Small molecule antiviral therapy for human papillomavirus infection

Abstract

The present invention provides compositions and methods for treating HPV infections, including pre-malignant and cancerous infections. Compounds that specifically bind to and inactivate the HPV E6 protein are disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 507,895 filed June 13, 2023, the disclosure of which is expressly incorporated herein by reference.

[0002] Federal government-supported research and development This invention was made with government support under CA268137, granted by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Human papillomavirus (HPV) is a very common infection. While most infections are benign, standard destructive treatments are painful and carry potential complications and comorbidities. Furthermore, persistent infections with certain HPV types can develop into invasive and metastatic cancers. These malignancies progress slowly over several years from benign to pre-malignant and invasive lesions, giving ample time for antiviral treatment.

[0004] HPV type 16 (HPV16) is the prototype of "high-risk" HPV for neoplastic transformation, accounting for approximately 50% of all cervical cancers worldwide, and is also present in the majority of precancerous (dysplasia) and cancers of the anus, vulva, and vagina. Globally, 600,000 cases of cervical cancer are diagnosed each year, and an estimated 380,000 women die from them annually. In the United States, approximately 12,000 new cases of cervical cancer caused by HPV infection are diagnosed each year. 44 million cases of cervical cancer are projected to occur worldwide in the next 50 years, a number that would be reduced by only 15% through a robust vaccination program (KTSimms et al Lancet Oncol. 2019;20(3):394-407). A majority of vulvar, vaginal, and penile cancers, as well as anal squamous cell carcinomas, are caused by HPV infection.

[0005] Recently, many oropharyngeal cancers (OPCs) have been identified as being caused by HPV, and this malignancy now exceeds the incidence of cervical cancer in the United States (MMWR Aug 23, 2019, Vol 68 p724). HPV16 can be detected by oral swab, but clinical identification of precursor lesions is unreliable, and surgical approaches carry significant complications within the oropharynx. HPV-associated OPCs develop gradually over more than 10 years and are largely asymptomatic until the patient has an advanced tumor. Destructive surgical treatment, radiation, and chemotherapy are routinely used, with high complication rates and significant financial burdens.

[0006] Existing HPV capsids are highly effective as preventive vaccines, but they are expensive and have not achieved sufficient adoption in the United States to achieve herd immunity. More importantly, this vaccine is not therapeutic for women and men with pre-existing HPV infections, including those who have progressed to pre-malignant or malignant disease. This vaccine does not alter the clinical course after viral infection has been established.

[0007] Studies suggest that the HPV E6 protein is essential for stable viral genome replication and epithelial cell transformation. E6 binds to the ubiquitin ligase E6AP (UBE3A), but does not bind to p53 in the absence of E6AP. E6AP is an early member of the HECT domain ubiquitin ligases, transferring ubiquitin to the tumor suppressor protein p53, leading to its destruction by the proteasome. Subsequently, a conformational change of E6 bound to E6AP exposes a large p53 interaction surface, forming the E6·E6AP·p53 trimer complex. The region of E6AP that complexes with HPV E6 contains an HPV E6 binding motif with the consensus sequence LxxLL, where L is leucine and x is any amino acid folded within an α-helix.

[0008] Trave and his collaborators have elucidated the trimer crystal structure of HPV16 E6 that forms a complex with a peptide containing the LxxLL E6 binding motif and the core domain of p53. The LxxLL motif employs an α-helix structure that docks into a clearly defined large pocket. Substitution of any of the leucines within the helix disrupts the binding to E6. This E6AP binding pocket of E6 functions as a “hotspot” for association with cellular proteins encoding the LxxLL motif. The compounds disclosed herein covalently bind to this pocket, or “hotspot,” of HPV E6, such as HPV16 E6, irreversibly blocking its interaction with E6AP and interfering with other binding partners encoding the HPV E6 binding motif.

[0009] High-risk HPV E7 inhibits the Rb tumor suppressor pathway, leading to sustained stimulation of cell division and induction of the p53 pathway. Both HPV16 E6 and E7 are expressed in HPV-induced tumors. HPV E6 counteracts p53 activation by forming a complex with E6AP and by binding to other cellular partners via the same binding pocket. Studies have shown that inhibiting E6 restores levels and function of wild-type p53 protein (E. Cukuroglu, et al., Prog. Biophys. Mol. Biol. 116, 165-173 (2014)). Inhibition of HPV E6 activity leads to growth arrest or cell death in HPV cervical cancer cell lines.

[0010] There is a great medical need for the treatment of HPV infection. Our strategy was to use structure-based drug design to select drug-like molecules that specifically covalently bind to E6 and disrupt its protein-protein interaction (PPI). The applicant anticipates that administration of agents that block the function of HPV E6, including those applied topically to the cervix, anus, penis, vagina, vagina, or oropharynx, will help effectively eliminate HPV DNA, treat pre-malignant infected tissue, and treat HPV-induced cancer. In one embodiment, an agent is provided that blocks the function of HPV E6 and is applied topically to the cervix, anus, penis, vulva, vagina, or oropharynx to treat pre-malignant infected tissue. In one embodiment, a systemically delivered E6-binding compound is used to treat HPV-induced cancer and pre-malignant HPV infection. [Overview of the project]

[0011] In one embodiment, the present disclosure relates to an HPV E6-binding compound having the following structure of formula I,

[0012] [ka]

[0013] In the formula, Y is either C or N;

[0014] X4 is either N or C;

[0015] W is -(CH2) n- Or -CH=CR 32 -and;

[0016] R 32 is H or -(CH2) n -and,

[0017] n is an integer selected from the range 0 to 4;

[0018] R 31 -CH=CH2, -CR 51 =CH2、 Selected from the group consisting of -CH=CHCH2N(CH3)2, -CH=CHCH3, CH2(halo), and CH3, wherein R 51 is H or halo, and optionally, R 51 is H or F, and optionally, R 31 is -CH=CH2;

[0019] R 33 and R 34 together with the atoms to which they are attached form a ring structure selected from the group consisting of

[0020]

Chemical formula

[0021] X3 is C or N;

[0022] R 38 is selected from the group consisting of H, -OCH3, -OCF3, -OCH2CH3, and -OCH2CH2OCH3;

[0023] R 39 is H, halo, or CH3;

[0024] R<​​​​​​​​​​​​​​​​​​​​​​​​​​is H or C1-C4 alkyl;

[0028] R 42 is H, -CN, C1-C4 alkyl, OH, CO2H, ester, amide, or R 41 and R 42 They form a 4-6 membered ring together with the atoms to which they are bonded (as part of a bridging bicyclic ring); however, R 41 and R 42 If both are H, then R 36 is -OCH2CH3, or R 38 It is anything other than H.

[0029] In one embodiment, an HPV E6-binding compound having the following general structure is provided,

[0030] [ka]

[0031] During the ceremony,

[0032] Y is either C or N;

[0033] W is -(CH2) n and;

[0034] n is an integer selected from the range of 2 to 4;

[0035] R 31 -CH=CH2, -CR 51 Selected from the group consisting of =CH2, -CH=CHCH2N(CH3)2, -CH=CHCH3, CH2(halo), and CH3, in the formula, R 51 is H or halo, and optionally R 51 is H or F, and optionally R 31 -CH=CH2;

[0036] R 37 and R 38 Each is either H or R 37 and R38 Together with the atoms to which they are bonded, they form the following ring structure:

[0037] [ka]

[0038] R 38 is H, halo, or CH3;

[0039] R 35 This is a halo:

[0040] R 36 It is selected from the group consisting of halo, -OCH3, and -OCH2CH3;

[0041] R 41 It is H or C1-C4 alkyl.

