MDM2 TARGETING PROTACS
20260053931 ยท 2026-02-26
Assignee
- The Wistar Institute Of Anatomy And Biology (Philadelphia, PA)
- Thomas Jefferson University (Philadelphia, PA)
Inventors
Cpc classification
C07D401/12
CHEMISTRY; METALLURGY
C07K5/06026
CHEMISTRY; METALLURGY
A61K31/496
HUMAN NECESSITIES
C07K5/06034
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
A61K31/496
HUMAN NECESSITIES
Abstract
Disclosed herein are compounds of the formulas: (I) as well as analogs thereof, wherein the variables are defined herein. Also provided are pharmaceutical compositions of these compounds. In some aspects, the compounds and compositions provided herein may be used to degrade Mdm2. Also provided are methods of administering compounds and compostions provided herein to a patient in need thereof, for example, for the treatment of cancers.
##STR00001##
Claims
1. A compound of the formula: ##STR00048## wherein: A is a mdm2 inhibitor; wherein the mdm2 inhibitor comprises an imidazole core; B is a ubiquitin ligase ligand; and L is a linker group, wherein the linker group forms a covalent bond between the linker group and the mdm2 inhibitor and a covalent bond between the ubiquitin ligase ligand; wherein the linker group comprises at least one heteroatom selected from O, N, and S; or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, wherein the mdm2 inhibitor is further defined by the formula: ##STR00049## wherein: R.sub.1 and R.sub.1 are each independently a group of the formula: ##STR00050## wherein: X is -alkanediyl.sub.(C8)C(O) or substituted -alkanediyl.sub.(C8)C(O); and x or y are each independently 1, 2, or 3; provided that either R.sub.1 or R.sub.1 is absent when the nitrogen to which it is attached is a part of a double bond; R.sub.2 and R.sub.2 are each independently hydrogen or alkyl.sub.(C8), cycloalkyl.sub.(C8), alkenyl.sub.(C8), alkynyl.sub.(C8), aryl.sub.(C12), aralkyl.sub.(C12), or a substituted version of any of these groups; R.sub.3 and R.sub.3 are each independently alkyl.sub.(C8), cycloalkyl.sub.(C8), alkenyl.sub.(C8), alkynyl.sub.(C8), aryl.sub.(C12), heteroaryl.sub.(C12), aralkyl.sub.(C12), or a substituted version of any of these groups; and R.sub.4 is cycloalkyl.sub.(C8), aryl.sub.(C8), heteroaryl.sub.(C8), heterocycloalkyl.sub.(C8), or a substituted version thereof, wherein the group may be further substituted by one or more alkyl.sub.(C8), alkoxy.sub.(C8), alkylamino.sub.(C8), dialkylamino.sub.(C8), or a substituted version of these groups; or a pharmaceutically acceptable salt thereof.
3. The compound of claim 2, wherein the mdm2 inhibitor is further defined: ##STR00051## wherein: R.sub.1 and R.sub.1 are each independently a group of the formula: ##STR00052## wherein: X is -alkanediyl.sub.(C8)C(O) or substituted -alkanediyl.sub.(C8)C(O); and x or y are each independently 1, 2, or 3; provided that either R.sub.1 or R.sub.1 is absent when the nitrogen to which it is attached is a part of a double bond; R.sub.2 and R.sub.2 are each independently hydrogen or alkyl.sub.(C8), cycloalkyl.sub.(C8), alkenyl.sub.(C8), alkynyl.sub.(C8), aryl.sub.(C12), aralkyl.sub.(C12), or a substituted version of any of these groups; R.sub.3 and R.sub.3 are each independently alkyl.sub.(C8), cycloalkyl.sub.(C8), alkenyl.sub.(C8), alkynyl.sub.(C8), aryl.sub.(C12), heteroaryl.sub.(C12), aralkyl.sub.(C12), or a substituted version of any of these groups; and R.sub.4 is cycloalkyl.sub.(C8), aryl.sub.(C8), heteroaryl.sub.(C8), heterocycloalkyl.sub.(C8), or a substituted version thereof, wherein the group may be further substituted by one or more alkyl.sub.(C8), alkoxy.sub.(C8), alkylamino.sub.(C8), dialkylamino.sub.(C8), or a substituted version of these groups; or a pharmaceutically acceptable salt thereof.
4. The compound of claim 2 further comprising: ##STR00053## wherein: R.sub.2 and R.sub.2 are each independently hydrogen or alkyl.sub.(C8), cycloalkyl.sub.(C8), alkenyl.sub.(C8), alkynyl.sub.(C8), aryl.sub.(C12), aralkyl.sub.(C12), or a substituted version of any of these groups; R.sub.3 and R.sub.3 are each independently alkyl.sub.(C8), cycloalkyl.sub.(C8), alkenyl.sub.(C8), alkynyl.sub.(C8), aryl.sub.(C12), heteroaryl.sub.(C12), aralkyl.sub.(C12), or a substituted version of any of these groups; and R.sub.4 is cycloalkyl.sub.(C8), aryl.sub.(C8), heteroaryl.sub.(C8), heterocycloalkyl.sub.(C8), or a substituted version thereof, wherein the group may be further substituted by one or more alkyl.sub.(C8), alkoxy.sub.(C8), alkylamino.sub.(C8), dialkylamino.sub.(C8), or a substituted version of these groups; or a pharmaceutically acceptable salt thereof.
5. The compound of claim 2, wherein R.sub.1 is ##STR00054## wherein: X is -alkanediyl.sub.(C8)C(O) or substituted -alkanediyl.sub.(C8)C(O); and x or y are each independently 1, 2, or 3.
6. The compound of claim 5, wherein X is -alkanediyl.sub.(C8)C(O).
7-11. (canceled)
12. The compound of claim 2, wherein R.sub.2 and/or R.sub.2 are each independently alkyl.sub.(C8) or substituted alkyl.sub.(C8).
13-17. (canceled)
18. The compound of claim 2, wherein R.sub.3 and/or R.sub.3 are each independently aryl.sub.(C12) or substituted aryl.sub.(C12).
19-25. (canceled)
26. The compound of claim 2, wherein R.sub.4 is aryl.sub.(C8) or substituted aryl.sub.(C8), wherein the group is further substituted with one or more alkyl.sub.(C8), alkoxy.sub.(C8), alkylamino.sub.(C8), dialkylamino.sub.(C8), or a substituted version of these groups.
27-35. (canceled)
36. The compound of claim 2, wherein the mdm2 inhibitor is further defined as: ##STR00055##
37. The compound of claim 1, wherein B is a Cereblon (CRBN) ligand, a Von-Hippel Lindau (VHL) ligand, a mouse double minute 2 homolog (MDM2) ligand, or a cellular inhibitor of apoptosis protein 1 (cIAP1) ligand.
38-46. (canceled)
47. The compound of claim 1, wherein L is a linker group of the formula: ##STR00056## wherein: Y.sub.1 and Y.sub.2 are each independently selected from O, NR.sub.a, C(O), C(O)O, OC(O), C(O)NR.sub.a, NR.sub.aC(O), S(O).sub.a, S(O).sub.aO, OS(O).sub.a, OS(O).sub.aO, wherein: R.sub.a is hydrogen, alkyl(c>6), or substituted alkyl(c>6); and a is 0, 1, or 2; and Z is alkanediyl.sub.(C12), one or more amino acid residues, or a group of the formula: ((Z.sub.1)O).sub.z, wherein: Z.sub.1 is an alkanediyl.sub.(C1-4) or a substituted alkanediyl.sub.(C1-4) and z is 1-10 repeating units; or a combination thereof.
48-53. (canceled)
54. The compound according of claim 47, wherein Z is alkanediyl.sub.(C12) or a substituted alkanediyl.sub.(C12).
55-56. (canceled)
57. The compound of claim 47, wherein Z is ((Z.sub.1)O).sub.z.
58-65. (canceled)
66. The compound of claim 1, wherein the compound is further defined as: ##STR00057## or a pharmaceutically acceptable salt thereof.
67. (canceled)
68. A pharmaceutical composition comprising: (A) a compound of claim 1; and (B) a pharmaceutically acceptable excipient.
69-70. (canceled)
71. A method of treating a disease or disorder in a patient in need thereof comprising administering to the patient in need thereof a therapeutically effective amount of a compound or of claim 1.
72-74. (canceled)
75. The method of claim 71, wherein the disease or disorder is a cancer comprises a mutated p53 gene or a deleted p53 gene.
76-95. (canceled)
96. A method of inhibiting cell replication by modulating mdm2 comprising contacting the cell with a compound of claim 1.
97. A method of modulating the activity of mdm2 in a cell comprising contacting the cell with a compound of claim 1.
98-100. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
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[0084] Proliferation (MTS assay, quadruplicate), B. Annexin-V C. sub-G1 DNA D. measured (B-D, triplicates); *p<0.01.
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[0086] Kaplan-Meier survival curves of the mice in A. Arrow indicates day tamoxifen or oil administration began. C. Evaluation of tumors harvested 48 hours after Tam or vehicle control administration showed loss of Mdm2, increased Annexin V positivity, and fragmented (sub-G1) DNA.
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[0088] MeanSD (B, C). *P<0.0001, comparing PROTAC to control compounds and DMSO vehicle control. For B and C, *P<0.00051, comparing MDM2 shRNA to non-targeting control shRNA. 2-way ANOVA (B, C (left)), 1-way ANOVA (C (right)), and unpaired 2-tailed 1-test.
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[0097] The proteasome inhibitor MG132. H. RG7112. I. VHL-Amine. J. The VHL small molecule inhibitor VH298. K. DMSO vehicle control ().
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[0099] Western blots for MDM2 using multiple antibodies in MDA-MB-231 cells treated with MDM2-PROTAC (6 M, 16 hr). D. qRT-PCR (quadruplicate) for MDM2 expression in MDA-MB-231 and MDA-MB-436 cells treated with MDM2-PROTAC (6 M) or DMSO vehicle control for the indicated times; meanSEM. MCF7 breast cancer cells treated with etoposide (10 M, 24 hr) served as a positive control for MDM2 expression. Data from each cell line was made relative to its own DMSO control; *P<0.0001 (1-way ANOVA), comparing MDM2-PROTAC or etoposide to DMSO vehicle control.
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[0106] Normal breast epithelium from a patient that formed loose aggregates was also treated with MDM2-PROTAC YX-02-030. For comparison, values for TNBC-4 and the normal breast mammospheres are relative to DMSO vehicle control (E, right graph). Representative images shown (RG, RG7112 and RG-D, RG7112D); scale bar 300 m. MeanSD (C-E). For C, *P=0.0477 (unpaired two-tailed i-test), comparing YX-02-030 to DMSO. For D and E, *P<0.0001 (2-way and 1-way ANOVA, respectively), comparing YX-02-030 to control compounds and DMSO vehicle control.
