples of excipients that may enhance disintegration and cover the affected area include, but are not limited to, sodium starch glycolate, pregelatinized starch, crospovidone, and croscarmellose sodium. Mucoadhesive excipients useful in the present invention include, but are not limited to, microcrystalline cellulose, polycarbophil, hydroxymethyl cellulose, hypromellose, hydroxypropyl cellulose, and PVP.

The following table lists combinations of excipients that have desirable characteristics for tablet formulations. Tablet formulations contain the active pharmaceutical ingredient, microcrystalline cellulose, and may contain mannitol. In certain non-limiting embodiments, the tablet formulation comprises one or more excipients selected from the class of fast disintegrants (left column of table 58). In certain non-limiting embodiments, the tablet formulation comprises one or more excipients selected from the class of disintegration enhancers (middle column of table 58). In certain non-limiting embodiments, the tablet formulation comprises one or more excipients selected from the group of mucoadhesive excipient classes (right column of table 58).

TABLE 58 excipient of tablet

(percentages are expressed in weight/wt.%)

Example 32 exemplary excipients for reconstitution of powder or Dry powder formulations

Reconstituting a powder or dry powder formulation may improve the shelf stability of the agent or formulation. In certain non-limiting embodiments, the dry powder formulation may be mixed with saline, propylene glycol, or other aqueous carrier shortly before administration, minimizing degradation time. In certain non-limiting embodiments, the dry powder formulation is mixed with an oil, cream, or other non-aqueous carrier shortly before administration.

In certain embodiments, the reconstituted powder or dry powder formulation rapidly covers the affected or diseased tissue in the cervix, vulva, vagina, perianal area, penis, or anus. Excipients that enhance rapid coverage of the cervix, vulva, vagina, perianal region, penis, or anus include, but are not limited to, mannitol, lactose, sucrose, calcium phosphate, and microcrystalline cellulose. In certain embodiments, the excipient used to rapidly cover the cervix, vulva, vagina, perianal region, penis, or anus is mannitol.

In certain embodiments, the reconstituted powder or dry powder formulation has 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 region, penis, or anus include sodium starch glycolate, pregelatinized starch, crospovidone, and croscarmellose sodium.

In certain embodiments, the reconstituted powder or dry powder formulation, once reconstituted, comprises mucoadhesive properties. This prevents smearing of the dosage form or otherwise exposing healthy tissue to the active pharmaceutical ingredient. Excipients that improve the mucoadhesive properties of the reconstituted powder or dry powder formulation include, but are not limited to, xanthan gum, polycarbophil, polyethylene oxide, hydroxyethyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, PVP, and microcrystalline cellulose. In certain embodiments, the mucoadhesive improving excipient is xanthan gum.

The following table lists combinations of excipients that have desirable characteristics for reconstituted powders or dry powder formulations. Dry powder or reconstituted powder formulations contain the active pharmaceutical ingredient and may contain mannitol and/or xanthan gum. In certain non-limiting embodiments, the dry powder or reconstituted powder formulation comprises one or more excipients selected from the class of quick-acting coating (left column of table 59). In certain non-limiting embodiments, the dry powder or reconstituted powder formulation comprises one or more excipients selected from the coverage enhancing variety (middle column of table 59). In certain non-limiting embodiments, the dry powder or reconstituted powder formulation comprises one or more excipients selected from the group of mucoadhesive excipient classes (right column of table 58).

TABLE 59 excipients for reconstitution of powder or dry powder dosage forms

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(percentages are expressed in weight/wt.%)

Example 33 exemplary excipients for semisolid formulations

The semi-solid formulation is selected to exhibit mucoadhesive properties and to facilitate penetration of the drug into the tissue. Semi-solid formulations may contain excipients that have solubilizing, lipophilic (to aid in dissolving the lipophilic compound), penetration enhancing (higher activity) and mucoadhesive (to maintain the drug at the target site) properties.

In certain embodiments, the semi-solid formulation has mucoadhesive properties. Excipients that aid in mucoadhesive properties include, but are not limited to, carbomers, polyethylene glycols, crospovidone, polycarbophil, hypromellose, and hydroxyethyl cellulose.

