# Patent

## Title
Compounds and their methods of use

## Abstract

Compounds and compositions comprising compounds that inhibit glutaminase are described herein. Also described herein are methods of using the compounds that inhibit glutaminase in the treatment of cancer.

## Background
CLAIM OF PRIORITY
This application claims priority from U.S. Ser. No. 61/729,321, filed Nov. 21, 2012 which is incorporated herein by reference in its entirety.


BACKGROUND OF INVENTION
Cancer cells rely primarily on glycolysis to generate cellular energy and biochemical intermediates for biosynthesis of lipids and nucleotides, while the majority of “normal” cells in adult tissues utilize aerobic respiration. This fundamental difference in cellular metabolism between cancer cells and normal cells is termed the Warburg Effect. As a result of this difference, pyruvate generated via the glycolytic pathway is converted to lactic acid, rather than being used to acetyl-CoA and eventually, the citrate utilized in a normal citric acid cycle. To compensate for these energetic changes and to maintain a citric acid cycle, cancer cells rely on glutamine metabolism which is achieved through an elevation of glutaminase activity. Exploitation of this phenomenon can be achieved by inhibition of this elevated glutaminase activity.
SUMMARY OF INVENTION
Described herein are heterocyclic containing, pharmaceutically acceptable salts, solvates, and hydrates thereof. The compounds can be used to treat a disorder described herein, for example, by inhibiting glutaminase in a patient. Also provided are compositions (e.g., pharmaceutical compositions) comprising a compound provided herewith and the use of such compositions in methods of treating diseases and conditions, for example, that are associated with the aberrant function of glutaminase or elevated activity of glutaminase, including, e.g., cancer.
In one embodiment, provided is a compound of formula (I) or a pharmaceutically acceptable salt thereof:




wherein
X is a bond, —S—, —S(O)—, —SO2—, —CH═CH—, or —C(O)—;
each W, Y and Z is independently —S—, —CH═, —O—, —N═, or —NH—, provided that (1) at least one of W, Y and Z is not —CH═ and (2) when one of W is —S— and the Y in the same ring is N, then the Z in the same ring is not —CH═;
each R1 and R2 is independently C1-6 alkylene-R4, —N(R3)—R4, —N(R3)—C(O)—R4, —C(O)—N(R3)—R4, —N(R3)—C(O)—O—R4, —N(R3)—C(O)—N(R3)—R4, —O—C(O)—N(R3)—R4, —N(R3)—C(O)—C1-6 alkylene-C(O)—R4, —N(R3)—C(O)—C1-6 alkylene-N(R3)—C(O)—R4 or —N(R3a)—C(O)—CH2—N(R3)—C(O)—R4;
each R3 is independently hydrogen, C1-6 alkyl or aryl;
each R4 is independently C1-6 alkyl, C1-6 alkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl, cycloalkyl or cycloalkylalkyl, each of which is substituted with 0-3 occurrences of R5, or two adjacent R5 moieties, taken together with the atoms to which they are attached form a heterocyclyl, heteroaryl, cycloalkyl or aryl;
each R5 is independently oxo (═O), C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, cyano, halo, —OH, —SH, —OCF3, —SO2—C1-6 alkyl, —NO2, —N(R7)—C(O)—C1-6 alkyl, —N(R6)2, —O—C(O)—C1-6 alkyl, C3-7 cycloalkyl, (C3-7 cycloalkyl)alkyl, aryl, aryloxy, —C(O)-aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl or heterocyclyl, wherein each aryl, heteroaryl or heterocyclyl is further substituted with 0-3 occurrences of R7;
each R6 is independently hydrogen, fluoro, OH or C1-6 alkyl;
each R7 is independently hydrogen, C1-6 alkyl, —OH, —SH, cyano, halo, —CF3, —OCF3, —SO2—C1-6 alkyl, —NO2, —N(R7)—C(O)—C1-6 alkyl, —N(R6)2 or C1-6 alkoxy;
m is 1, 2 or 3;
n is 1, 2 or 3; provided that when X is bond, the sum of m and n is from 3 to 6 and when X is —S—, —S(O)—, —SO2—, —CH═CH—, or —C(O)—, the sum of m and n is from 2 to 4;
o is 1, 2 or 3; and
p is 1, 2 or 3;
with the proviso that: (1) when X is —S—, m and n are both 2, each R6 is H, then (i) R1 and R2 are not both —NHC(O)—R4, wherein R4 is C1-6 alkyl, monocyclic aryl, monocyclic heteroaryl, monocyclic aralkyl, monocyclic heteroaralkyl and each member of R4 is substituted with 0-3 occurrences of R5; and (ii) R1 and R2 are not both —NHC(O)O-methyl, —NHC(O)O-ethyl, —NHC(±)-6-pyrimidine-2,4(1H,3H)-dionyl, or —NHC(O)NH-phenyl wherein said phenyl of the —NHC(O)NH-phenyl moiety is optionally substituted with 1 or 2 groups selected from methyl, nitro, and halo;
(2) when X is —S—, m and n are both 1, each R6 is H, then (i) R1 and R2 are not both —NH-phenyl or —NH-4-methoxy-phenyl;
(3) when X is a bond, the sum of m and n is 3, each R6 is H, then R1 and R2 are not both NHC(O)-phenyl;
(4) when X is a bond, m and n are both 2, each R6 is H, then R1 and R2 are not both —NHC(O)-furanyl, —NHC(O)-phenyl, —NHC(O)-o-methoxy-phenyl, —NHC(O)—C1-6 alkyl, —NH-benzyl, or —NH-phenyl wherein said phenyl of the —NH-phenyl moiety is substituted with 0-3 occurrences of R5;
(5) when X is a bond, the sum of m and n is 5, each R6 is H, then R1 and R2 are not both —NHC(O)—C1-6 alkyl, —NHC(O)-cyclohexyl, or —NH-phenyl wherein said phenyl of the —NH-phenyl moiety is optionally substituted with methyl; and
(6) when X is a bond, m and n are both 3, each R6 is H, then R1 and R2 are not both NH-phenyl.
In another embodiment, provided is a composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the composition is a pharmaceutical composition.
In another embodiment, provided herein is a method for treating or preventing a disease, condition or disorder as described (e.g., treating) herein comprising administering a compound described herein, a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising a compound described herein or a pharmaceutically acceptable salt thereof.
In another embodiment, provided herein is a method of inhibiting glutaminase, e.g, in a patient in need thereof. In some embodiments, provided here is reducing the level of the product of glutaminase in a subject, e.g., a patient in need thereof. The methods include administering an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof to a subject in need thereof, thereby inhibiting the level of glutaminase in the subject.
In another embodiment, provided herein is a method of treating a subject suffering from or susceptible to a disease or disorder associated with the aberrant function of glutaminase or elevated activity of glutaminase in a subject in need thereof. The method comprises the step of administering an effective amount of a compound described herein to the subject in need thereof, thereby treating, preventing or ameliorating the disease or disorder in the subject. In certain embodiments, the compound is provided in a pharmaceutical composition. In certain embodiments, the method includes identifying or selecting a subject who would benefit from inhibiting glutaminase or decreasing the level of glutaminase. E.g., the subject can be identified on the basis of the level of glutaminase activity in a cell or tissue sample of the subject for treatment of cancer associated with aberrant glutaminase function or activity. In another embodiment, the selected subject is a patient suffering from or susceptible to a disorder or disease identified herein, e.g., a disorder characterized by unwanted cell growth or proliferation, e.g., cancer or other neoplastic disorders.
In another embodiment, provided herein is a method for treating cancer in a subject, the method comprising: optionally, acquiring a subject sample; acquiring an evaluation of or evaluating the subject sample, wherein the subject sample is characterized by i) a low level of E-cadherin expression compared to a reference standard, ii) a high level of vimentin expression compared to a reference standard, or iii) a low or decreased level of pyruvate carboxylase expression; and administering to the subject in need thereof a therapeutically effective amount of a compound described here. In some embodiments, the subject sample is characterized by i) a low level of E-cadherin expression compared to a reference standard and ii) a high level of vimentin expression compared to a reference standard. In some embodiments, the subject sample is characterized or further characterized by low or decreased levels of pyruvate carboxylase expression compared to a reference standard.
In another embodiment, provided herein is a method for treating cancer in a subject characterized by i) a low level of E-cadherin expression compared to a reference standard, ii) a high level of vimentin expression compared to a reference standard, or iii) a low or decreased level of pyruvate carboxylase expression; comprising administering to the subject in need thereof a therapeutically effective amount of a compound described here. In some embodiments, the subject is characterized by i) a low level of E-cadherin expression compared to a reference standard and ii) a high level of vimentin expression compared to a reference standard. In some embodiments, the subject is characterized or further characterized by low or decreased levels of pyruvate carboxylase expression compared to a reference standard.


DETAILED DESCRIPTION
The details of construction and the arrangement of components set forth in the following description or illustrated in the drawings are not meant to be limiting. Embodiments can be practiced or carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
Compounds
Described herein are compounds and compositions that inhibit glutaminase. Compounds that inhibit glutaminase, can be used to treat disorders such as neoplastic disorders (e.g., cancer).
In one embodiment, provided is a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:




wherein
X is a bond, —S—, —S(O)—, —SO2—, —CH═CH—, or —C(O)—;
each W, Y and Z is independently —S—, —CH═, —O—, —N═, or —NH—, provided that (1) at least one of W, Y and Z is not —CH═ and (2) when one of W is —S— and the Y in the same ring is N, then the Z in the same ring is not —CH═;
each R1 and R2 is independently C1-6 alkylene-R4, —N(R3)—R4, —N(R3)—C(O)—R4, —C(O)—N(R3)—R4, —N(R3)—C(O)—O—R4, —N(R3)—C(O)—N(R3)—R4, —O—C(O)—N(R3)—R4, —N(R3)—C(O)—C1-6 alkylene-C(O)—R4, —N(R3)—C(O)—C1-16 alkylene-N(R3)—C(O)—R4 or —N(R3a)—C(O)—CH2—N(R3)—C(O)—R4;
each R3 is independently hydrogen, C1-6 alkyl or aryl;
each R4 is independently C1-6 alkyl, C1-6 alkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl, cycloalkyl or cycloalkylalkyl, each of which is substituted with 0-3 occurrences of R5, or two adjacent R5 moieties, taken together with the atoms to which they are attached form a heterocyclyl, heteroaryl, cycloalkyl or aryl;
each R5 is independently oxo (═O), C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, cyano, halo, —OH, —SH, —OCF3, —SO2—C1-6 alkyl, —NO2, —N(R7)—C(O)—C1-6 alkyl, —N(R6)2, —O—C(O)—C1-6 alkyl, C3-7 cycloalkyl, (C3-7 cycloalkyl)alkyl, aryl, aryloxy, —C(O)-aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl or heterocyclyl, wherein each aryl, heteroaryl or heterocyclyl is further substituted with 0-3 occurrences of R7;
each R6 is independently hydrogen, fluoro, OH or C1-6 alkyl;
each R7 is independently hydrogen, C1-6 alkyl, —OH, —SH, cyano, halo, —CF3, —OCF3, —SO2—C1-6 alkyl, —NO2, —N(R7)—C(O)—C1-6 alkyl, —N(R6)2 or C1-6 alkoxy;
m is 1, 2 or 3;
n is 1, 2 or 3; provided that when X is bond, the sum of m and n is from 3 to 6 and when X is —S—, —S(O)—, —SO2—, —CH═CH—, or —C(O)—, the sum of m and n is from 2 to 4;
o is 1, 2 or 3; and
p is 1, 2 or 3;
with the proviso that: (1) when X is —S—, m and n are both 2, each R6 is H, then (i) R1 and R2 are not both —NHC(O)—R4, wherein R4 is C1-6 alkyl, monocyclic aryl, monocyclic heteroaryl, monocyclic aralkyl, monocyclic heteroaralkyl and each member of R4 is substituted with 0-3 occurrences of R5; and (ii) R1 and R2 are not both —NHC(O)O-methyl, —NHC(O)O-ethyl, —NHC(±)-6-pyrimidine-2,4(1H,3H)-dionyl, or —NHC(O)NH-phenyl wherein said phenyl of the —NHC(O)NH-phenyl moiety is optionally substituted with 1 or 2 groups selected from methyl, nitro, and halo;
(2) when X is —S—, m and n are both 1, each R6 is H, then (i) R1 and R2 are not both —NH-phenyl or —NH-4-methoxy-phenyl;
(3) when X is a bond, the sum of m and n is 3, each R6 is H, then R1 and R2 are not both NHC(O)-phenyl;
(4) when X is a bond, m and n are both 2, each R6 is H, then R1 and R2 are not both —NHC(O)-furanyl, —NHC(O)-phenyl, —NHC(O)-o-methoxy-phenyl, —NHC(O)—C1-6 alkyl, —NH-benzyl, or —NH-phenyl wherein said phenyl of the —NH-phenyl moiety is substituted with 0-3 occurrences of R5;
(5) when X is a bond, the sum of m and n is 5, each R6 is H, then R1 and R2 are not both —NHC(O)—C1-6 alkyl, —NHC(O)-cyclohexyl, or —NH-phenyl wherein said phenyl of the —NH-phenyl moiety is optionally substituted with methyl; and
(6) when X is a bond, m and n are both 3, each R6 is H, then R1 and R2 are not both NH-phenyl.
In some embodiments, X is —S—. In some embodiments, X is —S(O)—. In some embodiments, X is —SO2—. In some embodiments, X is a bond. In some embodiments, X is —CH═CH—.
In some embodiments, each Y is —N═. In some embodiments, each Z is —N═. In some embodiments, each Z is —O—. In some embodiments, each W is —S—. In some embodiments, each W is —CH═. In some aspects of these embodiments, each W is —S—, each Y is —N═ and each Z is —N═. In other aspects of these embodiments, each W is —CH═, each Z is —O— and each Y is —N═.
In some embodiments, o is 1. In some embodiments, p is 1. In some embodiments, o is 1 and p is 1.
In some embodiments, m is 1. In some embodiments, n is 1. In some embodiments, n is 1 and m is 1. In some aspects of these embodiments, each R6 is hydrogen. In some embodiments, R1 and R2 are the same. In some embodiments, R1 and R2 are different.
In some embodiments, m is 1. In some embodiments, n is 2. In some embodiments, n is 2 and m is 1. In some aspects of these embodiments, each R6 is hydrogen. In some embodiments, R1 and R2 are the same. In some embodiments, R1 and R2 are different.
In some embodiments, m is 2. In some embodiments, n is 1. In some embodiments, n is 1 and m is 2. In some aspects of these embodiments, each R6 is hydrogen. In some embodiments, R1 and R2 are the same. In some embodiments, R1 and R2 are different.
In some embodiments, m is 2. In some embodiments, n is 2. In some embodiments, n is 2 and m is 2. In some aspects of these embodiments, each R6 is hydrogen. In some embodiments, R1 and R2 are the same. In some embodiments, R1 and R2 are different.
In some embodiments, m is 2. In some embodiments, n is 3. In some embodiments, n is 3 and m is 2. In some aspects of these embodiments, each R6 is hydrogen. In some embodiments, two R6 on the same carbon are C1-6 alkyl (e.g., methyl) and the remaining R6 are each hydrogen. In some embodiments, R1 and R2 are the same. In some embodiments, R1 and R2 are different.
In some embodiments, m is 3. In some embodiments, n is 2. In some embodiments, n is 3 and m is 2. In some aspects of these embodiments, each R6 is hydrogen. In some embodiments, two R6 on the same carbon are C1-6 alkyl (e.g., methyl) and the remaining R6 are each hydrogen. In some embodiments, R1 and R2 are the same. In some embodiments, R1 and R2 are different.
In some embodiments, m is 3. In some embodiments, n is 3. In some embodiments, n is 3 and m is 3. In some aspects of these embodiments, each R6 is hydrogen. In some embodiments, R1 and R2 are the same. In some embodiments, R1 and R2 are different.
In some embodiments, R1 and R2 are each —N(R3)—C(O)—O—R4, wherein R3 is hydrogen. In some aspects of these embodiments, each R4 is C1-6 alkyl (e.g., isopropyl, isobutyl or tert-butyl) substituted with 0-3 occurrences of R5. In some aspects of these embodiments, each R4 is C1-6 alkyl (e.g., isopropyl, isobutyl or tert-butyl) substituted with 0 occurrences of R5. In other aspects of these embodiments, each R4 is aryl (e.g., phenyl) substituted with 0-3 occurrences of R5. In other aspects of these embodiments, each R4 is aryl (e.g., phenyl) substituted with 0 occurrences of R5. In other aspects of these embodiments, each R4 is C3-7 cycloalkyl substituted with 0-3 occurrences of R5. In other aspects of these embodiments, each R4 is C3-7 cycloalkyl substituted with 0 occurrences of R5 (e.g., cyclohexyl). In other aspects of these embodiments, each R4 is aralkyl substituted with 0-3 occurrences of R5. In still other aspects of these embodiments, each R4 is aralkyl substituted with 0 occurrences of R5 (e.g., benzyl). In some aspects of these embodiments, R1 and R2 are the same. In other aspects of these embodiments, R1 and R2 are different.
In some embodiments, R1 and R2 are each —N(R3)—C(O)—R4, wherein each R3 is hydrogen. In some embodiments, R1 and R2 are each —N(R3)—C(O)—R4, wherein each R3 is C1-6 alkyl (e.g., methyl). In some aspects of these embodiments, R1 and R2 are the same. In other aspects of these embodiments, R1 and R2 are different.
In some aspects of these embodiments, each R4 is aralkyl substituted with 0-3 occurrences of R5. In other aspects of these embodiments, each R4 is aralkyl substituted with 0 occurrences of R5 (e.g., benzyl, 2-phenylethyl, 1-phenylethyl, 2-phenylpropyl, 3-phenylpropyl or naphthylmethyl). In other aspects of these embodiments, each R4 is aralkyl (e.g., benzyl, 2-phenethyl, 1-phenethyl or phenpropyl) substituted with one occurrence of R5. In further aspects of these embodiments, each R5 is heterocyclyl (e.g., isoindolinone) and is represented by the following structure:




In further aspects of these embodiments, each R5 is C1-6 alkoxy (e.g., methoxy). In other further aspects of these embodiments, each R5 is —OH. In other further aspects of these embodiments, each R5 is C1-6 alkyl (e.g., methyl). In other further aspects of these embodiments, each R5 is —N(Me)2. In other further aspects of these embodiments, each R5 is halo (e.g., chloro). In other further aspects of these embodiments, each R5 is C1-6 alkoxy (e.g., methoxy) substituted with one R6 wherein R6 is aryl (e.g., phenyl).

In other aspects of these embodiments, each R4 is aralkyl (e.g., benzyl) substituted with two occurrences of R5. In further aspects of these embodiments, each R5 is C1-6 alkoxy (e.g., methoxy). In other further aspects of these embodiments, two R5 are halo (e.g., fluoro) and the other two R5 are haloalkyl (e.g., trifluoromethyl). In other further aspects of these embodiments, two R5 are C1-6 alkyl (e.g., methyl) and two R5 are —O—C(O)—C1-6 alkyl (e.g., —OC(O)—CH3), each of which is further substituted with 0 occurrences of R7. In other further aspects, of these embodiments, two adjacent R5 moieties are taken together with the atoms to which they are attached to form a heterocyclyl ring resulting in a moiety of the following structure:




In other further aspects of these embodiments, each R5 is halo (e.g., fluoro). In other aspects of these embodiments, each R4 is aralkyl (e.g., 2-phenylethyl) substituted with 3 occurrences of R5. In further aspects of these embodiments, each R5 is C1-6 alkoxy (e.g., methoxy).

In other aspects of these embodiments, one R4 is aralkyl (e.g., benzyl) substituted with 0 occurrences of R5 and the other R4 is aralkyl (e.g., benzyl) substituted with 1 occurrence of R5. In a further aspect of this embodiment, R5 is C1-6 alkoxy (e.g., ethoxy) which is further substituted with one R6, wherein R6 is —NH2. In other aspects of these embodiments, one R4 is aralkyl (e.g., benzyl) substituted with 0 occurrences of R5 and the other R4 is C1-6 alkyl (e.g., methyl) substituted with 0 occurrences of R5. In other aspects of these embodiments, one R4 is C1-6 alkyl (e.g., methyl) substituted with 0 occurrences of R5 and the other R4 is aralkyl (e.g., benzyl) substituted with 2 occurrences of R5, wherein each R5 is C1-6 alkoxy (e.g., methoxy).
In other aspects of these embodiments, each R4 is cycloalkylalkyl substituted with 0-3 occurrences of R5. In still other aspects of these embodiments, each R4 is cycloalkylalkyl substituted with 0 occurrences of R5 (e.g., cyclohexylmethyl or 3-cyclohexylpropyl). In still other aspects of these embodiments, each R4 is heteroaralkyl substituted with 0-3 occurrences of R5. In other aspects of these embodiments, each R4 is cycloalkylalkyl (e.g., cyclopentylalkyl) substituted with 3 occurrences of R5 wherein two occurrences are oxo and each other occurrence is taken together with the atoms to which they are attached to form an aryl ring substituted with one R7, wherein each R7 is C1-6 alkoxy (e.g., methoxy). In further aspects of these embodiments each R4 is represented by the following structure:




In other aspect of these embodiments, each R4 is heteroaralkyl substituted with 0 occurrences of R5 (e.g., 2-pyridinylmethyl, 3-pyridinylmethyl, 3-pyridinylethyl, 3-benzoisoxazolylmethyl, 3-indolylmethyl, 5-tetrazolylmethyl 2-thiophenylmethyl or 2-thiophenylethyl). In other aspects of these embodiments, each R4 is heteroaralkyl (e.g., 3-pyridinylmethyl, 3-indolylmethyl or 5-isoxazolylmethyl) substituted with one occurrence of R5. In some further aspects of these embodiments, each R5 is C1-6 alkyl (e.g., methyl). In other further aspects of these embodiments, each R5 is C1-6 alkoxy (e.g., methoxy). In other further aspects of these embodiments, each R5 is halo (e.g., chloro). In other aspects of these embodiments, each R4 is heteroaralkyl substituted with 2 occurrences of R5. In some further aspects of these embodiments, each R5 is C1-6 alkyl (e.g., methyl) and the heteroaralkyl has the following structure:




In other aspects of these embodiments, each R4 is C1-6 alkyl (e.g., methyl, isobutyl or tert-pentyl) substituted with 0 occurrences of R5. In other aspects of these embodiments, each R4 is C1-6 alkyl (e.g., isopropyl or n-propyl) substituted with one occurrence of R5, wherein R5 is aryl (e.g., phenyl) substituted with 0 occurrences of R7. In other aspects of these embodiments, each R4 is C1-6 alkyl (e.g., isopropyl) substituted with one occurrence of R5, wherein R5 is aryloxy (e.g., phenoxy) substituted with one occurrence of R7 wherein R7 is halo (e.g., chloro).
In other aspects of these embodiments, each R4 is C3-7 cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) substituted with 0 occurrences of R5. In other aspects of these embodiments, each R4 is C1-6 alkyl (e.g., methyl or ethyl) substituted with 1 occurrence of R5. In further aspects of these embodiments, each R5 is aryloxy (e.g., phenoxy) substituted with 0 occurrences of R7. In other further aspects of these embodiments, each R5 is aryloxy (e.g., phenoxy) substituted with one occurrence of R7 wherein each R7 is C1-6 alkyl (e.g., methyl or isopropyl). In other further aspects of these embodiments, each R5 is aryloxy (e.g., phenoxy) substituted with one occurrence of R7 wherein each R7 is C1-6 alkoxy (e.g., methoxy). In other further aspects of these embodiments, each R5 is aryloxy (e.g., phenoxy) substituted with two occurrences of R6, wherein each R7 is C1-6 alkyl (e.g., methyl). In other aspects of these embodiments, one R4 is C1-6 alkyl (e.g., methyl) and the other R4 is aralkyl (e.g., benzyl), each of which is substituted with 0 occurrences of R5.
In other aspects of these embodiments, each R4 is heterocyclylalkyl (e.g., 2-tetrahydropyranylmethyl, N-piperidinylethyl or 5-benzo[1,3]dioxolylmethyl) substituted with 0 occurrences of R5. In some further aspects of these embodiments, each R4 is heterocyclylalkyl substituted with 0 occurrences of R5 and is represented by the following structure:




In some further aspects of these embodiments, each R4 is heterocyclylalkyl (e.g., 2-tetrahydrofuranyl) substituted with 3 occurrences of R5 wherein two occurrences of R5 are oxo and the remaining taken together with the atoms to which they are attached form an aryl moiety (e.g., phenyl). In some further aspects of these embodiments, each R4 is represented by the following structure:





In other aspects of these embodiments, each R4 is cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) substituted with 0-3 occurrences of R5. In other aspects of these embodiments, each R4 is cycloalkyl (e.g., cyclobutyl, cyclopentyl or cyclohexyl) substituted with 0 occurrences of R5. In other aspects of these embodiments, each R4 is cycloalkyl (e.g., cyclopropyl) substituted with one occurrence of R5. In some further aspects of these embodiments, each R5 is aryl (e.g., phenyl) further substituted with 0 occurrences of R7.
In other aspects of these embodiments, each R4 is C3-7 cycloalkenyl (e.g., cyclopentenyl) substituted with 0-3 occurrences of R5. In other aspects of these embodiments, each R4 is C3-7 cycloalkenyl (e.g., cyclopentenyl) substituted with 0 occurrences of R5.
In other aspects of these embodiments, each R4 is aryl (e.g., phenyl or naphthyl) substituted with 0-3 occurrences of R5. In other aspects of these embodiments, each R4 is aryl (e.g., phenyl or naphthyl) substituted with 0 occurrences of R5.
In other aspects of these embodiments, each R4 is heteroaryl (e.g., 6-benzimidazolyl) substituted with 0-3 occurrences of R5. In other aspects of these embodiments, each R4 is heteroaryl (e.g., 6-benzimidazolyl) substituted with one occurrence of R5. In some further aspects of these embodiments, each R5 is C1-6 alkyl (e.g., methyl).
In other aspects of these embodiments, each R4 is C2-6 alkenyl (e.g., propenyl) substituted with 0-3 occurrences of R5. In other aspects of these embodiments, each R4 is C2-6 alkenyl (e.g., propenyl) substituted with one occurrence of R5. In some further aspects of these embodiments, each R5 is aryl (e.g., phenyl) substituted with 0 occurrences of R7.
In some embodiments, R1 and R2 are each —N(R3a)—C(O)—N(R3)—R4, wherein each R3a and R3 are hydrogen. In some aspects of these embodiments, each R4 is aryl substituted with 0-3 occurrences of R5. In some aspects of these embodiments, each R4 is aryl substituted with 0 occurrences of R5 (e.g., phenyl). In some aspect of these embodiments, each R4 is aryl substituted with one occurrence of R5 (e.g., 3-tolyl or 4-tolyl). In some further aspects of these embodiments, each R5 is C1-6 alkyl (e.g., methyl). In some aspects of these embodiments, R1 and R2 are the same. In other aspects of these embodiments, R1 and R2 are different.
In some embodiments, R1 and R2 are each —N(R3a)—C(O)—N(R3)—R4, wherein each R3a is hydrogen and each R3 is C1-6 alkyl (e.g., methyl). In some aspects of these embodiments, each R4 is aryl substituted with 0-3 occurrences of R5. In some aspects of these embodiments, each R4 is aryl substituted with 0 occurrences of R5 (e.g., phenyl).
In some embodiments, R1 and R2 are each —N(R3a)—C(O)—C1-6 alkylene-N(R3)—C(O)—R4, wherein each R3a is hydrogen and each R3 is C1-6 alkyl (e.g., methyl). In some aspects of these embodiments, R4 is C1-6 alkoxy (e.g., tert-butoxy). In some aspects of these embodiments, R1 and R2 are the same. In other aspects of these embodiments, R1 and R2 are different.
In some embodiments, R1 and R2 are each —N(R3a)—C(O)—C1-6 alkylene-N(R3)—C(O)—R4, wherein each R3a is hydrogen and each R3 is hydrogen. In some aspects of these embodiments, each R4 is C1-6 alkyl (e.g., methyl). In some aspects of these embodiments, each R4 is aryl (e.g., phenyl) substituted with 0 occurrences of R5. In some aspects of these embodiments, R1 and R2 are the same. In other aspects of these embodiments, R1 and R2 are different.
In some embodiments, R1 and R2 are each —N(R3)—C(O)—C1-6 alkylene-C(O)—R4, wherein each R3 is hydrogen. In some aspects of these embodiments, R1 and R2 are each —N(H)—C(O)—CH2CH(CH3)—C(O)—R4 or —N(H)—C(O)—CH2CH2CH2—C(O)—R4. In some aspects of these embodiments, each R4 is C1-6 alkoxy (e.g., ethoxy). In some aspects of these embodiments, R1 and R2 are each —N(H)—C(O)—CH2CH2CH2CH2—C(O)—R4. In some aspects of these embodiments, each R4 is C1-6 alkyl (e.g., methyl). In some aspects of these embodiments, R1 and R2 are each —N(H)—C(O)—CH2CH2—C(O)—R4 or —N(H)—C(O)—CH(CH3)CH2—C(O)—R4. In some aspects of these embodiments, each R4 is aryl (e.g., phenyl) substituted with 0 occurrences of R5. In some aspects of these embodiments, R1 and R2 are the same. In other aspects of these embodiments, R1 and R2 are different.
In some embodiments, R1 and R2 are each —N(R3)—C(O)O—R4, wherein each R3 is hydrogen. In some aspects of these embodiments, each R4 is aryl (e.g., phenyl) substituted with 0 occurrences of R5. In other aspects of these embodiments, each R4 is cycloalkyl (e.g., cyclohexyl) substituted with 0 occurrences of R5. In other aspects of these embodiments, each R4 is C1-6 alkyl (e.g., isopropyl, isobutyl or tert-butyl) substituted with 0 occurrences of R5. In other aspects of these embodiments, each R4 is aralkyl (e.g., benzyl) substituted with 0 occurrences of R5. In some aspects of these embodiments, R1 and R2 are the same. In other aspects of these embodiments, R1 and R2 are different.
In some embodiments, R1 is —N(R3)—C(O)—R4, wherein R3 is hydrogen and R4 is aralkyl (e.g., phenethyl) substituted with one occurrence of R5 and R2 is —N(R3)R4, wherein R3 and R4 are both hydrogen. In some aspects of these embodiments, R5 is —OH.
In some embodiments, a compound of Formula (I) is represented by a compound of Formula (II):




wherein R1, R2, R3, R4, R5, R6, R7, m, n and X are as defined in Formula (I).

In some embodiments, a compound of Formula (I) is represented by a compound of Formula (III):




wherein R3, R4, R5, R6, R7, m, n and X are as defined in Formula (I). In some aspects of these embodiments, each R4 is the same. In other aspects of these embodiments, each R4 is different.

In certain embodiments, exemplary compounds of Formula I include the compounds described in Table 1 and in the Examples. A compound described herein may be tested for its ability to inhibit glutaminase, e.g., by an assay as described in the Examples. For simplicity, the inhibition activity of these compounds is represented as an IC50 tested in an assay of Example A or Example B in Table 1. Exemplary compounds are shown in Table 1 below. As shown, “A” refers to an inhibitor of glutaminase with an IC50<100 nM. “B” refers to inhibitor of glutaminase with an IC50 between 100 nM and 500 nM. “C” refers to inhibitor of glutaminase with an IC50 between 500 nM and 1000 nM. “D” refers to inhibitor of glutaminase with an IC50 between 1 μM and 10 μM. “E” refers to data that is not available.









TABLE 1





Cmpd #
Structure
AC50

















1




D


 


2




B


 


3




D


 


4




A


 


5




C


 


6




C


 


7




B


 


8




C


 


9




D


 


10




B


 


11




B


 


12




D


 


13




A


 


14




A


 


15




B


 


16




D


 


17




B


 


18




D


 


19




A


 


20




D


 


21




A


 


22




B


 


23




C


 


24




D


 


25




D


 


26




B


 


27




A


 


28




A


 


29




A


 


30




A


 


31




A


 


32




D


 


33




B


 


34




A


 


35




B


 


36




A


 


37




A


 


38




B


 


39




A 


 


40




A


 


41




D


 


42




B


 


43




B


 


44




D


 


45




A


 


46




B


 


47




A


 


48




A


 


49




B


 


50




B


 


51




B


 


52




B


 


53




A


 


54




C


 


55




A


 


56




B


 


57




A


 


58




D


 


59




D


 


60




A


 


61




A


 


62




B


 


63




A


 


64




B


 


65




D


 


66




B


 


67




D


 


68




D


 


69




B


 


70




D


 


71




B


 


72




B


 


73




C


 


74




B


 


75




B


 


76




B


 


77




D


 


78




C


 


79




D


 


80




C


 


81




B


 


82




B


 


83




A


 


84




D


 


85




B


 


86




A


 


87




A


 


88




C


 


89




B


 


90




B


 


91




B


 


92




D


 


93




B


 


94




C


 


95




C


 


96




D


 


97




D


 


98




B


 


99




D


 


100




B


 


101




C


 


102




C


 


103




C


 


104




D


 


105




D


 


106




B


 


107




B


 


108




D


 


109




D


 


110




D


 


111




B


 


112




B


 


113




B


 


114




B


 


115




C


 


116




A


 


117




C


 


118




B


 


119




A


 


120




A


 


121




B









The compounds described herein can be made using a variety of synthetic techniques such as those described in the examples provided herein. As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the 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 the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); 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.
The compounds provided herein may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included within the scope. Unless otherwise indicated when a compound is named or depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound. The compounds provided herewith may also contain linkages (e.g., carbon-carbon bonds) or substituents that can restrict bond rotation, e.g. restriction resulting from the presence of a ring or double bond. Accordingly, all cis/trans and E/Z isomers are expressly included.
The compounds provided herein (e.g. of Formula I) may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D or deuterium), and 3H (T or tritium); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like. The compounds provided herein may also be represented in multiple tautomeric forms, in such instances, expressly includes all tautomeric forms of the compounds described herein, even though only a single tautomeric form may be represented (e.g., alkylation of a ring system may result in alkylation at multiple sites; all such reaction products are expressly included). All such isomeric forms of such compounds are expressly included. All crystal forms of the compounds described herein are expressly included.
The compounds provided herein include the compounds themselves, as well as their salts and their prodrugs, if applicable. A salt, for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a compound described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt can also be formed between a cation and a negatively charged substituent (e.g., carboxylate) on a compound described herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives, which, upon administration to a subject, are capable of providing active compounds.
The compounds provided herein may be modified by appending appropriate functionalities to enhance selected biological properties, e.g., targeting to a particular tissue. Such modifications are known in the art and include those which increase biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
In an alternate embodiment, the compounds described herein may be used as platforms or scaffolds that may be utilized in combinatorial chemistry techniques for preparation of derivatives and/or chemical libraries of compounds. Such derivatives and libraries of compounds have biological activity and are useful for identifying and designing compounds possessing a particular activity. Combinatorial techniques suitable for utilizing the compounds described herein are known in the art as exemplified by Obrecht, D. and Villalgrodo, J. M., Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the “split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, A. W., Curr. Opin. Chem. Bio., (1997) 1, 60. Thus, one embodiment relates to a method of using the compounds described herein for generating derivatives or chemical libraries comprising: 1) providing a body comprising a plurality of wells; 2) providing one or more compounds identified by methods described herein in each well; 3) providing an additional one or more chemicals in each well; 4) isolating the resulting one or more products from each well. An alternate embodiment relates to a method of using the compounds described herein for generating derivatives or chemical libraries comprising: 1) providing one or more compounds described herein attached to a solid support; 2) treating the one or more compounds identified by methods described herein attached to a solid support with one or more additional chemicals; 3) isolating the resulting one or more products from the solid support. In the methods described above, “tags” or identifier or labeling moieties may be attached to and/or detached from the compounds described herein or their derivatives, to facilitate tracking, identification or isolation of the desired products or their intermediates. Such moieties are known in the art. The chemicals used in the aforementioned methods may include, for example, solvents, reagents, catalysts, protecting group and deprotecting group reagents and the like. Examples of such chemicals are those that appear in the various synthetic and protecting group chemistry texts and treatises referenced herein.
DEFINITIONS
The term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine.
The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term “alkyl” includes alkenyl, which contains unsaturation in the hydrocarbon chain. The term “haloalkyl” refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). The terms “arylalkyl” or “aralkyl” refer to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “arylalkyl” or “aralkyl” include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.
The term “alkylene” or “cycloalkylene” refers to a divalent alkyl or cycloalkyl, e.g., —CH2—, —CH2CH2—, and —CH2CH2CH2—.
The term “alkoxy” refers to an —O-alkyl radical. The term “haloalkoxy” refers to an alkoxy in which one or more hydrogen atoms are replaced by halo, and includes alkoxy moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkoxy).
The term “aryl” refers to a monocyclic, bicyclic, or tricyclic aromatic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted (e.g., by one or more substituents). Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl. Unless otherwise specified, any ring atom in an aryl can be substituted by one or more substituents.
The term “cycloalkyl” as employed herein includes cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon groups having 3 to 12 carbons. Any substitutable ring atom can be substituted (e.g., by one or more substituents). The term “cycloalkyl” includes cycloalkenyl, which contains unsaturation in the ring. The cycloalkyl groups can contain fused or spiro rings. Fused rings are rings that share a common carbon atom. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclohexyl, methylcyclohexyl, adamantyl, and norbornyl.
The term “cycloalkylalkyl” as employed herein refers to an alkyl group substituted with a cycloalkyl group.
The terms “heterocyclyl” or “heterocyclic group” refer to 3- to 14-membered non-aromatic ring structures (e.g., 3- to 14-membered rings, more preferably 3- to 7-membered rings), whose ring structures include one to four heteroatoms independently selected from O, N and S. The heterocyclyl or heterocyclic groups can contain fused or spiro rings. Heterocycles can also be polycycles, with each group having, e.g., 5-7 ring members. The term “heterocyclyl” or “heterocyclic group” includes saturated and partially saturated heterocyclyl structures. The heteroatom may optionally be the point of attachment of the heterocyclyl substituent.
The term “heteroaryl” refers to a 5-14 membered (i.e., a 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic) aromatic ring system having 1-3 ring heteroatoms if monocyclic, 1-6 ring heteroatoms if bicyclic, or 1-9 ring heteroatoms if tricyclic, said ring heteroatoms independently selected from O, N, and S (e.g., 1-3, 1-6, or 1-9 ring heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Any substitutable ring atom can be substituted (e.g., by one or more substituents).
Bicyclic and tricyclic ring systems containing one or more heteroatoms and both aromatic and non-aromatic rings wherein the point of attachment from the ring system to the rest of the molecule is through a non-aromatic ring are considered to be heterocyclyl groups. Bicyclic or tricyclic ring systems where an aryl or a heteroaryl is fused to a cycloalkyl or heterocyclyl and the point of attachment from the ring system to the rest of the molecule is through an aromatic ring are considered to be aryl or heteroaryl groups.
The term “heterocyclylalkyl”, as used herein, refers to an alkyl group substituted with a heterocycle group.
The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to an alkyl group substituted with a heteroaryl group. The ring heteroatoms of the compounds provided herein include N—O, S(O), and S(O)2.
Aryl, heteroaryl, cycloalkyl, and heterocyclyl groups, either alone or a part of a group (e.g., the aryl portion of an aralkyl group), are optionally substituted at one or more substitutable atoms with, unless specified otherwise, substituents independently selected from: halo, —C≡N, C1-C4 alkyl, ═O, —ORb, —ORb′, —SRb, —SRb′, —(C1-C4 alkyl)-N(Rb)(Rb), —(C1-C4 alkyl)-N(Rb)(Rb′), —N(Rb)(Rb), —N(Rb)(Rb′), —O—(C1-C4 alkyl)-N(Rb)(Rb), —O—(C1-C4 alkyl)-N(Rb)(Rb), —(C1-C4 alkyl)-O—(C1-C4 alkyl)-N(Rb)(Rb), —(C1-C4 alkyl)-O—(C1-C4 alkyl)-N(Rb)(Rb′), —C(O)—N(Rb)(Rb), —(C1-C4 alkyl)-C(O)—N(Rb)(Rb), —(C1-C4 alkyl)-C(O)—N(Rb)(Rb′), —ORb′, Rb′, —C(O)(C1-C4 alkyl), —C(O)Rb′, —C(O)N(Rb′)(Rb), —N(Rb)C(O)(Rb), —N(Rb)C(O)(Rb), N(Rb)SO2(Rb), —SO2N(Rb)(Rb), —N(Rb)SO2(Rb′), and —SO2N(Rb)(Rb′), wherein any alkyl substituent is optionally further substituted with one or more of —OH, —O—(C1-C4 alkyl), halo, —NH2, —NH(C1-C4 alkyl), or —N(C1-C4 alkyl)2;

