4,9-DIOXO-4,9-DIHYDRONAPHTHO[2,3-B]FURAN-3-CARBOXAMIDE DERIVATIVES AND USES THEREOF FOR TREATING PROLIFERATIVE DISEASES AND INFECTIOUS DISEASES
20230010562 · 2023-01-12
Inventors
- Chi-Huey Wong (Rancho Santa Fe, CA)
- Pan-Chyr Yang (Taipei, TW)
- Rong-Jie Chein (Taipei, TW)
- Szu-Hua Pan (Taipei, TW)
- Ting-Jen R. Cheng (New Taipei City, TW)
Cpc classification
A61P31/00
HUMAN NECESSITIES
C07D405/12
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
C07D413/12
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07D405/12
CHEMISTRY; METALLURGY
C07D403/12
CHEMISTRY; METALLURGY
Abstract
The present disclosure provides compounds of Formulas (I), (II), and pharmaceutically acceptable salts thereof. The compounds described herein are useful in treating proliferative diseases, for example, cancer (e.g., lung cancer), and infectious diseases (e.g., bacterial infections).
##STR00001##
Claims
1. A method of treating a proliferative disease or an infectious disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound, or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the compound is of Formula (I): ##STR00119## or a pharmaceutically acceptable salt thereof, wherein: n is 2, 3 or 4; p is 1; X is independently —O—, or —NR.sup.X—; R.sup.X is hydrogen; R.sup.1 is optionally substituted C.sub.1-6 alkyl, or optionally substituted aryl; R.sup.2 is optionally substituted aryl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R.sup.3 is hydrogen or halogen.
2. The method of claim 1, wherein the compound is of Formula (I-A): ##STR00120## or a pharmaceutically acceptable salt thereof.
3. The method of claim 1, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl.
4. The method of claim 3, wherein R.sup.1 is selected from the group consisting of methyl, ethyl, and isopropyl.
5. The method of claim 1, wherein R.sup.1 is phenyl.
6. The method of claim 1, wherein R.sup.2 is: ##STR00121##
7. The method of claim 1, wherein R.sup.2 is phenyl. ##STR00122##
8. The method of claim 1, wherein R.sup.2 is:
9. The method of claim 1, wherein R.sup.3 is hydrogen.
10. The method of claim 1, wherein the compound is of the formula: ##STR00123## or a pharmaceutically acceptable salt thereof.
11. The method of claim 1, wherein the proliferative disease is cancer.
12. The method of claim 1, wherein the infectious disease is a bacterial infection.
13. The method of claim 12, wherein the bacterial infection is Staphylococcus infection, Streptococcus infection, Enterococcus infection, or gram negative bacterial infection
14. The method of claim 11, wherein the cancer is lung cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer, or cervical cancer.
15. The method of claim 14, wherein the lung cancer is non-small cell lung cancer.
16. A method of modulating the activity of a minichromosomal maintenance replicative helicase (MCM) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound, or a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the compound is of Formula (I): ##STR00124## or a pharmaceutically acceptable salt thereof, wherein: n is 2, 3 or 4; p is 1; X is independently —O—, or —NR.sup.X—; R.sup.X is hydrogen; R.sup.1 is optionally substituted C.sub.1-6 alkyl, or optionally substituted aryl; R.sup.2 is optionally substituted aryl, optionally substituted heterocyclyl, or optionally substituted heteroaryl; and R.sup.3 is hydrogen or halogen.
17. The method of claim 16, wherein the compound is of Formula (I-A): ##STR00125## or a pharmaceutically acceptable salt thereof.
18. The method of claim 16, wherein R.sup.1 is optionally substituted C.sub.1-6 alkyl.
19. The method of claim 18, wherein R.sup.1 is selected from the group consisting of methyl, ethyl, and isopropyl.
20. The method of claim 16, wherein R.sup.1 is phenyl.
21. The method of claim 16, wherein R.sup.2 is: ##STR00126##
22. The method of claim 16, wherein R.sup.2 is phenyl.
23. The method of claim 16, wherein R.sup.2 is: ##STR00127##
24. The method of claim 16, wherein R.sup.3 is hydrogen.
25. The method of claim 16, wherein the compound is of the formula: ##STR00128## ##STR00129## or a pharmaceutically acceptable salt thereof.
26. The method of claim 16, wherein the compound is in an amount effective to inhibit a MCM selected from the group consisting of MCM2-MCM7.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0097]
[0098]
[0099]
[0100]
[0101]
[0102]
[0103]
[0104]
DETAILED DESCRIPTION
[0105] The present disclosure provides inhibitors of minichromosome maintenance eukaryote replicative helicase subunit 2 (MCM2), for example, the compounds of Formulas (I) and (II), which bind MCM2 and inhibit its activity. The compounds described herein are useful in inhibiting MCM2 activity, thereby benefiting the treatment of diseases associated with MCM2, for example, proliferative diseases such as cancer or infectious diseases such as bacterial infection. Also provided in the present disclosure are pharmaceutical compositions, kits, methods of using the MCM2 inhibitors described herein for treating any of the target diseases described herein.
Minichromosome Maintenance Eukaryote Replicative Helicase (MCM) Inhibiting Compounds
[0106] One aspect of the present disclosure relates to the MCM inhibitors (e.g., inhibitors for any of MCM2-MCM7) as described herein, as well as their pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, or prodrugs. These compounds are useful in treating and/or preventing proliferative and/or infectious diseases in a subject.
[0107] In certain embodiments, a compound described herein is of Formula (I):
##STR00017##
in which R.sup.1-R.sup.3, X, n, and p are as described herein, or pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.
[0108] In certain embodiments, a compound described herein is of Formula (II):
##STR00018##
in which R.sup.1-R.sup.3, X, n, and p are as described herein, or pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.
[0109] In Formulas (I) and (II), X is in an aromatic ring. In some embodiments, X can be —O—. In some embodiments, X can be —S—. In some embodiments, X can be —NR.sup.X—, in which R.sup.X is as defined herein. In some embodiments, R.sup.X can be hydrogen. In some embodiments, R.sup.X can be optionally substituted C.sub.1-6 alkyl (e.g., methyl, ethyl, or propyl). In some embodiments, R.sup.X can be optionally substituted C.sub.2-C.sub.6 alkenyl. In some embodiments, R.sup.X can be optionally substituted C.sub.2-C.sub.10 alkynyl (e.g., optionally substituted, propynyl or butynyl). In certain embodiments, R.sup.X can be optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In some embodiments, R.sup.X can be optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In some embodiments, R.sup.X can be optionally substituted aryl (e.g., phenyl, or benzyl). In some embodiments, R.sup.X can be optionally substituted 5- to 7-membered monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur. In some embodiments, R.sup.X can be optionally substituted 8- to 10-membered bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur. In some embodiments, R.sup.X can be a nitrogen protecting group (e.g., benzyl (Bn), t-butyl carbonate (BOC or Boc), benzyl carbamate (Cbz), 9-fluorenylmethyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)). In one example, X can be —NH—. In another example, X can be —NMe—.
[0110] In some embodiments, R.sup.1 in Formulas (I) and/or (II) can be hydrogen. In some embodiments, R.sup.1 can be halogen (e.g., F, Cl, Br, or I). In some embodiments, R.sup.1 can be substituted or unsubstituted C.sub.1-6 alkyl (e.g., substituted or unsubstituted, methyl, ethyl, propyl or butyl). In some embodiments, R.sup.1 can be methyl. In some embodiments, R.sup.1 can be ethyl. In some embodiments, R.sup.1 can be propyl. In some embodiments, R.sup.1 can be isopropyl. In some embodiments, R.sup.1 can be optionally substituted C.sub.2-C.sub.6 alkenyl. In some embodiments, R.sup.1 can be optionally substituted C.sub.2-C.sub.10 alkynyl (e.g., optionally substituted, propynyl or butynyl). In certain embodiments, R.sup.1 can be optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In some embodiments, R.sup.1 can be optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In some embodiments, R.sup.1 can be optionally substituted aryl (e.g., phenyl, or benzyl). In some embodiments, R.sup.1 can be phenyl. In some embodiments, R.sup.1 can be benzyl. In some embodiments, R.sup.1 can be optionally substituted 5- to 7-membered monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 can be optionally substituted 8- to 10-membered bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur. In some embodiments, R.sup.1 can be —CN. In some embodiments, R.sup.1 can be —NO.sub.2. In some embodiments, R.sup.1 can be —N.sub.3.
[0111] In Formulas (I) and/or (II), in some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5.
[0112] In Formulas (I) and/or (II), in some embodiments, R.sup.2 can be hydrogen. In some embodiments, R.sup.2 can be halogen (e.g., F, Cl, Br, or T). In some embodiments, R.sup.2 can be substituted or unsubstituted C.sub.1-6 alkyl (e.g., substituted or unsubstituted, methyl, ethyl, propyl or butyl). In some embodiments, R.sup.2 can be optionally substituted C.sub.2-C.sub.6 alkenyl. In some embodiments, R.sup.2 can be optionally substituted C.sub.2-C.sub.10 alkynyl (e.g., optionally substituted, propynyl or butynyl). In certain embodiments, R.sup.2 can be optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In some embodiments, R.sup.2 can be optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In some embodiments, R.sup.2 can be
##STR00019##
In some embodiments, R.sup.2 can be
##STR00020##
In some embodiments, R.sup.2 can be optionally substituted aryl (e.g., phenyl, or benzyl). In some embodiments, R.sup.2 can be phenyl. In some embodiments, R.sup.2 can be benzyl. In some embodiments, R.sup.2 can be optionally substituted 5- to 7-membered monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur. In some embodiments, R.sup.2 can be
##STR00021##
In some embodiments, R.sup.2 can be
##STR00022##
In some embodiments, R.sup.2 can be optionally substituted 8- to 10-membered bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur.
[0113] Formulas (I) and/or (II) include one or more instances of R.sup.3. In some embodiments, at least one instance of R.sup.3 can be hydrogen. In some embodiments, at least one instance of R.sup.3 can be halogen (e.g., F, Cl, Br, or I). In some embodiments, at least one instance of R.sup.3 can be substituted or unsubstituted C.sub.1-6 alkyl (e.g., substituted or unsubstituted, methyl, ethyl, propyl or butyl). In some embodiments, at least one instance of R.sup.3 can be optionally substituted C.sub.2-C.sub.6 alkenyl. In some embodiments, at least one instance of R.sup.3 can be optionally substituted C.sub.2-C.sub.10 alkynyl (e.g., optionally substituted, propynyl or butynyl). In certain embodiments, at least one instance of R.sup.3 can be optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In some embodiments, at least one instance of R.sup.3 can be optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In some embodiments, at least one instance of R.sup.3 can be optionally substituted 5- to 7-membered monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur. In some embodiments, at least one instance of R.sup.3 can be optionally substituted 8- to 10-membered bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur. In some embodiments, at least one instance of R.sup.3 can be —CN. In some embodiments, at least one instance of R.sup.3 can be —NO.sub.2. In some embodiments, at least one instance of R.sup.3 can be —N.sub.3. In some embodiments, at least one instance of R.sup.3 can be —OR.sup.A, in which R.sup.A is as defined herein, (e.g., —OH, —O(substituted or unsubstituted C.sub.1-6 alkyl) (e.g., —OMe, —OCF.sub.3, —OEt, —OPr, or —OBu). In certain embodiments, at least one instance of R.sup.3 can be —N(R.sup.B).sub.2, in which R.sup.B is as defined herein, (e.g., —NMe.sub.2). In certain embodiments, at least one instance of R.sup.3 can be —C(═O)R.sup.A, in which R.sup.A is as defined herein, (e.g., acetyl). In certain embodiments, at least one instance of R.sup.3 is —SO.sub.2R.sup.A, in which R.sup.A is as defined herein.
