PU.1 inhibitors
10774049 · 2020-09-15
Assignee
- Albert Einstein College Of Medicine (Bronx, NY)
- Georgia State University Research Foundation, Inc. (Atlanta, GA)
Inventors
- W. David Wilson (Atlanta, GA, US)
- David W. Boykin (Atlanta, GA)
- Gregory Poon (Lawrenceville, GA, US)
- Ulrich Steidl (New Rochelle, NY, US)
- Iléana Anthony-Debré (Paris, FR)
Cpc classification
C07D235/20
CHEMISTRY; METALLURGY
C07D235/18
CHEMISTRY; METALLURGY
C07D409/04
CHEMISTRY; METALLURGY
International classification
C07D235/20
CHEMISTRY; METALLURGY
C07D235/18
CHEMISTRY; METALLURGY
C07D409/04
CHEMISTRY; METALLURGY
Abstract
Disclosed herein are inhibitors of PU.1. The inhibitors are useful for treating disorders associated with abnormal PU.1 levels and function.
Claims
1. A compound having the formula: ##STR00033## or a pharmaceutically acceptable salt thereof, wherein: x and x are each 3; R is in each case independently selected from the group consisting of R.sup.a, OR.sup.a,, N(R.sup.a).sub.2, SR.sup.a, SO.sub.2R.sup.a, SO.sub.2N(R.sup.a).sub.2, COOR.sup.a, C(O)N(R.sup.a).sub.2, OC(O)N(R.sup.a).sub.2, N(R.sup.a)C(O)N(R.sup.a).sub.2, F, Cl, Br, I, cyano, and nitro, wherein R.sup.a is in each case independently selected from the group consisting of hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl, wherein any two or more of R and R.sup.a may together form a ring; G and G are independently selected from C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl, wherein each C.sub.6-12 aryl is optionally and independently substituted with R.sup.8 or R.sup.9; A and A are independently selected from the group consisting of NR.sup.1, O, S, and Se, wherein R.sup.1, when present, is in each case independently selected from the group consisting of R.sup.b, SO.sub.2R.sup.b, SO.sub.2N(R.sup.b).sub.2, COOR.sup.b, and C(O)N(R.sup.b).sub.2, wherein R.sup.b is in each case independently selected from the group consisting of hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl, wherein any two or more of R and R.sup.1 may together form a ring; B and B are independently selected from the group consisting of N and CR; has the formula: ##STR00034## wherein Q.sup.a is O or NR.sup.1a, wherein R.sup.1a, R.sup.2a, and R.sup.3a are independently selected from the group consisting of hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, and C.sub.2-8 heterocyclyl; wherein any two or more of R.sup.1a, R.sup.2a, R.sup.3a, R and R.sup.1 can together form a ring; has the formula: ##STR00035## wherein Q.sup.b is O or NR.sup.1b, wherein R.sup.1b, R.sup.2b, and R.sup.3b are independently selected from hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, and C.sub.2-8 heterocyclyl; wherein any two or more of R.sup.1b, R.sup.2b, and R.sup.3b, R and R.sup.1 can together form a ring; wherein Q.sup.a and Q.sup.b are not both O; Z is (a) a linking group having the formula:
X(C(R.sup.2).sub.2).sub.mC.sup.z(C(R.sup.2).sub.2).sub.nY; wherein X and Y are independently selected from the group consisting of O, S, Se, and NR.sup.4; wherein R.sup.4, when present, is in each case independently selected from the group consisting of R.sup.c, SO.sub.2R.sup.c, SO.sub.2N(R.sup.c).sub.2, COOR.sup.c, and C(O)N(R.sup.c).sub.2, wherein R.sup.c is in each case independently selected from the group consisting of hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl; R.sup.2 is in each case independently H or F; m and n are each an integer independently selected from 0-4; C.sup.z is selected from O, S, Se, NR.sup.4, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl; or (b) X(C(R.sup.5).sub.2).sub.mC.sup.z(C(R.sup.5).sub.2).sub.nY, wherein X and Y are independently selected from the group consisting of O, S, Se, and NR.sup.4, wherein R.sup.4, m and n are as defined in (a), wherein C.sup.z is a chemical bond, wherein R.sup.5 is in each case independently H or F and at least one R.sup.5 is F, and wherein the sum of m and n is greater than 2; R.sup.8H or R.sup.9; R.sup.9O(CH.sub.2)n.sup.aN(R.sup.10).sub.2 or O(CH.sub.2)n.sup.aNH (CNH)NH.sub.2; R.sup.10C.sub.1-C.sub.6 alkyl or cyclo-alkyl; and n.sup.a=2-8.