    
    
        each Rb is independently selected from hydrogen, and —C1-C4 alkyl; or
        two Rbs are taken together with the nitrogen atom to which they are bound to form a 4- to 8-membered heterocyclyl optionally comprising one additional heteroatom selected from N, S, and O; and
        each Rb′ is independently selected from C3-C7 carbocylyl, phenyl, heteroaryl, and heterocyclyl, wherein one or more substitutable positions on said phenyl, cycloalkyl, heteroaryl or heterocycle substituent is optionally further substituted with one or more of —(C1-C4 alkyl), —(C1-C4 fluoroalkyl), —OH, —O—(C1-C4 alkyl), —O—(C1-C4 fluoroalkyl), halo, —NH2, —NH(C1-C4 alkyl), or —N(C1-C4 alkyl)2.
    
    


Heterocyclyl groups, either alone or as part of a group, are optionally substituted on one or more any substitutable nitrogen atom with oxo (═O), —C1-C4 alkyl, or fluoro-substituted C1-C4 alkyl.
The term “substituted” refers to the replacement of a hydrogen atom by another group.
The term “selective” is meant at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or 10-fold greater inhibition of glutaminase than other targets.
The term “inhibitor” as used herein means an agent that measurably slows, stops, decreases or inactivates the enzymatic activity of glutaminase to decrease to a level that is less than the glutaminase normal levels of activity. Inhibitors of glutaminase may be peptides or nucleic acids (e.g., glutamate). An agent can be evaluated to determine if it is an inhibitor by measuring either directly or indirectly the activity of glutaminase when subjected to the agent. The activity of the agent can be measured, for example, against a control substance. In some instances, the activity measured of the agent is for inhibition of glutaminase.
“Acquire” or “acquiring” as the terms are used herein, refer to obtaining possession of a physical entity, or a value, e.g., a numerical value, by “directly acquiring” or “indirectly acquiring” the physical entity or value. “Directly acquiring” means performing a process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. “Indirectly acquiring” refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as “physical analysis”), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the reagent.
“Acquiring a sample” as the term is used herein, refers to obtaining possession of a sample, e.g., a tissue sample or nucleic acid sample, by “directly acquiring” or “indirectly acquiring” the sample. “Directly acquiring a sample” means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample. “Indirectly acquiring a sample” refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample). Directly acquiring a sample includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue, e.g., a tissue in a human patient or a tissue that has was previously isolated from a patient. Exemplary changes include making a physical entity from a starting material, dissecting or scraping a tissue; separating or purifying a substance (e.g., a sample tissue or a nucleic acid sample); combining two or more separate entities into a mixture; performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a sample includes performing a process that includes a physical change in a sample or another substance, e.g., as described above.
As used herein a “low” of E-cadherin expression compared to a reference standard refers a low, decreased, or absent level of E-cadherin expression compared to the level of E-cadherin expression in an epithelial cell as characterized by methods known in the art, e.g., on any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442).
As used herein, a “high” level of vimentin compared to a reference standard refers to a high or increased level of vimentin expression compared to the level of expression of vimentin in an epithelial cell as characterized by methods known in the art, e.g., on any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442).
As used herein, a “low” or “decreased”, level of pyruvate carboxylase expression compared to a reference standard refers to a low, decreased, or absent level of E-cadherin expression compared to the level of E-cadherin expression in an epithelial cell as characterized by methods known in the art, e.g., on any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442).
As used herein, “cancer” and “tumor” are synonymous terms. The term “cancer” or “tumor” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. The cells can possess characteristics typical of a mesenchymal cell, such as characterized on any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442).
The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.
Methods of Evaluating Compounds
Glutaminase activity can be monitored by detecting production of either of the products of the reaction, glutamate or ammonia. In some embodiments, glutamate production is measured because ammonia is a product of any of a number of biological reactions.
Glutamate production can be measured by any of a number of standard methods known in the art, e.g., chemical and chromatographic detection methods and coupled enzyme assays that utilize NADH and glutamate dehydrogenase. Extracellular glutamate concentrations can also be measured in vivo, using microdialysis methods known in the art. One suitable method for measuring glutamate is a microtiter-based two-step assay in which glutamate formed in the initial step is quantitatively deaminated by glutamate dehydrogenase to yield an equivalent amount of NADH (Godfrey et al., 1977; Kvamme et al., 1985), which can then be detected spectrophotometrically.
Methods of Treatment
In one embodiment, provided is a method for treating or preventing a disease, condition or disorder as described herein (e.g., treating) comprising administering a compound, a pharmaceutically acceptable salt of a compound or pharmaceutical composition comprising a compound described herein (e.g., a compound of formula (I) or in Table 1).
The compounds and compositions described herein can be administered to cells in culture, e.g. in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, prevent, and/or diagnose a variety of disorders, including those described herein below.
As used herein, the term “treat” or “treatment” is defined as the application or administration of a compound, alone or in combination with, a second compound to a subject, e.g., a patient, or application or administration of the compound to an isolated tissue or cell, e.g., cell line, from a subject, e.g., a patient, who has a disorder (e.g., a disorder as described herein), a symptom of a disorder, or a predisposition toward a disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, one or more symptoms of the disorder or the predisposition toward the disorder (e.g., to prevent at least one symptom of the disorder or to delay onset of at least one symptom of the disorder).
As used herein, an amount of a compound effective to treat a disorder, or a “therapeutically effective amount” refers to an amount of the compound which is effective, upon single or multiple dose administration to a subject, in treating a cell, or in curing, alleviating, relieving or improving a subject with a disorder beyond that expected in the absence of such treatment.
As used herein, an amount of a compound effective to prevent a disorder, or a “a prophylactically effective amount” of the compound refers to an amount effective, upon single- or multiple-dose administration to the subject, in preventing or delaying the occurrence of the onset or recurrence of a disorder or a symptom of the disorder.
The term “patient” and “subject” are synonymous, and as used herein, refer to an animal, typically a human (i.e., a male or female of any age group, e.g., a pediatric patient or adult patient or other mammal, such as primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys, that will be or has been the object of treatment, observation, and/or experiment. When the term is used in conjunction with administration of a compound or drug, then the patient has been the object of treatment, observation, and/or administration of the compound or drug.
Cancers
The methods described herein can be used with any cancer, for example those described by the National Cancer Institute. A cancer can be evaluated to determine whether it is using a method described herein. Exemplary cancers can include but are not limited to, lung cancer, e.g., non-small cell lung cancer; breast cancer; or hepatocellular carcninoma, osteosarcoma, lipomas, chondrosarcoma, or mesothelioma.
The cancer can be a primary tumor, i.e., located at the anatomical site of tumor growth initiation. The cancer can also be metastatic, i.e., appearing at least a second anatomical site other than the anatomical site of tumor growth initiation. The cancer can be a recurrent cancer, i.e., cancer that returns following treatment, and after a period of time in which the cancer was undetectable. The recurrent cancer can be anatomically located locally to the original tumor, e.g., anatomically near the original tumor; regionally to the original tumor, e.g., in a lymph node located near the original tumor; or distantly to the original tumor, e.g., anatomically in a region remote from the original tumor.
Cancer Combination Therapies
In some embodiments, a compound described herein is administered together with one or more additional cancer treatments. Exemplary cancer treatments include, for example: chemotherapy, targeted therapies such as antibody therapies, immunotherapy, and hormonal therapy. Examples of each of these treatments are provided below.
Chemotherapy
In some embodiments, a compound described herein is administered with one or morechemotherapies. Chemotherapy is the treatment of cancer with drugs that can destroy cancer cells. “Chemotherapy” usually refers to cytotoxic drugs which affect rapidly dividing cells in general, in contrast with targeted therapy. Chemotherapy drugs interfere with cell division in various possible ways, e.g., with the duplication of DNA or the separation of newly formed chromosomes. Most forms of chemotherapy target all rapidly dividing cells and are not specific for cancer cells, although some degree of specificity may come from the inability of many cancer cells to repair DNA damage, while normal cells generally can.
Examples of chemotherapeutic agents used in cancer therapy include, for example, antimetabolites (e.g., folic acid, purine, and pyrimidine derivatives) and alkylating agents (e.g., nitrogen mustards, nitrosoureas, platinum, alkyl sulfonates, hydrazines, triazenes, aziridines, spindle poison, cytotoxic agents, toposimerase inhibitors and others). Exemplary agents include Aclarubicin, Actinomycin, Alitretinon, Altretamine, Aminopterin, Aminolevulinic acid, Amrubicin, Amsacrine, Anagrelide, Arsenic trioxide, Asparaginase, Atrasentan, Belotecan, Bexarotene, endamustine, Bleomycin, Bortezomib, Busulfan, Camptothecin, Capecitabine, Carboplatin, Carboquone, Carmofur, Carmustine, Celecoxib, Chlorambucil, Chlormethine, Cisplatin, Cladribine, Clofarabine, Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin, Decitabine, Demecolcine, Docetaxel, Doxorubicin, Efaproxiral, Elesclomol, Elsamitrucin, Enocitabine, Epirubicin, Estramustine, Etoglucid, Etoposide, Floxuridine, Fludarabine, Fluorouracil (5FU), Fotemustine, Gemcitabine, Gliadel implants, Hydroxycarbamide, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Irofulven, Ixabepilone, Larotaxel, Leucovorin, Liposomal doxorubicin, Liposomal daunorubicin, Lonidamine, Lomustine, Lucanthone, Mannosulfan, Masoprocol, Melphalan, Mercaptopurine, Mesna, Methotrexate, Methyl aminolevulinate, Mitobronitol, Mitoguazone, Mitotane, Mitomycin, Mitoxantrone, Nedaplatin, Nimustine, Oblimersen, Omacetaxine, Ortataxel, Oxaliplatin, Paclitaxel, Pegaspargase, Pemetrexed, Pentostatin, Pirarubicin, Pixantrone, Plicamycin, Porfimer sodium, Prednimustine, Procarbazine, Raltitrexed, Ranimustine, Rubitecan, Sapacitabine, Semustine, Sitimagene ceradenovec, Satraplatin, Streptozocin, Talaporfin, Tegafur-uracil, Temoporfin, Temozolomide, Teniposide, Tesetaxel, Testolactone, Tetranitrate, Thiotepa, Tiazofurin, Tioguanine, Tipifarnib, Topotecan, Trabectedin, Triaziquone, Triethylenemelamine, Triplatin, Tretinoin, Treosulfan, Trofosfamide, Uramustine, Valrubicin, Verteporfin, Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, Vorinostat, Zorubicin, and other cytostatic or cytotoxic agents described herein.
Because some drugs work better together than alone, two or more drugs are often given at the same time. Often, two or more chemotherapy agents are used as combination chemotherapy. In some embodiments, the chemotherapy agents (including combination chemotherapy) can be used in combination with a compound described herein.
Targeted Therapy
In some embodiments, a compound described herein is administered with one or more targeted therapies. Targeted therapy constitutes the use of agents specific for the deregulated proteins of cancer cells. Small molecule targeted therapy drugs are generally inhibitors of enzymatic domains on mutated, overexpressed, or otherwise critical proteins within the cancer cell. Prominent examples are the tyrosine kinase inhibitors such as Axitinib, Bosutinib, Cediranib, dasatinib, erlotinib, imatinib, gefitinib, lapatinib, Lestaurtinib, Nilotinib, Semaxanib, Sorafenib, Sunitinib, and Vandetanib, and also cyclin-dependent kinase inhibitors such as Alvocidib and Seliciclib. Monoclonal antibody therapy is another strategy in which the therapeutic agent is an antibody which specifically binds to a protein on the surface of the cancer cells. Examples include the anti-HER2/neu antibody trastuzumab (HERCEPTIN®) typically used in breast cancer, and the anti-CD20 antibody rituximab and Tositumomab typically used in a variety of B-cell malignancies. Other exemplary anbibodies include Cetuximab, Panitumumab, Trastuzumab, Alemtuzumab, Bevacizumab, Edrecolomab, and Gemtuzumab. Exemplary fusion proteins include Aflibercept and Denileukin diftitox. In some embodiments, the targeted therapy can be used in combination with a compound described herein.
Exemplary additional therapeutic agents can also include epidermal growth factor receptor (EGFR) inhibitors, e.g., cetuximab, panitumumab, gefitinib, erlotinib, nimotuzamab, matuzamab, zalutumumab, or lapatinib. Resistance to EGFR inhibitors can occur as a result of the transition of a cell to a mesenchymal phenotype or a mesenchymal phenotype, and tumors with EGFR mutations and mesenchymal phenotype can be less sensitive to EGFR inhibitors (see for example, Sequist et al., (2011) Sci Transl Med. 3:75. Buck et al., (2007) Mol Cancer Ther. 6: 532; Thomson et al., (2008) Clin Exp Metastasis 25: 843).
Targeted therapy can also involve small peptides as “homing devices” which can bind to cell surface receptors or affected extracellular matrix surrounding the tumor. Radionuclides which are attached to these peptides (e.g., RGDs) eventually kill the cancer cell if the nuclide decays in the vicinity of the cell. An example of such therapy includes BEXXAR®.
Immunotherapy
In some embodiments, a compound described herein is administered with one or more immunotherapies. Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the patient's own immune system to fight the tumor. Contemporary methods for generating an immune response against tumors include intravesicular BCG immunotherapy for superficial bladder cancer, and use of interferons and other cytokines to induce an immune response in renal cell carcinoma and melanoma patients.
Allogeneic hematopoietic stem cell transplantation can be considered a form of immunotherapy, since the donor's immune cells will often attack the tumor in a graft-versus-tumor effect. In some embodiments, the immunotherapy agents can be used in combination with a compound described herein.
Hormonal Therapy