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0110] Provided herein are compounds that may be used to degrade Mdm2 and thus may be used in the treatment of cancers including cancers associated with overexpression of Mdm2 and/or cancers which may be treated by modulating the activity of p53. These compounds may show improved tolerability and stability compared to traditional inhibitors of Mdm2. These and more details will be described below.
I. Compounds and Formulations Thereof
A. Compounds of the Present Disclosure The compounds of the present disclosure are shown, for example, in the summary section above and in Table 1 and the claims below. They may be made using the synthetic methods outlined in the Examples section. These methods can be further modified and optimized using the principles and techniques of organic chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, (2013), which is incorporated by reference herein. In addition, the synthetic methods may be further modified and optimized for preparative, pilot- or large-scale production, either batch or continuous, using the principles and techniques of process chemistry as applied by a person skilled in the art. Such principles and techniques are taught, for example, in Anderson, Practical Process Research & DevelopmentA Guide for Organic Chemists (2012), which is incorporated by reference herein.
TABLE-US-00001 TABLE 1 Compounds of the Present Disclosure Com- pound ID Structure YX-02- 020
[0111] All the compounds of the present disclosure may in some embodiments be used for the prevention and treatment of one or more diseases or disorders discussed herein or otherwise. In some embodiments, one or more of the compounds characterized or exemplified herein as an intermediate, a metabolite, and/or prodrug, may nevertheless also be useful for the prevention and treatment of one or more diseases or disorders. As such unless explicitly stated to the contrary, all the compounds of the present disclosure are deemed active compounds and therapeutic compounds that are contemplated for use as active pharmaceutical ingredients (APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting the public health by assuring the safety, effectiveness, quality, and security of human and veterinary drugs, vaccines and other biological products, and medical devices.
[0112] In some embodiments, the compounds of the present disclosure have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, more metabolically stable than, more lipophilic than, more hydrophilic than, and/or have a better pharmacokinetic profile (e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the indications stated herein or otherwise.
[0113] Compounds of the present disclosure may contain one or more asymmetrically-substituted carbon or nitrogen atom and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a chemical formula are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the present disclosure can have the S or the R configuration. In some embodiments, the present compounds may contain two or more atoms which have a defined stereochemical orientation.
[0114] Chemical formulas used to represent compounds of the present disclosure will typically only show one of possibly several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given compound, and regardless of which one is most prevalent, all tautomers of a given chemical formula are intended.
[0115] In addition, atoms making up the compounds of the present disclosure are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include .sup.13C and .sup.14C.
[0116] In some embodiments, compounds of the present disclosure exist in salt or non-salt form. With regard to the salt form(s), in some embodiments the particular anion or cation forming a part of any salt form of a compound provided herein is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.
B. Pharmaceutical Formulations and Routes of Administration
[0117] In another aspect, for administration to a patient in need of such treatment, pharmaceutical formulations (also referred to as a pharmaceutical preparations, pharmaceutical compositions, pharmaceutical products, medicinal products, medicines, medications, or medicaments) comprise a therapeutically effective amount of a compound disclosed herein formulated with one or more excipients and/or drug carriers appropriate to the indicated route of administration. In some embodiments, the compounds disclosed herein are formulated in a manner amenable for the treatment of human and/or veterinary patients. In some embodiments, formulation comprises admixing or combining one or more of the compounds disclosed herein with one or more of the following excipients: lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol. In some embodiments, e.g., for oral administration, the pharmaceutical formulation may be tableted or encapsulated. In some embodiments, the compounds may be dissolved or slurried in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and/or various buffers. In some embodiments, the pharmaceutical formulations may be subjected to pharmaceutical operations, such as sterilization, and/or may contain drug carriers and/or excipients such as preservatives, stabilizers, wetting agents, emulsifiers, encapsulating agents such as lipids, dendrimers, polymers, proteins such as albumin, nucleic acids, and buffers.
[0118] Pharmaceutical formulations may be administered by a variety of methods, e.g., orally or by injection (e.g. subcutaneous, intravenous, and intraperitoneal). Depending on the route of administration, the compounds disclosed herein may be coated in a material to protect the compound from the action of acids and other natural conditions which may inactivate the compound. To administer the active compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. In some embodiments, the active compound may be administered to a patient in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.
[0119] The compounds disclosed herein may also be administered parenterally, intraperitoneally, intraspinally, or intracerebrally. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
[0120] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
[0121] The compounds disclosed herein can be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compounds and other ingredients may also be enclosed in a hard or soft-shell gelatin capsule, compressed into tablets, or incorporated directly into the patient's diet. For oral therapeutic administration, the compounds disclosed herein may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the therapeutic compound in the compositions and preparations may, of course, be varied. The amount of the therapeutic compound in such pharmaceutical formulations is such that a suitable dosage will be obtained.
[0122] The therapeutic compound may also be administered topically to the skin, eye, ear, or mucosal membranes. Administration of the therapeutic compound topically may include formulations of the compounds as a topical solution, lotion, cream, ointment, gel, foam, transdermal patch, or tincture. When the therapeutic compound is formulated for topical administration, the compound may be combined with one or more agents that increase the permeability of the compound through the tissue to which it is administered. In other embodiments, it is contemplated that the topical administration is administered to the eye. Such administration may be applied to the surface of the cornea, conjunctiva, or sclera. Without wishing to be bound by any theory, it is believed that administration to the surface of the eye allows the therapeutic compound to reach the posterior portion of the eye. Ophthalmic topical administration can be formulated as a solution, suspension, ointment, gel, or emulsion. Finally, topical administration may also include administration to the mucosa membranes such as the inside of the mouth. Such administration can be directly to a particular location within the mucosal membrane such as a tooth, a sore, or an ulcer. Alternatively, if local delivery to the lungs is desired the therapeutic compound may be administered by inhalation in a dry-powder or aerosol formulation.
[0123] In some embodiments, it may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some embodiments, the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of a selected condition in a patient. In some embodiments, active compounds are administered at a therapeutically effective dosage sufficient to treat a condition associated with a condition in a patient. For example, the efficacy of a compound can be evaluated in an animal model system that may be predictive of efficacy in treating the disease in a human or another animal.
[0124] In some embodiments, the effective dose range for the therapeutic compound can be extrapolated from effective doses determined in animal studies for a variety of different animals. In some embodiments, the human equivalent dose (HED) in mg/kg can be calculated in accordance with the following formula (see, e.g., Reagan-Shaw et al., FASEB J., 22(3):659-661, 2008, which is incorporated herein by reference):
Use of the K.sub.m factors in conversion results in HED values based on body surface area (BSA) rather than only on body mass. K.sub.m values for humans and various animals are well known. For example, the K.sub.m for an average 60 kg human (with a BSA of 1.6 m.sup.2) is 37, whereas a 20 kg child (BSA 0.8 m.sup.2) would have a K.sub.m of 25. K.sub.m for some relevant animal models are also well known, including: mice K.sub.m of 3 (given a weight of 0.02 kg and BSA of 0.007); hamster K.sub.m of 5 (given a weight of 0.08 kg and BSA of 0.02); rat K.sub.m of 6 (given a weight of 0.15 kg and BSA of 0.025) and monkey K.sub.m of 12 (given a weight of 3 kg and BSA of 0.24).
[0125] Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are specific to each individual. Nonetheless, a calculated HED dose provides a general guide. Other factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment and the potency, stability and toxicity of the particular therapeutic formulation.
[0126] The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a patient may be determined by physical and physiological factors such as type of animal treated, age, sex, body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. These factors may be determined by a skilled artisan. The practitioner responsible for administration will typically determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual patient. The dosage may be adjusted by the individual physician in the event of any complication.
[0127] In some embodiments, the therapeutically effective amount typically will vary from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100 mg/kg to about 500 mg/kg, from about 1 mg/kg to about 250 mg/kg, from about 10 mg/kg to about 150 mg/kg in one or more dose administrations daily, for one or several days (depending of course of the mode of administration and the factors discussed above). Other suitable dose ranges include 1 mg to 10,000 mg per day, 100 mg to 10,000 mg per day, 500 mg to 10,000 mg per day, and 500 mg to 1,000 mg per day. In some embodiments, the amount is less than 10,000 mg per day with a range of 750 mg to 9,000 mg per day.
[0128] In some embodiments, the amount of the active compound in the pharmaceutical formulation is from about 2 to about 75 weight percent. In some of these embodiments, the amount is from about 25 to about 60 weight percent.
[0129] Single or multiple doses of the agents are contemplated. Desired time intervals for delivery of multiple doses can be determined by one of ordinary skill in the art employing no more than routine experimentation. As an example, patients may be administered two doses daily at approximately 12-hour intervals. In some embodiments, the agent is administered once a day.
[0130] The agent(s) may be administered on a routine schedule. As used herein a routine schedule refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical, or which differ in length, as long as the schedule is predetermined. For instance, the routine schedule may involve administration twice a day, every day, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between. Alternatively, the predetermined routine schedule may involve administration on a twice daily basis for the first week, followed by a daily basis for several months, etc. In other embodiments, the disclosure provides that the agent(s) may be taken orally and that the timing of which is or is not dependent upon food intake. Thus, for example, the agent can be taken every morning and/or every evening, regardless of when the patient has eaten or will eat.
II. Methods of Treatment and Combination Therapies
A. Methods of Treatment
[0131] In particular the compositions that may be used in treating a disease or disorder in a subject (e.g., a human subject) are disclosed herein. The compositions described above are preferably administered to a mammal (e.g., rodent, human, non-human primates, canine, bovine, ovine, equine, feline, etc.) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., slowing, stopping, reducing or eliminating one or more symptoms or underlying causes of disease). Toxicity and therapeutic efficacy of the compositions utilized in methods of the disclosure can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, body weight, age, the particular composition to be administered, time and route of administration, general health, the clinical symptoms and other drugs being administered concurrently. In some embodiments, the amount of the compounds used is calculated to be from about 0.01 mg to about 10,000 mg/day. In some embodiments, the amount is from about 1 mg to about 1,000 mg/day. In some embodiments, the compounds may be administered for 1 day to 20 days. In further embodiments, it is contemplated that the compounds may be administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days, or any range derivable therein. In some embodiments, the compounds may be administered for between 3 and 5 days, inclusive. In some embodiments, the compounds may be administered once. It is also contemplated that in some embodiments, the compounds disclosed herein may be administered two or more times. In some embodiments, these dosings may be reduced or increased based upon the biological factors of a particular patient such as increased or decreased metabolic breakdown of the drug or decreased uptake by the digestive tract if administered orally. Additionally, the compounds may be more efficacious and thus a smaller dose is required to achieve a similar effect. Such a dose is typically administered once a day for a few weeks or until sufficient achieve clinical benefit.