2. The compound according to claim 1, wherein C.sup.z in (a) of Z is a group of the formula: ##STR00036## wherein A is O, S, Se, or NR.sup.6; wherein R.sup.6 is hydrogen, C.sub.1-8alkyl; C.sub.3-8 cycloalkyl, and C.sub.2-8 heterocyclyl.
3. The compound according to claim 1, wherein Z is as defined in (a), and X and Y are both O.
4. The compound according to claim 1, wherein B and B are both N.
5. The compound according to claim 1, wherein A and A are both NR.sup.4.
6. The compound according to claim 1, having the formula: ##STR00037##
7. The compound according to claim 1, wherein is in the 7 position.
8. The compound according to claim 1, wherein is in the 4, 5 or 6 position.
9. The compound according to claim 1, wherein is in the 7 position.
10. The compound according to claim 1, wherein is in the 4, 5 or 6 position.
11. The compound according to claim 1, wherein C.sup.z in (a) of Z is: ##STR00038## wherein R is selected from the group consisting of hydrogen, F, Cl, Br, I, cyano and nitro, wherein A is selected from the group consisting of O, S, Se, and NR.sup.6; wherein R.sup.6 is selected from the group consisting of hydrogen, C.sub.1-8alkyl; C.sub.3-8 cycloalkyl, and C.sub.2-8 heterocyclyl; and wherein R.sup.7 is selected from the group consisting of R, hydrogen, F, Cl, Br, I, cvano and nitro.
12. The compound according to claim 1, wherein C.sup.z is O or NR.sup.4.
13. The compound according to claim 1, wherein C.sup.z is NR.sup.4, wherein R.sup.4 is C.sub.1-4 alkyl.
14. The compound according to claim 1, wherein C.sub.z is a chemical bond.
15. The compound according to claim 1, wherein Z is selected from the group consisting of OCH.sub.2CF.sub.2CF.sub.2CF.sub.2CH.sub.2O, OCH.sub.2CF.sub.2CF.sub.2O, OCF.sub.2CF.sub.2CF.sub.2O, and OCH.sub.2CF.sub.2CH.sub.2O.
16. The compound according to claim 1, wherein has the formula: ##STR00039##
17. The compound according to claim 1, wherein has the formula: ##STR00040##
18. The compound according to claim 1, wherein the compound is selected from the group consisting of: ##STR00041## ##STR00042## ##STR00043## or a pharmaceutically acceptable salt thereof.
19. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.
20. A method of inhibiting PU.1, comprising contacting one or more cells with a compound according to claim 1.
21. A method of ameliorating a symptom of a disease associated with abnormal PU.1 function in a patient, comprising administering to the patient in need thereof the compound of claim 1 in an amount effective to inhibit PU.1.
22. The method according to claim 21, wherein the disease is selected from the group consisting of hematologic cancer, bone cancer, inflammatory disease, autoimmune disorders, endotoxemia neurodegenerative disease, leukemia, acute myeloid leukemia, rheumatoid arthritis, contact dermatitis, asthma, inflammatory bowel disease, chronic inflammatory disease, pediatric atropy, giant cell arteritis, Alzheimer's disease, amyotrophic lateral sclerosis and systemic lupus.
23. The method according to claim 21, wherein the patient is a human at least sixty years old.
24. The compound according to claim 1, wherein Z is as defined in (b), and X and Y are both O.
25. The compound of claim 1, which represented by the formula: ##STR00044## wherein A and A are each NH; B and B are each N; has the formula: ##STR00045## wherein R.sup.2a and R.sup.3a are each hydrogen; has the formula: ##STR00046## wherein R.sup.2b and R.sup.3b are each hydrogen; wherein and groups are each in the 5 or 6 position; G and G are independently phenyl substituted with R.sup.9.