[0042] In one embodiment, an HPV E6-binding compound having the following general structure is provided,

[0043] [ka]

[0044] During the ceremony,

[0045] Y and Z are independently C or N;

[0046] X3 is either C or N;

[0047] W is -(CH2) n Or -CH=CH-;

[0048] n is an integer selected from the range 0 to 4;

[0049] R 31 -CH=CH2, -CR 51Selected from the group consisting of =CH2, -CH=CHCH2N(CH3)2, -CH=CHCH3, CH2(halo), and CH3, in the formula, R 51 is H or halo, and optionally R 51 is H or F, and optionally R 31 -CH=CHCH2N(CH3)2;

[0050] R 35 The group consisting of H and halo is selected:

[0051] R 36 This is selected from the group consisting of H, halo, -OCH3, -OCH2CH3, -OCH2CH2OCH3, and CONHCH3;

[0052] R 40 It is selected from the group consisting of H, -OCH3, -OCH2CH3, and -OCH2CH2OCH3;

[0053] R 41 is H or C1-C4 alkyl;

[0054] R 42 is H, -CN, C1-C4 alkyl, OH, CO2H, ester, amide, or R 41 and R 42 They form a 4-6 membered ring together with the atoms to which they are bonded (as part of a bridging bicyclic ring); however, R 41 and R 42 If both are H, then R 36 is -OCH2CH3, or R 38 It is anything other than H.

[0055] According to one embodiment, any of the HPV E6-binding compounds disclosed herein may be used to treat HPV infections, including non-malignant, pre-malignant, and HPV-induced tumors. In one embodiment, the HPV E6-binding compounds disclosed herein are used to inhibit the binding of E6 to the ubiquitin ligase E6AP. In one embodiment, the HPV E6-binding compounds disclosed herein are used to inhibit E6 activity and restore the levels and function of wild-type p53 protein.

[0056] Additional embodiments, features, and advantages in this disclosure will become apparent through the following detailed description and practice of this disclosure. The compounds of this disclosure can be described as embodiments in any of the embodiments listed below. It will be understood that any of the embodiments described herein can be used in conjunction with any other embodiments described herein, to the extent that the embodiments are not inconsistent with each other. [Brief explanation of the drawing]

[0057] [Figure 1A] A and B are total protein mass spectrometry results for MBP (maltose-binding protein) fusion proteins of wild-type (WT) HPV-16 E6 (A) or HPV-16 E6 (C51S, B) with a serine mutation replacing cysteine ​​51, incubated at 4°C for 24 hours in the presence of DMSO (dimethyl sulfoxide). [Figure 1B] Same as above. [Figure 2A] A and B are the total protein mass spectrometry results for MBP (maltose-binding protein) fusion proteins of wild-type (WT) HPV-16 E6 (A) or HPV-16 E6 (C51S, B) with a serine mutation at cysteine ​​51, incubated at 4°C for 24 hours in the presence of 10 μM (top) or 100 μM (bottom) of compound 1. [Figure 2B] Same as above. [Figure 3A]A and B are total protein mass spectrometry results for MBP (maltose-binding protein) fusion proteins of wild-type (WT) HPV-16 E6 (A) or HPV-16 E6 (C51S, B) with a serine mutation replacing cysteine ​​51, incubated at 4°C for 24 hours in the presence of 100 μM of compound 2. [Figure 3B] Same as above. [Figure 4A] A and B are the total protein mass spectrometry results for MBP (maltose-binding protein) fusion proteins of wild-type (WT) HPV-16 E6 (A) or HPV-16 E6 (C51S, bottom) with a serine mutation at cysteine ​​51, incubated at 4°C for 24 hours in the presence of 10 μM (top) or 100 μM (bottom) of compound 3. [Figure 4B] Same as above. [Figure 5A] A and B are total protein mass spectrometry results for MBP (maltose-binding protein) fusion proteins of wild-type (WT) HPV-16 E6(A) or HPV-16 E6(C51S, B) with a serine mutation replacing cysteine ​​51, incubated at 4°C for 24 hours in the presence of 100 μM compound 4. [Figure 5B] Same as above. [Figure 6A] A and B are total protein mass spectrometry results for MBP (maltose-binding protein) fusion proteins of wild-type (WT) HPV-16 E6(A) or HPV-16 E6(C51S, B) with a serine mutation replacing cysteine ​​51, incubated at 4°C for 24 hours in the presence of 100 μM compound 7. [Figure 6B] Same as above. [Figure 7A] A and B are the total protein mass spectrometry results for MBP (maltose-binding protein) fusion proteins of wild-type (WT) HPV-16 E6(A) or HPV-16 E6(C51S, B) with a serine mutation replacing cysteine ​​51, incubated at 4°C for 24 hours in the presence of 100 μM compound 9. [Figure 7B] Same as above. [Figure 8A]A and B are total protein mass spectrometry results for MBP (maltose-binding protein) fusion proteins of wild-type (WT) HPV-16 E6 (A) or HPV-16 E6 (C51S, B) with a serine mutation replacing cysteine ​​51, incubated at 4°C for 24 hours in the presence of 10 μM (A) or 100 μM (B) of compound 15. [Figure 8B] Same as above. [Figure 9] A synthesis scheme for compound 29 is provided. [Figure 10] A synthesis scheme for compound 30 is provided. [Figure 11] A synthesis scheme for compound 31 is provided. [Figure 12] A synthesis scheme for compound 32 is provided. [Figure 13] A synthesis scheme for compound 33 is provided. [Figure 14] A synthesis scheme for compound 34 is provided. [Figure 15] A synthesis scheme for compound 35 is provided. [Figure 16] A synthesis scheme for compound 36 is provided. [Figure 17] A synthesis scheme for compound 37 is provided. [Figure 18] A synthesis scheme for compound 38 is provided. [Figure 19] This provides synthesis schemes for compounds 39-42. [Figure 20] A synthesis scheme for compound 43 is provided. [Figure 21] A synthesis scheme for compounds 44 and 45 is provided. [Figure 22] A synthesis scheme for compound 46 is provided. [Figure 23] A synthesis scheme for compound 47 is provided. [Figure 24A]HPV-negative (RPE-1) and HPV-positive cervical cancer cell lines (SiHa) were incubated for 48 hours with increasing concentrations of 20, 1, or DMSO (D1; 0.1% v / v). Cells were treated with etoposide (ETO, 25 μM) as a positive control for p53 induction. Cells were lysed, proteins were separated by SDS-PAGE, and p53 and GAPDH protein levels were determined using immunoblotting. Band intensity was measured by density measurement and expressed as a magnification change relative to DMSO. * indicates statistical significance of P<0.05. Data are represented by SEM, n≧3. [Figure 24B] HPV-negative (RPE-1) and HPV-positive cervical cancer cell lines (SiHa) were incubated for 48 hours with increasing concentrations of 20, 1, or DMSO (D1; 0.1% v / v). Cells were treated with etoposide (ETO, 25 μM) as a positive control for p53 induction. Cells were lysed, proteins were separated by SDS-PAGE, and p53 and GAPDH protein levels were determined using immunoblotting. Band intensity was measured by density measurement and expressed as a magnification change relative to DMSO. * indicates statistical significance of P<0.05. Data are represented by SEM, n≧3. [Figure 25A] HPV-negative cervical cancer cell lines (RPE-1) were incubated for 48 hours with increasing concentrations of 1, 3, or DMSO (D1; 0.1% v / v). Cells were treated with etoposide (ETO, 25 μM) as a positive control for p53 induction. Cells were lysed, proteins were separated by SDS-PAGE, and p53 and GAPDH protein levels were determined using immunoblotting. Band intensity was measured by density measurement and expressed as a magnification change relative to DMSO. * indicates statistical significance of P<0.05. Data are represented by SEM, n≧3. [Figure 25B]HPV-positive cervical cancer cell lines (SiHa) were incubated for 48 hours with increasing concentrations of 1, 3, or DMSO (D1; 0.1% v / v). Cells were treated with etoposide (ETO, 25 μM) as a positive control for p53 induction. Cells were lysed, proteins were separated by SDS-PAGE, and p53 and GAPDH protein levels were determined using immunoblotting. Band intensity was measured by density measurement and expressed as a magnification change relative to DMSO. * indicates statistical significance of P<0.05. Data are represented by SEM, n≧3. [Figure 26A] HPV-negative (RPE-1) and HPV-positive cervical cancer cell lines (SiHa) were incubated for 24 hours with increasing concentrations of 4, 15, or DMSO (D1, D2; 0.1% v / v). Cells were treated with etoposide (ETO, 25 μM) as a positive control for p53 induction. Cells were lysed, proteins were separated by SDS-PAGE, and p53 and GAPDH protein levels were determined using immunoblotting. Band intensity was measured by density measurement and expressed as a magnification change relative to DMSO. * indicates statistical significance of P<0.05. Data are represented by SEM, n≧3. [Figure 26B] HPV-negative (RPE-1) and HPV-positive cervical cancer cell lines (SiHa) were incubated for 24 hours with increasing concentrations of 4, 15, or DMSO (D1, D2; 0.1% v / v). Cells were treated with etoposide (ETO, 25 μM) as a positive control for p53 induction. Cells were lysed, proteins were separated by SDS-PAGE, and p53 and GAPDH protein levels were determined using immunoblotting. Band intensity was measured by density measurement and expressed as a magnification change relative to DMSO. * indicates statistical significance of P<0.05. Data are represented by SEM, n≧3. [Figure 27A] HPV-positive cervical cancer cell lines (CaSki) were incubated for 24 hours at increasing concentrations of 1, 3, or 15. Viable cells were quantified using the calcein AM cell viability assay. Data are presented as SEM. [Figure 27B]Oral cancer cell line (UM-SCC-47) was incubated for 24 hours at increasing concentrations of 1, 3, or 15. Live cells were quantified using the calcein AM cell viability assay. Data are presented as SEM. [Figure 27C] HPV-positive cervical cancer cell lines (SiHa) were incubated for 24 hours at increasing concentrations of 1, 3, or 15. Viable cells were quantified using the calcein AM cell viability assay. Data are presented as SEM. [Figure 27D] Oral cancer cell line (UM-SCC-104) was incubated for 24 hours at increasing concentrations of 1, 3, or 15. Live cells were quantified using the calcein AM cell viability assay. Data are presented as SEM. [Figure 27E] HPV-negative cells (RPE-1) were incubated for 24 hours at increasing concentrations of 1, 3, or 15. Viable cells were quantified using the calcein AM cell viability assay. Data are presented as SEM results.