In certain embodiments, the semi-solid formulation enhances penetration and/or solubility of the active pharmaceutical ingredient. Excipients that enhance penetration and/or solubility of the active pharmaceutical ingredient include, but are not limited to, polyethylene glycol 6 stearate type I, ethylene glycol stearate, polyethylene glycol 32 stearate type I, and propylene glycol.

The following table lists combinations of excipients that have desirable properties for semi-solid formulations. The semi-solid formulation comprises the active pharmaceutical ingredient and one or more excipients from each column in table 60. In certain non-limiting embodiments, the semi-solid formulation comprises one or more excipients selected from the class of mucoadhesive polymers (left column of table 60). In certain non-limiting embodiments, the tablet formulation comprises one or more excipients selected from the group consisting of solubility and penetration enhancer classes (second column of table 60). In certain non-limiting embodiments, the semi-solid formulation comprises one or more excipients selected from the class of lipophilic solubilizing agents (third column of table 60). In certain non-limiting embodiments, the semi-solid formulation comprises one or more excipients selected from the class of penetration enhancers (right column of table 60).

TABLE 60 excipient for semisolid dosage forms

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(percentages are expressed in weight/wt.%)

Example 34 exemplary excipients for semisolid formulations

The vaginal pessary and film-forming formulation are selected to be solid at room temperature, but to soften at body temperature to release the active pharmaceutical ingredient. This allows for convenient handling and storage of the formulation, as well as achieving the desired tissue coverage at the cervix, vulva, vagina, perianal area, penis or anus. In a non-limiting example of a film-forming formulation, one or more excipients from the left column in table 61 provide the desired characteristics. In a non-limiting example of a vaginal suppository formulation, one or more excipients from the right column in table 61 provide the desired characteristics.

TABLE 61 excipient for films and vaginal suppositories

(percentages are expressed in weight/wt.%)

Example 35 exemplary tablet formulation

In certain non-limiting embodiments, the formulation of the tablet dosage form comprises the ingredients in table 62. In certain non-limiting embodiments, the formulation of the tablet dosage form comprises the ingredients in table 63. An illustrative process for combining these ingredients into a tablet dosage form can be found in example 29.

TABLE 62 example tablet formulations

TABLE 63 example tablet formulations

Example 36 exemplary semisolid formulation

In certain non-limiting embodiments, the formulation for the cream semi-solid dosage form comprises the ingredients in table 64. In certain non-limiting embodiments, the formulation of the gel semi-solid dosage form comprises the ingredients in table 65. An illustrative process for combining these ingredients into a cream or gel semi-solid dosage form can be found in example 27.

Table 64 example semisolid formulation (cream)

Table 65 example semisolid preparation (gel)

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Example 37 exemplary film Forming formulation

Film dosage forms may be prepared by solvent casting or hot melt extrusion. For example, to prepare a film dosage form, the active pharmaceutical ingredient is dissolved into a solution of the excipient and water. The solution is then optionally degassed and cast into a film and dried in an oven. Film dosage forms can also be prepared by hot melt extrusion. In certain 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 is extruded through a die onto a roll where it cools. In certain embodiments, the composition of the film dosage form may be found in table 66.

TABLE 66 examples of film formulations

Example 38 exemplary Dry powder or reconstituted powder formulations

In certain 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 apparatus (e.g., V-blender) and then separated into sterile vials for reconstitution.

TABLE 67 examples of formulations of dry or reconstituted powders

Example 39 exemplary vaginal suppository formulation

In certain non-limiting embodiments, the vaginal suppository formulation comprises the ingredients listed in table 68 or table 69. For example, vaginal suppository formulations may be prepared by mixing the active pharmaceutical ingredient with excipients. In one non-limiting embodiment, the excipient is heated in a mixing device while stirring until it softens or melts, and then the active pharmaceutical ingredient is added in portions. 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 placed into a vaginal suppository or suppository mold for solidification.