26. The compound of claim 25, wherein and groups are each in the 6 position.
27. A compound, which is ##STR00047##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
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(3)
(4)
(5)
(6)
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(8)
DETAILED DESCRIPTION OF THE INVENTION
(9) Before the present methods and systems are disclosed and described, it is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
(10) As used in the specification and the appended claims, the singular forms a, an and the include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about, it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
(11) Optional or optionally means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
(12) Throughout the description and claims of this specification, the word comprise and variations of the word, such as comprising and comprises, means including but not limited to, and is not intended to exclude, for example, other additives, components, integers or steps. Exemplary means an example of and is not intended to convey an indication of a preferred or ideal embodiment. Such as is not used in a restrictive sense, but for explanatory purposes.
(13) Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture.
(14) The term alkyl as used herein is a branched or unbranched hydrocarbon group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, and the like. The alkyl group can also be substituted or unsubstituted. Unless stated otherwise, the term alkyl contemplates both substituted and unsubstituted alkyl groups. The alkyl group can be substituted with one or more groups including, but not limited to, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol as described herein. An alkyl group which contains no double or triple carbon-carbon bonds is designated a saturated alkyl group, whereas an alkyl group having one or more such bonds is designated an unsaturated alkyl group. Unsaturated alkyl groups having a double bond can be designated alkenyl groups, and unsaturated alkyl groups having a triple bond can be designated alkynyl groups. Unless specified to the contrary, the term alkyl embraces both saturated and unsaturated groups.
(15) The term cycloalkyl as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term heterocycloalkyl is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, selenium or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. Unless stated otherwise, the terms cycloalkyl and heterocycloalkyl contemplate both substituted and unsubstituted cycloalkyl and heterocycloalkyl groups. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol as described herein. A cycloalkyl group which contains no double or triple carbon-carbon bonds is designated a saturated cycloalkyl group, whereas an cycloalkyl group having one or more such bonds (yet is still not aromatic) is designated an unsaturated cycloalkyl group. Unless specified to the contrary, the terms cycloalkyl and heterocycloalkyl embrace both saturated and partially unsaturated systems.
(16) The term aryl as used herein is an aromatic ring composed of carbon atoms. Examples of aryl groups include, but are not limited to, phenyl and naphthyl, etc. The term heteroaryl is an aryl group as defined above where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, selenium or phosphorus. The aryl group and heteroaryl group can be substituted or unsubstituted. Unless stated otherwise, the terms aryl and heteroaryl contemplate both substituted and unsubstituted aryl and heteroaryl groups. The aryl group and heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol as described herein.
(17) Exemplary heteroaryl and heterocyclyl rings include: benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cirmolinyl, decahydroquinolinyl, 2H,6H1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.
(18) The terms alkoxy, cycloalkoxy, heterocycloalkoxy, cycloalkoxy, aryloxy, and heteroaryloxy have the aforementioned meanings for alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, further providing said group is connected via an oxygen atom.
(19) As used herein, the term substituted is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms substitution or substituted with include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Unless specifically stated, a substituent that is said to be substituted is meant that the substituent is substituted with one or more of the following: alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol as described herein.
(20) Unless specified otherwise, the term patient refers to any mammalian animal, including but not limited to, humans.
(21) Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects. Examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids and the like; salts formed with organic acids such as acetic, oxalic, tartaric, succinic, maleic, fumaric, gluconic, citric, malic, methanesulfonic, p-toluenesulfonic, napthalenesulfonic, and polygalacturonic acids, and the like; salts formed from elemental anions such as chloride, bromide, and iodide; salts formed from metal hydroxides, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, and magnesium hydroxide; salts formed from metal carbonates, for example, sodium carbonate, potassium carbonate, calcium carbonate, and magnesium carbonate; salts formed from metal bicarbonates, for example, sodium bicarbonate and potassium bicarbonate; salts formed from metal sulfates, for example, sodium sulfate and potassium sulfate; and salts formed from metal nitrates, for example, sodium nitrate and potassium nitrate. Pharmaceutically acceptable and non-pharmaceutically acceptable salts may be prepared using procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid comprising a physiologically acceptable anion. Alkali metal (for example, sodium, potassium, or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be made.