[0132] The therapeutic methods of the disclosure (which include prophylactic treatment) in general include administration of a therapeutically effective amount of the compositions described herein to a subject in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects at risk can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like).
B. Hyperproliferative Diseases
[0133] While hyperproliferative diseases can be associated with any medical disorder that causes a cell to begin to reproduce uncontrollably, the prototypical example is cancer. One of the key elements of cancer is that the normal apoptotic cycle of the cell is interrupted and thus agents that lead to apoptosis of the cell are important therapeutic agents for treating these diseases. As such, the Md2 degraders described in this disclosure may be effective in treating cancers. In particular, the Mdm2 degraders may be used to treat one or more cancers that expresses a mutant p53 gene or that have deleted or truncated the p53 gene. The p53 gene may be mutated in such a way that it results in decreased expression, decreased activity, or is non-functional. Similarly, the p53 gene may also be the wild type gene. In another embodiments, the p53 gene in the cancer may be null. In particular, the present disclosure relates to the treatment of one or more cancers such as breast cancer including triple negative breast cancer (TNBC). The present disclosure also relates to the treatment of lymphoma such as Burkitt lymphoma, Diffuse large B cell lymphoma, and T cell lymphoma. The present disclosure also relates to the treatment of leukemia such as B cell acute lymphoblastic leukemia (B-ALL) or T cell acute lymphoblastic leukemia (T-ALL). The present disclosure also relates to the treatment of lung cancer such as lung adenocarcinoma. The present disclosure also relates to the treatment of ovarian cancer, head and neck cancer, or sarcoma.
[0134] Cancer cells that may be treated with the compounds according to the embodiments include but are not limited to cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In certain aspects, the tumor may comprise an osteosarcoma, angiosarcoma, rhabdosarcoma, leiomyosarcoma, Ewing sarcoma, glioblastoma, neuroblastoma, or leukemia.
C. Combination Therapies
[0135] In addition to being used as a monotherapy, the compounds of the present invention may also find use in combination therapies. Effective combination therapy may be achieved with a single composition or pharmacological formulation that includes both agents, or with two distinct compositions or formulations, administered at the same time, wherein one composition includes a compound of this invention, and the other includes the second agent(s). Alternatively, the therapy may precede or follow the other agent treatment by intervals ranging from minutes to months.
[0136] To treat diseases or disorders using the methods and compositions of the present disclosure, one would generally contact a cell or a subject with a compound and at least one other therapy. These therapies would be provided in a combined amount effective to achieve a reduction in one or more disease parameter. This process may involve contacting the cells/subjects with both agents/therapies at the same time, e.g., using a single composition or pharmacological formulation that includes both agents, or by contacting the cell/subject with two distinct compositions or formulations, at the same time, wherein one composition includes the compound and the other includes the other agent. In some embodiments, the compounds of the present disclosure or any therapies used in conjunction with the compounds of the present disclosure may be administered in a less than therapeutically effective dose when used either alone or in combination.
[0137] Non-limiting examples of such combination therapy include combination of one or more compounds of the invention with another pro-inflammatory agent, an immunosuppressant agent, a chemotherapeutic agent, radiation therapy, an antidepressant, an antipsychotic agent, an anticonvulsant, a mood stabilizer, an anti-infective agent, an antihypertensive agent, a cholesterol-lowering agent or other modulator of blood lipids, an agent for promoting weight loss, an antithrombotic agent, an agent for treating or preventing cardiovascular events such as myocardial infarction or stroke, an antidiabetic agent, an agent for reducing transplant rejection or graft-versus-host disease, an anti-arthritic agent, an analgesic agent, an anti-asthmatic agent or other treatment for respiratory diseases, or an agent for treatment or prevention of skin disorders. Compounds of the invention may be combined with agents designed to improve a patient's immune response to cancer, including (but not limited to) cancer vaccines. See Lu et al. (2011), which is incorporated herein by reference.
[0138] Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, Temazolomide (an aqueous form of DTIC), or any analog or derivative variant of the foregoing. The combination of chemotherapy with biological therapy is known as biochemotherapy. The present invention contemplates any chemotherapeutic agent that may be employed or known in the art for treating or preventing cancers.
[0139] Other factors that cause DNA damage and have been used extensively include what are commonly known as -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
[0140] The terms contacted and exposed, when applied to a cell, are used herein to describe the process by which a therapeutic agent and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
[0141] It is also conceivable that more than one administration of either the compound or the other therapy will be desired. Various combinations may be employed, where a compound of the present disclosure is A, and the other therapy is B, as exemplified below:
TABLE-US-00002 A/B/AB/A/BB/B/AA/A/BB/A/AA/B/BB/B/B/A B/B/A/BA/A/B/BA/B/A/BA/B/B/AB/B/A/AB/A/B/A B/A/A/BB/B/B/AA/A/A/BB/A/A/AA/B/A/AA/A/B/A A/B/B/BB/A/B/BB/B/A/B
Other combinations are also contemplated.
III. Chemical Definitions
[0142] When used in the context of a chemical group: hydrogen means H; hydroxy means OH; oxo means O; carbonyl means C(O); carboxy means C(O)OH (also written as COOH or CO.sub.2H); halo means independently F, Cl, Br or I; amino means NH.sub.2; hydroxyamino means NHOH; nitro means NO.sub.2; imino means NH; cyano means CN; isocyanyl means NCO; azido means N.sub.3; in a monovalent context phosphate means OP(O)(OH).sub.2 or a deprotonated form thereof; in a divalent context phosphate means OP(O)(OH)O or a deprotonated form thereof, mercapto means SH; and thio means S; thiocarbonyl means C(S); sulfonyl means S(O).sub.2; and sulfinyl means S(O).
[0143] In the context of chemical formulas, the symbol means a single bond, means a double bond, and means triple bond. The symbol represents an optional bond, which if present is either single or double. The symbol
represents a single bond or a double bond. Thus, the formula
##STR00026##
covers, for example
##STR00027##
And it is understood that no one such ring atom forms part of more than one double bond. Furthermore, it is noted that the covalent bond symbol , when connecting one or two stereogenic atoms, does not indicate any preferred stereochemistry. Instead, it covers all stereoisomers as well as mixtures thereof. The symbol , when drawn perpendicularly across a bond (e.g.,
##STR00028##
for methyl) indicates a point of attachment of the group. It is noted that the point of attachment is typically only identified in this manner for larger groups in order to assist the reader in unambiguously identifying a point of attachment. The symbol means a single bond where the group attached to the thick end of the wedge is out of the page. The symbol
means a single bond where the group attached to the thick end of the wedge is into the page. The symbol
means a single bond where the geometry
around a double bond (e.g., either E or Z) is undefined. Both options, as well as combinations thereof are therefore intended. Any undefined valency on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is oriented out of the plane of the paper.
[0144] When a variable is depicted as a floating group on a ring system, for example, the group R in the formula:
##STR00029## [0145] then the variable may replace any hydrogen atom attached to any of the ring atoms, including a depicted, implied, or expressly defined hydrogen, so long as a stable structure is formed. When a variable is depicted as a floating group on a fused ring system, as for example the group R in the formula:
##STR00030## [0146] then the variable may replace any hydrogen attached to any of the ring atoms of either of the fused rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g., the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a hydrogen of the formula above that is not shown but understood to be present), expressly defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring atom (e.g., a hydrogen attached to group X, when X equals CH), so long as a stable structure is formed. In the example depicted, R may reside on either the 5-membered or the 6-membered ring of the fused ring system. In the formula above, the subscript letter y immediately following the R enclosed in parentheses, represents a numeric variable. Unless specified otherwise, this variable can be 0, 1, 2, or any integer greater than 2, only limited by the maximum number of replaceable hydrogen atoms of the ring or ring system.
[0147] For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: Cn or C=n defines the exact number (n) of carbon atoms in the group/class. C n defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group/class in question. For example, it is understood that the minimum number of carbon atoms in the groups alkyl.sub.(C8), cycloalkanediyl.sub.(C8), heteroaryl.sub.(C8), and acyl.sub.(C8) is one, the minimum number of carbon atoms in the groups alkenyl.sub.(C8), alkynyl.sub.(C8), and heterocycloalkyl.sub.(C8) is two, the minimum number of carbon atoms in the group cycloalkyl.sub.(C8) is three, and the minimum number of carbon atoms in the groups aryl.sub.(C8) and arenediyl.sub.(C8) is six. Cn-n defines both the minimum (n) and maximum number (n) of carbon atoms in the group. Thus, alkyl.sub.(C2-10) designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms C5 olefin, C5-olefin, olefin.sub.(C=8), and olefin.sub.C5 are all synonymous. Except as noted below, every carbon atom is counted to determine whether the group or compound falls with the specified number of carbon atoms. For example, the group dihexylamino is an example of a dialkylamino.sub.(C=12) group; however, it is not an example of a dialkylamino.sub.(C=6) group. Likewise, phenylethyl is an example of an aralkyl.sub.(C=8) group. When any of the chemical groups or compound classes defined herein is modified by the term substituted, any carbon atom in the moiety replacing the hydrogen atom is not counted. Thus methoxyhexyl, which has a total of seven carbon atoms, is an example of a substituted alkyl.sub.(C1-6). Unless specified otherwise, any chemical group or compound class listed in a claim set without a carbon atom limit has a carbon atom limit of less than or equal to twelve.
[0148] The term saturated when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded. When the term saturated is used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.
[0149] The term aliphatic signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic compound or group. In aliphatic compounds/groups, the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds/groups can be saturated, that is joined by single carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/alkenyl) or with one or more carbon-carbon triple bonds (alkynes/alkynyl).
[0150] The term aromatic signifies that the compound or chemical group so modified has a planar unsaturated ring of atoms with 4n +2 electrons in a fully conjugated cyclic 11 system.