[0114] In some embodiments, R.sup.A and/or R.sup.B can be hydrogen. In some embodiments, R.sup.A and/or R.sup.B can be halogen (e.g., F, Cl, Br, or I). In some embodiments, R.sup.A and/or R.sup.B can be substituted or unsubstituted C.sub.1-6 alkyl (e.g., substituted or unsubstituted, methyl, ethyl, propyl or butyl). In some embodiments, R.sup.A and/or R.sup.B can be methyl. In some embodiments, R.sup.A and/or R.sup.B can be ethyl. In some embodiments, R.sup.A and/or R.sup.B can be propyl. In some embodiments, R.sup.A and/or R.sup.B can be isopropyl. In some embodiments, R.sup.A and/or R.sup.B can be optionally substituted C.sub.2-C.sub.6 alkenyl. In some embodiments, R.sup.A and/or R.sup.B can be optionally substituted C.sub.2-C.sub.10 alkynyl (e.g., optionally substituted, propynyl or butynyl). In certain embodiments, R.sup.A and/or R.sup.B can be optionally substituted carbocyclyl (e.g., substituted or unsubstituted, 3- to 7-membered, monocyclic carbocyclyl comprising zero, one, or two double bonds in the carbocyclic ring system). In some embodiments, R.sup.A and/or R.sup.B can be optionally substituted heterocyclyl (e.g., substituted or unsubstituted, 5- to 10-membered monocyclic or bicyclic heterocyclic ring, wherein one or two atoms in the heterocyclic ring are independently nitrogen, oxygen, or sulfur). In some embodiments, R.sup.A and/or R.sup.B can be optionally substituted aryl (e.g., phenyl, or benzyl). In some embodiments, R.sup.A and/or R.sup.B can be phenyl. In some embodiments, R.sup.A and/or R.sup.B can be benzyl. In some embodiments, R.sup.A and/or R.sup.B can be optionally substituted 5- to 7-membered monocyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur. In some embodiments, R.sup.A and/or R.sup.B can be optionally substituted 8- to 10-membered bicyclic heteroaryl, wherein one, two, three, or four atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur. In some embodiments, R.sup.A can be an oxygen protecting group if attached to an oxygen atom (e.g., t-butyloxycarbonyl (BOC or Boc), methoxylmethyl (MOM) or methylthiomethyl (MTM)). In some embodiments, R.sup.B can be a nitrogen protecting group if attached to an nitrogen atom (e.g., benzyl (Bn), t-butyl carbonate (BOC or Boc), benzyl carbamate (Cbz), 9-fluorenylmethyl carbonate (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl, or p-toluenesulfonamide (Ts)). In certain embodiments, two instances of R.sup.B can be taken together with the intervening atoms to form substituted or unsubstituted heterocyclyl or substituted or unsubstituted heteroaryl.
[0115] In some embodiments, the compound of Formula (I) can be of one of the following formulae: Formula (I-A), Formula (I-B), Formula (I-C), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.
[0116] In some embodiments, the compound of Formula (II) can be of one of the following formulae: Formula (II-A), Formula (II-B), Formula (II-C), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.
[0117] In some embodiments, the compound of Formulas (I) or (II) can be of the formula of compounds described herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.
[0118] Exemplary compounds of Formula (I) are provided herein. Below is an additional example:
##STR00023##
or a pharmaceutically acceptable salt thereof.
[0119] The compounds described herein can be prepared from readily available starting materials using methods known in the art. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, and pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures. The chemicals used in the above-described synthetic routes may include, for example, solvents, reagents, catalysts, and protecting group and deprotecting group reagents. The methods described above may also additionally include steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups in order to ultimately allow synthesis of the compounds. In addition, various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing applicable compounds are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3.sup.rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents, for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.
[0120] The compounds of Formulas (I) and (II) provided herein can be prepared from readily available starting materials using the following general methods and procedures. An exemplary schematic illustration for synthesizing the MCM2 inhibiting compounds described herein is provided in the Examples section below. Where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.
Pharmaceutical Compositions and Kits
[0121] The present disclosure provides pharmaceutical compositions comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient. In certain embodiments, a pharmaceutical composition described herein comprises a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. The pharmaceutical compositions described herein are useful in treating and/or preventing proliferative diseases (e.g., cancer) and/or infectious diseases (e.g., bacterial infections).
[0122] In certain embodiments, a subject being treated herein is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject is a human. In certain embodiments, the subject is a mammal (e.g., non-human mammal). In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, a subject being treated herein is a companion animal such as a dog or cat. In certain embodiments, a subject being treated herein is a livestock animal such as a cow, pig, horse, sheep, or goat. In certain embodiments, a subject being treated herein is a zoo animal. In another embodiment, a subject being treated herein is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic or genetically engineered animal (e.g., transgenic mice and transgenic pigs).
[0123] In certain embodiments, the cell contacted with an effective amount of a compound or pharmaceutical composition described herein is in vitro. In certain embodiments, the contacted cell is ex vivo. In certain embodiments, the cell described herein is in vivo. In certain embodiments, the cell described herein is a malignant cell (e.g., malignant blood cell).
[0124] In certain embodiments, the compound described herein is provided in an effective amount in the pharmaceutical composition. In certain embodiments, the effective amount is a therapeutically effective amount (e.g., amount effective for inhibiting MCM2 and/or treating a proliferative disease or an infectious disease in a subject in need thereof). In certain embodiments, the proliferative disease is cancer, e.g., breast cancer, colorectal cancer cancer, pancreatic cancer, gastric cancer, or cervical cancer. In certain embodiments, the disease is bacterial infection, such as Staphylococcus infection, Streptococcus infection, Enterococcus infection and gram negative bacterial infection. In certain embodiments, the effective amount is a prophylactically effective amount (e.g., amount effective for preventing a proliferative disease or an infectious disease in a subject in need thereof and/or for keeping a subject in need thereof in remission of a proliferative disease). In certain embodiments, the effective amount is a therapeutically effective amount (e.g., amount effective for treating an infectious disease or a proliferative disease in a subject in need thereof).
[0125] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the compound described herein (i.e., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.
[0126] Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.
[0127] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.
[0128] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
[0129] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof. In certain embodiments, the composition of the instant disclosure is encapsulated in a carrier vehicle, which may be rigid vesicles, elastic vesicles, monolayer vesicles, multi-layer vesicles, liposomes, niosomes, proniosomes, Transfersomes®, ethosomes, L-595-PEG-8-L vesicles, nanoemulsions, nanosomes, nanoparticles, or a combination thereof.
[0130] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
[0131] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[0132] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.
[0133] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
[0134] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.
[0135] Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
[0136] The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.
[0137] Dosage forms for topical and/or transdermal administration of a compound described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.
[0138] Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder/particle delivery devices which use compressed gas to accelerate the compound in powder form through the outer layers of the skin to the dermis are suitable.
[0139] Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
[0140] A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
[0141] Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
[0142] Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.
[0143] Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
[0144] Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
[0145] A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.
[0146] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.
[0147] The compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
[0148] Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.
[0149] In certain embodiments, a kit described herein includes a first container comprising a compound or pharmaceutical composition described herein. In certain embodiments, a kit described herein is useful in treating a disease associated with MCM2, for example, a proliferative disease (e.g., breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or cervical cancer) and/or infectious disease including but not limited to bacterial and viral infections. In certain embodiments, the bacterial infection is Staphylococcus infection, Streptococcus infection, Enterococcus infection or gram negative bacterial infection in a subject in need thereof. In some embodiments, the MCM2 inhibitory compounds described herein are useful in treating diseases associated with bacterial infections.
[0150] In certain embodiments, a kit described herein further includes instructions for using the compound or pharmaceutical composition included in the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating a proliferative disease and/or infectious disease in a subject in need thereof, and/or preventing a proliferative disease and/or infectious disease in a subject in need thereof. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.
Methods of Treatment
[0151] As shown in the Examples below, exemplary MCM inhibitors described herein successfully induce protein degradation of MCM (e.g., MCM2-MCM7), may target MCM, and contribute to cell apoptosis in cells such as lung cancer cells.
[0152] Accordingly, the present disclosure provides methods of treating a proliferative disease, or an infectious disease in a subject in need thereof, the methods comprising administering to the subject an effective amount (e.g., therapeutically effective amount) of a compound, or pharmaceutical composition thereof, described herein.
[0153] Another aspect of the present disclosure relates to methods of preventing proliferative disease and/or infectious disease in a subject in need thereof, the methods comprising administering to the subject an effective amount (e.g., prophylactically effective amount) of a compound, or pharmaceutical composition thereof, described herein.
[0154] The compounds and pharmaceutical compositions described herein are useful in treating diseases associated with MCM, for example, proliferative diseases and/or infectious diseases. In certain embodiments, the proliferative disease is cancer. In certain embodiments, the proliferative disease is lung cancer. In certain embodiments, the proliferative disease is non-small cell lung cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or cervical cancer. In certain embodiments, the infectious disease is bacterial infection, such as Staphylococcus infection, Streptococcus infection, Enterococcus infection or gram negative bacterial infection.
[0155] In certain embodiments, the method described herein further includes administering to the subject an additional pharmaceutical agent. In certain embodiments, the method described herein further includes contacting the biological sample with an additional pharmaceutical agent. In certain embodiments, the method described herein further includes contacting the tissue with an additional pharmaceutical agent. In certain embodiments, the method described herein further includes contacting the cell with an additional pharmaceutical agent. In certain embodiments, the method described herein further includes radiotherapy, immunotherapy, and/or transplantation (e.g., bone marrow transplantation).
[0156] The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intracranial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), by any means that facilitate in vivo or ex vivo transport of the compound or composition as described herein in, into, or through tissue/skin of a subject (such as iontophoresis), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), transfusion, perfusion, regional administration via blood and/or lymph supply, and/or direct administration to an affected site, such as intra-tumoral. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.
[0157] The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, biological sample, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, biological sample, tissue, or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein.
[0158] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower, higher, or the same as that administered to an adult.