[0058] Detailed description of the invention definition In describing and claiming the present invention, the following terms will be used in accordance with the definitions set forth below.

[0059] As used herein, the terms “including,” “containing,” and “comprising” are used in an open, non-restrictive sense.

[0060] As used herein, the term “about” means 10 percent greater than or less than the value or range of values ​​described, but no value or range of values ​​is intended to be limited solely to this broader definition. Each value or range of values ​​preceded by the term “about” is also intended to encompass embodiments of the absolute value or range of values ​​described.

[0061] As used herein, the terms “purified” and similar terms refer to the isolation of a molecule or compound in a form substantially free of contaminants normally associated with the molecule or compound in its natural or natural environment. As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.

[0062] As used herein, the term “to treat” includes the relief of symptoms associated with a particular disorder or condition, and / or the prevention or elimination of such symptoms.

[0063] As used herein, the “effective” dose or “therapeutic dose” of a drug refers to a dose that is non-toxic but sufficient to produce the desired effect. The “effective” dose varies among subjects depending on the individual’s age and general condition, the mode of administration, etc. Therefore, it is not always possible to determine the exact “effective dose.” However, the appropriate “effective dose” in each case can be determined by a person skilled in the art using standard experiments.

[0064] As used herein, the term “patient” unless otherwise specified is intended to encompass any human being, including individuals not under the direct care of a physician.

[0065] The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, assists or enhances the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristics of the compound or composition for its intended use or purpose. For example, a carrier may be selected to minimize any degradation of the active ingredient and minimize any side effects in the subject.

[0066] The term "inhibit" refers to a reduction in activity, response, state, disease, or other biological parameter. This may include, but is not limited to, the complete elimination of such activity, response, state, or disease. It may also include, for example, a 10% reduction in such activity, response, state, or disease compared to natural or control levels. Thus, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, compared to natural or control levels.

[0067] As used herein, the term "alkyl" includes a chain of carbon atoms, which is optionally branched and contains 1 to 20 carbon atoms. In certain embodiments, alkyl is advantageously C1 to C20. 12 , C1~C 10 It will be understood that alkyl groups can be of limited lengths, including C1-C9, C1-C8, C1-C7, C1-C6, and C1-C4. For example, alkyl groups of particularly limited lengths, including C1-C8, C1-C7, C1-C6, and C1-C4 and similar groups, may be referred to as “lower alkyl groups.” Exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, heptyl, and octyl. Alkyl groups may be substituted or unsubstituted. Typical substituents include cycloalkyl, aryl, heteroalicyclic, alkoxy, halo, carbonyl, oxo, (=O), C-carboxy, O-carboxy, nitro, and amino, or those described in the various embodiments provided herein. It will be understood that "alkyl" can be combined with other groups, such as those provided above, to form functionalized alkyl groups. For example, the combination of "alkyl" and "aryl" groups described herein is sometimes called an "alkylaryl" group.

[0068] As used herein, the term “alkenyl” includes a chain of carbon atoms, which is optionally branched and contains 2 to 20 carbon atoms, and also contains at least one carbon-carbon double bond (i.e., C=C). In certain embodiments, the alkenyl is advantageously C2-C 12 It will be understood that the alkenyl groups may be of limited lengths, including C2-C9, C2-C8, C2-C7, C2-C6, and C2-C4. The alkenyl may be unsubstituted or substituted, as described for alkyl groups or as described in the various embodiments provided herein. At least one carbon-carbon double bond may be present internally or at the end. Exemplary alkenyl groups include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl.

[0069] As used herein, the term “alkynyl” includes a chain of carbon atoms, which is optionally branched and contains 2 to 20 carbon atoms, and also contains at least one carbon-carbon triple bond (i.e., C≡C). In certain embodiments, the alkynyl is advantageously a C2-C chain of limited length. 12 It will be understood that the alkynyl groups may be of limited lengths, including C2-C9, C2-C8, C2-C7, C2-C6, and C2-C4. The alkynyl may be unsubstituted or substituted, as described for alkyls or as described in the various embodiments provided herein. At least one carbon-carbon triple bond may be present internally or at the end. Exemplary alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl.

[0070] As used herein, the term "aryl" refers to an all-carbon monocyclic or fused polycyclic group of 6 to 12 carbon atoms having a fully conjugated π-electron system. In certain embodiments, the aryl is advantageously C6-C 10It will be understood that these groups may be of limited size, such as aryl groups. Examples of aryl groups include, but are not limited to, phenyl, naphthyrenyl, and anthracenyl. The aryl group may be unsubstituted or substituted, as described for alkyl groups or as described in the various embodiments provided herein.

[0071] As used herein, the term "cycloalkyl" refers to a 3- to 15-membered all-carbon monocyclic ring, including a 5-membered / 6-membered or 6-membered / 6-membered fused bicyclic ring, or a polycyclic fused ring ("fused" ring system means that each ring in the system shares a pair of adjacent carbon atoms with each other ring in the system), or another group, for example, a carbocyclic ring fused to a heterocyclic ring, such as a 5- or 6-membered cycloalkyl fused to a 5- to 7-membered heterocyclic ring, where one or more of the rings may contain one or more double bonds, but the cycloalkyl does not contain a fully conjugated π-electron system. In certain embodiments, the cycloalkyl is advantageously C3-C 13 It will be understood that these can be limited in size, such as C3-C9, C3-C6, and C4-C6. Cycloalkyls may be unsubstituted or substituted, as described for alkyls or as described in the various embodiments provided herein. Examples of cycloalkyls, but not limited to, include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, and cycloheptyl. Illustrative examples of cycloalkyls shown graphically, in the form of appropriately bonded moieties, include the following substances:

[0072] [ka]