Table 68 examples of formulations for vaginal suppositories

Table 69 examples of formulations for vaginal suppositories

Example 40 in vitro cytotoxicity test

A compound:

three compounds (compound I, compound II and compound III) were dissolved in DMSO at 40mM and stored at-20 ℃. Test compounds were evaluated using a high test concentration of 50 μm. Serial semilog 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 40mM and used as a positive control compound at high test concentrations of 100 μm for cytotoxicity assays.

In vitro cytotoxicity assessment：

The cells listed in table 70 were counted by Trypan Blue Dye exclusion and seeded at 100 μl/well in the wells of 96-well flat bottom microtiter plates. The proliferating cells were incubated overnight at 37 ℃/5% CO2 to allow the cells to adhere to the plates at about 70% confluency. 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 cells at six concentrations. Table 70 lists the cell lines; cell type; a source of cell stock; basic tissue culture medium supplemented with 10% fetal bovine serum, 2mM L-glutamine, 100U/mL penicillin, and 100 μg/mL streptomycin; microtiter plate inoculation density.

Table 70:

cytotoxic XTT:

at 37℃at 5% CO ₂ Five days after incubation in the incubator, the tetrazolium dye XTT (2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5- [ (phenylamino) carbonyl]-2H-tetrazolium hydroxide) staining the test plate. XTT-tetrazolium is metabolized by mitochondrial enzymes of metabolically active cells into soluble formazan products. XTT solutions were prepared daily as stock solutions of 1mg/ml in RPMl 1640. A Phenazine Methosulfate (PMS) solution was prepared at 0.15mg/ml in PBS and stored in the dark at-20 ℃. By adding 40 μl PMS/ml XTT solution, XTT/PMS stock was prepared immediately prior to use. 50 microliters of XTT/PMS was added to each well of the plate and the plate was incubated at 37 ℃ for an additional 4 hours. The plates were sealed with an adhesive plate sealant and gently shaken or inverted several times to mix the soluble formazan product and spectrophotometrically performed on plates at 450/650nm using a Molecular Devices Vmax plate readerAnd (5) reading by a method.

Data analysis and evaluation:

microsoft Excel 2010 was used to analyze and map the raw data. CCso (50% reduction in cell viability) values are tabulated and provided. Raw data for cytotoxicity with graphically represented data are provided as printouts summarizing the compound effects on cell viability.

In vitro cytotoxicity assessment:

cytotoxicity of compounds I, II and III against Hs27, heLa, C33A and HEK293 cell proliferation was assessed by measuring cell viability after 5 days of culture using XTI tetrazolium dye (table 71). CC calculated from these assays ₅₀ The values are summarized in the following table.

Tamoxifen citrate was evaluated in parallel as a control compound. CC of tamoxifen citrate in proliferation of C33A, heLa, hs27 and HEK293 cells ₅₀ The values were 17.2, 19.9, 21.2 and 21.3. Mu.M, respectively. When evaluated simultaneously, compound I and its two isoforms are similar in cytotoxicity against each of the four cell lines. CC of three test compounds in C33A cells ₅₀ Values range from about 0.1 to 0.28 μm. CC of three test compounds in HeLa cells ₅₀ Values range from 15.1 to 18.6 μm. CC of three test compounds in Hs27 cells ₅₀ Values range from about 7.62 to 23.2 μm. CC for three test compounds in HEK293 cells ₅₀ The value was about 0.1. Mu.M.

Table 71: in vitro cytotoxicity

EXAMPLE 41 ex vivo penetration and penetration of antiviral drugs in porcine vaginal tissue

Preparation of pig vaginal tissue

Freshly harvested pig vaginal tissue was purchased from a local slaughterhouse and placed in ice boxes. Incision of vaginal tissue to expose mucosa Surface and wash tissue with a gentle stream of PBS pH 7.4. With the aid of telemetry perforation, pig vaginal tissue was cut into circular portions (approximately 2cm ² )。

Fixing pig vaginal tissue to Franz diffusion cell

A circular portion of tissue is sandwiched between two chambers of a Franz diffusion cell with an active diffusion area of 1cm ² And exposing the mucosal layer to the drug delivery lumen. The resistance in porcine vaginal tissue was measured using a waveform generator to ensure the integrity of the tissue segment used for penetration studies. Resistance is not less than 3KΩ cm ² Is used for the study. The receiving chamber was filled with 8ml5%solutol PBS 7.4pH, stirred with a 3mm magnetic stirrer bar at 600rpm, and maintained at 37℃with a circulating water bath.