(22) Human Transcription factor PU.1 protein (product of the SPI1 gene is) has the following amino acid sequence (SEQ ID NO:1) (UniProtKB-P17947):
(23) TABLE-US-00001 10203040 MLQACKMEGFPLVPPPSEDLVPYDTDLYQRQTHEYYPYLS 50607080 SDGESHSDHYWDFHPHHVHSEFESFAENNFTELQSVQPPQ 90100110120 LQQLYRHMELEQMHVLDTPMVPPHPSLGHQVSYLPRMCLQ 130140150160 YPSLSPAQPSSDEEEGERQSPPLEVSDGEADGLEPGPGLL 170180190200 PGETGSKKKIRLYQFLLDLLRSGDMKDSIWWVDKDKGTFQ 210220230240 FSSKHKEALAHRWGIQKGNRKKMTYQKMARALRNYGKTGE 250260270 VKKVKKKLTYQFSGEVLGRGGLAERRHPPH.
A second isoform has been reported where at position 15, P.fwdarw.PQ (SEQ ID NO:2) (UniProtKB-P17947):
(24) TABLE-US-00002 10203040 MLQACKMEGFPLVPPQPSEDLVPYDTDLYQRQTHEYYPYL 50607080 SSDGESHSDHYWDFHPHHVHSEFESFAENNFTELQSVQPP 90100110120 QLQQLYRHMELEQMHVLDTPMVPPHPSLGHQVSYLPRMCL 130140150160 QYPSLSPAQPSSDEEEGERQSPPLEVSDGEADGLEPGPGL 170180190200 LPGETGSKKKIRLYQFLLDLLRSGDMKDSIWWVDKDKGTF 210220230240 QFSSKHKEALAHRWGIQKGNRKKMTYQKMARALRNYGKTG 250260270 EVKKVKKKLTYQFSGEVLGRGGLAERRHPPH.
(25) Disclosed herein are bis-heterocyclic PU.1 inhibiting compounds. The compounds can be represented by the following Formula 1:
(26) ##STR00004##
or a pharmaceutically acceptable salt thereof, wherein: x and x are each 3; R is in each case independently selected from R.sup.a, OR.sup.a, N(R.sup.a).sub.2, SR.sup.a, SO.sub.2R.sup.a, SO.sub.2N(R.sup.a).sub.2; COOR.sup.a, C(O)N(R.sup.a).sub.2, OC(O)N(R.sup.a).sub.2, N(R.sup.a)C(O)N(R.sup.a).sub.2, F, Cl, Br, I, cyano, and nitro, wherein R.sup.a is in each case independently selected from hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl, wherein any two or more of R and R.sup.a may together form a ring; G and G are independently selected from C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, OC.sub.6-12 aryl, and C.sub.3-12 heteroaryl, wherein each C.sub.6-12 aryl or OC.sub.6-12 aryl is optionally and independently substituted with R.sup.8 or R.sup.9; A and A are independently selected from NR.sup.1, O, S, and Se, wherein R.sup.1, when present, is in each case independently selected from R.sup.b, SO.sub.2R.sup.b, SO.sub.2N(R.sup.b).sub.2; COOR.sup.b, C(O)N(R.sup.b).sub.2, wherein R.sup.b is in each case independently selected from hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl, wherein any two or more of R and R.sup.1 may together form a ring B and B are independently selected from N and CR; has the formula:
(27) ##STR00005## wherein Q.sup.a is O or NR.sup.1a, wherein R.sup.1a, R.sup.2a, and R.sup.3a are independently selected from hydrogen, C.sub.1-8alkyl, C.sub.3-8 cycloalkyl, or C.sub.2-8 heterocyclyl; wherein any two or more of R.sup.1a, R.sup.2a, R.sup.3a, R and R.sup.1 can together form a ring; has the formula:
(28) ##STR00006## wherein Q.sup.b is O or NR.sup.1b, wherein R.sup.1b, R.sup.2b, and R.sup.3b are independently selected from hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, and C.sub.2-8 heterocyclyl; wherein any two or more of R.sup.1b, R.sup.2b, and R.sup.3b, R and R.sup.1 can together form a ring; wherein Q.sup.a and Q.sup.b are not both O; Z is a linking group having the formula:
X(CR.sub.2).sub.mC.sup.z(CR.sub.2).sub.nY; wherein X and Y are independently selected from: a chemical bond; O, S, Se, and NR.sup.4; wherein R.sup.4, when present, is in each case independently selected from R.sup.c, SO.sub.2R.sup.c, SO.sub.2N(R.sup.c).sub.2; COOR.sup.c, C(O)N(R.sup.c).sub.2, wherein R.sup.c is in each case independently selected from hydrogen, C.sub.1-s alkyl, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl R.sub.2 is in each case independently H or F or mixtures thereof; m and n are each an integer independently selected from 0-4; C.sup.z is selected from a chemical bond, O, S, Se, NR.sup.4, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl; wherein when X and Y are both O, C.sup.z is not a chemical bond, and when C.sup.z is a selenium containing heterocycle, X and Y are not both a chemical bond; R.sup.8=H or R.sup.9; R.sup.9O(CH.sub.2)n.sup.aN(R.sup.10).sub.2 or O(CH.sub.2)n.sup.aNH(CNH)NH.sub.2; R.sup.10C.sub.1-C.sub.6 alkyl or cyclo-alkyl; and n.sup.a=2-8.
(29) In some embodiments, one or both of X and Y are O.
(30) In some instances, B and B are both N.
(31) In some instances A and A are both NR.sup.4, in which R.sup.4 is either hydrogen or C.sub.1-4 alkyl. In certain embodiments, both of X and Y are O, B and B are both N, and A and A are both NR.sup.4, in which R.sup.4 is either hydrogen or C.sub.1-4 alkyl.
(32) In certain embodiments, one or both of the and groups can be in the 4 position, e.g.:
(33) ##STR00007##
(34) Likewise, in some embodiments, one or both of the and groups can be in the 5, 6 or 7 position. In certain embodiments, the phenyl portion of the bicyclic heterocycle portion of the compound may be substituted only with and , that is, each of the R groups in those rings is hydrogen, for instance a compound having the formula:
(35) ##STR00008##
(36) In other embodiments, it is preferred that at least one R group is not hydrogen. For instance, one of the R groups can be an electron donating group in ortho, meta or para position relative to the or substituent. Exemplary electron donating groups include R.sup.a, OR.sup.a, N(R.sup.a).sub.2. In other embodiments, one of the R groups can be an electron withdrawing group in the ortho, meta or para relative to the or substituent. Exemplary electron withdrawing groups include SO.sub.2R.sup.a, SO.sub.2N(R.sup.a).sub.2; COOR.sup.a, C(O)N(R.sup.a).sub.2, OC(O)N(R.sup.a).sub.2, N(R.sup.a)C(O)N(R.sup.a).sub.2, F, Cl, Br, I, cyano, and nitro.
(37) In some cases, G and G can be an optionally substituted phenyl group or an optionally substituted heteroaryl. G and G can be independently selected from:
(38) ##STR00009##
wherein R.sup.6 is independently selected from R.sup.d, OR.sup.d, N(R.sup.d).sub.2, SR.sup.d, SO.sub.2R.sup.d, SO.sub.2N(R.sup.d).sub.2; COOR.sup.d, C(O)N(R.sup.d).sub.2, OC(O)N(R.sup.d).sub.2, N(R.sup.d)C(O)N(R.sup.d).sub.2, F, Cl, Br, I, cyano, and nitro, wherein R.sup.d is in each case independently selected from hydrogen, C.sub.1-8 alkyl, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl, wherein any two or more of R and R.sup.a may together form a ring. In certain embodiments, R.sup.6 is in each case hydrogen. In other embodiments, G or G can be a phenyl or heteroaryl having one or two non-hydrogen groups. In certain cases, the non-hydrogen R.sup.6 group can be selected from R.sup.d, OR.sup.d, COOR.sup.d, F, Cl, Br, I, cyano, and nitro.