[0073] As used herein, the terms “heterocycloalkyl” or “heterocyclic” define a monocyclic or fused ring group having 3 to 12 ring atoms in a ring(s), where at least one ring atom is a heteroatom such as nitrogen, oxygen, or sulfur, and the remaining ring atoms are carbon atoms. Heterocycloalkyls may optionally contain 1, 2, 3, or 4 heteroatoms. Heterocycloalkyls may also be fused to another group, e.g., another heterocycloalkyl or heteroaryl group. Heterocycloalkyls may also have one or more double bonds, including a double bond to nitrogen (e.g., C=N or N=N), but do not contain a fully conjugated π-electron system. In certain embodiments, it will be understood that heterocycloalkyls are advantageously limited in size, such as 3-8 membered heterocycloalkyls, 5-7 membered heterocycloalkyls, or 3, 4, 5, 6, or 7 membered heterocycloalkyls. Heterocycloalkyls may be unsubstituted or substituted as described for alkyls or as described in the various embodiments provided herein. Examples of heterocycloalkyl groups include, but are not limited to, oxyranil, thianalil, azetidinil, oxetanil, tetrahydrofuranil, pyrrolidinil, tetrahydropyranil, piperidinil, 1,4-dioxanil, morpholinil, 1,4-dithianil, piperazinil, oxepanil, 3,4-dihydro-2H-pyranil, 5,6-dihydro-2H-pyranil, 2H-pyranil, and 1,2,3,4-tetrahydropyridinil. Examples of heterocycloalkyl groups shown graphically, in the form of appropriately bonded moieties, include the following substances:

[0074] [ka]

[0075] As used herein, "halo" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0076] The term "oxo" refers to the carbonyl oxygen. For example, cyclopentyl, when substituted with oxo, is cyclopentanone.

[0077] As used herein, “bond” refers to a covalent bond unless otherwise specified.

[0078] The term "substituted" indicates that the specified group or part supports one or more substituents. The term "unsubstituted" means that the specified group does not support substituents. When the term "substituted" is used to describe a structural system, substitution means that it occurs at any position on the system where any valence is permissible. In some embodiments, the term "substituted" means that the specified group or part supports one, two, or three substituents. In another embodiment, the term "substituted" indicates that the specified group or part supports one or two substituents. In yet another embodiment, the term "substituted" indicates that the specified group or part supports one substituent.

[0079] As used herein, “optional” or “optionally” means that the following events or circumstances may or may not occur, and that such statements include both the occurrence and non-occurrence of the events or circumstances. For example, “C6~C 10 Each hydrogen atom of the aryl group, D "Can be arbitrarily substituted by R" means D However, each R D By substituting the hydrogen atoms of the base, C6~C 10 This means that it can exist at any position on the aryl, but does not need to exist. This explanation is from C6~C 10 aryl is R D This includes situations where the base is not substituted.

[0080] As used herein, “independently” means that the event or situation described thereafter can be read independently of other similar events or situations. For example, in a situation where several equivalent hydrogen groups are optionally substituted with another group described in that situation, the use of “independently and optionally” means that each of the hydrogen atoms on the group may be substituted with another group, and the group substituting each hydrogen atom may be the same or different. Or, for example, if there are multiple groups and all of them may be selected from a set of possibilities, the use of “independently” means that each of the groups may be selected from a set of possibilities separately from any other group, and the groups selected in that situation may be the same or different.

[0081] As used herein, the phrases “together with the atoms to which they are bonded” or “together with the carbon atoms to which they are bonded” or “together formed by combination” mean two substituents (e.g., R 1a and R 1b) Each of these atoms bonds to an additional atom to form a structure defined by the claim, such as a C3-C5 cycloalkyl group. For example, in the context of a compound of formula I, "R 1a and R 1b The expression "they form C3-C8 cycloalkyl groups together with the atoms to which they are bonded" includes, but is not limited to, the following compounds:

[0082] [ka]

[0083] As used herein, the term “pharmaceutically acceptable salt” refers to a salt having a counterion that can be used in pharmaceuticals. See, in general, SMBerge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977, 66, 1-19. A preferred pharmaceutically acceptable salt is one that is pharmacologically effective and suitable for contact with the target tissue without excessive toxicity, irritation, or allergic response. The compounds described herein have a sufficiently acidic group, a sufficiently basic group, both types of functional groups, or two or more of each type, and therefore react with several inorganic or organic bases, as well as inorganic and organic acids, to form pharmaceutically acceptable salts. Examples of such salts include:

[0084] (1) Acid addition salts that can be obtained by reacting the free base of the parent compound with an inorganic acid such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, or with an organic acid such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, or malonic acid; or

[0085] (2) Salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion; or when it coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimhamine, or N-methylglucamine.

[0086] Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutically acceptable salt may be contemplated in connection with the embodiments described herein.

[0087] In the case of the compounds of this disclosure containing basic nitrogen, pharmaceutically acceptable salts can be prepared by any suitable method available in the art, for example, by treatment of a free base with an inorganic acid.

[0088] This disclosure also relates to pharmaceutically acceptable prodrugs of the compounds of the present invention and methods of treatment using such pharmaceutically acceptable prodrugs. The term “prodrug” means a precursor of a given compound that, after administration to a subject, brings the compound in vivo via chemical or physiological processes such as solvolysis or enzymatic cleavage, or under physiological conditions. A “pharmaceutically acceptable prodrug” is a prodrug that is nontoxic to a subject, biologically tolerable, or otherwise biologically appropriate. Exemplary procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs,” ed. H. Bundgaard, Elsevier, 1985.

[0089] Any formula shown herein is also intended to represent both the unlabeled and isotopically labeled forms of the compounds. The isotopically labeled compounds have the structure represented by the formulas given herein, except that one or more atoms are replaced by atoms having a selected atomic mass or mass number. Examples of isotopes that may be incorporated into the compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, for example, respectively. 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, 36 Cl, and 125I is included. Such isotope-labeled compounds are useful in metabolic studies (preferably 14C), reaction kinetic studies (e.g., 2H or 3H), detection or imaging techniques [such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT)] (including tissue distribution assays of drugs or substrates), or in radiotherapy for patients. Furthermore, substitution with heavier isotopes such as deuterium (i.e., 2H) may result in certain therapeutic benefits due to higher metabolic stability, such as increased in vivo half-life or reduced required dose. The isotope-labeled compounds and their prodrugs of this disclosure can generally be prepared by using readily available isotope-labeling reagents in place of non-isotope-labeling reagents, according to the schemes or examples and preparations disclosed below.

[0090] Embodiment This disclosure relates to compositions and methods for treating human papillomavirus (HPV) infection. In one embodiment, a composition comprising an HPV E6-binding compound disclosed herein is formulated for topical application to the cervix, anus, or oropharynx. The compounds disclosed herein have been shown to bind to an amino acid residue in the E6AP-binding pocket of the HPV E6 protein and interfere with the activity of HPV E6, including its ability to interact with E6AP. More specifically, the HPV E6-binding compounds disclosed herein form a covalent bond with a cysteine ​​residue (e.g., Cys51) in the E6AP-binding pocket of the human papillomavirus (HPV) E6 protein, thereby preventing the E6 protein from binding to the E6AP protein. Inhibition of HPV E6 activity has been shown to result in cessation of proliferation of HPV-infected cells and / or cell death of HPV cervical cancer cell lines.

[0091] According to one embodiment, compounds that directly and irreversibly bind to the HPV-16 E6 protein are provided. In one embodiment, these compounds are used in methods relating to the treatment of HPV infections, including HPV E6 infection, such as suppression, prevention, or improvement, using one or more of the HPV E6 binding compounds disclosed herein, or a mixture thereof. In one embodiment, the small molecules described herein interfere with the interaction of E6 with E6AP, thereby restoring the function of p53 in HPV-infected cells. In one embodiment, the disclosure relates to methods for treating HPV infections in subjects requiring HPV treatment (e.g., reduction of HPV E6 levels, reduction of the total number of infectious particles, or reduction of the number of infected cells). In one embodiment, a method for reducing HPV includes administering to a subject one or more of the E6 binding compounds disclosed herein, or a mixture thereof. In another embodiment, the disclosure relates to methods for improving HPV, for example HPV E6, in subjects requiring improvement thereof, the method including administering to the subject any of the E6 binding compounds disclosed herein, or a mixture thereof.

[0092] In one embodiment, the disclosure relates to a method for prophylactic treatment of HPV infection in subjects requiring prophylactic treatment. The method includes administering a compound comprising any or a mixture thereof of the E6-conjugated compounds disclosed herein as prophylactically to a subject.