[0151] An aromatic compound or chemical group may be depicted as a single resonance structure; however, depiction of one resonance structure is taken to also refer to any other resonance structure. For example:
##STR00031##
is also taken to refer to
##STR00032##
[0152] Aromatic compounds may also be depicted using a circle to represent the delocalized nature of the electrons in the fully conjugated cyclic a system, two non-limiting examples of which are shown below;
##STR00033##
[0153] The term alkyl refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups CH.sub.3 (Me), CH.sub.2CH.sub.3 (Et), CH.sub.2CH.sub.2CH.sub.3 (n-Pr or propyl), CH(CH.sub.3).sub.2 (i-Pr, .sup.iPr or isopropyl), CH.sub.2CH.sub.2CH.sub.2CH.sub.3 (n-Bu), CH(CH.sub.3)CH.sub.2CH.sub.3 (sec-butyl), CH.sub.2CH(CH.sub.3).sub.2 (isobutyl), C(CH.sub.3).sub.3 (tert-butyl, t-butyl, t-Bu or .sup.tBu), and CH.sub.2C(CH.sub.3).sub.3 (neo-pentyl) are non-limiting examples of alkyl groups. The term alkanediyl refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups CH.sub.2 (methylene), CH.sub.2CH.sub.2, CH.sub.2C(CH.sub.3).sub.2CH.sub.2, and CH.sub.2CH.sub.2CH.sub.2 are non-limiting examples of alkanediyl groups. The term alkylidene refers to the divalent group CRR in which R and R are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include: CH.sub.2, CH(CH.sub.2CH.sub.3), and C(CH.sub.3).sub.2. An alkane refers to the class of compounds having the formula H-R, wherein R is alkyl as this term is defined above.
[0154] The term cycloalkyl refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, said carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: CH(CH.sub.2).sub.2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to a carbon atom of the non-aromatic ring structure. The term cycloalkanediyl refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group
##STR00034##
is a non-limiting example of cycloalkanediyl group. A cycloalkane refers to the class of compounds having the formula H-R, wherein R is cycloalkyl as this term is defined above.
[0155] The term alkenyl refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: CHCH.sub.2 (vinyl), CHCHCH.sub.3, CHCHCH.sub.2CH.sub.3, CH.sub.2CHCH.sub.2 (allyl), CH.sub.2CHCHCH.sub.3, and CHCHCHCH.sub.2. The term alkenediyl refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups CHCH, CHC(CH.sub.3)CH.sub.2, CHCHCH.sub.2, and CH.sub.2CHCHCH.sub.2 are non-limiting examples of alkenediyl groups. It is noted that while the alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. The terms alkene and olefin are synonymous and refer to the class of compounds having the formula H-R, wherein R is alkenyl as this term is defined above. Similarly, the terms terminal alkene and -olefin are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.
[0156] The term alkynyl refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups CCH, CCCH.sub.3, and CH.sub.2CCCH.sub.3 are non-limiting examples of alkynyl groups. The term alkynediyl refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon triple bond, no carbon-carbon double bonds, and no atoms other than carbon and hydrogen. The groups CC, CCCH.sub.2, and CH.sub.2CCCH.sub.2 are non-limiting examples of alkynediyl groups. It is noted that while the alkynediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure. An alkyne refers to the class of compounds having the formula H-R, wherein R is alkynyl.
[0157] The term aryl refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said carbon atom forming part of a one or more aromatic ring structures, each with six ring atoms that are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. As used herein, the term aryl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, C.sub.6H.sub.4CH.sub.2CH.sub.3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl (e.g., 4-phenylphenyl). The term arenediyl refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, said carbon atoms forming part of one or more six-membered aromatic ring structures, each with six ring atoms that are all carbon, and wherein the divalent group consists of no atoms other than carbon and hydrogen. As used herein, the term arenediyl does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings are connected with a covalent bond. Non-limiting examples of arenediyl groups include:
##STR00035##
An arene refers to the class of compounds having the formula H-R, wherein R is aryl as that term is defined above. Benzene and toluene are non-limiting examples of arenes.
[0158] The term aralkyl refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.
[0159] The term heteroaryl refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings are fused; however, the term heteroaryl does not preclude the presence of one or more alkyl or aryl groups (carbon number limitation permitting) attached to one or more ring atoms. Non-limiting examples of heteroaryl groups include benzoxazolyl, benzimidazolyl, furanyl, imidazolyl (Im), indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, oxadiazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term N-heteroaryl refers to a heteroaryl group with a nitrogen atom as the point of attachment. A heteroarene refers to the class of compounds having the formula H-R, wherein R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes.
[0160] The term heterocycloalkyl refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures, each with three to eight ring atoms, wherein at least one of the ring atoms of the non-aromatic ring structure(s) is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings are fused. As used herein, the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to one or more ring atoms. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term N-heterocycloalkyl refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group.
[0161] The term acyl refers to the group C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, or aryl as those terms are defined above. The groups, CHO, C(O)CH.sub.3 (acetyl, Ac), C(O)CH.sub.2CH.sub.3, C(O)CH(CH.sub.3).sub.2, C(O)CH(CH.sub.2).sub.2, C(O)C.sub.6H.sub.5, and C(O)C.sub.6H.sub.4CH.sub.3 are non-limiting examples of acyl groups. A thioacyl is defined in an analogous manner, except that the oxygen atom of the group C(O)R has been replaced with a sulfur atom, C(S)R. The term aldehyde corresponds to an alkyl group, as defined above, attached to a CHO group.
[0162] The term alkoxy refers to the group OR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: OCH.sub.3 (methoxy), OCH.sub.2CH.sub.3 (ethoxy), OCH.sub.2CH.sub.2CH.sub.3, OCH(CH.sub.3).sub.2 (isopropoxy), or OC(CH.sub.3).sub.3 (tert-butoxy). The terms cycloalkoxy, alkenyloxy, alkynyloxy, aryloxy, aralkoxy, heteroaryloxy, heterocycloalkoxy, and acyloxy, when used without the substituted modifier, refers to groups, defined as OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term alkylthio and acylthio refers to the group SR, in which R is an alkyl and acyl, respectively. The term alcohol corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group. The term ether corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group.
[0163] The term alkylamino refers to the group NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples include: NHCH.sub.3 and NHCH.sub.2CH.sub.3. The term dialkylamino refers to the group NRR, in which R and R can be the same or different alkyl groups. Non-limiting examples of dialkylamino groups include: N(CH.sub.3).sub.2 and N(CH.sub.3)(CH.sub.2CH.sub.3). The terms cycloalkylamino, alkenylamino, alkynylamino, arylamino, aralkylamino, heteroarylamino, heterocycloalkylamino, and alkoxyamino when used without the substituted modifier, refers to groups, defined as NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkoxy, respectively. A non-limiting example of an arylamino group is NHC.sub.6H.sub.5. The terms dicycloalkylamino, dialkenylamino, dialkynylamino, diarylamino, diaralkylamino, diheteroarylamino, diheterocycloalkylamino, and dialkoxyamino, refers to groups, defined as NRR, in which R and R are both cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and alkoxy, respectively. Similarly, the term alkyl(cycloalkyl)amino refers to a group defined as NRR, in which R is alkyl and R is cycloalkyl. The term amido (acylamino), when used without the substituted modifier, refers to the group NHR, in which R is acyl, as that term is defined above. A non-limiting example of an amido group is NHC(O)CH.sub.3.
[0164] An amine protecting group or amino protecting group is well understood in the art. An amine protecting group is a group which prevents the reactivity of the amine group during a reaction which modifies some other portion of the molecule and can be easily removed to generate the desired amine. Amine protecting groups can be found at least in Greene and Wuts, 1999, which is incorporated herein by reference. Some non-limiting examples of amino protecting groups include formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; alkoxy- or aryloxycarbonyl groups (which form urethanes with the protected amine) such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxy-benzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, ,-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl (Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Additionally, the amine protecting group can be a divalent protecting group such that both hydrogen atoms on a primary amine are replaced with a single protecting group. In such a situation the amine protecting group can be phthalimide (phth) or a substituted derivative thereof wherein the term substituted is as defined above. In some embodiments, the halogenated phthalimide derivative may be tetrachlorophthalimide (TCphth). When used herein, a protected amino group, is a group of the formula PG.sub.MANH or PG.sub.DAN wherein PG.sub.MA is a monovalent amine protecting group, which may also be described as a monvalently protected amino group and PG.sub.DA is a divalent amine protecting group as described above, which may also be described as a divalently protected amino group.
[0165] When a chemical group is used with the substituted modifier, one or more hydrogen atom has been replaced, independently at each instance, by OH, F, Cl, Br, I, NH.sub.2, NO.sub.2, CO.sub.2H, CO.sub.2CH.sub.3, CO.sub.2CH.sub.2CH.sub.3, CN, SH, OCH.sub.3, OCH.sub.2CH.sub.3, C(O)CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3, N(CH.sub.3).sub.2, C(O)NH.sub.2, C(O)NHCH.sub.3, C(O)N(CH.sub.3).sub.2, OC(O)CH.sub.3, NHC(O)CH.sub.3, S(O).sub.2OH, or S(O).sub.2NH.sub.2. For example, the following groups are non-limiting examples of substituted alkyl groups: CH.sub.2OH, CH.sub.2Cl, CF.sub.3, CH.sub.2CN, CH.sub.2C(O)OH, CH.sub.2C(O)OCH.sub.3, CH.sub.2C(O)NH.sub.2, CH.sub.2C(O)CH.sub.3, CH.sub.2OCH.sub.3, CH.sub.2OC(O)CH.sub.3, CH.sub.2NH.sub.2, CH.sub.2N(CH.sub.3).sub.2, and CH.sub.2CH.sub.2C1. The term haloalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. F, Cl, Br, or I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, CH.sub.2Cl is a non-limiting example of a haloalkyl. The term fluoroalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups CH.sub.2F, CF.sub.3, and CH.sub.2CF.sub.3 are non-limiting examples of fluoroalkyl groups. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl. The groups, C(O)CH.sub.2CF.sub.3, C02H (carboxyl), CO.sub.2CH.sub.3 (methylcarboxyl), CO.sub.2CH.sub.2CH.sub.3, C(O)NH.sub.2 (carbamoyl), and CON(CH.sub.3).sub.2, are non-limiting examples of substituted acyl groups. The groups NHC(O)OCH.sub.3 and NHC(O)NHCH.sub.3 are non-limiting examples of substituted amido groups.
[0166] As used herein, the term functional group refers to any chemical group or substituent covalently bound to a core structure. Functional groups may include but are not limited to hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, OH, F, Cl, Br, I, NH.sub.2, NO.sub.2, CO.sub.2H, CO.sub.2CH.sub.3, CO.sub.2CH.sub.2CH.sub.3, CN, SH, OCH.sub.3, OCH.sub.2CH.sub.3, C(O)CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3, N(CH.sub.3).sub.2, C(O)NH.sub.2, C(O)NHCH.sub.3, C(O)N(CH.sub.3).sub.2, OC(O)CH.sub.3, NHC(O)CH.sub.3, S(O).sub.2OH, S(O).sub.2NH.sub.2 or a combination or substituted version of any of these groups.
[0167] The use of the word a or an, when used in conjunction with the term comprising in the claims and/or the specification may mean one, but it is also consistent with the meaning of one or more, at least one, and one or more than one.