(39) The four G and G systems described above can be designated 1,4 systems by virtue that the connectivity pattern in the para configuration. In other embodiments, G and G can be optionally substituted phenyl, pyridinyl or 1,3 pyrazine group in the 1,3 or 1,2 configuration. When G or G is a 1,4 pyrazine, the substitution pattern can also be in the 1,2 configuration.
(40) In certain embodiment, and are both at the 6 position, and in further embodiments can have an electron withdrawing group at the 4, 5 or 7 position. In some embodiments, and are both at the 6 position, and in further embodiments have an electron donating group at the 4, 5, or 7 position.
(41) In certain instances, Z has the formula:
X((CR.sup.5).sub.2).sub.mC.sup.z(C(R.sup.5).sub.2).sub.nY;
wherein each R.sup.5 is independently selected from hydrogen and F, or mixtures thereof. For example, Z can be OCH.sub.2CF.sub.2CF.sub.2CF.sub.2CH.sub.2O; OCH.sub.2CF.sub.2CF.sub.2O; OCF.sub.2CF.sub.2CF.sub.2O; or OCH.sub.2CF.sub.2CH.sub.2O.
(42) In some embodiments, C.sup.z can be an optionally substituted phenyl or heteroaryl. For instance, C.sup.z can have the formula:
(43) ##STR00010##
wherein A is O, S, Se, or NR.sup.6; wherein R.sup.6 is hydrogen, C.sub.1-8alkyl; C.sub.3-8 cycloalkyl, and C.sub.2-8 heterocyclyl; and in other instances C.sup.z can have the formula:
(44) ##STR00011##
wherein R and A have the meanings given above. However, when A is Se, X and Y are not both a chemical bond. In some embodiments, A can be O. In some embodiments, C.sup.z can be an optionally substituted phenyl or heteroaryl. For instance, C.sub.z can have the formula:
(45) ##STR00012##
wherein R.sup.7 is independently selected from hydrogen, F, Cl, Br, I, cyano or nitro. In some embodiments, C.sup.z can have the formula:
(46) ##STR00013##
wherein R has the meaning given above. In some embodiments, R can be R.sup.7. In certain embodiments, R can be R.sup.7, in which R.sup.7 is in each case hydrogen. In other embodiments, C.sup.z can be a phenyl group having the formula:
(47) ##STR00014##
wherein R has the meaning given above. In some embodiments, R can be R.sup.7. In certain embodiments, R can be R.sup.7, in which R.sup.7 is in each case hydrogen. In some cases, C.sup.z can be a phenyl group having the formula:
(48) ##STR00015##
wherein R.sup.7 can be R, or can be selected from hydrogen, F, Cl, Br, I, cyano or nitro, preferably F.
(49) C.sup.z can be selected from a chemical bond, O, S, Se, NR.sup.4, C.sub.3-8 cycloalkyl, C.sub.2-8 heterocyclyl, C.sub.6-12 aryl, and C.sub.3-12 heteroaryl or a group of the formula:
(50) ##STR00016##
wherein A is O, S, Se, or NR.sup.6; wherein R.sup.6 is hydrogen, C.sub.1-8alkyl; C.sub.3-8 cycloalkyl, and C.sub.2-8 heterocyclyl, wherein when X and Y are both O, C.sup.z is not a chemical bond, and when A is Se, X and Y are not both a chemical bond.
(51) In some embodiments, has the formula:
(52) ##STR00017##
(53) In some embodiments, has the formula:
(54) ##STR00018##
(55) In some embodiments, The compound has the formula
(56) ##STR00019##
wherein R.sup.8H or R.sup.9; R.sup.9O(CH.sub.2)n.sup.aN(R.sup.10).sub.2 or O(CH.sub.2)n.sup.aNH(CNH)NH.sub.2; R.sup.10C.sub.1-C.sub.6 alkyl or cyclo-alkyl; and n.sup.a=2-8.
In one embodiment, R.sup.8H and R.sup.9O(CH.sub.2)n.sup.aN(R.sup.10).sub.2. In one embodiment, both R.sup.8 and R.sup.9O(CH.sub.2)n.sup.aN(R.sup.10).sub.2. In one embodiment, R.sup.8H and R.sup.9O(CH.sub.2)n.sup.aNH(CNH)NH.sub.2. In one embodiment, both R.sup.8 and R.sup.9O(CH.sub.2)n.sup.aNH(CNH)NH.sub.2.