[0093] Appropriate subjects requiring treatment include subjects who have (or are suspected of having based on symptoms or known exposure) an HPV infection. In one embodiment, a subject known to be exposed to HPV is administered a composition comprising the E6 conjugate compound of the Disclosure, even before the subject shows any symptoms of infection.

[0094] According to Embodiment 1, a compound that specifically binds to HPV E6 is provided, wherein the compound has the following general structure of formula I,

[0095] [ka]

[0096] Wherein, Y is C or N;

[0097] X4 is N or C;

[0098] W is -(CH2) n- or -CH=CR 32 -;

[0099] R 32 is H or -(CH2) n -;

[0100] n is an integer selected from the range of 0 to 4;

[0101] R 31 is selected from the group consisting of -CH=CH2, -CR 51 =CH2, -CH=CHCH2N(CH3)2, -CH=CHCH3, CH2(halo) and CH3, wherein R 51 is H or halo, optionally, R 51 is H or F, optionally, R 31 is -CH=CH2;

[0102] R 33 and R 34 together with the atom to which they are attached form a ring structure selected from the group consisting of:

[0103]

Chemical formula

[0104] X3 is C or N;

[0105] R 38 is selected from the group consisting of H, -OCH3, -OCF3, -OCH2CH3, and -OCH2CH2OCH3;

[0106] R 39is H, halo, or CH3;

[0107] R 35 The group consisting of H and halo is selected:

[0108] R 36 It is selected from the group consisting of H, halo, -OCH3, -OCH2CH3, and CONHCH3;

[0109] R 41 is H or C1-C4 alkyl;

[0110] R 42 is either H, -CN, C1-C4 alkyl, or R 41 and R 42 They form a 4-6 membered ring together with the atoms to which they are bonded; however, R 41 and R 42 If both are H, then R 36 is -OCH2CH3, or R 38 It is anything other than H.

[0111] According to Embodiment 2, an HPV E6-binding compound having the general structure of Formula I is provided, in which

[0112] Y is either C or N;

[0113] X4 is either N or C;

[0114] W is -(CH2) n Or -CH=CH-;

[0115] n is an integer selected from the range of 2 to 4;

[0116] R 31 The group is selected from -CH=CH2, -CH=CHCH2N(CH3)2, -CH=CHCH3, CH2(halo), and CH3, and R is arbitrarily selected. 31 -CH=CH2;

[0117] R33 and R 34 Together with the atoms to which they are bonded, they form a ring structure selected from the following group:

[0118] [ka]

[0119] X3 is either C or N;

[0120] X4 is N;

[0121] R 38 It is selected from the group consisting of H, -OCH3, -OCF3, -OCH2CH3, and -OCH2CH2OCH3;

[0122] R 39 is H, halo, or CH3;

[0123] R 35 The group consisting of H and halo is selected:

[0124] R 36 It is selected from the group consisting of H, halo, -OCH3, -OCH2CH3, and CONHCH3;

[0125] R 41 is H or C1-C4 alkyl;

[0126] R 42 is either H, -CN, C1-C4 alkyl, or R 41 and R 42 They form a 4-6 membered ring together with the atoms to which they are bonded; however, R may be optional. 41 and R 42 If both are H, then R 36 is -OCH2CH3, or R 38 It is anything other than H.

[0127] According to Embodiment 3, an HPV E6-binding compound according to Embodiment 1 or 2 is provided, having the general structure of Formula I, in

[0128] X4 is N;

[0129] W is a bond (i.e., n=0);

[0130] R 31 The group is selected from -CH=CH2, -CH=CHCH2N(CH3)2, -CH=CHCH3, CH2(halo), and CH3, and R is arbitrarily selected. 31 -CH=CH2;

[0131] R 33 and R 34 Together with the atoms to which they are bonded, they form the following ring structure:

[0132] [ka]

[0133] R 38 It is selected from the group consisting of H, -OCH3, -OCH2CH3, and -OCH2CH2OCH3;

[0134] R 39 H is;

[0135] R 35 The group consisting of H and halo is selected:

[0136] R 36 It is selected from the group consisting of H, halo, -OCH3, and -OCH2CH3;

[0137] R 41 is H or C1-C4 alkyl;

[0138] R 42 is H, -CN, or C1-C4 alkyl, but optionally R 41 and R 42If both are H, then R 36 is -OCH2CH3, or R 38 It is anything other than H.

[0139] According to Embodiment 4, an HPV E6-binding compound described in any one of Embodiments 1 to 3 is provided, having the general structure of Formula I, in

[0140] R 41 and R 42 They, together with the atoms to which they are bonded, form a 4-6 membered ring (as part of a bridging bicyclic ring); optionally, R 41 and R 42 These atoms, together with the atoms to which they are bonded, form a four-membered ring.

[0141] According to Embodiment 5, an HPV E6-binding compound according to any one of Embodiments 1 to 4 is provided, having the following general structure of Formula II:

[0142] [ka]

[0143] During the ceremony,

[0144] Y is either C or N;

[0145] W is -(CH2) n and;

[0146] n is 2;

[0147] R 31 The group is selected from -CH=CH2, -CH=CHCH2N(CH3)2, -CH=CHCH3, CH2(halo), and CH3, and R is arbitrarily selected. 31 -CH=CH2;

[0148] R 37 and R 38 Each is either H or R 37 and R 38Together with the atoms to which they are bonded, they form the following ring structure:

[0149] [ka]

[0150] R 38 is H, halo, or CH3, and optionally R 38 H is,

[0151] R 35 This is a halo:

[0152] R 36 It is selected from the group consisting of halo, -OCH3, and -OCH2CH3;

[0153] R 41 It is H or C1-C4 alkyl.

[0154] Embodiment 6 provides an HPV E6-binding compound according to any one of Embodiments 1 to 4 having the following general structure of Formula III:

[0155] [ka]

[0156] During the ceremony,

[0157] Y is either C or N;

[0158] X3 is either C or N;

[0159] W is -(CH2) n Or -CH=CH-;

[0160] n is an integer selected from the range 0 to 4;

[0161] R 31 -CH=CH2, -CR 51Selected from the group consisting of =CH2, -CH=CHCH2N(CH3)2, -CH=CHCH3, CH2(halo), and CH3, in the formula, R 51 is H or halo, and optionally R 51 is H or F, and optionally R 31 -CH=CH2;

[0162] R 35 The group consisting of H and halo is selected:

[0163] R 36 It is selected from the group consisting of H, halo, -OCH3, -OCH2CH3, and CONHCH3;

[0164] R 40 It is selected from the group consisting of H, -OCH3, -OCH2CH3, and -OCH2CH2OCH3;

[0165] R 41 is H or C1-C4 alkyl;

[0166] R 42 is either H, -CN, C1-C4 alkyl, or R 41 and R 42 They form a 4-6 membered ring together with the atoms to which they are bonded (as part of a bridging bicyclic ring); however, R can be optionally 41 and R 42 If both are H, then R 36 is -OCH2CH3, or R 40 It is anything other than H.

[0167] According to Embodiment 7, an HPV E6-binding compound described in any one of Embodiments 1 to 6 is provided, in the formula

[0168] X3 is either C or N;

[0169] W is -(CH2)2 or -CH=CH-;

[0170] R 31is selected from the group consisting of -CH=CH2, -CH=CHCH2N(CH3)2, -CH=CHCH3, CH2(halo), and CH3, and optionally R 31 is -CH=CH2;

[0171] R 38 or R 40 is selected from the group consisting of H, -OCH3, -OCH2CH3, and -OCH2CH2OCH3;

[0172] R 35 is selected from the group consisting of H and halo:

[0173] R 36 is selected from the group consisting of H, halo, -OCH3, and -OCH2CH3;

[0174] R 41 is H;

[0175] R 42 is H, -CN, or C1-C4 alkyl, provided that optionally when R 41 and R 42 are both H, then R 36 is -OCH2CH3 or R 38 is other than H.