Loading a formulation in a drug delivery chamber

About 200mg of gel was injected into the dephenolized 1mL syringe and the gel was dispensed into the drug delivery chamber. The gel was applied to the mucosal surface with a pre-weighed applicator. After the gel was loaded and spread onto the surface of the mucosal surface, the weight of the 1mL syringe and applicator was recorded to determine the exact amount of gel loaded into the drug delivery chamber.

Penetration and penetration studies

Time course (2 h, 4h and 8 h) pig vaginal penetration studies were performed. After loading the gel, 500 μl of sample was removed from the receiving chamber at different time intervals, and an equal volume of fresh receiving medium was used to replace the removed sample each time. Samples taken at each time interval were immediately stored at-20 ℃ until analysis. After 2, 4, 8 hours, the formulation was removed from the dosing chamber with the aid of a syringe and cleaned with a cotton swab. The tissue was removed and gently washed 5 times with a wash solution (50% methanol in water) and alternately with a cotton swab.

TABLE 72 time course IVPT study design

Study of	Liquid sampling time point of receiving cavity	During the study period
			2h IVPT	0 and 2h	2h
4h IVPT	0. 2 and 4h	4h
			8h IVPT	0. 2, 4, 6 and 8h	8h

Pig vaginal tissue (active diffusion region) was minced after IVPT

The 8mm perforations of the active diffusion areas in the washed pig vaginal tissue were removed, weighed and transferred into a test tube. The tube was immediately placed in dry ice for about 15min. After a specified time, the tissue is removed and placed in a pre-chilled tray. The tissue was minced into small pieces on a dish with a pre-chilled surgical blade. The minced tissue was transferred to a sample tube and the dish was rinsed with 1ml of 5% solutol in PBS 7.4pH and transferred to the same tissue sample tube. These tubes were stored at-70 ℃ until analysis.

Preparation of receiving chamber liquid for analysis

Samples stored at-20℃were removed and thawed at room temperature for 30min. The drug from the receiving chamber liquid was centrifuged at 13000rpm for 5min, and an equal volume of extraction solvent was added to 200 μl of supernatant. These samples were centrifuged at 13000rpm for 5min and the supernatant was transferred to a vial for analysis.

Extraction of medicine from pig vagina tissue

The minced tissue stored at-70 ℃ was removed and thawed at room temperature for about 90min. The samples were kept shaking in a Bioshaker for 4h at room temperature. After 4h, the sample was centrifuged at 13000rpm for 5min. To 100. Mu.L of the supernatant, 400. Mu.L of the extraction solvent was added, and vortexed for 2min. These samples were centrifuged at 13000rpm for 5min and the supernatant was transferred to a vial for analysis.

All publications and patent applications cited in this specification are herein incorporated 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 purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the invention as defined in the embodiments and/or claims.

Claims

1. A compound having the formula:

2. a compound having the formula:

or a pharmaceutically acceptable salt thereof.

3. A compound having the formula:

or a pharmaceutically acceptable salt thereof.

4. The compound of claim 2, having the formula:

5. a compound according to claim 3 having the formula:

6. an isolated crystalline form of a compound having the formula:

wherein the isolated crystalline form is characterized by a ZRPD pattern comprising at least 5 2θ values selected from the group consisting of: 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. An isolated crystalline form of a compound having the formula:

wherein the isolated crystalline form is characterized by a ZRPD pattern comprising at least 7 2θ values selected from the group consisting of: 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. An isolated crystalline form of a compound having the formula:

wherein the isolated crystalline form is characterized by an XRPD pattern comprising at least 7 2Θ values selected from: 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. An isolated crystalline form of a compound having the formula:

wherein the isolated crystalline form is characterized by an XRPD pattern comprising at least 5 2Θ values selected from: 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 of any one of claims 1-5 and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition of claims 10-11, which is in a solid dosage form.