[0159] A compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents) useful in treating and/or preventing a proliferative disease disease and/or infectious disease. The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a proliferative disease disease and/or infectious disease in a subject in need thereof, and/or in preventing a proliferative disease disease and/or infectious disease in a subject in need thereof), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject, biological sample, tissue, or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, for different disorders, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a compound described herein is administered to a patient in need thereof, to advantageously treat one or more diseases. In certain embodiments, said one or more disease is a proliferative disease, an infectious disease, or a combination thereof. In a preferred embodiment, said one or more disease is cancer, a bacterial infection, or a combination thereof. It is believed that an MCM2-inhibitory compound described herein would act synergistically with at least one additional therapeutic agent in inhibiting target cell growth (e.g., inhibiting the growth of cancer cells or bacterial cells, or cells infected with a pathogen such as a virus).
[0160] The compound or composition may be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies in treating and/or preventing a proliferative disease and/or infectious disease. In certain embodiments, the compound or composition described herein can be administered to a patient in need thereof, wherein the proliferative disease or the infectious disease of the patient is resistant to at least one pharmaceutical agent. In certain embodiments, the proliferative disease is cancer and said cancer is resistant to one or more anti-cancer agents including but not limited to tyrosine kinase inhibitors (TKIs) such as Gefitinib or elrotinib. In certain embodiments, the infectious disease is bacterial infection and said bacterial strain is resistant to one or more antibiotics including but not limited to methicillin and ciprofloxacin. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in treating a proliferative disease disease and/or infectious disease. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in preventing a proliferative disease disease and/or infectious disease. In certain embodiments, the additional pharmaceutical agent is Gefitinib (Iressa). In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent approved by a regulatory agency (e.g., the US FDA) for treating and/or preventing a proliferative disease and/or infectious disease. Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
[0161] In certain embodiments, the additional pharmaceutical agent is an anti-proliferative agent (e.g., anti-cancer agent). In certain embodiments, the additional pharmaceutical agent is an anti-angiogenesis agent, anti-inflammatory agent, immunosuppressant, anti-bacterial agent, anti-viral agent, cardiovascular agent, cholesterol-lowering agent, anti-diabetic agent, anti-allergic agent, pain-relieving agent, or a combination thereof. In certain embodiments, the compounds described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, transplantation (e.g., bone marrow transplantation, stem cell transplantation), surgery, radiation therapy, immunotherapy, and chemotherapy.
Methods of Synthesis
[0162] An exemplary synthetic scheme for making the compounds described herein is provided below:
##STR00024##
[0163] In one aspect, the present invention provides methods for preparing compounds of Formulas (I) and (II). In certain embodiments, the method for preparing a compound of Formula (I) comprises coupling a compound of Formula (I-D):
##STR00025##
or a salt thereof, with a compound of the formula:
##STR00026##
or a salt thereof, under conditions sufficient to form a compound of Formula (I):
##STR00027##
or a salt thereof, wherein n, p, X, R.sup.X, R.sup.1, R.sup.2, and R.sup.3 are as defined herein.
[0164] In certain embodiments, the method for preparing a compound of Formula (II) comprises coupling a compound of Formula (II-D):
##STR00028##
or a salt thereof, with a compound of the formula:
##STR00029##
or a salt thereof, under conditions sufficient to form a compound of Formula (II):
##STR00030##
or a salt thereof, wherein n, p, X, R.sup.X, R.sup.1, R.sup.2, and R.sup.3 are as defined herein.
[0165] In certain embodiments, the step of coupling a compound of Formula (I-D) with a compound of the formula:
##STR00031##
to form a compound of Formula (I) is carried out in the presence of a coupling reagent.
[0166] In certain embodiments, the step of coupling a compound of Formula (II-D) with a compound of the formula:
##STR00032##
to form a compound of Formula (II) is carried out in the presence of a coupling reagent.
[0167] In certain embodiments, the step of coupling is carried out in the presence of a coupling reagent and a base. In certain embodiments, the coupling reagent is a reagent that promotes the formation of an amide bond (e.g., a peptide coupling reagent). Any coupling reagent (e.g., peptide coupling reagent) known in the art may be used. In certain embodiments, the coupling reagent is N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU). In certain embodiments, the coupling reagent is 1,1′-Carbonyldiimidazole (CDI). In certain embodiments, the step of coupling is carried out in the presence of a base. In certain embodiments, the base is a pyridine. In certain embodiments, the base is an aminopyridine. In certain embodiments, the base is 4-dimethylaminopyridine (DMAP). In certain embodiments, the step of coupling is carried out in the presence of a coupling reagent and a base. In certain embodiments, the step of coupling is carried out in the presence of HBTU and DMAP. In certain embodiments, the step of coupling is carried out in a solvent. In certain embodiments, the solvent is N,N-dimethylformamide (DMF).
[0168] In certain embodiments, the method of preparing a compound of Formula (I) comprises hydrolyzing a compound of Formula (I-E):
##STR00033##
or a salt thereof, under conditions sufficient to form a compound of Formula (I-D):
##STR00034##
or a salt thereof, wherein X, R.sup.1, R.sup.3, and p are as defined herein; and
[0169] R.sup.4 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group.
[0170] In certain embodiments, the method of preparing a compound of Formula (II) comprises hydrolyzing a compound of Formula (II-E):
##STR00035##
or a salt thereof, under conditions sufficient to form a compound of Formula (II-D):
##STR00036##
or a salt thereof, wherein X, R.sup.1, R.sup.3, and p are as defined herein; and
[0171] R.sup.4 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group.
[0172] In certain embodiments, the step of hydrolyzing a compound of Formula (I-E) to form a compound of Formula (I-D) is carried out in the presence of a base. In certain embodiments, the step of hydrolyzing a compound of Formula (II-E) to form a compound of Formula (II-D) is carried out in the presence of a base. In certain embodiments, the base is hydroxide or an alkoxide. In certain embodiments, the base is hydroxide. In certain embodiments, the base is selected from the group consisting of sodium hydroxide, potassium hydroxide, and lithium hydrozide. In certain embodiments, the base is lithium hydroxide (LiOH). In certain embodiments, the base is sodium hydroxide (NaOH). In certain embodiments, the step of hydrolyzing is carried out in the presence of water. In certain embodiments, the step of hydrolyzing is carried out in the presence of a base and water. In certain embodiments, the step of hydrolyzing is carried out in the presence of lithium hydrozide (LiOH) and water. In certain embodiments, the step of hydrolyzing is carried out in the presence of a solvent. In certain embodiments, the solvent is tetrahydrofuran (THF). In certain embodiments, the solvent is a mixture of THF and water.
[0173] As generally defined herein, R.sup.4 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group. In certain embodiments, R.sup.4 is optionally substituted alkyl. In certain embodiments, R.sup.4 is optionally substituted alkenyl. In certain embodiments, R.sup.4 is optionally substituted alkynyl. In certain embodiments, R.sup.4 is optionally substituted carbocyclyl. In certain embodiments, R.sup.4 is optionally substituted heterocyclyl. In certain embodiments, R.sup.4 is optionally substituted aryl. In certain embodiments, R.sup.4 is optionally substituted heteroaryl. In certain embodiments, R.sup.4 is an oxygen protecting group. In certain embodiments, R.sup.4 is optionally substituted C.sub.1-6 alkyl. In certain embodiments, R.sup.4 is unsubstituted C.sub.1-6 alkyl. In certain embodiments, R.sup.4 is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl. In certain embodiments, R.sup.4 is ethyl. In certain embodiments, R.sup.4 is methyl.
[0174] In certain embodiments, the method of preparing a compound of Formula (I) comprises contacting a compound of Formula (I-G):
##STR00037##
or a salt thereof, with a compound of formula:
##STR00038##
or a salt thereof, under conditions sufficient to form a compound of Formula (I-F):
##STR00039##
or a salt thereof, wherein R.sup.1, R.sup.3, R.sup.4, and p are as defined herein; and
[0175] each instance of R.sup.5 is independently optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; or optionally two R.sup.5 are joined together with the intervening atoms to form optionally substituted carbocyclyl or optionally substituted heterocyclyl.
[0176] In certain embodiments, the method of preparing a compound of Formula (II) comprises contacting a compound of Formula (II-G):
##STR00040##
or a salt thereof, with a compound of formula:
##STR00041##
or a salt thereof, under conditions sufficient to form a compound of Formula (II-F):
##STR00042##
or a salt thereof,
wherein:
[0177] R.sup.4 is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group; and
[0178] each instance of R.sup.5 is independently optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; or optionally two R.sup.5 are joined together with the intervening atoms to form optionally substituted carbocyclyl or optionally substituted heterocyclyl.
[0179] In certain embodiments, the step of contacting a compound of Formula (I-G) with a compound of formula:
##STR00043##
to form a compound of Formula (I-F) is carried out in the presence of a base. In certain embodiments, the step of contacting a compound of Formula (II-G) with a compound of formula:
##STR00044##
to form a compound of Formula (II-F) is carried out in the presence of a base. In certain embodiments, a compound of Formula (II-G) is contacted with a compound of formula:
##STR00045##
to form a compound of Formula (II-F). In certain embodiments, the base is an amine base. In certain embodiments, the base is a trialkylamine. In certain embodiments, the base is triethylamine. In certain embodiments, the base is N,N-Diisopropylethylamine (DIPEA). In certain embodiments, the step of contacting is carried out in the presence of a solvent. In certain embodiments, the solvent is tetrahydrofuran (THF).
[0180] As generally defined herein, each instance of R.sup.5 is independently optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; or optionally two R.sup.5 are joined together with the intervening atoms to form optionally substituted carbocyclyl or optionally substituted heterocyclyl. In certain embodiments, at least one instance of R.sup.5 is optionally substituted alkyl. In certain embodiments, at least one instance of R.sup.5 is optionally substituted alkenyl. In certain embodiments, at least one instance of R.sup.5 is optionally substituted alkynyl. In certain embodiments, at least one instance of R.sup.5 is optionally substituted carbocyclyl. In certain embodiments, at least one instance of R.sup.5 is optionally substituted heterocyclyl. In certain embodiments, at least one instance of R.sup.5 is optionally substituted aryl. In certain embodiments, at least one instance of R.sup.5 is optionally substituted heteroaryl. In certain embodiments, at least one instance of R.sup.5 is optionally substituted C.sub.1-6 alkyl. In certain embodiments, at least one instance of R.sup.5 is unsubstituted C.sub.1-6 alkyl. In certain embodiments, at least one instance of R.sup.5 is selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl. In certain embodiments, at least one instance of R.sup.5 is n-butyl. In certain embodiments, each instance of R.sup.5 is n-butyl. In certain embodiments, at least one instance of R.sup.5 is phenyl. In certain embodiments, each instance of R.sup.5 is phenyl.