[0168] Throughout this application, the term about is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects or patients.
[0169] An active ingredient (AI) or active pharmaceutical ingredient (API) (also referred to as an active compound, active substance, active agent, pharmaceutical agent, agent, biologically active molecule, or a therapeutic compound) is the ingredient in a pharmaceutical drug that is biologically active.
[0170] The terms comprise, have and include are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as comprises, comprising, has, having, includes and including, are also open-ended. For example, any method that comprises, has or includes one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.
[0171] The term effective, as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. Effective amount, Therapeutically effective amount or pharmaceutically effective amount when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to effect such treatment or prevention of the disease.
[0172] An excipient is a pharmaceutically acceptable substance formulated along with the active ingredient(s) of a medication, pharmaceutical composition, formulation, or drug delivery system. Excipients may be used, for example, to stabilize the composition, to bulk up the composition (thus often referred to as bulking agents, fillers, or diluents when used for this purpose), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.
[0173] Excipients include pharmaceutically acceptable versions of antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. The main excipient that serves as a medium for conveying the active ingredient is usually called the vehicle. Excipients may also be used in the manufacturing process, for example, to aid in the handling of the active substance, such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The suitability of an excipient will typically vary depending on the route of administration, the dosage form, the active ingredient, as well as other factors.
[0174] As used herein, the term IC.sub.50 refers to an inhibitory dose which is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half.
[0175] An isomer of a first compound is a separate compound in which each molecule contains the same constituent atoms as the first compound, but where the configuration of those atoms in three dimensions differs.
[0176] As used herein, the term patient or subject refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants and fetuses.
[0177] As generally used herein pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
[0178] Pharmaceutically acceptable salts means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
[0179] A pharmaceutically acceptable carrier, drug carrier, or simply carrier is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and/or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and/or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to the specific target sites. Examples of carriers include: liposomes, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.
[0180] A pharmaceutical drug (also referred to as a pharmaceutical, pharmaceutical preparation, pharmaceutical composition, pharmaceutical formulation, pharmaceutical product, medicinal product, medicine, medication, medicament, or simply a drug, agent, or preparation) is a composition used to diagnose, cure, treat, or prevent disease, which comprises an active pharmaceutical ingredient (API) (defined above) and optionally contains one or more inactive ingredients, which are also referred to as excipients (defined above).
[0181] Prevention or preventing includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
[0182] A stereoisomer or optical isomer is an isomer of a given compound in which the same atoms are bonded to the same other atoms, but where the configuration of those atoms in three dimensions differs. Enantiomers are stereoisomers of a given compound that are mirror images of each other, like left and right hands. Diastereomers are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also referred to as a stereocenter or stereogenic center, which is any point, though not necessarily an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a stereoisomer. In organic compounds, the chiral center is typically a carbon, phosphorus or sulfur atom, though it is also possible for other atoms to be stereocenters in organic and inorganic compounds. A molecule can have multiple stereocenters, giving it many stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), the total number of hypothetically possible stereoisomers will not exceed 2.sup.n, where n is the number of tetrahedral stereocenters. Molecules with symmetry frequently have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched so that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and/or diastereomers can be resolved or separated using techniques known in the art. It is contemplated that that for any stereocenter or axis of chirality for which stereochemistry has not been defined, that stereocenter or axis of chirality can be present in its R form, S form, or as a mixture of the R and S forms, including racemic and non-racemic mixtures. As used herein, the phrase substantially free from other stereoisomers means that the composition contains <15%, more preferably <10%, even more preferably <5%, or most preferably <1% of another stereoisomer(s).
[0183] Treatment or treating includes (1) inhibiting a disease in a subject or patient experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting further development of the pathology and/or symptomatology), (2) ameliorating a disease in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any measurable decrease in a disease or symptom thereof in a subject or patient that is experiencing or displaying the pathology or symptomatology of the disease.
[0184] The term ubiquitin ligase ligand or E3 ligase ligand refers to a chemical group capable of binding ubiquitin ligase. Ubiquitin ligase (also called an E3 ubiquitin ligase) is a protein that recruits an E2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin, recognizes a protein substrate, and assists or directly catalyzes the transfer of ubiquitin from the E2 to the protein substrate. The ubiquitin is attached to a lysine on the target protein by an isopeptide bond. E3 ligases interact with both the target protein and the E2 enzyme, and so impart substrate specificity to the E2. Commonly, E3s polyubiquitinate their substrate with Lys48-linked chains of ubiquitin, targeting the substrate for destruction by the proteasome.
[0185] However, one of skill in the art recognizes that many other types of linkages are possible and that each may alter a protein's activity, interactions, or localization. Ubiquitination by E3 ligases regulates diverse areas such as cell trafficking, DNA repair, and signaling and is of profound importance in cell biology. E3 ligases are also key players in cell cycle control, mediating the degradation of cyclins, as well as cyclin dependent kinase inhibitor proteins. The human genome encodes over 600 putative E3 ligases, allowing for tremendous diversity in substrates. Non-limiting examples of ubiquitin ligase ligands include the von Hippel-Lindau (VHL) ligand, a cIAP1 ligand, a MDM2 ligand, a CRBN ligand, a CUL2 ligand, or other ligand which binds to one or more of the proteins of the ubiquitin protein complex especially the E3 component of this complex.
[0186] Ubiquitin ligase (also called an E3 ubiquitin ligase or E3 ligase) is a protein that recruits an E2 ubiquitin-conjugating enzyme that has been loaded with ubiquitin, recognizes a protein substrate, and assists or directly catalyzes the transfer of ubiquitin from the E2 to the protein substrate. The ubiquitin is attached to a lysine on the target protein by an isopeptide bond. E3 ligases interact with both the target protein and the E2 enzyme, and so impart substrate specificity to the E2. Commonly, E3s polyubiquitinate their substrate with Lys48-linked chains of ubiquitin, targeting the substrate for destruction by the proteasome. However, one of skill in the art recognizes that many other types of linkages are possible and that each may alter a protein's activity, interactions, or localization. Ubiquitination by E3 ligases regulates diverse areas such as cell trafficking, DNA repair, and signaling and is of profound importance in cell biology. E3 ligases are also key players in cell cycle control, mediating the degradation of cyclins, as well as cyclin dependent kinase inhibitor proteins. The human genome encodes over 600 putative E3 ligases, allowing for tremendous diversity in substrates. Non-limiting examples of ubiquitin ligase ligands include the von Hippel-Lindau (VHL) ligand, a cIAP1 ligand, a MDM2 ligand, a CRBN ligand, a CUL2 ligand, or other ligand which binds to one or more of the proteins of the ubiquitin protein complex especially the E3 component of this complex.
[0187] The term unit dose refers to a formulation of the compound or composition such that the formulation is prepared in a manner sufficient to provide a single therapeutically effective dose of the active ingredient to a patient in a single administration. Such unit dose formulations that may be used include but are not limited to a single tablet, capsule, or other oral formulations, or a single vial with a syringeable liquid or other injectable formulations.
[0188] The above definitions supersede any conflicting definition in any reference that is incorporated by reference herein. The fact that certain terms are defined, however, should not be considered as indicative that any term that is undefined is indefinite. Rather, all terms used are believed to describe the disclosure in terms such that one of ordinary skill can appreciate the scope and practice the present disclosure.
I. Examples
[0189] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Example 1Synthetic Methodology and Characterization Data
A. Synthesis of MDM2 PROTACs
##STR00036##
[0190] N-((2S,3R)-3-amino-2,3-bis(4-chlorophenyl)butan-2-yl)-4-(tert-butyl)-2-ethoxybenzamide (YX-02-014): 4-(tert-butyl)-2-ethoxy-benzoyl acid (666 mg, 3.0 mmol) was dissolved in toluene and treated with oxalyl chloride (320 L). After it was stirred at 50 C. for 1 h, the reaction solvent was removed and used directly for next step. A mixture of the obtained product and triethylamine (823 L, 6.0 mmol) in DCM (15 mL) was reacted with 2,3-bis-(4-chlorophenyl)-2,3-butanediamine (840 mg, 3.0 mmol) for 30 min at room temperature. The mixture was partitioned between 10% sodium bicarbonate solution and dichloromethane. The organic phase was separated and washed with water, dried over anhydrous sodium sulfate, concentrated and purified by chromatography on silica gel to afford YX-02-014 (600 mg, 43% yield).
##STR00037##
[0191] (4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole (YX-02-018): N-((2S,3R)-3-amino-2,3-bis(4-chlorophenyl)butan-2-yl)-4-(tert-butyl)-2-ethoxybenzamide (YX-02-014) (300 mg, 0.58 mmol) was dissolved in Tol (15 mL) and treated with TsOH (20 mg, 0.116 mmol) and heat to reflux for 4 h, then remove the solvent, purified by chromatography on silica gel to afford YX-02-018 (220 mg, 73% yield).
##STR00038##
[0192] (4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl chloride (YX-02-019): To a solution of (4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole (YX-02-018) (220 mg, 0.44 mmol) and triethylamine (300p L, 2.2 mmol) in DCM (15 mL) at 0 C. was added triphosgene (600 mg, 2.0 mmol). After stirred for 30 min the mixture was taken up and washed with water, dried over anhydrous sodium sulfate, concentrated and purified by chromatography on silica gel to afford YX-02-019 (200 mg, 81% yield).
##STR00039##
[0193] Tert-butyl-2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetate (YX-02-020): To a solution of (4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl chloride (YX-02-019) (120 mg, 0.214 mmol) and tert-butyl 2-(piperazin-1-yl) acetate (80 mg, 0.428 mmol) in DCM (10 mL) at 0 C. was added triethylamine (240 L, 2 mmol). The reaction was allowed to react for 24 hour. The mixture was partitioned between 10% sodium bicarbonate solution and dichloromethane. The organic phase was separated and washed with water, dried over anhydrous sodium sulfate, concentrated, and purified by chromatography on silica gel to afford YX-02-020 (150 mg, 99% yield).
##STR00040##
[0194] 2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetic acid (YX-02-023): Tert-butyl-2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetate (YX-02-020) (150 mg, 0.2 mmol) was dissolved in DCM (5 mL) and treated with TFA (1 mL), after stirred for 18 hour, remove the reaction solvent and use directly for next step.