In some embodiments, a compound described herein is administered with one or more hormonal therapies. The growth of some cancers can be inhibited by providing or blocking certain hormones. Common examples of hormone-sensitive tumors include certain types of breast and prostate cancers. Removing or blocking estrogen or testosterone is often an important additional treatment. In certain cancers, administration of hormone agonists, such as progestogens may be therapeutically beneficial. In some embodiments, the hormonal therapy agents can be used in combination with a compound described herein.
Neuronal Disorders
A compound or composition described herein can be used to treat or prevent neuronal cell death as a result of an injury to neuronal tissue, e.g., nervous tissue exposed to an ischemic or hypoxic event, to trauma or to a chronic neurodegenerative disorder. A “neuronal disorder” is a neurological disease or disorder that is associated with glutamate excitotoxicity, e.g., cerebral ischemia or hypoxia resulting from an neurological event such as a stroke or ischemic event. Treatment with the compound may be in an amount effective to provide a neuroprotective effect, e.g., to prevent neuronal cell death.
Compositions and Routes of Administration
The compositions delineated herein include the compounds delineated herein (e.g., a compound described herein), as well as additional therapeutic agents if present, in amounts effective for achieving a modulation of disease or disease symptoms, including those described herein.
The term “pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a subject, together with a compound provided herewith, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions provided herewith include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.
The pharmaceutical compositions provided herewith may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection. The pharmaceutical compositions provided herewith may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution 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 may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
The pharmaceutical compositions provided herewith may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
The pharmaceutical compositions provided herewith may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound provided herewith with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
Topical administration of the pharmaceutical compositions provided herewith is useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds provided herewith include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions provided herewith may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included.
The pharmaceutical compositions provided herewith may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
When the compositions provided herewith comprise a combination of a compound of the formulae described herein and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen. The additional agents may be administered separately, as part of a multiple dose regimen, from the compounds provided herewith. Alternatively, those agents may be part of a single dosage form, mixed together with the compounds provided herewith in a single composition.
The compounds described herein can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, with a dosage ranging from about 0.5 to about 100 mg/kg of body weight, alternatively dosages between 1 mg and 1000 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug. The methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect. Typically, the pharmaceutical compositions provided herewith will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations contain from about 20% to about 80% active compound.
Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.
Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination provided herewith may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
Patient Selection and Monitoring
The compounds described herein can inhibit glutaminase. Accordingly, a patient and/or subject can be selected for treatment using a compound described herein by first evaluating the patient and/or subject to determine whether the subject is in need of inhibition of glutaminase, and if the subject is determined to be in need of glutaminase inhibition, then administering to the subject a compound described herein.
A subject can be evaluated as being in need of glutaminase inhibition using methods known in the art, e.g., by measuring the presence and/or activity of glutaminase in the patient. In some embodiments, the activity and/or level of glutaminase is evaluated in the cancer.
A patient receiving a compound described herein can be monitored, for example, for improvement in the condition and/or adverse effects. Improvement of a patient's condition can be evaluated, for example, by monitoring the growth, absence of growth, or regression of the cancer (e.g., a tumor). In some embodiments, the patient is evaluated using a radiological assay or evaluation of hemolytic parameters.
A patient and/or subject can be selected for treatment using a compound described hereby by optionally, acquiring a patient sample; evaluating the sample to determine whether the sample is characterized by i) a low level of E-cadherin expression compared to a reference standard, ii) a high level of vimentin expression compared to a reference standard, and/or iii) a low or decreased level of pyruvate carboxylase expression compared to a reference standard; and if the patient is determined to have a low level of E-cadherin expression compared to a reference standard, or a high level of vimentin expression compared to a reference standard, then the patient is administered a compound described herein.
In some embodiments, the level of E-cadherin expression is compared to a reference standard, wherein the reference standard is the level of E-cadherin expression in an epithelial cell as characterized on any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442). In some embodiments, the level of E-cadherin expression is low, decreased, or absent compared to the reference standard. In some embodiments, the level of E-cadherin expression is measured by the evaluation of the level of RNA that encodes E-cadherin. In some embodiments, the level of E-cadherin expression is evaluated by the level of E-cadherin protein expression. In some embodiments the level of E-cadherin expression is at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 90% less than the reference standard. In some embodiments the level of E-cadherin expression is at least a 1.5, 2, 5, 10, 15, 20, 25, 50, 75, 100 fold decrease in expression compared to the reference standard.
In some embodiments, the level of vimentin expression is compared to a reference standard, wherein the reference standard is the level of vimentin expression in an epithelial cell as characterized on any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442). In some embodiments, the level of vimentin expression is measured by the evaluation of the level of RNA that encodes vimentin. In some embodiments, the level of vimentin expression is evaluated by the level of vimentin protein expression. In some embodiments the level of vimentin expression is at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 90% greater than the reference standard. In some embodiments the level of vimentin expression is at least a 1.5, 2, 5, 10, 15, 20, 25, 50, 75, 100 fold increase in expression compared to the reference standard.
In some embodiments, the level of pyruvate carboxylase expression is low or decreased compared to a reference standard, wherein the reference standard is the level of pyruvate carboxylase expression in an epithelial cell as characterized on any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442). In some embodiments, the level of pyruvate carboxylase expression is high or increased compared to the reference standard. In some embodiments, the level of pyruvate carboxylase expression is measured by the evaluation of the level of RNA that encodes pyruvate carboxylase. In some embodiments, the level of vimentin expression is evaluated by the level of pyruvate carboxylase protein expression. In some embodiments the level of pyruvate carboxylase expression is at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or 90% greater than the reference standard. In some embodiments the level of pyruvate carboxylase expression is at least a 1.5, 2, 5, 10, 15, 20, 25, 50, 75, 100 fold increase in expression compared to the reference standard.
Patient Sample
The terms “patient sample”, “subject sample”, and “sample” are used interchangeably herein. The patient sample can be a tissue, or bodily fluid, or bodily product. Tissue samples can include fixed, paraffin embedded, fresh, or frozen samples. For example, the tissue sample can include a biopsy, cheek swab. Exemplary tissues include lung, breast, brain, nervous tissue, kidney, ovary, thyroid, pancreas, colon, prostate, lymph node, skin, hair follicles and nails. Exemplary samples include samples derived from solid tumors. Exemplary bodily fluids include blood, plasma, urine, lymph, tears, sweat, saliva, semen, and cerebrospinal fluid. Exemplary bodily products include exhaled breath.
The tissue, fluid or product can be removed from the patient and analyzed. The evaluation can include one or more of: performing the analysis of the tissue, fluid or product; requesting analysis of the tissue fluid or product; requesting results from analysis of the tissue, fluid or product; or receiving the results from analysis of the tissue, fluid or product.
The sample tissue, fluid, or product can be analyzed for the expression level of a gene described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase. The sample tissue, fluid, or product can be analyzed for the expression level of a protein described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase. The sample tissue, fluid or product can further be analyzed for the level of gene expression of a gene or plurality of genes of a preselected signaling pathway or phenotypic pathway, e.g., the epithelial to mesenchymal transition pathway, E-cadherin pathway, vimentin pathway, or the pyruvate carboxylase pathway. The sample tissue, fluid or product can further be analyzed for the level of protein expression of a protein or plurality of proteins of a preselected signaling pathway or phenotypic pathway, e.g., the epithelial to mesenchymal transition pathway, E-cadherin pathway, vimentin pathway, or the pyruvate carboxylase pathway.
Methods of Evaluating Samples
The expression level of a gene described herein, e.g., E-cadherin, vimentin, and pyruvate carboxylase, can be assessed using any of a wide variety of well known methods for detecting expression of a transcribed molecule, gene, protein, mRNA, genomic DNA, or cDNA. Gene expression can be measured or monitored by measure of a gene transcript, e.g., mRNA, by a measure of the quantity of a translated protein, or by a measure of gene product activity; any of which can be measured using standard techniques known to one of skill in the art. Non-limiting examples of such methods include nucleic acid hybridization methods, nucleic acid reverse transcription methods, nucleic acid amplification methods, immunological methods for detection of proteins, protein purification methods, protein function or activity assays.
E-Cadherin
The E-cadherin gene is located on human chromosome 16. E-cadherin is a classical cadherin of the cadherin superfamily. The encoded E-cadherin protein is a calcium dependent cell-cell adhesion glycoprotein comprised of five extracellular cadherin repeats, a transmembrane region and a highly conserved cytoplasmic tail. Mutations in this gene have been correlated with cancer, including gastric, breast, colorectal, thyroid and ovarian cancers. Loss of function of E-cadherin is contemplated to contribute to cancer progression by increasing proliferation, invasion, and/or metastasis. The ectodomain of this protein mediates bacterial adhesion to mammalian cells and the cytoplasmic domain is required for internalization. Identified E-cadherin transcript variants arise from mutation at consensus splice sites.
Vimentin
The vimentin gene is located on human chromosome 10 and encodes a member of the intermediate filament family of proteins. Intermediate filaments, along with microtubules and actin microfilaments, make up the cellular cytoskeleton, which helps maintain cell shape and integrity of the cytoplasm, as well as stabilizing cytoskeletal interactions. Vimentin also functions in mediating immune responses, control of the transport of low-density lipoprotein derived cholesterol from lysosomes to the sites of esterification, and as an organizer of a number of critical proteins involved in attachment, migration, and cell signaling.
Pyruvate Carboxylase (PC)
The PC gene is located on human chromosomes 11 and encodes the protein pyruvate carboxylase, which catalyzes the carboxylation of pyruvate to oxaloacetate. The active enzyme is a homotetramer arranged in a tetrahedron which is located exclusively in the mitochondrial matrix. Pyruvate carboxylase is involved in multiple cellular processes including gluconeogenesis, lipogenesis, insulin secretion and synthesis of the neurotransmitter glutamate. Mutations in this gene have been associated with pyruvate carboxylase deficiency. Alternatively spliced transcript variants with different 5′ UTRs, but encoding the same protein, have been identified.
Nucleic Acid Molecules
The methods described herein can pertain to the evaluation of a sample for the expression of a gene described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase; based on isolated nucleic acids which correspond to the gene described herein, e.g., the mRNA level of E-cadherin; the mRNA level of vimentin; the mRNA level of pyruvate carboxylase. As used herein, the term “nucleic acid” or “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded.
An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. An “isolated” nucleic acid molecule can be free of sequences (such as protein-encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. An “isolated” nucleic acid molecule, such mRNA, can be substantially free of other cellular material cellular material or other contaminating proteins from the cell or tissue source from which the nucleic acid is derived.
A nucleic acid molecule described herein can be isolated using standard molecular biology techniques and the sequence information available in database records known to those of skill in the art. Using all or a portion of such nucleic acid sequences, nucleic acid molecules described herein can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
A nucleic acid molecule described herein can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecules so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
An isolated nucleic acid molecule can comprise a nucleic acid molecule which has a nucleotide sequence complementary to the nucleotide sequence of a nucleic acid corresponding to gene described herein, or to the nucleotide sequence of a nucleic acid encoding a protein which corresponds to the gene described herein. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.
A nucleic acid molecule described herein can comprise only a portion of a nucleic acid sequence. Such nucleic acid molecules can be used, for example, as a probe or primer. The probe/primer can be one or more substantially purified oligonucleotides. Probes based on the sequence of a nucleic acid molecules described herein can be used to detect transcripts or genomic sequences corresponding to the genes described herein. The probe can contain comprise a label group, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in a sample of cells from a patient, e.g., detecting mRNA levels.
Methods for Detection of Gene Expression