(57) The compounds disclosed herein may be prepared according to the process depicted in
(58) The compounds disclosed herein may be formulated in a wide variety of compositions for administration to a patient, for instance, a human patient. The compounds disclosed herein are especially useful in treating disease states in elderly patients, i.e., those of sixty years of age or greater. The compounds can be delivered, for example, orally, intravenously, topically, parentally, subcutaneously, intradermally, or by inhalation. Exemplary routes of administration include buccal, oral, intravenous, intramuscular, topical, subcutaneous, rectal, vaginal, parenteral, pulmonary, intranasal, ophthalmic, and the like.
(59) Also provided is a pharmaceutical composition comprising any of the compounds disclosed herein and a pharmaceutically acceptable carrier. The term carrier is used in accordance with its art-understood meaning, to refer to a material that is included in a pharmaceutical composition but does not abrogate the biological activity of pharmaceutically active agent(s) that are also included within the composition. Typically, carriers have very low toxicity to the animal to which such compositions are to be administered. In some embodiments, carriers are inert. Pharmaceutically acceptable carriers and diluents that can be used herewith encompasses any of the standard pharmaceutical carriers or diluents, such as, for example, a sterile isotonic saline, phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsions.
(60) Also provided is a medicament comprising any of the compounds disclosed herein or any of the pharmaceutical compositions disclosed herein, wherein the compound is in an amount effective to inhibit PU.1.
(61) Also provided is a method of inhibiting PU.1, comprising contacting one or more cells with any of the compounds disclosed herein.
(62) The compounds and compositions disclosed herein may be used to treat diseases associated with abnormal PU.1 levels and activity. The compounds can be used to treat consisting of hematologic cancer, bone cancer, inflammatory disease, inflammatory disorders, autoimmune disorders, endotoxemia and neurodegenerative disease. Exemplary such conditions include leukemia, acute myeloid leukemia, rheumatoid arthritis, contact dermatitis, asthma, inflammatory bowel disease, chronic inflammatory disease, pediatric atrophy, giant cell arteritis, Alzheimer's disease, amyotrophic lateral sclerosis, and systemic lupus. The invention provides a method of treating a patient with a disease associated with abnormal PU.1 function, comprising administering to the patient in need thereof any of the compounds or compositions disclosed herein in an amount effective to inhibit PU.1. The patient can be a human patient or a veterinary patient. The human patient can be, for example, at least 60 years old. As used herein, treat a disease means to ameliorate a sign or symptom of the disease, or to cure the patient of the disease.
(63) Useful dosages of the compounds of the invention for inclusion in the pharmaceutical compositions of the invention can be determined by comparing in vitro activity and in vivo activity of the compounds in appropriate animal models. Generally, the concentration of the compound(s) of the invention in a liquid composition will range from about 0.1% to about 95% by weight, preferably from about 0.5% to about 25% by weight. The concentration in a semi-solid or solid composition will range from about 0.1% to 100% by weight, preferably about 0.5% to about 5% by weight. Single doses for intravenous injection, subcutaneous, intramuscular or topical administration, infusion, ingestion or suppository will generally be from about 0.001 to about 5000 mg, and be administered from about 1 to about 3 times daily, to yield levels of about 0.01 to about 500 mg/kg, for adults.
(64) The compounds can be co-administered with one or more other agents for the treatment of any of the aforementioned diseases. In certain embodiments, the one or more other agents is a transcription modulator. The one or more other agents can be immunosuppressants. The other agents can be formulated separately, and administered either at the same or different time as the compounds of the instant invention. The other agents can be co-formulated with the compounds of the instant invention to give a combination dosage form.
(65) Disclosed are components that can be used to perform the disclosed methods. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
EXAMPLES
(66) The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.