[0176] According to Embodiment 8, there is provided an HPV E6 binding compound according to any one of Embodiments 1 to 4, 6 or 7 having the general structure of Formula III, wherein

[0177] ​​​​​​​​​​​​​​​​The group is selected from the group consisting of -OCH3, -OCH2CH3, and -OCH2CH2OCH3.

[0181] Embodiment 10 provides an HPV E6-binding compound according to any one of Embodiments 1 to 4, 6, 7, or 8 having the general structure of Formula III, where X3 is N.

[0182] Embodiment 11 provides an HPV E6-binding compound according to any one of Embodiments 1 to 4, 6, 7, or 8 having the general structure of Formula III, where X3 is C.

[0183] Embodiment 12 provides an HPV E6-binding compound according to any one of Embodiments 1 to 4, 6, 7, 8, 9, 10, or 11 having the general structure of Formula III, in which

[0184] R 35 It is a halo,

[0185] R 36 The group is selected from H, halo, -OCH3, -OCH2CH3, and CONHCH3, and R is optionally selected. 36 These are -OCH3, -OCH2CH3, and R is arbitrary. 36 It is a halo.

[0186] Embodiment 13 provides an HPV E6-binding compound according to any one of Embodiments 1 to 4, 6, 7, 8, 9, 10, 11, or 12 having the general structure of Formula III, wherein

[0187] R 41 H is,

[0188] R 42 It is -CN or C1-C4 alkyl.

[0189] Embodiment 13 provides an HPV E6-binding compound according to any one of Embodiments 1 to 4, 6, 7, 8, 9, 10, 11, or 12 having the general structure of Formula III, wherein R 41 and R42 Together with the atoms to which they are attached, they form a 4- to 5-membered cycloalkyl ring (as part of a bridged bicyclic ring).

[0190] According to Embodiment 14, there is provided an HPV E6 binding compound having the general structure of Formula I below,

[0191]

Chemical formula

[0192] In the formula, Y is C or N;

[0193] X4 is N;

[0194] W is -(CH2)2 or -CH=CH-;

[0195] R 31 is selected from the group consisting of -CH=CH2 or -CH=CHCH3;

[0196] R 33 and R<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ R 36 It is selected from the group consisting of H, halo, -OCH3, and -OCH2CH3;

[0203] R 41 H is;

[0204] R 42 is H, -CN, or C1-C4 alkyl, but optionally R 41 and R 42 If both are H, then R 36 is -OCH2CH3, or R 38 It is anything other than H.

[0205] Embodiment 15 provides the HPV E6-binding compound of Embodiment 14, where W is -(CH2)2.

[0206] Embodiment 16 provides the HPV E6-binding compound of Embodiment 14, where W is -CH=CH-.

[0207] Embodiment 17 provides an HPV E6 conjugate compound described in any one of Embodiments 14 to 16, wherein X3 is C and R 38 The group is selected from the group consisting of -OCH3, -OCH2CH3, and -OCH2CH2OCH3.

[0208] Embodiment 18 provides an HPV E6 conjugated compound described in any one of Embodiments 14 to 16, wherein X3 is N and R 38 The group is selected from the group consisting of -OCH3, -OCH2CH3, and -OCH2CH2OCH3.

[0209] According to Embodiment 19, an HPV E6-binding compound having the following general structure of formula VI is provided, [ka] During the ceremony, X3 is either C or N; R 40is H, halo, or CH3; R 41 is H or halo; R 42 CH3 or [ka] and; R 43 These are CH3, CH2OCH3, or C1-C3 cycloalkyl groups; R 44 and R 45 These are independently H or -(CH2) n is or R 44 and R 45 However, together with the atoms to which they are bonded, they form a bridging bicyclic ring; R 46 This is CH3, CH2NCH3CH3, N-methylpyrrolidine, or CH2R 47 And in the formula, R 47 It is an N-linked morpholine or piperidine ring, n is an integer selected from the range of 2 to 4.

[0210] Example 20 provides the HPV E6-binding compound of Embodiment 21, in which

[0211] X3 is N;

[0212] R 40 H is;

[0213] R 41 is H or F,

[0214] R 42 This is CH3.

[0215] According to Embodiment 21, the following general structure [ka] [ka] [ka] Alternatively, HPV E6-binding compounds having the structure of any one of compounds 29 to 47 are provided.

[0216] Embodiment 22 provides a pharmaceutical composition comprising any of the HPV E6 binding compounds described in Embodiments 1 to 21 and an acceptable carrier, which is optionally formulated for topical application, for example, as a cream.

[0217] Embodiment 23 provides a formulation comprising any of the HPV E6-binding compounds described in Embodiments 1 to 21 and a pharmaceutically acceptable adjuvant, diluent, or carrier.

[0218] Embodiment 24 provides a method for treating HPV infection, the method comprising the step of delivering the pharmaceutical composition described in Embodiment 22 or the formulation described in Embodiment 23 to a patient in need of treatment.

[0219] Embodiment 25 provides a method for treating HPV infection as described in Embodiment 24, wherein the formulation is delivered orally, percutaneously, topically, subcutaneously, intramuscularly, or intravenously.

[0220] Embodiment 26 provides a method for treating HPV infection as described in Embodiment 24 or 25, wherein the formulation is formulated for topical application to the cervix, anus, or oropharynx.

[0221] Embodiment 27 provides a method for treating an HPV infection as described in any one of Embodiments 24 to 26, wherein the formulation comprises about 0.01% to about 10% of the compound of Embodiment 1, which is an effective dose for transdermal delivery.

[0222] Embodiment 28 provides a method for treating HPV infection as described in any one of Embodiments 24 to 27, wherein the formulation is a sustained-release formulation.

[0223] Embodiment 29 provides a method for treating an HPV infection as described in any one of Embodiments 24 to 28, wherein the formulation inhibits E6AP binding to HPV E6 and prevents p53 ubiquitination.

[0224] Embodiment 30 provides a method for treating an HPV infection as described in any one of Embodiments 24 to 29, wherein the formulation further comprises a compound selected from the group consisting of fatty acids, glucose, amino acids, cholesterol, lipids, glycosides, alkaloids, and natural phenols.

[0225] Each of the following compounds represents and is based on one embodiment of the general structure provided above. In each example, a synthetic route yielding a preferred compound is shown. The numbering of the synthetic routes does not reflect the numbering of the compounds below.

[0226] Compound 29 is shown below: [ka]

[0227] Figure 9 shows the synthesis route used to prepare compound 29.

[0228] Compound 30 is shown below: [ka]

[0229] Figure 10 shows the synthesis route from which compound 30 was obtained.

[0230] Compound 31 is shown below: [ka]

[0231] The synthesis route for obtaining compound 31 is shown in Figure 11.

[0232] Compound 32 is shown below: [ka]

[0233] The synthesis pathway leading to compound 32 is shown in Figure 12.

[0234] Compound 33 is shown below: [ka]

[0235] The synthetic route for obtaining compound 33 is shown in Figure 13.

[0236] Compound 34 is shown below: [ka]

[0237] The synthesis route leading to compound 34 is shown in Figure 14.

[0238] Compound 35 is shown below: [ka]

[0239] The synthesis route for obtaining compound 35 is shown in Figure 15.

[0240] Compound 36 is shown below: [ka]

[0241] The synthesis route to obtain compound 36 is shown in Figure 16.

[0242] Compound 37 is shown below: [ka]

[0243] The synthesis route to obtain compound 37 is shown in Figure 17.

[0244] Compound 38 is shown below: [ka]

[0245] The synthetic route for obtaining compound 38 is shown in Figure 18.

[0246] Compounds 39-42 are shown below: [ka]

[0247] Figure 19 shows the synthesis scheme for obtaining compounds 39-42.

[0248] Compound 43 is shown below: [ka]

[0249] The synthesis route to obtain compound 43 is shown in Figure 20.

[0250] Compounds 44 and 45 are shown below: [ka]

[0251] The synthesis scheme for obtaining compounds 44 and 45 is shown in Figure 21.

[0252] Compound 46 is shown below: [ka]

[0253] The synthesis scheme for obtaining compound 46 is shown in Figure 22.

[0254] Compound 47 is shown below: [ka]

[0255] The synthesis scheme for obtaining compound 47 is shown in Figure 23.