Methods of detecting and/or quantifying a gene transcript, e.g., mRNA or cDNA made therefrom, can include but are not limited to Southern Blot analysis, Northern Blot analysis, polymerase chain reaction (PCR) analyses and probe arrays. Methods of detecting and/or quantifying a gene transcript, e.g., mRNA or cDNA made therefrom, can include but are not limited to hybridization based methods, e.g., hybridization with a probe that is specific for the gene transcript, e.g., mRNA or cDNA made therefrom. The level of a gene transcript, e.g., mRNA or cDNA made therefrom, can be assayed by applying the sample, or the mRNA or cDNA made therefrom, or amplified from; to a nucleic acid microarray, or chip array.
The level of a gene transcript, e.g., mRNA or cDNA made therefrom, can be assayed by a polymerase chain reaction (PCR) based method, e.g., quantitative PCR, quantitative real time PCR, real time PCR, reverse transcription PCR, real time reverse transcription PCR. The level of a gene transcript, e.g., mRNA or cDNA made therefrom, can be assayed by a sequencing based method, e.g., quantitative RNA sequencing.
The level of a gene transcript, e.g., mRNA, can be determined by in situ or by in vitro methods known in the art. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from a sample, e.g., from cells of a sample (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155). For in situ methods, mRNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample can be prepared/processed using known histological methods. The sample can then be immobilized on a support, and then contacted with a probe that can hybridize to mRNA that encodes the gene transcript of interest.
Determinations can be based on absolute expression level; normalized expression level, or relative expression level; of a gene transcript, e.g., mRNA. Expression levels can be normalized by correcting the absolute expression level of a gene transcript by comparing its expression level to the expression level of another gene which is stably expressed, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as histone H3 gene or the actin gene. This normalization allows the comparison of the expression level in one sample to another sample, e.g., a first sample taken from a patient to a second sample taken from the same patient, e.g., from another tissue or at a different time point; or between samples from different sources, e.g., a patient sample from one patient to a patient sample from another patient.
The expression level can be provided as a relative expression level. The relative expression level can be determined by comparing the absolute level of expression of the gene transcript, e.g., mRNA, to a reference standard. The reference standard can include the level of expression of the gene transcript of interest in a genotypically or phenotypically defined sample. The reference standard can be the level of expression of the gene transcript of interest, e.g., E-cadherin, vimentin, pyruvate carboxylase, in a cell genotypically or phenotypically characterized as an epithelial cell. An epithelial cell can be characterized as in any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442).
The expression level of a gene transcript described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase, can be measured at least at two time-points to determine if a change in the level of expression has occurred. For example, the level of expression can be measured pre- and post-treatment with a compound described herein, or at one or more time-points while treatment with a compound described herein is ongoing. If the expression level is found to be decreased, e.g., decreased expression of E-cadherin compared to a reference standard and/or increased expression of vimentin compared to a reference standard; the subject may be administered treatment with a compound described herein. The reference standard can be the level of expression of the gene transcript of interest in an epithelial cell characterized. An epithelial cell can be characterized by methods known in the art, e.g., as in any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442).
Proteins
The methods described herein can pertain to the evaluation of a sample for the expression of a gene described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase; based on isolated proteins which correspond to the gene described herein, e.g., the protein level of E-cadherin; the protein level of vimentin; the protein level of pyruvate carboxylase. This can also include the evaluation of biologically active portions, variants, isoforms, or splice variants thereof. The native polypeptide corresponding to the protein of interest can be isolated from the sample by an appropriate purification scheme using standard protein purification techniques known to those of skill in the art.
An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived. The language “substantially free of cellular material” includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated. Biologically active portions of a polypeptide can include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein, which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein.
Methods for Detection of Protein Expression
The level of expression of a protein or polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. Methods of detecting and/or quantifying a protein or polypeptide described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase; can include but are not limited to biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunoassays such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, immunohistochemistry, in situ hybridization, fluorescence-activated cell sorting (FACS) and the like. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express the protein or polypeptide described herein.
A protein or polypeptide can be detected using an immunoassay. As used herein, immunoassays include assays that utilize an antibody to specifically bind to a protein or polypeptide. An immunoassay can be characterized by the detection of specific binding of a protein or polypeptide to an antibody as opposed to the use of other physical or chemical properties to isolate, target, and quantify the polypeptide. The polypeptide can be detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York; Stites & Terr (1991) Basic and Clinical Immunology 7th Edition. Immunoassays for the detection and/or quantification of a protein or polypeptide can take a wide variety of formats well known to those of skill in the art.
An antibody capable of binding to a protein or polypeptide, e.g., an antibody with a detectable label (either directly or indirectly labeled), corresponding to a protein or polypeptide described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase, can be used to detect the protein or polypeptide. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof, e.g., Fab or F(ab′)2 can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling, i.e., physically linking a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The antibody can also be labeled, e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody. An antibody derivative, e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair, e.g., biotin-streptavidin, or an antibody fragment, e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc, which binds specifically with a protein described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase, such as the protein encoded by the open reading frame corresponding to the gene transcript of a protein or polypeptide described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase, or such a protein or polypeptide which has undergone all or a portion of its normal post-translational modification, is used.
Proteins from cells can be isolated using techniques that are well known to those of skill in the art. The protein isolation methods employed can, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
The expression level can be provided as a relative expression level. The relative expression level can be determined by comparing the absolute level of expression of the protein, to a reference standard. The reference standard can include the level of expression of the protein of interest in a genotypically or phenotypically defined sample. The reference standard can be the level of expression of the protein of interest, e.g., E-cadherin, vimentin, pyruvate carboxylase, in a cell genotypically or phenotypically characterized as an epithelial cell. An epithelial cell can be characterized by methods known in the art, e.g., as described in on any one of the following references: (Yauch et al., (2005) Clin Cancer Res 11:24; Savagner et al., (2010) Ann Oncol. 21(suppl 7): vii89; Thiery et al., (2002) Nature Reviews Cancer 2(6):442).
The expression level of a protein or polypeptide described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase, can be measured at least at two time-points to determine if a change in the level of expression has occurred. For example, the level of expression can be measured pre- and post-treatment with a compound described herein, or at one or more time-points while treatment with a compound described herein is ongoing. If the expression level is found to be decreased, e.g., decreased expression of E-cadherin compared to a reference standard and/or increased expression of vimentin compared to a reference standard; the subject may be administered treatment with a compound described herein.
Kits
Also described herein are kits comprising a means to assay the level of gene expression of a gene described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase. For example, the kit can include an agent capable of interacting with a gene expression product of a gene described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase. The kit can include a plurality of agents capable of interacting with gene expression products of a plurality of genes described herein, e.g., E-cadherin, vimentin, pyruvate carboxylase. The agent can include, but is not limited to, an antibody, a plurality of antibodies, an oligonucleotide, or a plurality of oligonucleotides. The gene expression product can include, but is not limited to, a transcribed molecule, a RNA molecule, a polypeptide, a protein, genomic DNA, or cDNA.
The kit can further optionally include reagents for performing the assays described herein. For example, the kit can include buffers, solvents, stabilizers, preservatives, purification columns, detection reagents, and enzymes, which may be necessary for isolating nucleic acids from a patient sample, amplifying the samples, e.g., by qRT-PCR, and applying the samples to the agent described above; or for isolating proteins from a subject sample, and applying the samples to the agent described above; or reagents for directly applying the subject sample to the agent described above. A kit can also include positive and negative control samples, e.g., control nucleic acid samples (e.g., nucleic acid sample from a non-cancer subject, or a non-tumor tissue sample, or a subject who has not received treatment for cancer, or other test samples for testing at the same time as subject samples. A kit can also include instructional material, which may provide guidance for collecting and processing patient samples, applying the samples to the level of gene expression assay, and for interpreting assay results.
The components of the kit can be provided in any form, e.g., liquid, dried, semi-dried, or in lyophilized form, or in a form for storage in a frozen condition. Typically, the components of the kit are provided in a form that is sterile. When reagents are provided in a liquid solution, the liquid solution generally is an aqueous solution, e.g., a sterile aqueous solution. When reagents are provided in a dried form, reconstitution generally is accomplished by the addition of a suitable solvent. The solvent, e.g., sterile buffer, can optionally be provided in the kit.
The kit can include one or more containers for the kit components in a concentration suitable for use in the level of gene expression assays or with instructions for dilution for use in the assay. The kit can contain separate containers, dividers or compartments for the assay components, and the informational material. For example, the positive and negative control samples can be contained in a bottle or vial, the clinically compatible classifier can be sealed in a sterile plastic wrapping, and the informational material can be contained in a plastic sleeve or packet. The kit can include a plurality (e.g., a pack) of individual containers, each containing one or more unit forms (e.g., for use with one assay) of an agent. The containers of the kits can be air tight and/or waterproof. The container can be labeled for use.
The kit can include informational material for performing and interpreting the assay. The kit can also provide guidance as to where to report the results of the assay, e.g., to a treatment center or healthcare provider. The kit can include forms for reporting the results of a gene activity assay described herein, and address and contact information regarding where to send such forms or other related information; or a URL (Uniform Resource Locator) address for reporting the results in an online database or an online application (e.g., an app). In another embodiment, the informational material can include guidance regarding whether a patient should receive treatment with an anti-cancer stem cell agent, depending on the results of the assay.
The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as computer readable material, video recording, or audio recording. The informational material of the kit can be contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about the gene activity assay and/or its use in the methods described herein. The informational material can also be provided in any combination of formats.
A subject sample can be provided to an assay provider, e.g., a service provider (such as a third party facility) or a healthcare provider that evaluates the sample in an assay and provides a read out. For example, an assay provider can receive a sample from a subject, such as a tissue sample, or a plasma, blood or serum sample, and evaluate the sample using an assay described herein, and determines that the subject is a candidate to receive treatment with an inhibitor as described herein. The assay provider can inform a healthcare provider that the subject is a candidate for treatment with an inhibitor as described herein, and the candidate is administered the inhibitor as described herein. The assay provider can provide the results of the evaluation, and optionally, conclusions regarding one or more of diagnosis, prognosis, or appropriate therapy options to, for example, a healthcare provider, or patient, or an insurance company, in any suitable format, such as by mail or electronically, or through an online database. The information collected and provided by the assay provider can be stored in a database.
EXAMPLES
Example A
In this example, the enzymatic activity of glutaminase is measured through a coupled endpoint assay. Glutamine and phosphate are supplied to GAC at a concentration equal to Km and AC50, respectively, and GAC concentration is adjusted to give a linear reaction for 60 minutes. The glutamate produced is converted to 2-OG by a kinetic excess of glutamate dehydrogenase. This second step is configured for 2× Km for NAD, since excess NAD is inhibitory. However, a kinetic excess of the third coupling enzyme, diaphorase, recycles NAD from NADH to keep the NAD concentration constant during the timecourse of the assay. Diaphorase, also supplied in kinetic excess, oxidizes NADH produced by GDH back to NAD with the concomitant reduction of rezasurin to the highly fluorescent resorufin. Resorufin in measured after the assay is stopped with SDS by Ex544/Em590. A reduction in the signal indicates inhibition of some component of the coupled enzyme system. Prospective hits are counterscreened against GDH/diaphorase alone to remove hits to the coupling enzyme system in a second assay.
1. Materials