##STR00041##
[0195] (2S,4R)-1-((S)-2-(tert-butyl)-17-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)-4,16-dioxo-6,9,12-trioxa-3,15-diazaheptadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YX-02-030): A mixture of VHL-Amine (31 mg, 0.05 mmol) and 2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetic acid (YX-02-020) (36 mg, 0.05 mmol) in DMF (3 mL) was treated with DIPEA (16 L, 0.1 mmol) and then HATU (20 mg, 0.05 mmol) and stirred at ambient temperature for 1 hour. After which time the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The organic layer was dried by Na.sub.2SO.sub.4 and concentrated in vacuum. The resulting material was purified by chromatography on silica to afford YX-02-030 (28 mg, 50% yield). .sup.1H NMR (400 MHz, DMSO) 8.98 (s, 1H), 8.59 (s, 1H), 7.58 (d, J=8.2 Hz, 2H), 7.48-7.32 (m, 5H), 7.05 (dd, J=24.6, 15.3 Hz, 9H), 5.15 (s, 1H), 4.56 (d, J=9.5 Hz, 1H), 4.48-4.38 (m, 2H), 4.35 (s, 2H), 4.25 (d, J=11.5 Hz, 2H), 3.96 (s, 3H), 3.67 (d, J=7.6 Hz, 1H), 3.59 (s, 3H), 3.56 (s, 2H), 3.51 (d, J=3.7 Hz, 2H), 3.49 (s, 2H), 3.36 (d, J=6.1 Hz, 2H), 3.17 (d, J=6.2 Hz, 3H), 2.60 (s, 2H), 2.44 (s, 4H), 2.21 (s, 1H), 2.04 (d, J=8.6 Hz, 1H), 1.88 (s, 4H), 1.59 (s, 4H), 1.38 (d, J=16.7 Hz, 9H), 1.28 (dd, J=15.2, 8.1 Hz, 8H), 0.94 (s, 9H), 0.90-0.77 (m, 5H). LC-MS RT=1.81, [(M+2H)/2]=634.
##STR00042##
[0196] 2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)-N-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butyl)acetamide (YX-02-041): A mixture of 2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetic acid (YX-02-023) (36 mmg, 0.05 mmol) and Celebron-amine (20 mg, 0.05 mmol) in DMF (3 mL) was treated with DIPEA (16 L, 0.1 mmol) and then HATU (20 mg, 0.05 mmol) and stirred at ambient temperature for 24 hour. After which time the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The organic layer was dried by Na.sub.2SO.sub.4 and concentrated in vacuum. The resulting material was purified by chromatography on silica to afford YX-02-041 (12 mg, 23% yield). .sup.1H NMR (400 MHz, DMSO) 11.11 (s, 1H), 7.94 (d, J=5.7 Hz, 1H), 7.80 (t, J=7.9 Hz, 1H), 7.58 (d, J=7.7 Hz, 2H), 7.49 (d, J=7.3 Hz, 1H), 7.38 (d, J=8.4 Hz, 1H), 7.05 (dd, J=24.7, 15.7 Hz, 9H), 5.12 (dd, J=13.0, 5.4 Hz, 1H), 4.75 (s, 2H), 4.23 (s, 1H), 3.95 (s, 1H), 3.13 (s, 3H), 3.02 (s, 2H), 2.98-2.80 (m, 4H), 2.65-2.54 (m, 3H), 2.21 (s, 1H), 2.03 (d, J=11.7 Hz, 1H), 1.88 (s, 3H), 1.59 (s, 3H), 1.29 (t, J=6.7 Hz, 4H). LC-MS RT=2.17, [(M+2H)/2]=526.
##STR00043##
[0197] N,N-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-(4-((4R,5S)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetamide) (YX-02-052): A mixture of 2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetic acid (36 mmg, 0.05 mmol) and 2,2-(ethane-1,2-diylbis(oxy))diethanamine (YX-02-023) (YX-02-023 (3.7 L, 0.025 mmol) in DMF (3 mL) was treated with DIPEA (16 L, 0.1 mmol) and then HATU (20 mg, 0.05 mmol) and stirred at ambient temperature for 24 hour. After which time the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The organic layer was dried by Na.sub.2SO.sub.4 and concentrated in vacuum. The resulting material was purified by chromatography on silica to afford YX-02-052 (30 mg, 84% yield). .sup.1H NMR (400 MHz, DMSO) 7.55 (dd, J=15.1, 7.1 Hz, 4H), 7.03 (d, J=31.7 Hz, 18H), 4.22 (s, 2H), 4.01-3.86 (m, 2H), 3.44 (s, 5H), 3.36 (t, J=5.8 Hz, 5H), 3.18 (d, J=5.1 Hz, 6H), 3.00-2.83 (m, 7H), 2.60 (s, 4H), 2.44 (d, J=10.1 Hz, 2H), 2.22 (d, J=9.4 Hz, 2H), 1.88 (s, 6H), 1.58 (s, 6H), 1.35 (s, 18H), 1.28 (t, J=6.8 Hz, 7H). LC-MS RT=2.09, [(M+2H)/2]=724.
##STR00044##
[0198] 2-(4-((4R,5S)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)-N-(13-(4-((4R,5S)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)-12-oxo-2,5,8-trioxa-11-azatridecyl)acetamide (YX-02-062): A mixture of 2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetic acid (YX-02-023) (36 mmg, 0.05 mmol) and 2,2-(2,2-oxybis(ethane-2,1-diyl) bis(oxy))diethanamine (4.8 mg, 0.025 mmol) in DMF (3 mL) was treated with DIPEA (16 L, 0.1 mmol) and then HATU (20 mg, 0.05 mmol) and stirred at ambient temperature for 24 hour. After which time the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The organic layer was dried by Na.sub.2SO.sub.4 and concentrated in vacuum. The resulting material was purified by chromatography on silica to afford YX-02-062 (35 mg, 99% yield). .sup.1H NMR (400 MHz, DMSO) 7.95 (s, 1H), 7.57 (t, J=7.5 Hz, 4H), 7.13-6.92 (m, 18H), 4.22 (s, 2H), 4.00-3.89 (m, 2H), 3.46 (s, 7H), 3.37 (t, J=5.7 Hz, 4H), 3.18 (d, J=5.8 Hz, 6H), 2.92 (d, J=26.8 Hz, 9H), 2.73 (s, 3H), 2.69 (s, 2H), 2.58 (d, J=16.5 Hz, 4H), 2.44 (d, J=9.3 Hz, 2H), 2.22 (s, 2H), 1.88 (s, 6H), 1.59 (s, 6H), 1.36 (s, 17H), 1.29 (t, J=6.8 Hz, 7H). LC-MS RT=2.00, [(M+2H)/2]=745.
##STR00045##
[0199] 1-(2-(2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (YX-02-230): A mixture of 2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetic acid (YX-02-023) (36 mmg, 0.05 mmol) and VHL-Ligand (25 mmg, 0.05 mmol) in DMF (3 mL) was treated with DIPEA (16 L, 0.1 mmol) and then HATU (20 mg, 0.05 mmol) and stirred at ambient temperature for 24 hour. After which time the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The organic layer was dried by Na.sub.2SO.sub.4 and concentrated in vacuum. The resulting material was purified by chromatography on silica to afford YX-02-230 (30 mg, 56% yield). .sup.1H NMR (400 MHz, DMSO) 8.99 (s, 1H), 8.56 (d, J=5.2 Hz, 1H), 7.57 (dd, J=12.0, 6.6 Hz, 2H), 7.48-7.33 (m, 4H), 7.03 (d, J=32.2 Hz, 9H), 5.11 (s, 1H), 4.48-4.36 (m, 2H), 4.33 (s, 2H), 4.31-4.15 (m, 2H), 3.95 (s, 1H), 3.60 (dd, J=30.0, 9.4 Hz, 2H), 3.16 (s, 1H), 2.90 (s, 3H), 2.78 (d, J=15.4 Hz, 1H), 2.65 (d, J=15.8 Hz, 1H), 2.44 (d, J=2.6 Hz, 4H), 2.30 (d, J=31.8 Hz, 2H), 2.10-1.97 (m, 1H), 1.97-1.81 (m, 4H), 1.59 (d, J=7.2 Hz, 3H), 1.35 (s, 8H), 1.28 (dd, J=16.0, 9.3 Hz, 4H), 1.02-0.73 (m, 11H). LC-MS RT=1.85, ES+ve 1079.
##STR00046##
[0200] N-(3-aminopropyl)-2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetamide (YX-02-219): A mixture of 2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetic acid (60 mg, 0.083 mmol) (YX-02-023) and tert-butyl 3-aminopropylcarbamate (20 mg, 0.12 mmol) in DMF (4 mL) was treated with DIPEA (30 L, 0.17 mmol) and then HATU (32 mg, 0.083 mmol) and stirred at ambient temperature for 24 hour. After which time the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The organic layer was dried by Na.sub.2SO.sub.4 and concentrated in vacuum. The resulting material was purified by chromatography on silica to afford the coupling product YX-02-219 (55 mg, 80% yield). Then treat the product with Dioxane/HCl solution (4.0 M, 0.1 mL) and stir for 2 hours, remove the solvent and use directly for next step.
##STR00047##
[0201] (R)-7-(2-((3-(2-(4-((4R,5S)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetamido)propyl)amino)-2-oxoethoxy)-2-((R)-3,3-dimethyl-2-((R)-2-(methylamino)propanamido)butanoyl)-N-((S)-1,2,3,4-tetrahydronaphthalen-1-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (YX-02-228): A mixture of N-(3-aminopropyl)-2-(4-((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazole-1-carbonyl)piperazin-1-yl)acetamide (YX-02-219) (18 mg, 0.025 mmol) and IAP-Ligand (20 mg, 0.025 mmol) in DMF (3 mL) was treated with DIPEA (20 L, 0.05 mmol) and then HATU (12 mg, 0.025 mmol) and stirred at ambient temperature for 24 hour. After which time the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The organic layer was dried by Na.sub.2SO.sub.4 and concentrated in vacuum. The resulting material was purified by chromatography on silica and then treated with Dioxane/HCl solution (4.0 M, 0.1 mL) for 2 hour, the reaction solvent was removed and purified by reverse-HPLC to afford YX-02-228 (10 mg, 31% yield). LC-MS RT=1.76, [(M+2H)/2]=642.