[0181] In certain embodiments, the method of preparing a compound of Formula (I) comprises contacting a compound of the formula:
##STR00046##
or a salt thereof, with a compound of the formula:
##STR00047##
or a salt thereof, and a compound of the formula:
##STR00048##
or a salt thereof, under conditions sufficient to form a compound of Formula (I-G):
##STR00049##
or a salt thereof, wherein R.sup.3, R.sup.4, R.sup.5, and p are as defined herein.
[0182] In certain embodiments, the method of preparing a compound of Formula (II) comprises contacting a compound of the formula:
##STR00050##
or a salt thereof, with a compound of the formula:
##STR00051##
or a salt thereof, and a compound of the formula:
##STR00052##
or a salt thereof, under conditions sufficient to form a compound of Formula (II-G):
##STR00053##
or a salt thereof, wherein R.sup.3, R.sup.4, R.sup.5, and p are as defined herein.
[0183] In certain embodiments, the step of contacting a compound of the formula:
##STR00054##
with a compound of the formula:
##STR00055##
and a compound of the formula:
##STR00056##
to form a compound of Formula (I-G) is carried out in the presence of an acid. In certain embodiments, the step of contacting a compound of the formula:
##STR00057##
with a compound of the formula:
##STR00058##
and a compound of the formula:
##STR00059##
to form a compound of Formula (I-G) is carried out in the presence of an acid. In certain embodiments, the acid is a carboxylic acid. In certain embodiments, the acid is trifluoroacetic acid (TFA). In certain embodiments, the step of contacting is carried out in the presence of a solvent.
[0184] In certain embodiments, the method of preparing a compound of Formula (I) comprises contacting a compound of the formula:
##STR00060##
or a salt thereof, with a compound of formula:
##STR00061##
or salt thereof, under conditions sufficient to form a compound of Formula (I-F):
##STR00062##
or a salt thereof, wherein R.sup.1, R.sup.3, R.sup.4, and p are as defined herein; and
[0185] each instance of Y is independently a halogen or a leaving group.
[0186] In certain embodiments, the step of contacting a compound of the formula:
##STR00063##
with a compound of formula:
##STR00064##
to form a compound of Formula (I-F) is carried out in the presence of a base. In certain embodiments, the base is carbonate. In certain embodiments, the base is sodium carbonate, lithium carbonate, potassium carbonate, or calcium carbonate. In certain embodiments, the base is potassium carbonate (K.sub.2CO.sub.3). In certain embodiments, the step of coupling is carried out in the presence of a solvent. In certain embodiments, the solvent is acetonitrile.
[0187] As generally defined herein, each instance of Y is independently halogen or a leaving group. In certain embodiments, at least one instance of Y is halogen. In certain embodiments, at least one instance of Y is a leaving group. In certain embodiments, at least one instance of Y is —Cl. In certain embodiments, at least one instance of Y is —Br. In certain embodiments, at least one instance of Y is —F. In certain embodiments, at least one instance of Y is —I. In certain embodiments, each instance of Y is —Cl.
[0188] The synthetic schemes and synthetic steps provided are not limiting, and the disclosure contemplates methods wherein additional steps are added, existing steps are omitted or substituted, starting materials or reagents are modified or substituted, or the order of steps is altered. For example, for certain functional groups, additional protection or deprotection steps may be necessary or desired to maintain compatibility with certain reactions or reagents.
[0189] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
EXAMPLES
[0190] In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.
Example 1: Preparation of Exemplary MCM Inhibitory Compounds
[0191] The compounds provided herein can be prepared from readily available starting materials using methods known in the art, such as the methods described in Mauger et al., Eur. Pat. Appl., 1746097, 24 Jan. 2007, and the methods described in Nitsche et al., Journal of Medicinal Chemistry, 56(21), 8389-8403; 2013. Where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.
[0192] The reactions were generally performed in flame-dried glassware under a positive pressure of nitrogen. Commercial grade reagents and solvents were used without further purification. CH.sub.2Cl.sub.2, CH.sub.3CN and THF were purified by PS-MD-5 solvent purification system (Innovative Technology, Inc). Magnetic bead was purchased from TAN Beads, Taiwan. The progress of all the reactions were monitored by TLC, using TLC glass plates precoated with silica gel 60 F.sub.254 (Merck). Flash column chromatography was performed with silica gel Geduran® Si 60 (Merck). IR spectra were recorded with Thermo Nicolet iS-5 FT-IR spectrophotometer, ν.sub.max in cm.sup.−1. .sup.1H and .sup.13C NMR spectra were recorded with Bruker AV-III 400 MHz or Bruker AV-400 MHz spectrometers and chemical shifts were measured in δ (ppm) with residual solvent peaks as internal standards (CDCl.sub.3, δ 7.24 ppm in .sup.1H NMR, δ 77.0 ppm in .sup.13C NMR; CD.sub.3OD, δ 3.31 ppm in .sup.1H NMR, δ 49.0 ppm in .sup.13C NMR). Coupling constants J, measured in Hz. HR FAB (LR FAB) and HR EJ (LR EJ)-mass spectra were recorded on a JMS-700 double focusing mass spectrometer (JEOL, Tokyo, Japan) with a resolution of 8000(3000) (5% valley definition) and HR (LR) ESI (Electrospray)-mass spectra were recorded using dual ionization ESCi® (ESI/APCi) source options, Waters LCT premier XE (Waters Corp., Manchester, UK). Melting points were recorded on Büchi M-565 apparatus.
##STR00065##
(1) Ethyl 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylate
[0193] ##STR00066##
[0194] A mixture of 2, 3-dichloro-1, 4-naphthoquione (500 mg, 2.20 mmol), K.sub.2CO.sub.3 (760 mg, 5.51 mmol) and ethyl acetoacetate (0.31 mL, 2.42 mmol) in MeCN (30 mL) was stirred at reflux temperature for 6 hr. After completion, the reaction mixture was diluted with EtOAc and filtered through Celite®, filtrate was evaporated in vacuo to yield the crude product. The residue was purified by flash column chromatography (EtOAc/hexanes, 1/4 to 2/3) to give 1 as a yellow solid (305 mg, 48% yield). R.sub.f (30% EtOAc/hexanes) 0.52; Mp 149.3-159.9° C.; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.42-7.99 (m, 2H), 7.98-7.53 (m, 2H), 4.43 (q, J=7.2 Hz, 2H), 2.70 (s, 3H), 1.43 (t, J=7.1 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 178.7, 173.5, 164.4, 162.0, 151.3, 134.1, 133.7, 133.6, 131.5, 128.2, 127.4, 126.5, 113.8, 61.5, 14.2, 14.2.
[0195] Exemplary syntheses of compounds of Formula (I), as depicted in Schemes 1 and 2 above, are also described in Hu et al, Synthesis (10):1605-1610 (2005).
(2) 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid
[0196] ##STR00067##
[0197] To a stirred solution of ethyl 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b]furan-3-carboxylate (378 mg, 1.33 mmol) in THF/H.sub.2O (0.2 M, 7/3 v/v) was added LiOH (167 mg, 3.99 mmol) at room temperature. After stirred at room temperature for 3 hr, the reaction mixture was quenched with 1N HCl (adjust to pH ˜1, aqueous layer will shows transparent), extracted with EtOAc (15 mL×3), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/9) to give 2 as a yellow solid (324 mg, 95% yield). R.sub.f (5% MeOH/CH.sub.2Cl.sub.2) 0.35; IR (neat) 3425, 3022, 2975, 2920, 2712, 1744, 1673, 1581, 1413, 1217, 1192, 995, 721 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.25 (td, J=7.4, 1.2 Hz, 2H), 7.84 (dtd, J=18.8, 7.5, 1.3 Hz, 2H), 2.88 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 184.9, 172.4, 169.0, 160.9, 150.9, 135.8, 134.5, 131.8, 131.7, 128.0, 127.5, 126.0, 112.8, 14.5.
[0198] Exemplary syntheses of compounds of Formula (I), as depicted in Schemes 1 and 2 above, is also described in Reynolds et al, J. Org. Chem., 30(11):3819-3824 (1965).
(3)N-(2-Azidoethyl)-2-methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxamide
[0199] ##STR00068##
[0200] To a stirred solution of 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid (25.6 mg, 0.1 mmol), HBTU (45.5 mg, 0.12 mmol) and DMAP (12.2 mg, 0.1 mmol) in DMF (0.5 mL) was added 2-azidoethanamine (25 μL, 0.3 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 4 days. Then the crude was washed by H.sub.2O, extracted with CH.sub.2Cl.sub.2 (10 mL×3), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/49) to give 3 as a yellow solid (18.3 mg, 57% yield). R.sub.f (CH.sub.2Cl.sub.2) 0.49; Mp 152.7-162.5° C.; IR (neat) 3245, 2914, 2845, 2090, 1644, 1574, 1210, 991, 713 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.95 (s, 1H), 8.19 (ddd, J=8.8, 6.3, 3.6 Hz, 2H), 7.98-7.55 (m, 2H), 3.93-3.22 (m, 4H), 2.86 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 183.0, 173.0, 166.7, 161.6, 151.1, 134.7, 134.2, 132.8, 131.5, 127.8, 126.8, 126.0, 115.0, 50.7, 38.9, 15.0; HRMS (ESI.sup.+, TOF) calculated for C.sub.16H.sub.12N.sub.4NaO.sub.4 [M+Na].sup.+ 347.0756, found 347.0757.
(4)N-(3-(1H-Imidazol-1-yl) propyl)-2-methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b]furan-3-carboxamide (Compound 1 or GRC4583)
[0201] ##STR00069##
[0202] An exemplary synthesis scheme of Compound 1 is provided below:
##STR00070##
[0203] To a stirred solution of 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid (256.2 mg, 1.0 mmol), DMAP (12.2 mg, 0.1 mmol) and HBTU (455 mg, 1.2 mmol) in DMF (5 mL) was added 1-(3-Aminopropyl)imidazole (180 μL, 1.5 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 1 day. Then the crude was washed by H.sub.2O, extracted with CH.sub.2Cl.sub.2 (10 mL×3), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/49) to give Compound 1 as a yellow solid (240 mg, 66% yield). R.sub.f (5% MeOH/CH.sub.2Cl.sub.2) 0.28; IR (neat) 3285, 3095, 2923, 2848, 1650, 1582, 1213, 991, 844, 715 cm.sup.−1; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.79 (s, 1H), 8.21 (qd, J=4.0, 1.7 Hz, 2H), 7.83-7.76 (m, 2H), 7.59 (s, 1H), 7.07 (s, 1H), 7.00 (s, 1H), 4.11 (t, J=7.0 Hz, 2H), 3.46 (dd, J=12.5, 6.2 Hz, 2H), 2.87 (s, 3H), 2.16 (quint, J=6.8 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 183.3, 172.9, 166.5, 161.5, 151.1, 137.2, 134.8, 134.2, 132.7, 131.4, 129.3, 127.8, 126.9, 125.9, 118.9, 115.1, 44.5, 36.2, 30.9, 14.9; HRMS (ESI.sup.+, TOF) calculated for C.sub.20H.sub.18N.sub.3O.sub.4 [M+H].sup.+ 364.1297, found 364.1289.