[0256] Pharmaceutical composition For therapeutic purposes, a pharmaceutical composition comprising the compounds described herein may further comprise one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient is a substance that is nontoxic to administration to a subject and otherwise biocompatible. Such excipients facilitate the administration of the compounds described herein and are compatible with the active ingredients. Examples of pharmaceutically acceptable excipients include stabilizers, lubricants, surfactants, diluents, antioxidants, binders, colorants, fillers, emulsifiers, or flavor modifiers. In preferred embodiments, the pharmaceutical composition according to the present invention is a sterile composition. The pharmaceutical composition can be prepared using known formulation techniques or those available to those skilled in the art.

[0257] The sterilization compositions also include compositions intended by the present invention that are in accordance with the regulations of the countries and regions governing such compositions.

[0258] The pharmaceutical compositions and compounds described herein may be formulated as solutions, emulsions, suspensions, or dispersants in a suitable pharmaceutical solvent or carrier, or as pills, tablets, lozenges, suppositories, pouches, sugar-coated tablets, granules, powders, reconstituted powders, or capsules with a solid carrier, according to conventional methods known in the art for the preparation of various dosage forms. The pharmaceutical compositions of the present invention may be administered by a suitable delivery route such as oral, parenteral, intravenous, subcutaneous injection, rectal, nasal, topical, or ocular routes, or by inhalation.

[0259] In one embodiment, the composition is formulated for topical administration. For topical use, the compounds of the present invention are preferably formulated as a cream, ointment, lotion, gel, or similar vehicle suitable for topical administration. For topical administration, the compounds of the present invention can be mixed with a pharmaceutical carrier at a drug concentration of about 0.1% to about 10% relative to the vehicle. Another mode of administering the drug of the present invention may utilize a patch formulation to result in transdermal delivery.

[0260] The pharmaceutical compositions of this disclosure can be used to alleviate or prevent the exacerbation of existing HPV disease symptoms, prevent the onset of additional symptoms, alleviate or prevent the underlying systemic cause of symptoms, suppress a disease or disorder (e.g., prevent the onset of HPV infection and / or related symptoms), alleviate a disease or disorder, reverse a disease or disorder, alleviate a condition caused by a disease or disorder, or halt the symptoms of a disease or disorder.

[0261] Exemplary diseases include, but are not limited to, HPV infections of the vagina, cervix, ileum, rectum, anus, penis, vulva, skin, and oropharynx. These are subclinical and can be detected by highly sensitive molecular diagnostic tests. Diseases include histologically benign infected epithelium, pre-malignant and dysplastic lesions, carcinoma in situ, invasive carcinoma, and HPV-induced metastatic carcinoma.

[0262] In one embodiment of the method of the present invention, an effective amount of HPV E6-binding compound disclosed herein is provided to inhibit a target protein. Measuring the modulation of the target can be performed by routine analytical methods, such as those described below. Such modulation is useful in a variety of settings, including in vitro assays. In this method, cells are infected with HPV.

[0263] In one embodiment, the treatment method provides one or more effective amounts of the active compounds disclosed herein that are generally sufficient to produce the desired therapeutic effect in a subject requiring such treatment. The effective amount or dose of the compounds of the present invention may be determined by conventional methods such as modeling, dose escalation, or clinical trials, taking into account conventional factors, such as the mode of administration or route of drug delivery, the pharmacokinetics of the drug, the severity and course of the infection, the health status, condition, and weight of the subject, and the judgment of the treating physician. Exemplary doses are in the range of about 0.1 mg to 1 g per day, or about 1 mg to 50 mg per day, or about 50 to 250 mg per day, or about 250 mg to 1 g per day. The total dose can be given in single or divided dose units (e.g., BID, TID, QID). Exemplary doses for topical administration may be formulations containing 0.01% to 10% of the E6 inhibitor compound.

Claims

1. An HPV-binding compound having the following general structure of formula I, 【Chemistry 1】 In the formula, Y is either C or N; X ４ is N or C; W is -(CH ２ ) ｎ－ or -CH=CR ３２ - and; R ３２ is H or -(CH ２ ) ｎ - and n is an integer selected from the range 0 to 4; R ３１ is -CH=CH ２ , -CR ５１ =CH ２ , -CH=CHCH ２ N(CH ３ ) ２ , -CH=CHCH ３ , CH ２ (halo) and CH ３ and is selected from the group consisting of; wherein R ５１ is H or halo, optionally, R ５１ is H or F, optionally, R ３１ is -CH=CH ２ ; R ３３ and R ３４ Together with the atoms to which they are bonded, they form a ring structure selected from the following group: 【Chemistry 2】 X ３ is C or N; R ３８ H, -OCH ３ , -OCF ３ , -OCH ２ CH ３ , and -OCH ２ CH ２ OCH ３ Selected from the group consisting of; R ３９ This is H, halo, or CH ３ And; R ３５ The group consisting of H and halo is selected: R ３６ H, Hal, -OCH ３ , -OCH ２ CH ３ and CONHCH ３ Selected from the group consisting of; R ４１ is H or C １ -C ４ It is alkyl; R ４２ H, -CN, C １ -C ４ Alkyl or R ４１ and R ４２ They form a 4-6 membered ring together with the atoms to which they are bonded; however, R ４１ and R ４２ If both are H, then R ３６ ha-OCH ２ CH ３ is or R ３８ The compound wherein the element is not H.

2. Y is either C or N: X ４ is N or C; W is -(CH ２ ) ｎ Or -CH=CH-; n is an integer selected from 2 to 4; R ３１ -CH=CH ２ ien-CH=CHCH ２ N(CH ３ ) ２ ien-CH=CHCH ３ ,CH ２ (Halo), and CH ３ Selected from the group consisting of, R ３１ -CH=CH ２ And; R ３３ and R ３４ Together with the atoms to which they are bonded, they form a ring structure selected from the following group: 【Transformation 3】 X ３ is C or N; X ４ is N; R ３８ H, -OCH ３ , -OCF ３ , -OCH ２ CH ３ , and -OCH ２ CH ２ OCH ３ Selected from the group consisting of; R ３９ This is H, halo, or CH ３ And; R ３５ The group consisting of H and halo is selected: R ３６ H, Hal, -OCH ３ , -OCH ２ CH ３ and CONHCH ３ Selected from the group consisting of; R ４１ is H or C １ -C ４ is alkyl; R ４２ is H, -CN, C １ -C ４ alkyl, or R ４１ and R ４２ together with the atom to which they are attached form a 4- to 6-membered ring; provided that optionally when both R ４１ and R ４２ are H, R ３６ is -OCH ２ CH ３ or R ３８ is other than H, the compound according to claim 1.

3. X ４ is N; W is a bond (i.e., n=0); R ３１ -CH=CH ２ ien-CH=CHCH ２ N(CH ３ ) ２ ien-CH=CHCH ３ ,CH ２ (Halo), and CH ３ Selected from the group consisting of, R ３１ -CH=CH ２ And; R ３３ and R ３４ Together with the atoms to which they are bonded, they form the following ring structure: 【Chemistry 4】 R ３８ H, -OCH ３ , -OCH ２ CH ３ , and -OCH ２ CH ２ OCH ３ Selected from the group consisting of; R ３９ H is; R ３５ The group consisting of H and halo is selected: R ３６ H, Hal, -OCH ３ , and -OCH ２ CH ３ Selected from the group consisting of; R ４１ H is; R ４２ is H, -CN, or C １ -C ４ It is alkyl, but optionally R ４１ and R ４２ If both are H, then R ３６ ha-OCH ２ CH ３ is or R ３８ The compound according to claim 1, wherein is other than H.

4. R ４１ and R ４２ Together with the atoms to which they are bonded, they form a 4-6 membered ring, and optionally R ４１ and R ４２ The compound according to claim 1, wherein the atoms to which they are bonded form a four-membered ring.