BSA
Sigma #3294 (protease-free)


diaphorase
Worthington Enzyme LS004330. Resuspend at



10 mg/ml in ddH20 and store at −80 C.


EDTA
Sigma E6758 or equivalent


glutamate dehydrogenase
Sigma G7882


glutamine
Sigma G3126 or equivalent


HEPES (pH8.5)
Sigma H3375 or equivalent, to pH 8.5 with


NaOH


NaCl
Sigma S7653 or equivalent


NAD
Sigma N7004; note: power will decompose to



inhibitor if stored outside dessicator. Purchase



small lots and prepare stocks in solution and



store at −80 C.


resazurin
Sigma 199303


sodium dodecyl sulfate
Sigma L4390 or equivalent


sodium phosphate
Prepare from Sigma monobasic (S8282) and


(pH8.5)
dibasic (S7907) solutions or equivalents; 1M



stock final concentration prepared from 1M



stocks of each of the dibasic and monobasic



solutions.









2. Buffers

2× Buffer (300 mM NaCl, 100 mM HEPES pH 8.5, 0.1% BSA, 0.5 mM EDTA, 100 mM sodium phosphate pH 8.5)

5× Substrate Mix (1× Buffer final concentration, with 13 mM glutamine, 100 μM resazurin, 50 μg/ml diaphorase)

1.2× Enzyme Mix (1× Buffer final concentration, with 0.875 μg/ml GAC, 1.56 mM NAD, 6.25 units/ml GDH)

Stop Mix (6% SDS in ddH2O)

Reaction Procedure

1. Add 1 μl compound in 100% DMSO

2. Add 40 μl of Enzyme Mix and incubate for 60 minute at room temperature

3. Add 10 μl of Substrate Mix to start reaction

4. Stop reaction with 25 μl of 6% SDS and read Ex544 Em 590

Example B
In this example, the potential for a compound to inhibit the coupled enzyme assay system of the glutaminase HTS method, which comprises glutamate dehydrogenase and diaphorase, is tested through a coupled endpoint assay. Glutamate is supplied at Km to GDH, which then performs a reductive deamidation to produce 2OG. NAD is supplied at 2× Km to the system, and its conversion to NADH is monitored by the activity of diaphorase. Diaphorase, supplied in large kinetic excess to GDH, converts NADH back to NAD to keep NAD levels constant in the reaction while at the same time reducing rezasurin to the highly fluorescent resorufin. Resorufin in measured after the assay is stopped with SDS by Ex544/Em590. A reduction in the signal indicates inhibition of some component of the coupled enzyme system.
3. Materials













BSA
Sigma #3294 (protease-free)


diaphorase
Worthington Enzyme LS004330. Resuspend at



10 mg/ml in ddH20 and store at −80 C.


EDTA
Sigma E6758 or equivalent


glutamate dehydrogenase
Sigma G7882


glutamic acid
Sigma G1251 or equivalent


HEPES (pH8.5)
Sigma H3375 or equivalent, to pH 8.5 with



NaOH


NaCl
Sigma S7653 or equivalent


NAD
Sigma N7004; note: powder will decompose to



inhibitor if stored outside dessicator. Purchase



small lots and prepare stocks in solution and



store at −80 C.


resazurin
Sigma 199303


sodium dodecyl sulfate
Sigma L4390 or equivalent









4. Buffers

2× Buffer (300 mM NaCl, 100 mM HEPES pH 8.5, 0.1% BSA, 0.5 mM EDTA, 100 mM phosphate pH 8.5)

2× Substrate Mix (1× Buffer final concentration, 40 μM resazurin, 1.8 mM glutamate, 20 μg/ml diaphorase)

10×NAD Mix (1× Buffer final concentration, 12.5 mM NAD)

2.5× Enzyme Mix (1× Buffer final concentration, GDH enzyme as determined for appropriate linearity; for example 0.05 units/ml as described here to get 0.02 units/ml final concentration)

Reaction Procedure

1. Add 1 μl compound in 100% DMSO

2. Add 20 μl of Enzyme Mix and incubate for 60 minutes at room temperature

3. Add 5 μl of NAD Mix

4. Add 25 μl of Substrate Mix to start reaction

5. Stop reaction with 25 μl of 6% SDS and read Ex544 Em 590

Example 1




5,5′-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazol-2-amine) (I-2)




A mixture of 3,3′-thiodipropanoic acid (1 equivalent) and thiosemicarbazide (2 equivalents) were taken in POCl3 (10 fold) and stirred at 90° C. for 3 h. The reaction mixture was cooled to room temperature and poured on to ice. The resulting mixture was filtered and then brought to pH 14 using KOH. The white solid that was formed was filtered, washed with water and dried to afford the desired product I-2 in 50-60% yield. This material was used as such for the next step.
General Procedure for the Synthesis of Compounds (I-3):
Diamine I-2 (1 equivalent) was taken in DMF and cooled to 0° C. PYBOP (3 equivalents) was then added followed by the addition of DIPEA (3 equivalents) and stirred for 10 min. The corresponding acid (3 equivalents) was added and the reaction mixture was stirred for overnight at room temperature. The progress of the reaction was monitored by LCMS. After completion of the reaction, water was added to the reaction mixture and stirred for 10 min. The obtained solid was filtered through Buchner funnel, washed with water and dried. The crude product was purified by recrystallization/column chromatography to afford the desired products (I-3) in 16-70% yield.
N,N′-(5,5′-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-cyclohexylacetamide) (5)




1H NMR (400 MHz, DMSO-d6) δ: 0.90-1.30 (m, 12H), 1.55-1.82 (m, 10H), 2.36 (t, 4H), 2.96 (t, 4H), 3.25 (t, 4H), 12.38 (brs, 2H); HPLC Purity: 92%; Mass (M++23): 559.30.
Example 2




General Procedure for the Carbamate Preparation (I-5a to I-5d)

Diamine I-2 (1 equivalent) was taken in DCM and pyridine (2 equivalents) was added followed by the addition of corresponding chloroformate (3 equivalents) and stirred at room temperature for overnight. Progress of the reaction was monitored by LCMS. After completion of the reaction, water was added to the mixture and extracted with DCM. The organic layer was washed with brine, dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography to afford the pure carbamate (I-5a to I-5d) in 10-20% yield.
Diphenyl(5,5′-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))dicarbamate (1)




1H NMR (400 MHz, DMSO-d6) δ: 2.95 (t, 4H), 3.24 (t, 4H), 7.22-7.32 (m, 6H), 7.40-7.50 (m, 4H), 12.62 (s, 2H); HPLC Purity: 92.21%; Mass (M++23): 551.15.
Dicyclohexyl(5,5′-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))dicarbamate (8)




1H NMR (400 MHz, DMSO-d6) a: 1.22-1.55 (m, 12H), 1.65-1.78 (m, 4H), 1.82-1.90 (m, 4H), 2.92 (t, 4H), 3.22 (t, 4H), 4.84-4.90 (m, 2H), 11.92 (s, 2H); HPLC Purity: 94.4%; Mass (M++1): 541.15.
Example 3




1,1′-(5,5′-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(3-methyl-3-phenylurea) (18)




Diamine I-2 (1 equivalent) was taken in THF, cooled to −50° C. and LiHMDS (2 equivalents) was added to the mixture and stirred for 30 min. Methyl(phenyl)carbamic chloride (4 equivalents) in THF was then added to the reaction mixture and brought to room temperature and stirred overnight. The progress of the reaction was monitored by TLC. After completion of the reaction, reaction mixture was quenched with saturated ammonium chloride solution and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by column chromatography using 2% MeOH-DCM to afford the desired product 18 in 5% yield.
1H NMR (400 MHz, DMSO-d6) δ: 2.85 (t, 4H), 3.15 (t, 4H), 3.28 (s, 6H), 7.22-7.30 (m, 6H), 7.34-7.40 (m, 4H); HPLC Purity: 86%; Mass (M++1): 555.25.
Example 4




5,5′-(thiobis(ethane-2,1-diyl))bis(N-phenethyl-1,3,4-thiadiazol-2-amine) (9)
N,N′-(5,5′-(thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide) 3 (1 equivalent) in THF was added to a stirred suspension of LiALH4 (2.5 equivalents) in THF at 0° C. and refluxed for 4 h. The progress of the reaction was monitored by LCMS. The reaction was quenched by careful addition of saturated NH4Cl solution followed by ethyl acetate and obtained solid which was filtered through a pad of celite. The celite layer was further washed with ethyl acetate. The filtrate obtained was evaporated under reduced pressure and purified by preparative HPLC to afford pure product 9 in 21% yield.




1H NMR (400 MHz, DMSO-d6+TFA) δ: 2.84 (q, 8H), 3.08 (t, 4H), 3.48 (q, 4H), 7.19-7.36 (m, 10H), 7.66 (m, 2H); HPLC Purity: 94%; Mass (M++23): 519.15.
Example 5




General Procedure for the Synthesis of Compound (I-13a to I-13b):

Corresponding sulfide analogue (1 equivalent) was taken in THF, m-CPBA (0.9 equivalents) in THF:H2O (6:1) was added drop wise at 0° C. and stirred at same temperature for 10 min. The progress of the reaction was monitored by TLC. After completion of the reaction, reaction mixture was quenched with aq. NaHCO3 solution and solvent was evaporated to dryness. Water was added to the residue, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude product was purified by column chromatography using 5-10% MeOH-DCM to afford the desired products (I-13a to I-13b) in 15-22% yield.
N,N′-(5,5′-(sulfinylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenyl acetamide) (7)




1H NMR (400 MHz, DMSO-d6) δ: 3.18 (t, 2H), 3.20-3.40 (m, 6H), 3.80 (s, 4H), 7.20-7.38 (m, 10H), 12.62 (brs, 2H); HPLC Purity: 96.85%; Mass (M++1): 541.20.
N,N′-(5,5′-(sulfinylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide) (17)




1H NMR (400 MHz, DMSO-d6) δ: 3.18 (t, 4H), 3.30-3.42 (m, 4H), 4.00 (s, 4H), 7.26 (t, 2H), 7.40 (d, 2H), 7.78 (t, 2H), 8.48-8.52 (m, 2H); HPLC Purity: 99.17%; Mass (M++1): 543.10.
Example 6




3,3′-sulfonyldipropanoic acid (I-14)




3,3′-thiodipropanoic acid (1 equivalent) was taken in THF and to it oxone (2 equivalents) taken in water was slowly added and stirred at room temperature for 3 h. The progress of the reaction was monitored by TLC. After completion of the reaction, water was added to the reaction mixture and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness to afford the crude product I-14 in 20-30% yield.
5,5′-(sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazol-2-amine) (I-15)




3,3′-sulfonyldipropanoic acid (1 equivalent) and thiosemicarbazide (2 equivalents) were taken in PPA and refluxed for overnight. The reaction mixture was then cooled to room temperature. The pH of the reaction mixture was adjusted to 14 using aq. KOH solution, stirred for 30 min and filtered off the solid. The Solid obtained was triturated with diethyl ether and dried to afford the title compound I-15 in 20-30% yield.
General Procedure for the Synthesis of Compound (I-16a to I-16d):
Diamine I-16 (1 equivalent) was taken in DMF cooled to 0° C. and PYBOP (3 equivalents) was added followed by the addition of DIPEA (3 equivalents) and stirred for 10 min. Corresponding acid (3 equivalents) was then added to the reaction mixture and stirred at room temperature for overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, water was added to the reaction mixture and stirred for 10 min. The solid obtained was filtered through Buchner funnel and dried. The crude product was purified either by silica gel column chromatography or preparative HPLC to obtain the pure products (I-16a to I-16d) in 10-30% yields.
N,N′-(5,5′-(sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide) (4)




1H NMR (400 MHz, DMSO-d6) δ: 3.45 (t, 4H), 3.70 (t, 4H), 3.80 (s, 4H), 7.20-7.38 (m, 10H), 12.75 (s, 2H); HPLC Purity: 94%; Mass (M++1): 557.1.
N,N′-(5,5′-(sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide) (13)




1H NMR (400 MHz, DMSO-d6) δ: 3.45 (t, 4H), 3.68 (t, 4H), 4.00 (s, 4H), 7.25 (t, 2H), 7.40 (d, 2H), 7.78 (t, 2H), 8.50 (s, 2H) 12.75 (s, 2H); HPLC Purity: 97.6%; Mass (M++1): 559.15.
N,N′-(5,5′-(sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-3-yl)acetamide) (14)




1H NMR (400 MHz, DMSO-d6) δ: 3.42 (t, 4H), 3.68 (t, 4H), 3.88 (s, 4H), 7.38 (t, 2H), 7.76 (d, 2H), 8.42-8.55 (m, 4H), 12.78 (brs, 2H); HPLC Purity: 90%; Mass (M++1): 559.1.
N,N′-(5,5′-(2,2′-sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(3-phenylpropanamide) (21)




1H NMR (400 MHz, DMSO-d6) δ: 2.80 (t, 4H), 2.95 (t, 4H), 3.45 (t, 4H), 3.70 (t, 4H), 7.10-7.32 (m, 10H), 12.5 (brs, 2H); HPLC Purity: 97%; Mass (M++1): 585.3.
General Procedure for the Carbamate Preparation (I-17a to I-17b)
5,5′-(sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazol-2-amine) (1 equivalent) was taken in DCM, and to it was added pyridine (2 equivalents) followed by the addition of corresponding chloroformate (2 equivalents) and stirred at room temperature for overnight. The progress of the reaction was monitored by LCMS. After completion of the reaction, water was added to the reaction mixture and extracted with DCM. The organic layer was washed with brine, dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography using 3% MeOH-DCM to afford pure carbamates (I-17a to I-17b) in 10-20% yield.
Diisopropyl(5,5′-(sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))dicarbamate (23)




1H NMR (400 MHz, DMSO-d6) δ: 1.25 (d, 12H), 3.42 (t, 4H), 3.65 (t, 4H), 4.88-4.98 (m, 2H), 12.00 (s, 2H); HPLC Purity: 94.47%; Mass (M++1): 493.10.
Diisobutyl(5,5′-(sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))dicarbamate (22)




1H NMR (400 MHz, DMSO-d6) a: 0.90 (d, 12H), 1.85-1.98 (m, 2H), 3.42 (t, 4H), 3.65 (t, 4H), 3.98 (d, 4H), 12.10 (s, 2H); HPLC Purity: 94.67%; Mass (M++1): 521.00.
Example 7




General Procedure for the Synthesis of Compounds (I-19a-I-19b):