13. The pharmaceutical composition of claims 10-11, which is a semi-solid dosage form.

14. The pharmaceutical composition according to claims 10-11, which is in the form of a reconstituted powder.

15. The pharmaceutical composition of claims 10-11, in the form of a dry powder dosage form.

16. The pharmaceutical composition of claims 10-11, which is in the form of a film.

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

18. The pharmaceutical composition of claim 12, which is in the form of a tablet.

19. The pharmaceutical composition of claim 13, which is in the form of a cream.

20. The pharmaceutical composition of claim 13, which is in the form of a gel.

21. The pharmaceutical composition of any one of claims 10-20, formulated for topical administration.

22. The pharmaceutical composition of any one of claims 10-21, for delivery to the cervix.

23. The pharmaceutical composition of any one of claims 10-21 for delivery to the vagina.

24. The pharmaceutical composition of any one of claims 10-21, for delivery to the vulva.

25. The pharmaceutical composition of any one of claims 10-21, for delivery to the perianal region.

26. The pharmaceutical composition of any one of claims 10-21, for delivery to the anus.

27. The pharmaceutical composition of 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 of any one of claims 10-30, comprising about 0.01mg to about 10mg of the compound.

32. The pharmaceutical composition of any one of claims 10-30, comprising about 0.01mg to about 5mg of the compound.

33. The pharmaceutical composition of any one of claims 10-30, comprising about 0.03mg to about 1mg of the compound.

34. The pharmaceutical composition of any one of claims 10-30, comprising about 0.03mg to about 0.07mg of the compound.

35. The pharmaceutical composition of any one of claims 10-30, comprising about 0.05mg to about 0.15mg of the compound.

36. The pharmaceutical composition of any one of claims 10-30, comprising about 0.15mg to about 0.45mg of the compound.

37. The pharmaceutical composition of any one of claims 10-30, comprising at least about 0.05mg of the compound.

38. The pharmaceutical composition of any one of claims 10-30, comprising at least about 0.1mg of the compound.

39. The pharmaceutical composition of any one of claims 10-30, comprising about 0.001% to about 10% of the compound.

40. The pharmaceutical composition of any one of claims 10-30, comprising about 0.01% to 0.5% of the compound.

41. The pharmaceutical composition of any one of claims 10-30, comprising about 0.1% to 5% of the compound.

42. The pharmaceutical composition of any one of claims 10-41, comprising a mucoadhesive polymer.

43. The pharmaceutical composition of claim 42, comprising about 5% to about 20% mucoadhesive polymer.

44. The pharmaceutical composition of claim 42, comprising about 10% to about 50% mucoadhesive polymer.

45. The pharmaceutical composition of claim 42, comprising about 50% to about 90% mucoadhesive 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 controlled release of an active compound.

49. The pharmaceutical composition of claim 19, wherein the pharmaceutically acceptable carrier comprises: light mineral oil, propyl parahydroxybenzoate, tefose 63, water, EDTA, methyl parahydroxybenzoate, 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 of treating a human papillomavirus infection comprising administering to a host in need thereof an effective amount of a compound of any one of claims 1-5, optionally in a pharmaceutically acceptable carrier.

53. A method of treating a disorder caused by human papillomavirus infection, the method comprising administering to a host in need thereof an effective amount of a compound of any one of claims 1-5, optionally in a pharmaceutically acceptable carrier.

54. The method of claim 53, wherein the condition caused by human papillomavirus infection is an intraepithelial neoplasia.

55. The method of claim 54, wherein the disorder caused by human papillomavirus is atypical squamous cell of unknown significance (ASC-US).

56. The method of claim 54, wherein the condition caused by human papillomavirus is atypical Adenovirus (AGC).

57. The method of claim 54, wherein the disorder caused by human papillomavirus is low grade squamous intraepithelial lesions (LSIL).

58. The method of claim 54, wherein the disorder caused by human papillomavirus is atypical squamous cells and high-grade squamous intraepithelial lesions (ASC-H) cannot be excluded.