(5)N-(2-(1H-imidazol-1-yl) ethyl)-2-methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b]furan-3-carboxamide (Compound 5)
[0204] ##STR00071##
[0205] To a stirred solution of 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid (44 mg, 0.17 mmol), DMAP (21 mg, 0.17 mmol) and HBTU (97 mg, 0.26 mmol) in DMF (0.85 mL) was added 2-Imidazol-1yl-ethylamine (47 mg, 0.43 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 1 day. Then the crude was washed by H.sub.2O, extracted with CH.sub.2Cl.sub.2 (10 mL×3), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/49) to give compound 5 as a yellow solid (42 mg, 70% yield). R.sub.f (5% MeOH/CH.sub.2Cl.sub.2) 0.28; .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.27-8.14 (m, 3H), 7.92-7.81 (m, 2H), 7.43 (s, 1H), 7.22 (s, 1H), 4.41 (t, J=6.0 Hz, 1H), 3.86 (t, J=6.0 Hz, 1H), 2.77 (s, 3H); .sup.13C NMR (100 MHz, CD.sub.3OD) δ 184.0, 174.2, 167.1, 163.7, 152.6, 138.8, 135.9, 135.4, 134.2, 132.9, 129.3, 128.6, 127.5, 127.2, 120.8, 115.8, 47.2, 41.3 14.7; HRMS (ESI.sup.+, TOF) calculated for C.sub.19H.sub.16N.sub.3O.sub.4 [M+H].sup.+ 350.1141, found 350.1135.
(6)N-(4-(1H-imidazol-1-yl)butyl)-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (Compound 13)
[0206] ##STR00072##
[0207] To a stirred solution of 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid (95 mg, 0.37 mmol), DMAP (45 mg, 0.37 mmol) and HBTU (154 mg, 0.41 mmol) in DMF (1.9 mL) was added 4-(1H-imidazol-1-yl)butan-1-amine (77 mg, 0.56 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 18 hr. Then the crude was washed by H.sub.2O, extracted with CH.sub.2Cl.sub.2 (10 mL×3), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/49) to give compound 13 as a yellow solid (97 mg, 70% yield). R.sub.f (5% MeOH/CH.sub.2Cl.sub.2) 0.25; Mp 158.6-164.1° C.; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.70 (s, 1H), 8.18 (d, J=7.5 Hz, 2H), 7.83-7.75 (m, 2H), 7.49 (s, 1H), 7.03 (s, 1H), 6.93 (s, 1H), 4.01 (t, J=7.1 Hz, 2H), 3.47 (dd, J=12.6, 6.4 Hz, 2H), 2.86 (s, 3H), 1.94 (dt, J=15.1, 7.4 Hz, 2H), 1.69 (dt, J=14.2, 6.9 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 183.1, 172.8, 166.4, 161.3, 151.0, 137.0, 134.7, 134.1, 132.7, 131.4, 129.3, 127.6, 126.7, 125.9, 118.7, 115.2, 46.6, 38.5, 28.5, 26.4, 14.9; HRMS (ESI.sup.+, TOF) calculated for C.sub.21H.sub.20N.sub.3O.sub.4[M+H].sup.+ 378.1454, found 378.1448.
(7)N-(3-(1H-imidazol-1-yl)propyl)-2-ethyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (Compound 11)
[0208] ##STR00073##
[0209] To a stirred solution of 2-Ethyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxylic acid (92 mg, 0.34 mmol), DMAP (42 mg, 0.34 mmol) and HBTU (142 mg, 0.37 mmol) in DMF (1.7 mL) was added 1-(3-Aminopropyl)imidazole (49 μL, 0.41 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 1 day. Then the crude was washed by H.sub.2O, extracted with CH.sub.2Cl.sub.2 (10 mL×3), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/19) to give compound 11 as a yellow solid (116 mg, 90% yield). R.sub.f (5% MeOH/CH.sub.2Cl.sub.2) 0.31; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.82 (s, 1H), 8.26-8.18 (m, 2H), 7.84-7.74 (m, 2H), 7.66 (s, 1H), 7.08 (s, 1H), 7.02 (s, 1H), 4.12 (t, J=7.0 Hz, 2H), 3.46 (dd, J=12.5, 6.2 Hz, 2H), 3.35 (q, J=7.5 Hz, 2H), 2.24-2.10 (m, 2H), 1.36 (t, J=7.5 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 183.5, 173.0, 171.2, 161.5, 151.3, 137.3, 134.8, 134.2, 132.8, 131.6, 129.5, 127.8, 126.9, 118.9, 114.4, 44.6, 36.3, 31.0, 22.1, 12.0; HRMS (MALDI*, TOF) calculated for C.sub.21H.sub.20N.sub.3O.sub.4 [M+H].sup.+ 378.1448, found 378.1457.
(8)N-(3-(1H-imidazol-1-yl)propyl)-2-isopropyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (Compound 10)
[0210] ##STR00074##
[0211] To a stirred solution of 2-Isopropyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxylic acid (41.8 mg, 0.15 mmol), DMAP (18 mg, 0.15 mmol) and HBTU (61 mg, 0.16 mmol) in DMF (0.74 mL) was added 1-(3-Aminopropyl)imidazole (21 μL, 0.18 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 1 day. Then the crude was washed by H.sub.2O, extracted with CH.sub.2Cl.sub.2 (10 mL×3), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 1/199 to 1/19) to give compound 10 as a yellow solid (52 mg, 90% yield). R.sub.f (5% MeOH/CH.sub.2Cl.sub.2) 0.32; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.83 (s, 1H), 8.20 (td, J=5.3, 1.8 Hz, 2H), 7.83-7.73 (m, 2H), 7.72 (s, 1H), 7.08 (s, 1H), 7.05 (s, 1H), 4.34-4.27 (m, 1H), 4.14 (t, J=7.0 Hz, 2H), 3.46 (dd, J=12.4, 6.2 Hz, 2H), 2.17 (quint, J=6.8 Hz, 2H), 1.37 (s, 3H), 1.36 (s, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 183.6, 174.1, 172.9, 161.6, 151.3, 134.8, 134.2, 132.8, 131.6, 128.7, 127.7, 126.8, 125.9, 113.5, 44.8, 36.2, 30.9, 27.7, 20.4; HRMS (ESI.sup.+, TOF) calculated for C.sub.22H.sub.22N.sub.3O.sub.4 [M+H].sup.+ 392.1610, found 392.1605.
(9)N-(3-(1H-imidazol-1-yl)propyl)-4,9-dioxo-2-phenyl-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (Compound 9)
[0212] ##STR00075##
[0213] To a stirred solution of 4,9-Dioxo-2-phenyl-4,9-dihydronaphtho[2,3-b]furan-3-carboxylic acid (50.9 mg, 0.16 mmol), DMAP (19.5 mg, 0.16 mmol) and HBTU (73 mg, 0.19 mmol) in DMF (0.8 mL) was added 1-(3-Aminopropyl)imidazole (29 μL, 0.24 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 1 day. Then the crude was washed by H.sub.2O, extracted with CH.sub.2Cl.sub.2 (10 mL×3), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/19) to give compound 9 as a yellow solid (38 mg, 56% yield). R.sub.f (5% MeOH/CH.sub.2Cl.sub.2) 0.32; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.22 (s, 1H), 8.26-8.22 (m, 1H), 8.12-8.10 (m, 1H), 7.92-7.89 (m, 2H), 7.84-7.80 (m, 2H), 7.62 (s, 1H), 7.50-7.44 (m, 3H), 7.06 (s, 1H), 7.01 (s, 1H), 4.14 (t, J=7.0 Hz, 2H), 3.48 (dd, J=12.5, 6.3 Hz, 2H), 2.18 (quint, J=6.8 Hz, 2H); HRMS (ESI.sup.+, TOF) calculated for C.sub.25H.sub.20N.sub.3O.sub.4 [M+H].sup.+ 426.1454, found 426.1451.
(10)N-(3-(1H-imidazol-1-yl)propyl)-2-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indole-3-carboxamide (Compound 45)
[0214] ##STR00076##
[0215] To a stirred solution of 2-methyl-4,9-dioxo-4,9-dihydro-1H-benzo[f]indole-3-carboxylic acid (23 mg, 0.09 mmol), DMAP (17 mg, 0.14 mmol) and HBTU (51 mg, 0.14 mmol) in DMF (0.45 mL) was added 1-(3-Aminopropyl)imidazole (13 μL, 0.11 mmol) at 0° C. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 2 days. The residue was purified by flash column chromatography to give compound 45 as a yellow solid (9.1 mg, 28% yield). R.sub.f (5% MeOH/CH.sub.2Cl.sub.2) 0.20; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.33 (t, J=5.4 Hz, 1H), 8.24-8.10 (m, 1H), 8.10-7.98 (m, 1H), 7.75-7.63 (m, 2H), 7.60 (s, 1H), 7.00 (s, 1H), 6.99 (s, 1H), 4.10 (t, J=7.1 Hz, 2H), 3.47 (q, J=6.1 Hz, 2H), 2.67 (s, 3H), 2.17-2.05 (m, 2H); HRMS (ESI.sup.+, TOF) calculated for C.sub.20H.sub.19N.sub.4O.sub.3 [M+H].sup.+ 363.1457, found 363.1454.
(11)N-(3-(1H-pyrrol-1-yl)propyl)-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (Compound 12)
[0216] ##STR00077##
[0217] To a stirred solution of 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid (100 mg, 0.39 mmol), DMAP (47 mg, 0.39 mmol) and HBTU (163 mg, 0.43 mmol) in DMF (1.95 mL) was added 3-(1H-pyrrol-1-yl)propan-1-amine (58 mg, 0.47 mmol) at room temperature. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 1 day. Then the crude was washed by H.sub.2O, extracted with CH.sub.2Cl.sub.2 (10 mL×3), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/49) to give compound 12 as a yellow solid (67 mg, 47% yield). R.sub.f (CH.sub.2Cl.sub.2) 0.07; Mp 143.5-150.7° C.; H NMR (400 MHz, CDCl.sub.3) δ 9.73 (s, 1H), 8.26-8.13 (m, 2H), 7.84-7.71 (m, 2H), 6.74 (t, J=2.1 Hz, 2H), 6.14 (t, J=2.1 Hz, 2H), 4.05 (t, J=6.9 Hz, 2H), 3.42 (dd, J=12.3, 6.6 Hz, 2H), 2.86 (s, 3H), 2.14 (quint, J=6.8 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 183.1, 172.9, 166.5, 161.3, 151.0, 134.7, 134.1, 132.8, 131.5, 127.7, 126.8, 126.0, 120.6, 115.3, 108.2, 47.0, 36.6, 31.2, 14.9; HRMS (MALDI.sup.+, TOF) calculated for C.sub.21H.sub.19N.sub.2O.sub.4 [M+H].sup.+ 363.1339, found 363.1325.