Example 2Analysis and Activity of Mdm2 Degrader
A. Mdm2 is Required for p53-Null T-Cell Lymphoma and Sarcoma Survival.
[0202] Previous work has shown that Mdm2 deletion inhibits growth and survival of both p53.sup./ T-cell lymphoma and sarcoma by inducing apoptosis (Feeley et al., 2017). CreER, a 4-Hydroxytamoxifen (4-OHT) inducible form of Cre.sup.61 was expressed (retrovirus) in the lymphoma and sarcoma cells derived from Mdm2.sup.fl/flp53.sup./ mice (Grier et al., 2002; Donehower et al., 1992). Loss of Mdm2 protein occurred with 4-OHT addition (
B. Mdm2 Deletion Induces Apoptosis of p53.sup./ T-Cell Lymphoma and Sarcoma In Vivo.
[0203] To test whether Mdm2 deletion would impact p53-null tumor growth and survival in vivo, CreER expressing Mdm2.sup.fl/flp53.sup./ T-cell lymphoma cells were injected subcutaneously into nude mice. Following detection of palpable tumors (day 15), tamoxifen (Tam) or corn oil (Oil) vehicle control was administered to all mice to activate CreER. Lymphomas in the vehicle control group continued to increase in volume, whereas the lymphomas in the mice given Tam to delete Mdm2 regressed and were undetectable at day 21 when the first control treated mouse was sacrificed (
[0204] Analogous results were obtained in vivo with the sarcoma cells (Feeley et al., 2017). Evaluation of tumors harvested 48 hours after Tam or vehicle control administration showed loss of Mdm2, increased Annexin V positivity, and fragmented (sub-G1) DNA (
C. Mdm2 Deletion Activates p53/p73 Transcriptional Target Genes which Induces Apoptosis in Murine p53-Null Cancer Cells.
[0205] RNA-sequencing was performed following Mdm2 deletion and compared this to vehicle control in the p53-null murine cancers, and this showed that Mdm2 deletion upregulated pro-apoptotic and cell cycle arrest genes known to be transcriptionally regulated by the p53 family (Feeley et al., 2017). p73, a p53 family member, can bind Mdm2 and is capable of mediating apoptosis and cell cycle arrest by transactivating p53 target genes (Ongkeko et al., 1999; Zeng et al., 1999; Wu & Leng, 2015). It was investigated whether p73 was involved in transcriptionally activating the p53 regulated genes and mediating the apoptosis observed. Mdm2 deletion increased p73 protein, and knockdown of p73 prevented the upregulation of the apoptotic target genes and the apoptosis induced by loss of Mdm2 in murine cancer cells lacking p53 (Feeley et al., 2017).
D. Mdm2 Knockdown in Human Cancer Cells with Mutated p53 Activates p73 and Induces Apoptosis
[0206] To determine whether the results in p53-null murine cancers translate to human cancers and in particular p53 mutant cancers, Mdm2 were knocked down with shRNAs in breast cancer cells with mutated p53. Mdm2 knockdown (
E. Development of Assays to Support Medicinal Chemistry Lead Optimization Efforts
i. Mdm2 Homogeneous Time-Resolved Fluorescence (HTRF) Binding Assays
[0207] An HTRF binding assay was developed using a biotinylated peptide containing the Mdm2-binding domain of p53 and a recombinant GST-tagged N-terminus of Mdm2 containing the p53-binding domain. HTRF is a proximity-based fluorescence energy transfer (FRET) assay that takes advantage of the unique time-resolved fluorescence of the lanthanide chelates, such as terbium and europium. HTRF IC.sub.50 values are provided for all key compounds (
ii. Mdm2 Surface Plasmon Resonance (SPR) Binding Assays.
[0208] SPR assays were developed to measure the direct label free binding to Mdm2. Assay optimization involved protein coupling to the sensor chip through non-specific amine coupling and directed coupling using a His tagged protein. The best results were obtained using the direct coupling method resulting in approximately 10,000 RU of His-SUMO-Mdm2 bound to the sensor chip. Control Mdm2-p53 inhibitors RG7112 (Kd=9.7 nM;
iii. HTRF Binding Assays for Cereblon, VHL, and cCbl.
[0209] Biochemical assays in 384-well format have established to assess the binding of newly synthesized PROTACs to the appropriate E3 ligase using HTRF technology. In these assays an anti-HIS antibody conjugated to terbium was used as the donor fluor, and either a Cy5 labeled small molecule probe, or a biotinylated peptide that is bound to a streptavidin acceptor. See TABLE 2.
TABLE-US-00003 TABLE 2 HTRF Binding Data for PROTACs Mdm2 VHL CRBN HTRF Binding Binding Ligand ID IC.sub.50 nM IC.sub.50 nM IC.sub.50 nM series AC-3-049 23 >10 uM Idasanutlin YX-2-228 13 >10 uM >10 uM RG7112 YX 2-230 18
>10 uM RG7112 YX-2-30 14
>10 uM RG7112 YX 2-41 3 >10 uM
RG7112
indicates data missing or illegible when filed
[0210] cCBL: Recombinant human cCBL protein with an N-terminal HIS tag was combined with anti-HIS-Tb Cryptate Gold (CisBio) in low volume 384-well plates. Test compounds are added, then biotinylated phospho-tyrosine containing peptide (IK(-Biotin)NTNEpYTEGPTV (SEQ ID NO: 1)). After incubation the HTRF signal is measured on the Envision. Data is normalized to % inhibition, where the baseline HTRF signal is determined in the absence of cCBL, but including all other assay components, and is defined as 100%. The total HTRF signal in the absence of test compound is defined as 0%. The typical signal-to-noise is 5:1. Potent hit ligands have been identified and will be evaluated as new E3 recruiting ligands.
[0211] Cereblon: Recombinant human cereblon (CRBN) complex, including CUL4A, RBX1, DDB1 and CRBN is purchased from R & D systems. This four-member protein complex has an N-terminal HIS tag on the CUL4A subunit. The CRBN complex is diluted in assay buffer containing anti-HIS-Tb Cryptate Gold (CisBio) and is added to low volume 384-well plates. Test compounds in 100% DMSO are added to the plates followed by the Cy5-labeled lenolidamide probe. After 1 hour the HTRF signal is measured on the Envision. Data are normalized to % inhibition. The typical signal-to-noise is 5:1. Bi-functional molecules containing the Cereblon ligands are tested in TABLE 2 (see YX-02-041).
F. ICW Assays to Screen for Protein Degradation.
i. Linearity Protocol
[0212] Cells were seeded at decreasing cell number from 15K cells to 2K cells. The Mdm2 primary antibody was tested at 1:200 and 1:300 dilution. The secondary antibody conjugated to IRDye800CW was used with 1:800 dilution, and CellTag 700 Stain was used at 1:1000 dilution as manufacture recommended. The linearity of cell number versus signal was generated with Prism. The signal over background ratio was also calculated. The seeding cell number and primary antibody dilution factor was determined based on these data.
ii. ICW Protocol
[0213] Cells are seeded in 96 half area well plates (black clear bottom) and cultured overnight. Then they are treated with compounds, fixed with 4% paraformaldehyde for 20 minutes, and permeabilized in 0.1% Triton. They are then treated with the primary antibody and secondary antibody conjugated to IRDye800CW or treated with CellTag 700 Stain. The plates are then scanned on the OdysseyCLX and the data processed to provide a dose response curve.
G. Design and Chemical Synthesis of Mdm2 Targeting PROTACs
[0214] Mdm2 targeting ligands were utilized that have advanced into human clinical trials since these have undergone a significant level of optimization, particularly for off-target selectivity and in vivo pharmacokinetic exposure. RG7112, developed by Hoffman LaRoche, is the first Mdm2 inhibitor to enter clinical trials (Ray-Coquard et al., 2012). RG7112 is an improved derivative over its precursor Nutlin-3a (Vassilev et al., 2004). AMG-232 is a potent sub-nanomolar piperidinone Mdm2 targeting ligand developed by Amgen, with excellent reported pharmacokinetic properties (Rew & Sun, 2014). RG7388 (idasanutlin) is a second-generation derivative of RG7112 developed by Hoffman LaRoche. Idasanutlin is reported to have improved potency and selectivity over RG7112 (Ding et al., 2013). It is also the first Mdm2 inhibitor to move into Phase 3 clinical trials (Mdm2 ligands shown in
H. In-Cell Western Assay to Confirm Degradation.
[0215] The potential PROTACs were evaluated in the wt p53 MCF7 cell line and in the mut p53 MDA-MB-231 cell line. Importantly these were compared to MD-224, which was recently reported to potently degrade Mdm2 in RS4; 11 leukemia cells (Li et al., 2019). Assay linearity was performed confirming a strong 4-fold signal to noise in the assay. A series of bi-functional molecules were then treated and evaluated for protein level in the assay over time from 2 h-48 h. In
[0216] Since genetic deletion of Mdm2 induces p73 and induces apoptosis in p53 mutant and deficient cancer cells, YX-02-030 and YX-02-228 were evaluated for their effect on cell viability. First, the CellTag700 Stain was utilized to count the number of live cells in the ICW format. Interestingly, YX-02-030 and YX-02-228 show potent cytotoxic effects. YX-02-030 and YX-02-228 reduced the number of live mut p53 MDA-MD-231 cells remaining after 24 h and 48 h treatment, whereas MD-224 had no cytotoxic effect (
I. YX-02-030 Targets Mdm2 for 26S Proteasome Degradation Via VHL-Dependent Ubiquitination.
[0217] Treatment with the proteasome inhibitor MG132 prevented YX-02-030 mediated Mdm2 degradation (
[0218] PROTACs work by forming a ternary complex consisting of the target protein, the PROTAC, and the recruited E3 ubiquitin ligase-E2 complex, causing ubiquitination of the target protein, marking it for 26S proteasome-mediated degradation (Nalawansha & Crews, 2020).
[0219] To determine whether the ternary complex was required for MDM2 degradation, competition for binding to MDM2 and VHL was carried out using adding excess RG7112 or VHL-Amine, respectively. PROTAC-induced MDM2 degradation was prevented with either RG7112 or VHL-Amine (
J. Reduced p53-Inactivated TNBC Clonogenic Potential and Increased Mammosphere Apoptosis with MDM2-PROTAC Treatment
[0220] The ability of the PROTAC-mediated degradation of MDM2 to alter the clonogenic potential of p53-inactivated TNBC cells was evaluated by performing colony formation assays with MDA-MB-231 and MDA-MB-436 cells. Following YX-02-030 treatment, there was a significant reduction in colony numbers with 1 M of YX-02-030 (
[0221] These were then evaluated in 3D culture as to when the YX-02-030 was effective at preventing TNBC cell mammosphere formation and eliminating already formed mammospheres. For both MDA-MB-231 and MDA-MB-436 cells, significantly fewer mammospheres formed at each YX-02-030 concentration tested, compared to the MDM2 inhibitors and vehicle control (
K. Mouse Liver Microsomal Stability and In Vivo Pharmacokinetic Exposures of Mdm2 Compounds.
[0222] To translate cell-based activity to in vivo efficacy, the Mdm2 PROTACs were evaluated for metabolic stability and plasma compound levels after IP injection in the mouse. Mouse liver microsome stability was obtained for lead compounds (Alliance Pharma, Inc; Malvern PA) as a predictive measure for suitable exposure in a mouse pharmacokinetic study. In brief, test compounds (0.5 M) are incubated with liver microsomes (0.5 g/mL) and an NADPH-regenerating system (cofactor solution) and samples are taken at various timepoints, quenched with an acetonitrile solution containing an internal standard, then analyzed by LC-MS/MS. These data provide half-life of parent remaining and intrinsic clearance (CLint) determined from the first-order elimination constant by nonlinear regression (
L. In Vivo Efficacy Study with Mdm2 PROTAC, YX-02-030.
[0223] Prior to testing YX-02-030 tumors in vivo, mouse liver microsome and pharmacokinetic (PK) studies were performed to determine its stability in vivo. YX-02-030 was moderately metabolically stable with a 25.9-minute half-life (
M. The Use of Proteomics to Evaluate Protein Degradation
[0224] A proteomics analysis was performed to assess the selectivity of degradation by YX-02-030. MDA-MB-231 cells were treated for 8 hours with 5 M of YX-02-030 or DMSO in triplicate and processed to provide 25 g of protein loaded on a 10% acrylamide gel. The entire protein-containing gel regions were excised, digested with trypsin and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using a 240-min gradient as described (Chae et al., 2016). Peptide sequences were identified using MaxQuant 1.6.3.3 (Cox & Mann, 2008). MS/MS spectra were searched against a UniProt human protein database. A total of 4237 significantly different protein groups were quantified. Under these experimental conditions Mdm2 was not able to be detected either in the treated or untreated samples.