59. The method of claim 54, wherein the disorder caused by human papillomavirus is High Squamous Intraepithelial Lesions (HSIL).

60. The method of claim 54, wherein the condition caused by human papillomavirus is in situ Adenocarcinoma (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 intravaginal intraepithelial neoplasia.

66. The method of claim 54, wherein the intraepithelial neoplasia is intraepithelial neoplasia of the vulva.

67. The method of claim 54, wherein the intraepithelial neoplasia is an intraanal 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 intrapenile intraepithelial neoplasia.

70. The method of any one of claims 52-69, wherein the host is a human.

71. The method of any one of claims 52-70, wherein the compound is administered 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 lubrication to epithelial tissue prior to inserting the dosage form into the affected area.

75. The method of any one of claims 52-73, further comprising applying lubrication to the dosage form prior to inserting the dosage form into the affected area.

76. The method of claim 74 or 75, wherein said lubricating means is selected from the group consisting of water, glycerol-based lubricants, and hydroxyethylcellulose-based lubricants.

77. The method of any one of claims 52-76, wherein the compound is administered daily.

78. The method of any one of claims 52-76, administered about once daily.

79. The method of any one of claims 52-76, administered about twice weekly.

80. The method of any one of claims 52-76, administered about three or more times per week.

81. The method of any one of claims 52-80, administered for about one week.

82. The method of any one of claims 52-80, administered for about two weeks.

83. The method of any one of claims 52-80, administered for about three weeks.

84. The method of any one of claims 52-80, administered for about four weeks.

85. The method of any one of claims 52-80, administered for about five weeks.

86. The method of any one of claims 52-80, administered for about six weeks.

87. The method of any one of claims 52-86, wherein the compound is administered in a treatment cycle comprising:

a. a treatment period comprising administration of the compound, and

b. a withdrawal period comprising a period of no treatment.

88. The method of claim 87, wherein the withdrawal period is about one week.

89. The method of claim 87, wherein the withdrawal period is about two weeks.

90. The method of claim 87, wherein the withdrawal period is about three weeks.

91. The method of claim 87, wherein the withdrawal period is about four weeks.

92. The method of claim 87, wherein the withdrawal period is about five weeks.

93. The method of claim 87, wherein the withdrawal period is about 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 per 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 weekly.

99. The method of any one of claims 52-98, wherein 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, other DNA polymerase inhibitors, inhibitors of E6 or E6AP, inhibitors of E7, inhibitors of E1, inhibitors of E2, inhibitors of E1-E2 protein interactions, inhibitors of L2 lipopeptides, inhibitors of L1, inhibitors of L2, degradants of L1, and degradants of L2.

103. The method of any one of claims 52-100, wherein the compound is administered in combination with an anti-cancer compound.

104. The method of claim 103, wherein said anti-cancer compound is selected from the group consisting of: HDAC inhibitors, degradants of four transmembrane proteins, immune checkpoint inhibitors, T cell therapies, and antiproliferative agents.

105. The method of any one of claims 52-100, wherein the compound is administered in combination with surgery.

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 administration of the compound.

109. The method of any one of claims 105-108, wherein the surgical procedure is excision of diseased tissue.

110. The method of claim 109, wherein the resection is a loop electro-resection (LEEP).

111. The method of claim 109, wherein the excision is a transformation zone macrocyclic excision (LLETZ).

112. The method of claim 109, wherein the resection is a knife and cone resection.

113. The method of claim 109, wherein the ablation is a laser cone ablation.

114. The method of any one of claims 105-108, wherein the surgical procedure is ablation of the diseased tissue.

115. The method of claim 114, wherein the ablating is laser ablating.

116. The method of claim 114, wherein the ablating is cryoablating.

117. Use of a compound according to claims 1-5, optionally in a pharmaceutically acceptable carrier, for the manufacture of a medicament for the treatment of human papillomavirus infection in a host in need thereof.

118. Use of a compound according to claims 1-5, optionally in a pharmaceutically acceptable carrier, for the manufacture of a medicament for the treatment of a disorder caused by human papillomavirus infection in a host in need thereof.