(12) 2-Methyl-N-(3-morpholinopropyl)-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (Compound 20)
[0218] ##STR00078##
[0219] To a stirred solution of 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid (23.8 mg, 0.093 mmol), DMAP (17 mg, 0.14 mmol) and HBTU (54 mg, 0.14 mmol) in DMF (0.47 mL) was added 3-morpholinopropylamine (20.4 μL, 0.14 mmol) at 0° C. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 2 days. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/9) to give compound 20 as a yellow solid. H NMR (400 MHz, CDCl.sub.3) δ 9.69 (s, 1H), 8.26-8.14 (m, 2H), 7.83-7.71 (m, 2H), 3.81-3.66 (m, 4H), 3.51 (dd, J=12.5, 6.8 Hz, 2H), 2.87 (s, 3H), 2.60-2.43 (m, 6H), 1.88 (quint, J=7.2 Hz, 2H); HRMS (ESI.sup.+, TOF) calculated for C.sub.21H.sub.23N.sub.2O.sub.5 [M+H].sup.+ 383.1607, found 383.1604.
(13) 2-Methyl-4,9-dioxo-N-(3-(pyrrolidin-1-yl)propyl)-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (Compound 18)
[0220] ##STR00079##
[0221] To a stirred solution of 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid (80 mg, 0.31 mmol), DMAP (57 mg, 0.47 mmol) and HBTU (177 mg, 0.47 mmol) in DMF (1.6 mL) was added 1-(3-Aminopropyl)pyrrolidine (59 μL, 0.60 mmol) at 0° C. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 36 hr. The residue was purified by flash column chromatography (MeOH/EtOAc/CH.sub.2Cl.sub.2, 0/1/1 to 1/4/5) to give compound 18 as a yellow solid (47 mg, 41% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.72 (s, 1H), 8.20-8.18 (m, 2H), 7.84-7.68 (m, 2H), 3.51 (dd, J=12.5, 6.7 Hz, 2H), 2.86 (s, 3H), 2.66 (t, J=7.6 Hz, 2H), 2.62 (brs, 4H), 1.96-1.86 (m, 2H); 1.81 (brs, 4H); HRMS (ESI.sup.+, TOF) calculated for C.sub.21H.sub.23N.sub.2O.sub.4 [M+H].sup.+ 367.1658, found 367.1657.
(14) 2-Methyl-4,9-dioxo-N-(4-phenylbutyl)-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (Compound 17)
[0222] ##STR00080##
[0223] To a stirred solution of 2-Methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b] furan-3-carboxylic acid (90 mg, 0.35 mmol), DMAP (43 mg, 0.35 mmol) and HBTU (146 mg, 0.39 mmol) in DMF (1.8 mL) was added 4-Phenylbutylamine (72 μL, 0.46 mmol) at 0° C. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 20 hr. The residue was purified by flash column chromatography (EtOAc/hexanes, 1/49 to 1/9) to give compound 17 as a yellow solid (78 mg, 58% yield). R.sub.f (20% EtOAc/hexanes) 0.35; Mp 151.4-152.7° C.; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.66 (s, 1H), 8.26-8.11 (m, 2H), 7.87-7.68 (m, 2H), 7.34-7.09 (m, 5H), 3.47 (dd, J=12.2, 6.6 Hz, 2H), 2.87 (s, 3H), 2.69 (t, J=7.3 Hz, 2H), 1.88-1.66 (m, 4H); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 182.9, 172.9, 166.3, 161.1, 150.9, 142.2, 134.5, 134.0, 132.8, 131.5, 128.4, 128.2, 127.6, 126.7, 126.0, 125.7, 115.5, 39.2, 35.5, 28.9, 28.8, 14.9; HRMS (ESI.sup.+, TOF) calculated for C.sub.24H.sub.21NO.sub.4Na [M+Na].sup.+ 410.1368, found 410.1374.
(15)N-(3-(1H-imidazol-1-yl)propyl)-2-methylbenzofuran-3-carboxamide (Compound 44)
[0224] ##STR00081##
[0225] To a stirred solution of 2-methylbenzofuran-3-carboxylic acid (25 mg, 0.14 mmol), DMAP (25.7 mg, 0.21 mmol) and HBTU (80 mg, 0.21 mmol) in DMF (0.7 mL) was added 1-(3-Aminopropyl)imidazole (19 μL, 0.16 mmol) at 0° C. The solvent was removed by vacuo after the reaction mixture was stirred at room temperature for 1 day. The residue was purified by flash column chromatography (MeOH/CH.sub.2Cl.sub.2, 0/100 to 1/9) to give compound 44 as a colorless oil (37 mg, 95% yield). R.sub.f (5% MeOH/CH.sub.2Cl.sub.2) 0.29; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.58-7.48 (m, 2H), 7.48-7.41 (m, 1H), 7.32-7.25 (m, 2H), 7.05 (s, 1H), 6.97 (s, 1H), 5.97 (s, 1H), 4.07 (t, J=7.0 Hz, 2H), 3.51 (dd, J=13.1, 6.7 Hz, 2H), 2.71 (s, 3H), 2.15 (quint, J=6.9 Hz, 2H); HRMS (ESI.sup.+, TOF) calculated for C.sub.16H.sub.18N.sub.3O.sub.2 [M+H].sup.+ 284.1399, found 284.1392.
(16)N-(3-(1H-imidazol-1-yl)propyl)-4,9-dioxo-2-(trifluoromethyl)-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (RJ-LC-01-47)
[0226] ##STR00082##
[0227] An exemplary synthetic scheme of Compound RJ-LC-01-47 is provided below:
##STR00083##
(17) N-(3-(1H-imidazol-1-yl)propyl)-7-fluoro-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide (RJ-LC-07-48)
[0228] ##STR00084##
[0229] An exemplary synthetic scheme of Compound RJ-LC-07-48 is provided below:
##STR00085##
[0230] An exemplary synthetic scheme of Compound RJ-LC-07-48 (compound 48) is provided below:
##STR00086##
7-Fluoro-2-hydroxynaphthalene-1,4-dione 2
[0231] ##STR00087##
[0232] To a solution of KO.sup.tBu (547 mg, 4.873 mmol) in anhydrous t-BuOH (7.6 mL) which had previously been saturated with O.sub.2 (bubbled for 10 min) was added a solution of 7-fluoro-1-tetralone 1 (100 mg, 0.609 mmol) in anhydrous t-BuOH (2.4 mL), and the resulting red reaction mixture was stirred at ambient temperature for 30 min under O.sub.2 atmosphere. Then, the reaction mixture was cooled down to 0° C. and acidified with 3M HCl until the pH of the mixture became 1-2. The volatiles were removed in vacuo, and the crude material was extracted with CH.sub.2Cl.sub.2. Combined organic layers were dried over anhydrous sodium sulfate then filtered and concentrated under vacuo to afford 7-fluoro-2-hydroxynaphthalene-1,4-dione 2 as a yellow solid (108 mg, 92% yield).
3-(2-Ethoxy-2-oxo-1-(tributylphosphonio)ethyl)-7-fluoro-1,4-dioxo-1,4-dihydronaphthalen-2-olate 3
[0233] ##STR00088##
[0234] To compound 2 (138 mg, 0.718 mmol) with THF (1.0 ml) in a dry and nitrogen-flushed sealed tube at 0° C. was sequentially added ethyl glyoxalate (50% solution in toluene) (263 μL, 1.8 equiv), trifluoroacetic acid (96 μL, 1.8 equiv) and Bu.sub.3P (210 μL, 1.2 equiv), then the reaction mixture was stirred for 3.5 h at 60-64° C. Solvent was removed under vacuo to obtain the crude, which then purified by flash chromatography in neutral Al.sub.2O.sub.3(EtOAc/n-Hexane, 1/1) to provide compound 3 as red gummy liquid (190.0 mg, 63% yield).
Ethyl 7-fluoro-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxylate 4
[0235] ##STR00089##
[0236] To compound 3 (393 mg, 0.821 mmol) with THE (4.1 ml) in a dry and nitrogen-flushed round bottom flask at 0° C. was slowly added acetyl chloride (76 μL, 1.3 equiv) and Et.sub.3N (171 μL, 1.5 equiv), then stirred for 15 min. Solvent was removed under vacuo to obtain the crude, which then purified by flash chromatography (EtOAc/n-Hexane, 1/9) to provide compound 4 as pale yellow solid (199.0 mg, 61% yield).
7-Fluoro-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxylic acid 5
[0237] ##STR00090##
[0238] To compound 4 (355 mg, 1.175 mmol) with THF (9.4 mL) and H.sub.2O (2.35 mL) in a round bottom flask at 0° C. was added 3M NaOH.sub.(aq) (1.17 ml, 4.5 eq), then allowed to room temperature for a period of 5 hr. Solvent was removed under vacuo then dilute with H2O and washed with CH.sub.2Cl.sub.2. The aqueous layer was added 3M HCl.sub.(aq) until pH=1˜2, then extracted with EA and Brine. Combined organic layers were dried over anhydrous sodium sulfate then filtered and concentrated under vacuo to afford the crude carboxylic acid 5 (313 mg, 97% yield).
N-(3-(1H-imidazol-1-yl)propyl)-7-fluoro-2-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-3-carboxamide 6 (RJ-LC-07-48)
[0239] ##STR00091##
[0240] To a solution of carboxylic acid 5 (282 mg, 1.028 mmol) with DMF (10.3 mL) in a round bottom flask at room temperature was added DMAP (126 mg, 1.028 mmol), HBTU (468 mg, 1.234 mmol), 3-(1H-imidazol-1-yl)propan-1-amine (184 μL, 1.542 mmol), then stirred for 6 h. Solvent was removed under vacuo to obtain the crude material, which then purified by flash chromatography (MeOH/CH.sub.2Cl.sub.2, 1/9) to provide product 6 (251 mg, 64% yield).
[0241] An exemplary synthetic scheme of Compound RJ-LC-07-49 (compound 49) is provided below:
##STR00092##
1,4-dioxo-3-(1-(tributylphosphonio)ethyl)-1,4-dihydronaphthalen-2-olate
[0242] ##STR00093##
[0243] To a dry and N2-flushed sealed tube equipped with a stir bar was sequentially added 2-hydroxy-pnaphthoquinone (100 mg, 1 eq.) in dry THF (0.38 M, 1.5 mL), acetaldehyde (240 μL, 7.2 eq.), TFA (160 μL, 1.8 eq.) and tributylphosphine (170 μL, 1.2 eq.). The resulting solution was stirred at 70° C. for 20 h. After completion of the reaction, the crude mixture was concentrated in vacuo and purified by column chromatography (50% EA/hex followed by 1-2% MeOH/DCM) to give a red oil (192 mg, 83%). R.sub.f (100% EA) 0.34. .sup.1H NMR (400 MHz, CDCl3) δ 7.92-8.01 (m, 2H), 7.57 (t, J=7.5 Hz, 1H), 7.45 (t, J=7.4 Hz, 1H), 4.16-4.27 (m, 1H), 2.01-2.28 (m, 6H), 1.32-1.55 (m, 15H), 0.85 (t, 9H). .sup.13C NMR (100 MHz, CDCl3) δ 184.5, 180.5, 170.4, 134.8, 133.6, 131.6, 130.9, 126.1, 125.7, 113.2, 24.3, 24.2, 24.1, 24.0, 20.0, 19.5, 13.3. .sup.3P NMR (162 MHz, CDCl3) δ 36.4.