[0225] However, this experiment demonstrates that protein degradation induced by YX-02-030 only affected <10% of the observable proteins. Only 31 proteins out of 4237 proteins were significantly changed, 4 changed greater than 100-fold, and 11 proteins were increased, and 19 proteins were decreased. Without wishing to be bound by any theory, it is believed that as Mdm2 is an E3 ubiquitin ligase, the decreases of these protein could be impacted and these would be detected in these methods. This experiment confirms the specificity of YX-02-030, and that it does not affect hundreds of proteins in a non-specific manner.
N. YX-02-030 and YX-02-228 Treatment is Well Tolerated
[0226] In vivo tolerability tests were conducted with the 2 lead Mdm2 PROTACs to evaluate tolerated doses and effects on normal tissues. Nude mice were treated with Mdm2 PROTACs, YX-02-030 (50 mg/kg; IP) and YX-02-228 (30 mg/kg; IP) once daily for 8 days. Results showed no adverse clinical observations for spleen and bone marrow (
O. Targeted MDM2 Degradation Induces Apoptosis in Patient-Derived TNBC Explants
[0227] To assess the effects of YX-02-030 on TNBC patient samples, fresh, surgically-resected tumor from five TNBC patients was obtained, and 80% were from Black/African American patients (
[0228] For three of the patient TNBC tumor samples there was enough tissue to also establish 3D mammosphere cultures, and for one of these patients, normal breast epithelial tissue was also received that generated loose cell clusters. YX-02-030 significantly increased Caspase-3/7 activity (
P. TAp73 is Induced and Required for Apoptosis Upon MDM2-Targeted Loss in p53-Inactivated TNBC Cells
[0229] To gain insight into the mechanism by which YX-02-030 was killing p53-mutant and deleted TNBC cells, RNA-sequencing data were evaluated on cells treated with YX-02-030 and cells expressing MDM2 shRNA and each of their controls. As expected, evaluation of gene signatures from the Hallmark database (Liberzon et al., 2015) that were significantly enriched showed genes linked to apoptosis in both cell lines whether they were treated with YX-02-030 or MDM2 was knocked down (>2 fold-change, FDR<0.05;
[0230] To independently validate the RNA-seq results, qRT-PCR was performed on a subset of the genes regulated by the p53 family that mediate apoptosis. Following MDM2 degradation or MDM2 knockdown in MDA-MB-231 and MDA-MB-436 cells, levels of apoptotic genes BAX, NOXA, PUMA, AEN, APAF1, TP53I3, and PIDD1 were significantly increased in both TNBC cell lines compared to controls (
[0231] MDM2 can bind and regulate p53 family members p73 (Balint et al., 1999; Dobbelstein et al., 1999; Zeng et al., 1999; Ongkeko et al., 1999) and p63 (Calabro et al., 2002; Kadakia et al., 2001), but the conditions in which this takes place, particularly in p53-inactivated cells remain unresolved. Because the transcriptionally active forms of p73 (TAp73) and p63 (TAp63) are capable of transactivating most of the same genes as p53 (Levine, 2020), we evaluated their expression. Upon MDM2-PROTAC treatment or MDM2 knockdown, levels of TAp73 protein increased in all five TNBC cell lines assessed (
[0232] To further understand how MDM2 loss would activate TAp73, YX-02-030 binding was assessed in the p53/p73 binding pocket of MDM2 to determine if it would disrupt interactions with TAp73 in p53-inactivated TNBC cells, allowing TAp73 to move to chromatin to transcriptionally upregulate its apoptotic target genes. Immunoprecipitations of both MDM2 and TAp73 were performed in the presence of YX-02-030 or vehicle control in p53-mutant and deleted TNBC cells. The proteasome inhibitor MG132 was included to prevent proteasomal degradation of MDM2. In the YX-02-030-treated samples, there was markedly less TAp73 associated with MDM2, in both MDM2 and TAp73 immunoprecipitations (
[0233] To assess the requirements of p73 in mediating the effects of YX-02-030, TAp73 was knocked down in p53-mutant and deleted TNBC cells (
Example 3MDM2 and VHL Binding
TABLE-US-00004 TABLE 3 MDM2 and VHL Binding Activity for Other Compounds MDM2-p53 VHL-HIFa peptide IC.sub.50, nM S.E.M. Nutlin-3 89 11 >10000 IK-01-186 2700 330 1630 169 IK-01-189 32 1.8 1560 318 JG-02-070 >10000 1600 168 YX-02-023 11 1.3 >10000
A. Inhibition of MDM2 Binding to p53 Using HTRF Technology
[0234] Both GST-MDM2 and HIS-p53 were expressed E Coli and purified by affinity chromatography. pGEX-4T MDM2 WT was a gift from Mien-Chie Hung (Addgene plasmid #16237; n2t.net/addgene:16237; RRID:Addgene_16237) and human p53-(1-393) was a gift from Cheryl Arrowsmith (Addgene plasmid #24859; http://n2t.net/addgene:24859; RRID:Addgene_24859). HTRF assays contained 1 nM GST-MDM2, 0.7 nM HIS-p53, 0.3 nM of anti-GST-Tb HTRF donor and 4 nM of anti-HIS-d2 HTRF acceptor antibodies (PerkinElmer) in 10 L of 10 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 5 mM DTT, 0.01% Triton X-100 in white low-volume 384-well plates. Both GST-MDM2 and HIS-p53 were pre-incubated with their respective HTRF antibodies for 15 min. Test compounds were solubilized at 10 mM in DMSO and concentration series was added to the assay plate using the Echo 650 acoustic liquid handling system by direct dilution. The final amount of DMSO added to the assay was 100 nL (0.1%). Compounds were pre-incubated with 5 L of GST-MDM2 for 15-30 min before 5 L of HIS-p53/anti-HIS-d2 was added to the assay. After a 2 hr incubation, the HTRF signal was measured using the ClarioStar plate reader (BMG Labtech).
[0235] Data were then normalized to % inhibition, where 0% is the HTRF signal in the absence of compound, and 100% is the HTRF signal in the absence of GST-MDM2. IC.sub.50 values were determined from nonlinear regression fits of a data to a four parameter dose-response equation using XLFit (IDBS). Data are the meansS.E.M. of three replications. The data is described in Table 3 above.
B. Inhibition of HIF-1 Peptide Binding to the VHL Complex Using HTRF Technology
[0236] Assays contained 2 nM of HIS-VHL complex (R&D systems, E3-655-025), 2 nM of biotin-hydroxyproline-HIF1 peptide (Biotin-DLDLEMLAXYIPMDDDFQL (SEQ ID NO: 2), X=hydroxyproline), 0.3 nM anti-HIS-Tb HTRF donor antibody and 4 nM streptavidin-d2 HTRF acceptor (PerkinElmer) in a final volume of 10 L of 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM DTT, 0.005% Tween20 in white low volume 384-well plates. Both HIS-VHL complex and biotin-HIF1 peptide were pre-incubated with their respective HTRF antibodies for 15 min. Test compounds were solubilized at 10 mM in DMSO and concentration series was added to the assay plate using the Echo 650 acoustic liquid handling system by direct dilution. The final amount of DMSO added to the assay was 100 nL (0.1%). Compounds were pre-incubated with HIS-VHL complex before the addition of the biotin-HIF1 peptide. After a 2 hr incubation, the HTRF signal was measured using the ClarioStar plate reader (BMG Labtech). Data were then normalized to % inhibition, where 0% is the HTRF signal in the absence of compound, and 100% is the HTRF signal in the absence of HIS-VHL. IC50 values were determined from nonlinear regression fits of a data to a four parameter dose-response equation using XLFit (IDBS). Data are the meansS.E.M. of three replications. The data is described in Table 3 above.
Example 4Activity in Other Cell Lines
[0237] The PROTACs prepared herein were tested for their use in other cancer cell lines to determine activity in multiple different types of cancer. See the data presented in Table 4.
TABLE-US-00005 TABLE 4 MDM2-PROTAC Induced Cell Death of Other Cancer Cell Lines YX-02- YX-02- 030 228 Cell line Cancer type Species IC50 (M) IC50 (M) Raji Burkitt lymphoma Human 2.8 5.9 Ramos Burkitt lymphoma Human 3.1 5.5 SU-DHL-6 Diffuse large B cell Human 3.6 2.9 lymphoma SU-DHL-8 Diffuse large B cell Human 1.9 4.6 lymphoma HH T cell lymphoma Human 6.3 5.5 Hut-78 T cell lymphoma Human 9.0 8.8 SEM B-ALL Human 3.2 4.2 RS4; 11 B-ALL Human 1.9 2.5 MOLT1 T-ALL Human 2.6 2.7 SUP-T1 T-ALL Human 5.7 6 H1299 Lung adenocarcinoma Human 4.2 5.3 PC9 Lung adenocarcinoma Human 4.5 4.6 SKOV3 Ovarian Human 5.7 3.2 OVCAR3 Ovarian Human 4.5 ** OVCAR4 Ovarian Human 4.6 4.8 KPCA Ovarian Mouse 2.9 3.4 Cal27 Head & Neck Human 2.3 ** SCC-25 Head & Neck Human 3.1 ** DE1069 Sarcoma Mouse 6.1 ** * Not that values may change up to 1-2 M depending on the cell density ** Not Tested
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