119. The use of claim 118, wherein the condition caused by human papillomavirus infection is intraepithelial neoplasia.

120. The use of claim 119, wherein the intraepithelial neoplasia is a vaginal intraepithelial neoplasia.

121. The use of claim 119, wherein the intraepithelial neoplasia is intraepithelial neoplasia of the vulva.

122. The use of claim 119, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia.

123. The use of claim 119, wherein the intraepithelial neoplasia is an intraanal neoplasia.

124. The use of claim 119, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia.

125. The use of claim 119, wherein the intraepithelial neoplasia is intrapenile intraepithelial neoplasia.

126. The use of claims 117-125, wherein the host is a human.

127. The use of claims 117-126 for topical administration.

128. A compound according to any one of claims 1-5 for use in the treatment of human papillomavirus infection in a host in need thereof, optionally in a pharmaceutically acceptable carrier.

129. A compound according to any one of claims 1-5 for use in the treatment of a disorder caused by human papillomavirus infection in a host in need thereof, optionally in a pharmaceutically acceptable carrier.

130. The compound for use of claim 129, wherein the disorder caused by human papilloma virus infection is intraepithelial neoplasia.

131. The compound for use of claim 130, wherein the intraepithelial neoplasia is intravaginal intraepithelial neoplasia.

132. The compound for use of claim 130, wherein the intraepithelial neoplasia is intraepithelial neoplasia of the vulva.

133. The compound for use of claim 130, wherein the intraepithelial neoplasia is cervical intraepithelial neoplasia.

134. The compound for use of claim 130, wherein the intraepithelial neoplasia is anal intraepithelial neoplasia.

135. The compound for use of claim 130, wherein the intraepithelial neoplasia is perianal intraepithelial neoplasia.

136. The compound for use of claim 130, wherein the intraepithelial neoplasia is intrapenile intraepithelial neoplasia.

137. The compound for use of claims 128-136, wherein the host is a human.

138. The compound for use of claims 128-137, wherein the compound is administered topically.

139. A process for preparing the crystalline form of claim 4, the process comprising:

a. r is R _P Compound I is dissolved in a solvent selected from methanol, ethanol and isopropanol;

b. Stirring at a temperature of about 20 ℃ to about 70 ℃;

c. 1.0 equivalent of fumaric acid was added;

d. adding a solvent selected from pentane, hexane and heptane;

e. cooling the mixture;

f. stirring the cooled solution; and is also provided with

g. The solid was isolated and dried.

140. The method of claim 139, wherein the alcoholic solvent of step (a) is ethanol or isopropanol.

141. The method of claims 139-140, wherein the alcohol solvent of step (a) is isopropyl alcohol.

142. The method of claim 139, wherein the solution of step (b) is stirred at about 45 ℃ to about 55 ℃.

143. The process 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 less than about 20 ℃.

146. The method of claim 139, wherein the mixture is cooled to less than about 10 ℃.

147. The method of claim 139, wherein the mixture is cooled to less than about 5 ℃.

148. The method of claim 139, wherein the mixture is cooled to about 5 ℃ to 0 ℃.

149. The pharmaceutical composition of claim 10 or 11, comprising microcrystalline cellulose.

150. The pharmaceutical composition of claim 10 or 11, comprising 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 starch glycolate.

156. The pharmaceutical composition of claim 10 or 11, comprising pregelatinized starch.

157. The pharmaceutical composition of claim 10 or 11, comprising crospovidone.

158. The pharmaceutical composition of claim 10 or 11, comprising croscarmellose sodium.

159. The pharmaceutical composition of claim 10 or 11, comprising polycarbophil.

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 hydroxyethylcellulose.

163. The pharmaceutical composition of claim 10 or 11, comprising hypromellose.

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, comprising 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, comprising a 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 a polysorbate.

176. The pharmaceutical composition of claim 10 or 11, comprising sodium lauryl sulfate.

177. The pharmaceutical composition of claim 10 or 11, comprising a light mineral oil or mineral oil.

178. The pharmaceutical composition of claim 10 or 11, comprising white wax.

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.