Ethyl 3-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-2-carboxylate
[0244] ##STR00094##
[0245] To a dry and N.sub.2-flushed 10 mL Schlenk tube equipped with a stir bar and septum was sequentially added SM (100 mg, 1 eq.) in dry THF (0.14 M, 1.8 mL), dry DIPEA (262 μL, 6 eq.) and ethyl oxalyl chloride (44.7 μL, 1.6 eq.). The resulting solution was stirred at 70° C. for 3 h and after completion of the reaction, the crude mixture was concentrated in vacuo and purified by column chromatography (0-40% DCM/hex) to give a pale yellow solid (40 mg, 57%). R.sub.f (20% EA/hex) 0.55. .sup.1H NMR (400 MHz, CDCl3) δ 8.14-8.24 (m, 2H), 7.74-7.79 (m, 2H), 4.44 (q, J=7.2 Hz, 2H), 2.70 (s, 3H), 1.43 (t, J=7.3 Hz, 3H); .sup.13C NMR (100 MHz, CDCl3) δ 181.1, 173.7, 158.6, 152.1, 144.6, 134.2, 134.0, 133.4, 132.4, 129.8, 128.9, 128.6, 126.9, 61.7, 14.2, 10.0
3-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-2-carboxylic acid
[0246] ##STR00095##
[0247] To a test tube equipped with a stir bar and septum was added SM (300 mg, 1 eq.), 7:3 THF/H.sub.2O (0.2 M, 0.63 mL) and NaOH (63 mg, 1.5 eq.). The resulting mixture was stirred at R.T for 12 h. After removal of THF in vacuo, the crude mixture extracted with DCM, then discard the organic layers. The aqueous layers was acidified to pH=2 by 1N HCl and extracted with EA, and the combined organic layers were dried over MgSO.sub.4, filtered, and concentrated to yield a dark yellow solid (269 mg, 99%). R.sub.f (5% MeOH/DCM, silica) 0.13. H NMR (400 MHz, d-MeOH/CDCl3) δ 8.11-8.17 (m, 2H), 7.72-7.78 (m, 2H), 2.66 (s, 3H)
N-(3-(1H-imidazol-1-yl)propyl)-3-methyl-4,9-dioxo-4,9-dihydronaphtho[2,3-b]furan-2-carboxamide (RJ-LC-07-49)
[0248] ##STR00096##
[0249] To a dry and N.sub.2-flushed 10 mL Schlenk flask equipped with stir bar and septum was added SM (11 mg, 1 eq.) and CDI (11.3 mg, 1.05 eq.) in dry DMF (0.05 M, 0.8 mL) and stirred at room temperature for 30 min. After added N-(3-aminopropyl)imidazole (7.7 μL, 1.Seq), the reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the DMF was removed in vacuo and the crude solid was washed with H.sub.2O and Et.sub.2O separately to remove impurities yield a yellow solid (14 mg, 86%). R.sub.f(20% MeOH/CHCl.sub.3) 0.52. .sup.1H NMR (600 MHz, CDCl3) δ 8.16-8.21 (m, 2H), 7.74-7.80 (m, 2H), 7.56 (s, 1H), 7.04-7.10 (m, 2H), 6.98 (s, 1H), 4.07 (t, J=6.9 Hz, 2H), 3.47-3.51 (q, 6.4 Hz, 2H), 2.75 (s, 3H), 2.13-2.19 (quintet, J=6.8 Hz, 2H); .sup.13C NMR (150 MHz, CDCl3) δ 181.1, 174.0, 158.7, 150.8, 146.2, 137.3, 134.4, 134.0, 133.5, 132.2, 129.8, 129.7, 127.5, 127.1, 126.8, 118.7, 118.7, 44.6, 36.5, 31.2, 9.75.
Example 2: Characterization of the Exemplary MCM Inhibitory Compounds
[0250] Select compounds described herein were evaluated for structure-activity analyses in vitro. The inhibitory activity of the compounds against the H1975 cell line was evaluated by Sulforhodamine B colorimetric (SRB) assay. 2×10.sup.3 cells were cultured in 96-well culture plates for 24 hours before use in the experiment. The culture medium was replaced with fresh medium containing the appropriate concentration of compound ranging from 0.005 μM to 10 μM for 72 hours. After an incubation period, the cells were fixed with 10% trichloroacetic acid and stained for 30 min, after which the excess dye was removed by washing repeatedly with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader.
TABLE-US-00001 TABLE 1 IC.sub.50 values and toxicity data of exemplary compounds described herein. Compound Structure IC.sub.50 (nM) 1
(I) Assay of Effect on Cell Cycle Progression, by Exemplary Compounds Described Herein
[0251] The effect of compound 1 on cell cycle progression in several lung cancer cell lines was determined. Panel (A) of
TABLE-US-00002 TABLE 2 IC.sub.50 of compound 1 in different lung cancer and normal cell lines. Cell line IC.sub.50 (uM) PC9 0.037 (human lung adenocarcinoma cells, EGFR del 19 mutation and sensitive to gefitinib) PC9/IR 0.048 (human lung adenocarcinoma cells, EGFR del 19 mutation but resistant to gefitinib) CL1-0 0.123 (human lung adenocarcinoma cells, EGFR wild type and resistant to gefitinib) CL1-5 0.280 (human lung adenocarcinoma cells, EGFR wild type and resistant to gefitinib) A549 0.053 (adenocarcinomic human alveolar basal epithelial cells, EGFR wild type and resistant to gefitinib) H1975 0.077 (non-small cell lung cancer cells, EGFR L858R and T790M mutation and resistant to gefitinib) HS68 >1 (human foreskin fibroblast cells, used as normal cells)
TABLE-US-00003 TABLE 3 Cell cycle analysis and quantification of H1975 cells treated with Compound 1 for 72 h at different dosages. Phase(%) Conc.(nM) subG1 G1 S G2/M 0 5 79 2 17 35 1 70 4 23 70 1 58 7 31 140 4 39 18 38
(II) In Vivo and In Vitro Effect of Exemplary Compounds Described Herein
[0252] To evaluate the anti-lung cancer potency of compound 1 in vivo (Ahn, et al. (2010), Molecular Cancer Therapeutics, 9(11):2859-2868), athymic nude mice that bear established subcutaneous H1975 tumors were daily treated intraperitoneal with compound at 1 mg/kg or 4 mg/kg in 50% PEG400 versus DMSO control for 4 weeks. Exemplary results are shown in Panel (C) of
(III) Effects on DNA Replication by Exemplary Compounds Described Herein
[0253] To evaluate the effects on DNA replication of Compound 1, the levels of BrdU (5′-bromo′-2′-deoxyuridine) incorporation were examined. Exemplary results are shown in Panel (A) of
[0254] In addition, to evaluate the effects of compound 1 on the progression of DNA replication, the number of replication forks was determined by MCM2 immunofluorescence staining in H1975 cells released from the G1 phase with DMSO and with compound 1 treatment. Exemplary results are shown in Panel (B) of
[0255] To evaluate the effects of compound Ion a single round of DNA replication in a cell cycle, H1975 cells were synchronized using a double thymidine block followed by release with DMSO or compound 1-containing medium for 2 to 14 hours. Exemplary results are shown in Panel (C) of
(IV) Effects on MCM2-7 Ubiquitination and Degradation in Lung Cancer Cells by Exemplary Compounds Described Herein
[0256] To evaluate the effects of Compound 1 on modulating the expression of different MCM protein components, immunoblotting of DMSO or compound 1-treated (at 140 nM) H1975 cell lysates was performed. Exemplary results are shown in Panels (A)-(C) of
[0257] To evaluate the effects of Compound 1 on triggering MCM2, MCM6, and MCM7 degradation through transcriptional or post-translational regulation, compound 1-treated H1975 cells were harvested at 48 hours with or without 10 μM MG132 treatment for 12 hours; and the protein expression levels of MCM2/6/7 were examined by immunoblotting. After treating H1975 cells with DMSO or compound 1 for 24 hours, it was observed that compound 1 induced a substantial decrease in MCM protein levels without a change in their messenger RNA levels, and this phenomenon was markedly reversed in the presence of the proteasome inhibitor MG132, especially in MCM2 expression (Panel (B) of
[0258] To determine whether Compound 1 triggers MCM2 degradation through promoting MCM2 ubiquitination, Myc-tagged ubiquitin (Myc-Ub) was transfected into H1975 cells treated with or without compound 1 and followed by MCM2 immunoprecipitation. The cells were transiently transfected with pcDNA3-Myc-Ub and treated with or without 140 nM of compound 1 combined with MG132. After 12 hours, the cells were harvested and MCM2 proteins were precipitated with specific anti-MCM2 antibodies. The ubiqiuitination pattern were detected by immunoblotting. IgG served as the antibody control in the immunoprecipitation experiment. Ubiquitylated MCM2 was detected in treated cells co-expressing Myc-Ub, and the ubiquitination extent was increased in the presence of MG132 (Panel (C) of
(V) Effects on DNA Binding Ability of MCM2 by Exemplary Compounds Described Herein
[0259] To explore whether Compound 1-induced protein degradation also interfered with the DNA binding ability of MCM2, HI 975 cells were synchronized using a double thymidine block followed by release with DMSO or compound 1 treatment for 3 to 9 hours. Exemplary results are shown in Panel (D) of
[0260] To determine whether manipulation of MCM2 expression can induce cell apoptosis, lentivirus-based short hairpin RNAs (shRNAs) were used to silence the expression levels of MCM2 in H1975 cell lines, and the effects on cell growth were examined. Exemplary results are shown in Panel (E) of
(VI) Target Identification Experiments of Exemplary Compounds Described Herein
[0261] To explore the mechanism of action of Compound 1, preliminary target identification experiments involving affinity-based pull downs were carried out. Panel (A) of
##STR00108##
[0262] In Scheme 3, a polyethylene glycol (PEG) reagent (carboxylate-PEG12-amine) was attached to beads to reduce nonspecific binding on the bead surface. Next, the carboxylic acid end of the tentacle was coupled with propargylamine to give alkynyl-PEG12-MBs, which was then conjugated with N-(2-Azidoethyl)-2-methyl-4, 9-dioxo-4, 9-dihydronaphtho [2, 3-b]furan-3-carboxamide by Cu catalyzed Click reaction.