Appropriate carbamate (1 equivalent) was taken in acetone, and to it oxone (2.5 equivalents) taken in water was added slowly and stirred at room temperature for 2 h. The progress of the reaction was monitored by TLC. After completion of the reaction, acetone was evaporated under reduced pressure. Water was added to the reaction mixture and extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and evaporated to dryness. The crude was purified by column chromatography using 10% MeOH-DCM to afford the desired products (I-19a-I-19b) in 20-50% yield.
Dibenzyl(5,5′-(sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))dicarbamate (19)




1H NMR (400 MHz, DMSO-d6) δ: 3.45 (t, 4H), 3.70 (t, 4H), 5.24 (s, 4H), 7.34-7.44 (m, 10H), 12.22 (s, 2H); HPLC Purity: 97%; Mass (M++1): 589.1.
Di-tert-butyl(5,5′-(sulfonylbis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))dicarbamate (15)




1H NMR (400 MHz, DMSO-d6) δ: 1.45 (s, 18H), 3.45 (t, 4H), 3.70 (t, 4H), 11.82 (s, 2H); HPLC Purity: 96%; Mass (M++1): 521.15.
Example 8




5,5′-(pentane-1,5-diyl)bis(1,3,4-thiadiazol-2-amine) (I-50)




The title compound was synthesized from pimelic acid I-49 following the general procedure described for compound I-2.
General Procedure for the Synthesis of Compounds (I-51a to I-51d):
Diamine I-50 (1 equivalent) was taken in DMF and cooled to 0° C. PYBOP (3 equivalents) was then added followed by the addition of DIPEA (3 equivalents) and stirred for 10 min. The corresponding acid (3 equivalents) was added and the reaction mixture and was stirred for overnight at room temperature. The progress of the reaction was monitored by LCMS. After completion of the reaction, water was added to the reaction mixture and stirred for 10 min. The obtained solid was filtered through Buchner funnel, washed with water and dried. The crude product was purified by recrystallization/column chromatography to afford the desired products (I-51a to I-51d) in 2-34% yield.
N,N′-(5,5′-(pentane-1,5-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide) (2)




1H NMR (400 MHz, DMSO-d6) δ: 1.40 (t, 2H), 1.70 (t, 4H), 2.94 (t, 4H), 3.80 (s, 4H), 7.22-7.40 (m, 10H), 12.64 (s, 2H); HPLC Purity: 90%; Mass (M++1): 507.25.
N,N′-(5,5′-(pentane-1,5-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-3-yl)acetamide) (10)




1H NMR (400 MHz, DMSO-d6) δ: 1.36 (t, 2H), 1.70 (m, 4H), 2.96 (t, 4H), 3.92 (s, 4H), 7.50 (t, 2H), 7.90 (d, 2H), 8.50-8.60 (m, 4H), 12.70 (s, 2H); HPLC Purity: 97.29%; Mass (M++23): 531.25.
N,N′-(5,5′-(pentane-1,5-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide) (11)




1H NMR (400 MHz, DMSO-d6) δ: 1.43 (t, 2H), 1.72 (m, 4H), 2.99 (t, 4H), 4.0 (s, 4H), 7.26 (t, 2H), 7.40 (d, 2H), 7.80 (t, 2H), 8.50 (d, 2H), 12.65 (s, 2H); HPLC Purity: 98%; Mass (M++23): 531.10.
N,N′-(5,5′-(pentane-1,5-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(3-phenylpropanamide) (20)




1H NMR (400 MHz, DMSO-d6) δ: 1.40 (t, 2H), 1.74 (t, 4H), 2.80 (t, 4H), 2.88-3.0 (m, 8H), 7.15-7.34 (m, 10H), 12.40 (brs, 2H); HPLC Purity: 93%; Mass (M++1): 535.05.
Example 9




Octanedioic acid (I-59):





10% Palladium on carbon (10% wt) was added to a solution of oct-4-ynedioic acid I-56 in ethanol. The mixture was degassed with argon and then stirred under an atmosphere of H2 for 14 h. The mixture was filtered through celite and concentrated to afford octanedioic acid in 68% yield.
5,5′-(hexane-1,6-diyl)bis(1,3,4-thiadiazol-2-amine) (I-60)




The title compound was synthesized from octanedioic acid I-59 and thiosemicarbazide by following the general procedure as described for compound I-2.
N,N′-(5,5′-(hexane-1,6-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide) (12)




The title compound was synthesized from 5,5′-(hexane-1,6-diyl)bis(1,3,4-thiadiazol-2-amine) I-60 by following the general procedure as described for compound I-4.
1H NMR (400 MHz, DMSO-d6) δ: 1.36 (t, 4H), 1.64 (t, 4H), 2.94 (t, 4H), 3.89 (s, 4H), 7.20-7.36 (m, 10H), 12.62 (s, 2H); HPLC Purity: 96%; Mass (M++23): 543.10.
N,N′-(5,5′-(hexane-1,6-diyl)bis(1,3,4-thiadiazole-5,2-diyl))bis(2-(pyridin-2-yl)acetamide) (16)




The title compound was synthesized from 5,5′-(hexane-1,6-diyl)bis(1,3,4-thiadiazol-2-amine) I-60 by following the general procedure as described for compound I-4.
1H NMR (400 MHz, DMSO-d6) δ: 1.38 (t, 4H), 1.70 (t, 4H), 2.95 (t, 4H), 4.0 (s, 4H), 7.30 (t, 2H), 7.41 (d, 2H), 7.78 (t, 2H), 8.50 (d, 2H), 12.68 (s, 2H); HPLC Purity: 93%; Mass (M++23): 545.20.
Having thus described several aspects of several embodiments, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

## Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein:



X is a bond, —S—, —S(O)—, —SO2—, —CH═CH—, or —C(O)—;
each W, Y and Z is independently —S—, —CH═, —O—, —N═, or —NH—, provided that (1) at least one of W, Y and Z is not —CH═ and (2) when one of W is —S— and the Y in the same ring is N, then the Z in the same ring is not —CH═;
each R1 and R2 is independently C1-6 alkylene-R4, N(R3)—C(O)—R4, —C(O)—N(R3)—R4, —N(R3)—C(O)—O—R4, —N(R3)—C(O)—N(R3)—R4, —O—C(O)—N(R3)—R4, —N(R3)—C(O)—C1-6 alkylene-C(O)—R4, —N(R3)—C(O)—C1-6 alkylene-N(R3)—C(O)—R4 or —N(R3a)—C(O)—CH2—N(R3)—C(O)—R4;
each R3 is independently hydrogen, C1-6 alkyl or aryl;
each R4 is independently C1-6 alkyl, C1-6 alkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl, cycloalkyl or cycloalkylalkyl, each of which is substituted with 0-3 occurrences of R5, or two adjacent R5 moieties, taken together with the atoms to which they are attached form a heterocyclyl, heteroaryl, cycloalkyl or aryl;
each R5 is independently oxo (═O), C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, cyano, halo, —OH, —SH, —OCF3, —SO2—C1-6 alkyl, —NO2, —N(R7)—C(O)—C1-6 alkyl, —N(R6)2, —O—C(O)—C1-6 alkyl, C3-7 cycloalkyl, (C3-7 cycloalkyl)alkyl, aryl, aryloxy, —C(O)-aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl or heterocyclyl, wherein each aryl, heteroaryl or heterocyclyl is further substituted with 0-3 occurrences of R7;
each R6 is independently hydrogen, OH or C1-6 alkyl;
each R7 is independently hydrogen, C1-6 alkyl, —OH, —SH, cyano, halo, —CF3, —OCF3, —SO2—C1-6 alkyl, —NO2, —N(R7)—C(O)—C1-6 alkyl, —N(R6)2 or C1-6 alkoxy;
m is 1, 2 or 3;
n is 1, 2 or 3; provided that when X is bond, the sum of m and n is from 3 to 6 and when X is —S—, —S(O)—, —SO2—, —CH═CH—, or —C(O)—, the sum of m and n is from 2 to 4;
o is 1, 2 or 3; and
p is 1, 2 or 3;
with the proviso that:
(1) when X is —S—, m and n are both 2, each R6 is H, then (i) R1 and R2 are not both —NHC(O)—R4, wherein R4 is C1-6 alkyl, monocyclic aryl, monocyclic heteroaryl, monocyclic aralkyl, monocyclic heteroaralkyl and each member of R4 is substituted with 0-3 occurrences of R5; and (ii) R1 and R2 are not both —NHC(O)O-methyl, —NHC(O)O-ethyl, —NHC(O)-6-pyrimidine-2,4(1H,3H)-dionyl, or —NHC(O)NH-phenyl wherein said phenyl of the —NHC(O)NH-phenyl moiety is optionally substituted with 1 or 2 groups selected from methyl, nitro, and halo;
(2) when X is —S—, m and n are both 1, each R6 is H, then (i) R1 and R2 are not both —NH-phenyl or —NH-4-methoxy-phenyl;
(3) when X is a bond, the sum of m and n is 3, each R6 is H, then R1 and R2 are not both NHC(O)-phenyl;
(4) when X is a bond, m and n are both 2, each R6 is H, then R1 and R2 are not both —NHC(O)-furanyl, —NHC(O)-phenyl, —NHC(O)-o-methoxy-phenyl, —NHC(O)—C1-6 alkyl, —NH-benzyl, or —NH-phenyl wherein said phenyl of the —NH-phenyl moiety is substituted with 0-3 occurrences of R5;
(5) when X is a bond, the sum of m and n is 5, each R6 is H, then R1 and R2 are not both —NHC(O)—C1-6 alkyl, —NHC(O)-cyclohexyl, or —NH-phenyl wherein said phenyl of the —NH-phenyl moiety is optionally substituted with methyl; and
(6) when X is a bond, m and n are both 3, each R6 is H, then R1 and R2 are not both NH-phenyl.

2. The compound of claim 1, wherein each W is —S—, each Y is —N═ and each Z is —N═.

3. The compound of claim 1, wherein each W is —CH═, each Z is —O— and each Y is —N═.

4. The compound of claim 1, wherein o is 1 and p is 1.

5. The compound of claim 1, wherein R1 and R2 are each —N(R3)—C(O)—O—R4, wherein R3 is hydrogen.

6. The compound of claim 5, wherein R1 and R2 are the same.

7. The compound of claim 1, wherein the compound is a compound of Formula (II):

8. The compound of claim 6, wherein R1 and R2 are the same.

9. The compound of claim 1, wherein the compound is a compound of Formula (III):

10. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or adjuvant.

11. The compound of claim 1, wherein the compound is selected from the group consisting of:

12. The compound of claim 1, wherein the compound is selected from the group consisting of:

13. The compound of claim 1, wherein the compound is selected from the group consisting of:

14. A compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein:



X is —S(O)— or —SO2—;
each W, Y and Z is independently —S—, —CH═, —O—, —N═, or —NH—, provided that (1) at least one of W, Y and Z is not —CH═ and (2) when one of W is —S— and the Y in the same ring is N, then the Z in the same ring is not —CH═;
each R1 and R2 is independently C1-6 alkylene-R4, N(R3)—C(O)—R4, —C(O)—N(R3)—R4, —N(R3)—C(O)—O—R4, —N(R3)—C(O)—N(R3)—R4, —O—C(O)—N(R3)—R4, —N(R3)—C(O)—C1-6 alkylene-C(O)—R4, —N(R3)—C(O)—C1-6 alkylene-N(R3)—C(O)—R4 or —N(R3a)—C(O)—CH2—N(R3)—C(O)—R4;
each R3 is independently hydrogen, C1-6 alkyl or aryl;
each R4 is independently C1-6 alkyl, C1-6 alkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl, heterocyclyl, cycloalkyl or cycloalkylalkyl, each of which is substituted with 0-3 occurrences of R5, or two adjacent R5 moieties, taken together with the atoms to which they are attached form a heterocyclyl, heteroaryl, cycloalkyl or aryl;
each R5 is independently oxo (═O), C1-6 alkyl, C1-6haloalkyl, C1-6 alkoxy, cyano, halo, —OH, —SH, —OCF3, —SO2—C1-6 alkyl, —NO2, —N(R7)—C(O)—C1-6 alkyl, —N(R6)2, —O—C(O)—C1-6 alkyl, C3-7 cycloalkyl, (C3-7 cycloalkyl)alkyl, aryl, aryloxy, —C(O)-aryl, heteroaryl, aralkyl, heteroaralkyl, heterocyclylalkyl or heterocyclyl, wherein each aryl, heteroaryl or heterocyclyl is further substituted with 0-3 occurrences of R7;
each R6 is independently hydrogen, fluoro, —OH or C1-6 alkyl;
each R7 is independently hydrogen, C1-6 alkyl, —OH, —SH, cyano, halo, —CF3, —OCF3, —SO2—C1-6 alkyl, —NO2, —N(R7)—C(O)—C1-6 alkyl, —N(R6)2 or C1-6 alkoxy;
m is 1, 2 or 3;
n is 1, 2 or 3; provided that when X is —S(O)— or —SO2—, the sum of m and n is from 2 to 4;
o is 1, 2 or 3; and
p is 1, 2or 3.

15. A pharmaceutical composition comprising a compound of claim 14 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or adjuvant.