5. A compound according to claim 1 having the following general structure of formula II, 【Transformation 5】 During the ceremony, Y is either C or N; W is -(CH ２ ) ｎ And; n is 2; R ３１ -CH=CH ２ ien-CH=CHCH ２ N(CH ３ ) ２ ien-CH=CHCH ３ ,CH ２ (Halo), and CH ３ Selected from the group consisting of, R ３１ -CH=CH ２ And; R ３７ and R ３８ These are either H or R, respectively. ３７ and R ３８ Together with the atoms to which they are bonded, they form the following ring structure: 【Transformation 6】 R ３８ This is H, halo, or CH ３ And, arbitrarily, R ３８ H is; R ３５ This is a halo: R ３６ Ha, -OCH ３ , and -OCH ２ CH ３ Selected from the group consisting of; R ４１ is H or C １ -C ４ The compound is alkyl.

6. A compound according to claim 1 having the following general structure of formula III, 【Transformation 7】 During the ceremony, Y is either C or N; X ３ is C or N; W is -(CH ２ ) ｎ Or -CH=CH-; n is an integer selected from the range 0 to 4; R ３１ -CH=CH ２ , -CR ５１ =CH ２ ien-CH=CHCH ２ N(CH ３ ) ２ ien-CH=CHCH ３ ,CH ２ (Halo) and CH ３ Selected from the group consisting of, in the formula, R ５１ is H or halo, and optionally R ５１ is H or F, and optionally R ３１ -CH=CH ２ And; R ３５ The group consisting of H and halo is selected: R ３６ H, Hal, -OCH ３ , -OCH ２ CH ３ and CONHCH ３ Selected from the group consisting of; R ４０ H, -OCH ３ , -OCH ２ CH ３ , and -OCH ２ CH ２ OCH ３ Selected from the group consisting of; R ４１ is H or C １ -C ４ It is alkyl; R ４２ H, -CN, C １ -C ４ Alkyl or R ４１ and R ４２ They form a 4-6 membered ring together with the atoms to which they are bonded; however, R may be optional. ４１ and R ４２ If both are H, then R ３６ ha-OCH ２ CH ３ is or R ４０ The compound wherein the element is not H.

7. A compound according to claim 1 having the following general structure of formula VI, 【Transformation 8】 During the ceremony, X ３ is C or N; R ４０ H, CH ３ It is a Halloween; R ４１ is H or halo; R ４２ CH ３ or 【Chemistry 9】 And; R ４３ CH ３ ,CH ２ OCH ３ , or C １ -C ３ It is a cycloalkyl; R ４４ and R ４５ These are independently H or -(CH ２ ) ｎ is or R ４４ and R ４５ However, together with the atoms to which they are bonded, they form a bridging bicyclic ring; and R ４６ CH ３ ,CH ２ NCH ３ CH ３ , N-methylpyrrolidine, or CH ２ R ４７ And in the formula, R ４７ This is an N-linked morpholine or piperidine ring, The compound, wherein n is an integer selected from the range of 2 to 4.

8. Y is either C or N; X ３ is C or N; W is -(CH ２ ) ２ Or -CH=CH-; R ３１ -CH=CH ２ ien-CH=CHCH ２ N(CH ３ ) ２ ien-CH=CHCH ３ ,CH ２ (Halo), and CH ３ Selected from the group consisting of, R ３１ -CH=CH ２ And; R ３８ or R ４０ H, -OCH ３ , -OCH ２ CH ３ , and -OCH ２ CH ２ OCH ３ Selected from the group consisting of; R ３５ The group consisting of H and halo is selected: R ３６ H, Hal, -OCH ３ , and -OCH ２ CH ３ Selected from the group consisting of; R ４１ H is; R ４２ is H, -CN, or C １ -C ４ It is alkyl, but optionally R ４１ and R ４２ If both are H, then R ３６ ha-OCH ２ CH ３ is or R ３８ The compound according to claim 1, wherein is other than H.

9. X ３ C is, R ４２ is -CN, or C １ -C ４ The compound according to claim 6, wherein it is alkyl.

10. R ４０ is, -OCH ３ , -OCH ２ CH ３ , and -OCH ２ CH ２ OCH ３ A compound according to claim 6, selected from the group consisting of the following.

11. A compound according to any one of claims 1 to 4, 6, 7, 8, or 9, wherein X ３ The compound wherein is N.

12. A compound according to any one of claims 1 to 4, 6, 7, 8, or 9, wherein X ３ The compound is C.

13. A compound according to any one of claims 1 to 4, 6, 8, 9, 10, 11 or 12, wherein the formula is R ３５ It is a halo, R ３６ H, Hal, -OCH ３ , -OCH ２ CH ３ and CONHCH ３ Selected from the group consisting of, and R arbitrarily in the formula ３６ is, -OCH ３ , -OCH ２ CH ３ And in the formula, R can be any ３６ The compound is a halo.

14. A compound according to any one of claims 6, 8, 9, 10, 11, 12, or 13, wherein, R ４１ H is, R ４２ is -CN, or C １ -C ４ The compound is alkyl.

15. R ４１ and R ４２ The compound according to any one of claims 6, 8, 9, 10, 11, 12, or 13, wherein they form a 4- to 5-membered cycloalkyl ring together with the atoms to which they are bonded.

16. A compound according to claim 1 having the following general structure of formula V, 【Chemistry 10】 During the ceremony, Y is either C or N; W is -(CH ２ ) ｎ And; n is 2; R ３１ -CH=CH ２ ien-CH=CHCH ２ N(CH ３ ) ２ ien-CH=CHCH ３ ,CH ２ (Halo), and CH ３ Selected from the group consisting of, R ３１ -CH=CH ２ And; R ３７ and R ３８ These are either H or R, respectively. ３７ and R ３８ Together with the atoms to which they are bonded, they form the following ring structure: 【Chemistry 11】 R ３８ This is H, halo, or CH ３ And, arbitrarily, R ３８ H is, R ３５ This is a halo: R ３６ Ha, -OCH ３ , and -OCH ２ CH ３ The compound selected from the group consisting of the above.

17. A compound having the following general structure of formula I, 【Chemistry 12】 In the formula, Y is either C or N; X ４ is N; W is -(CH ２ ) ２ Or -CH=CH-; R ３１ -CH=CH ２ , or -CH=CHCH ３ Selected from the group consisting of; R ３３ and R ３４ Together with the atoms to which they are bonded, they form the following ring structure: 【Chemistry 13】 X ３ is C or N; R ３８ H, -OCH ３ , -OCH ２ CH ３ , and -OCH ２ CH ２ OCH ３ Selected from the group consisting of; R ３９ H is; R ３５ The group consisting of H and halo is selected: R ３６ H, Hal, -OCH ３ , and -OCH ２ CH ３ Selected from the group consisting of; R ４１ H is; R ４２ is H, -CN, or C １ -C ４ It is alkyl, but optionally R ４１ and R ４２ If both are H, then R ３６ ha-OCH ２ CH ３ is or R ３８ The compound wherein the element is not H.

18. W is -(CH ２ ) ２ The compound according to claim 17.

19. The compound according to claim 17, wherein W is -CH=CH-.

20. The compound according to any one of claims 17 to 19, wherein Y is C or N.

21. X ３ C is R ３８ is, -OCH ３ , -OCH ２ CH ３ , and -OCH ２ CH ２ OCH ３ A compound according to claim 20, selected from the group consisting of the following.

22. X ３ N is R ３８ is, -OCH ３ , -OCH ２ CH ３ , and -OCH ２ CH ２ OCH ３ A compound according to any one of claims 17 to 19, selected from the group consisting of the following.

23. HPV E6-conjugated compounds having the following general structure 【Chemistry 14-1】 【Chemistry 14-2】 【Chemistry 14-3】 【Chemistry 14-4】 【Chemistry 14-5】 【Chemistry 14-6】

24. A pharmaceutical composition comprising an HPV E6 conjugated compound according to any one of claims 1 to 23 and an acceptable carrier, wherein the pharmaceutical composition is formulated for topical application, optionally including, for example, a cream.

25. A formulation comprising an HPV E6 conjugated compound according to any one of claims 1 to 23 and a pharmaceutically acceptable adjuvant, diluent, or carrier.

26. A method for treating an HPV infection, the method comprising the step of administering to a patient in need of treatment the pharmaceutical composition according to claim 24 or the formulation according to claim 25.

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