Carboxyl-PEG.sub.12-MBs.sup.57
##STR00109##
[0263] Carboxylate magnetic beads (10 mg) were dispersed into MES (50 mM, pH 6.0, 300 VL). N-hydroxysuccinimide (NHS, 3.5 mg, 0.03 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, 5.7 mg, 0.03 mmol) were added to the solution and stirred for 1 hr at room temperature. The resulting beads were washed with PBS (50 mM, pH 7.0, 300 μL x 2) to remove excess NHS and EDC.HCl. 200 μL of 1 mM carboxylated-PEG.sub.12-amine (pH 7.8 in 50 mM HEPES) was added to the beads and then stirred for 6 hr at room temperature. After separation with a magnet, the beads were washed with MES (50 mM, pH 6.0) to give carboxyl-PEG.sub.12-MBs.
[0264] Exemplary syntheses of carboxylate magnetic beads, as depicted in Scheme 3 above, are also described in Hung et al, J. Am. Chem. Soc., 135(16): 5934-5937.
Alkynyl-PEG.SUB.12.-MBs
[0265] ##STR00110##
[0266] Carboxyl-PEG.sub.12-MBs (10 mg) were dispersed into MES (50 mM, pH 6.0, 300 μL). N-hydroxysuccinimide (NHS, 3.5 mg, 0.03 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, 5.7 mg, 0.03 mmol) were added to the solution and stirred for 3 hr at room temperature. The resulting beads were washed with PBS (50 mM, pH 7.0, 300 μL×2) to remove excess NHS and EDC.HCl. 200 μL of 1 mM propargylamine (pH 7.0 in 50 mM PBS) was added to the beads and then stirred for 6 hr at room temperature. After separation with a magnet, the beads were washed with MES (50 mM, pH 6.0) to give alkynyl-PEG.sub.12-MBs.
Mag-Beads-Compound 1
[0267] ##STR00111##
[0268] To a solution of alkynyl-PEG.sub.12-MBs (10 mg), azide 3 (1.3 mg, 4.0 μmol), CuSO.sub.4 in 0.1 M H.sub.2O (5 μL, 0.5 μmol) and sodium ascorbate in 0.1 M H.sub.2O (2 μL, 0.2 μmol) was in t-Butanol/H.sub.2O (0.2 mL, 1/1, v/v) and in Microwave condition: 100 W, 60° C. for 30 min. After separation with a magnet, the beads were washed with MES (50 mM, pH 6.0) to give the Mag-beads-compound 1 complex.
[0269] Returning to
[0270] In
[0271] Panel (F) of
[0272] The results of the biological experiments suggest that compound 1, may target to the N-terminus of MCM2 and may contribute to the induction of ubiquitinated-MCM2 through proteasome degradation. The MCM2 degradation may further interfere the formation of MCM complex, cause inhibition of DNA replication and prolong the duration of S phase in cancer cell proliferation; may promote cell apoptosis in vitro; and may inhibit tumor growth in vivo.
Example 3: Anti-Bacterial Activity of Compound 1 Against MRSA
[0273] To investigate the antibacterial activity of Compound 1 against methicillin resistant Staphylococcus aureus (MRSA), a panel of clinical bacterial strains including 15 Staphylococcus, 4 Streptococcus, 2 Enterococcus, 5 gram negative and 15 clinical isolate strains were used to evaluate the antimicrobial efficacy by the disc diffusion method via determination of the surrounding inhibition zones, as well as by evaluating the MIC using the broth micro dilution method. In disk diffusion test, compound land well known anti-bacterial drug, ciprofloxacin were dissolved in DMSO and 10 μl was added to sterile 6 mm diameter paper disk. Bacteria were grown in Mueller Hinton agar (Difco) for 18 h at 37° C. in an incubator. After incubation, inhibition zones were visually measured along the edge of the disks and the plates were photographed. Compound 1 showed antimicrobial activity against all the MRSA strains at 5 μg concentration as determined by disc diffusion method, especially three of the tested bacteria strains are ciprofloxacin resistant strains (
TABLE-US-00004 TABLE 4 Qualitative antibacterial activity of 5 μg Ciprofloxacin and Compound 1 Inhibition zone (mm) Strain Ciprofloxacin Compound 1 Resistances or genotypes ATCC25923 28 26 MSSA NTUH-2102 6 26 MRSA (SCCmec II, ST5, FA-S) NTUH-9383 6 29 MRSA (SCCmec III, ST239, FA-R) NTUH-4578 30 27 MRSA (SCCmec IV, ST59) PM1 30, 23 25 MRSA (SCCmec V.sub.T, ST59) NTUH-104-2 6 24 MRSA (SCCmec V.sub.T, ST45) SA10451 26 24 SA10780 27 25 SAI 1863 28 25 MRSA14979 28 25 MRSA15211 28 26
TABLE-US-00005 TABLE 5 Quantitative Antibacterial activity of compound 1 by MIC determination assays MIC (μg/ml) Compound I Resistances or No. Species Strain 100% 50% genotypes Source 1 Staphylococcus ATCC2921 3 4 1 MSSA aureus 2 Staphylococcus ATCC12598 4 0.5 MSSA aureus 3 Staphylococcus ATCC25923 4 2 MSSA aureus 4 Staphylococcus ATCC33592 1 0.5 MRSA (SCCmec aureus III) 5 Staphylococcus ATCC49476 1 0.5 MRSA (SCCmec III) aureus 6 Staphylococcus TSHR17 4 0.5 MRSA (SCCmec aureus V.sub.T) 7 Staphylococcus ATCC12228 2 0.5 methicillin- epidermidis susceptible 8 Staphylococcus ATCC35984 1 0.25 methicillin-resistant epidermidis 9 Staphylococcus ATCC29970 4 2 methicillin- haemolyticus susceptible 10 Streptococcus ATCC43143 4 2 S. bovis gallolyticus subsp. gallolyticus 11 Streptococcus ATCC43144 4 2 S. bovis gallolyticus subsp. pasteurianus 12 Streptococcus ATCC BAA- 8 4 S. bovis infantarius subsp. 102 infantarius 13 Streptococcus ATCC BAA- 4 2 S. bovis infantarius subsp. 103 coli 14 Enterococcus ATCC19434 16 2 faecium 15 Enterococcus ATCC19433 32 16 faecalis 17 Escherichia coli ATCC33625 >32 >32 gram negative 18 Pseudomonas ATCC27853 >32 >32 gram negative aeruginosa 19 Shigella sonnei ATCC9290 >32 >32 gram negative 20 Klebsiella ATCC700603 >32 >32 gram negative pneumoniae 21 Acinetobacter ATCC19606 >32 >32 gram negative baumannii 22 Staphylococcus NTUH-4400 2 1 MRSA (SCCmec II, clinical isolate aureus ST5, FA-R) 23 Staphylococcus NTUH-2102 4 1 MRSA (SCCmec II, clinical isolate aureus ST5, FA-S) 24 Staphylococcus NTUH-560 4 1 MRSA (SCCmec II, clinical isolate aureus GM-S) 25 Staphylococcus NTUH-9383 1 0.5 MRSA (SCCmec clinical isolate aureus III, ST239, FA-R) 26 Staphylococcus NTUH-2417 1 0.5 MRSA (SCCmec clinical isolate aureus III, SXT-S) 27 Staphylococcus NTUH-1-2868 1 0.5 MRSA (SCCmec clinical isolate aureus III, ST239, FA-R) 28 Staphylococcus NTUH-4578 4 1 MRSA (SCCmec clinical isolate aureus IV, ST59) 29 Staphylococcus NTUH-406-2 4 0.5 MRSA (SCCmec clinical isolate aureus IV, spa type t008) 30 Staphylococcus NTUH-2643 4 1 MRSA (SCCmec clinical isolate aureus IV, spa type to 15) 31 Staphylococcus NTUH-8038 4 1 MRSA (SCCmec clinical isolate aureus IV, spa type t189) 32 Staphylococcus PM1 4 1 MRSA (SCCmec clinical isolate aureus V.sub.T, ST59) 33 Staphylococcus NTUH-104-2 1 0.5 MRSA (SCCmec clinical isolate aureus V.sub.T, ST45) 34 Staphylococcus NTUH-2967 4 1 MRSA (SCCmec clinical isolate aureus V.sub.T, ST1) 35 Staphylococcus NTUH-4179 4 0.5 methicillin-resistant clinical isolate lugdunensis 36 Staphylococcus NTUH-6767 4 0.5 methicillin-resistant clinical isolate lugdunensis
[0274] Thus, results of this study demonstrated that GRC4583 (Compound 1) showed stronger antibacterial activity against MRSA as compared to ciprofloxacin.
Example 4: Anti-Cancer and Anti-Bacterial Activity of Compound I and Analogues Thereof
[0275] Compound 1 and its analogues shown in Table 6 below were tested for their activities on cancer cells and bacterial cells using assays described herein. H1975, MDA-MB231, and MCF7 cells were incubated in the presence of the compounds in cell growth medium containing 10% fetal bovine serum for two days in a mammalian cell culture incubatoe. Cell viability were monitored by using Cell-TiterGlo. Staphylococcus aureus and Escherichia coli were incubated in the presence of the compounds in Muller-Hinton broth for 16-24 hours in a 37° C. incubator. Bacterial growth was monitored by using Alamar Blue (Invitrogen).
[0276] The results are provided in Table 7.
TABLE-US-00006 TABLE 6 Structures of Compound 1 Analogues. Compound Name Structure 00056034583 (compound 1) 00055727872
TABLE-US-00007 TABLE 7 Anti-Cancer and Anti-Bacterial Activities of Compound 1 and Analogues Thereof % of the activities in the presence of ~10 μM compound Lung Breast Cancer Breast Cancer Cells Cells (MDA- Cancer Staphylococcus Escherichia Compounds (H1975) MB231) Cells (MCF) aureus coli 00056034583 4.2% 13.7% 6.0% 38.1% >100% (compound 1) (MIC = 2 μg/ml) 00055727872 4.2% 72.0% 44.3% >100% >100% 00053891400 4.0% 12.2% 6.1% >100% >100% 00056034607 4.4% 61.2% 12.9% 75.8% >100% 00054506228 4.6% 54.4% 13.6% 65.4% >100% 00055422996 4.3% 20.9% 6.9% >100% >100% 00055422098 3.7% 69.8% 35.0% >100% >100% 00054811255 4.2% 13.4% 6.5% >100% >100%
Example 5: Anti-Tumor Efficacy of Exemplary Compounds
[0277] Exemplary compounds 48 and 49 were tested for their in vivo effect on tumor growth by measuring H1975 tumor growth following treatment of compounds 48 and 49. To explore the in vivo antitumor efficacy of exemplary compounds 48 and 49, athymic nude mice that bear established subcutaneous H1975 tumors were treated daily intraperitoneally with compounds 48 or 49 at 1 mg/kg or 4 mg/kg versus DMSO control for 4 weeks (n=6 for each group). Results of the in vivo anti-tumor effects of exemplary compounds 48 and 49 are shown in
EQUIVALENTS AND SCOPE
[0278] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
[0279] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
[0280] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
[0281] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.