(67) Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in C. or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
(68) All solvents and reagents were used without purification as acquired from commercial sources. Melting points were measured using a capillary melting point apparatus which are uncorrected. Progress of the chemical reactions were monitored by thin-layer chromatography on silica gel 60-F254 aluminum plates and detected under UV light. All NMR spectra were recorded employing a 400 MHz spectrometer, and chemical shifts () are in ppm relative to TMS as internal standard. Electrospray ionization (ESI) Q-T of and Orbitrap were used for the mass spectra measurements. Elemental analyses are within 0.4 of the theoretical values. Compounds reported as salts frequently analyzed for fractional moles of water; the proton NMR showed the presence of the indicated solvent.
Example 1: Synthesis of DB 2313
(69) ##STR00020##
1. 3-Bis (4-formylphenoxymethyl)-2-fluorobenzene
(70) A mixture of 1,3-bis(bromomethyl)-2-fluorobenzene (1.41 g, 0.005 mole), 4-hydroxybenzaldehyde (1.22 g, 0.01 mole) and anhydrous. K.sub.2CO.sub.3 (2.07 g, 0.015 mole) in 10 ml DMF was heated at 45 C. for 4 h [tlc (Hexane:EtOAc 8:2) monitored], diluted with ice water 70 ml, the precipitated white solid was filtered, washed with water, and dried. It was dissolved in DCM (75 ml), dried over anhydrous MgSO4, filtered, concentrated and triturated with cold hexane, filtered and dried under reduced pressure to yield a white solid 1.46 g (78%), mp 110-111 C.; .sup.1H NMR (DMSO-d.sub.6): 9.89 (s, 2H), 7.90 (d, 4H, J=8.4 Hz), 7.62 (t, 2H, J=7.6 Hz), 7.30 (t, 1H, J=7.6 Hz), 7.26 (d, 4H, J=8.4 Hz), 5.31 (s, 4H); .sup.13C NMR (DMSO-d.sub.6): 191.8, 163.5, 159.5 (J.sub.C-F=248.6 Hz), 132.3, 131.6 (J.sub.C-F=3.7 Hz) 130.5, 125.0 (J.sub.C-F=3.7 Hz), 123.9 (J.sub.C-F=14.7 Hz) 115.5, 64.4 (J.sub.C-F=4.0); MS: HRMS-ESIPOS.: Calcd. For: C.sub.22H.sub.17FO.sub.4 Na m/z 387.1009 (M.sup.++Na), found m/z 387.1537; Anal. calcd. for: C.sub.22H.sub.17FO.sub.4: C, 72.50; H, 4.70. Found: C, 72.49; H, 4.72.
3-Bis{4[4(5)-N-isopropylamidinobenzimidazolyl]phenoxymethyl]}-2-fluorobenzene tetrahydrochloride
(71) A well stirred solution of 1, 3-bis (4-formylphenoxymethyl)-2-fluorobenzene (0.182 g, 0.0005 mole), 4-(N-isopropylamidino)-1, 2-phenylenediamine hydrochloride. 0.2H.sub.2O (46) (0.232 g, 0.001 mole) and 1, 4-benzoquinone (0.108 g, 0.001 mole) in anhydrous ethanol (40 ml) (under nitrogen) was heated at reflux for 8-10 h. The reaction mixture was cooled, concentrated to 10 ml and stirred in 50 ml acetone, filtered, washed with dry ether and dried to yield a hydrochloride salt. This salt was dissolved in a 1:1 mixture of hot ethanol-methanol (50 ml) and filtered, volume reduced to 20 ml and acidified with HCl-saturated ethanol (3 ml). After stirring overnight and diluting with anhydrous ether, filtered, washed with ether, and dried under reduced pressure at 70 C. (12 h) yielding purple-bluish grey solid 0.33 g (75%); mp>320 C. dec.; .sup.1H NMR (DMSO-d.sub.6/65 C.): 9.68 (s, 1H), 9.67 (s, 1H), 9.53 (s, 2H), 9.12 (s, 2H), 8.41 (d, 4H, J=8.4 Hz), 8.08 (s, 2H), 7.86 (d, 2H, J=8.4 Hz), 7.67 (d, 2H, J=8 Hz), 7.65 (d, 2H, J=8 Hz), 7.37-7.32 (m, 5H), 5.34 (s, 4H), 4.10 (quintet, 2H, J=6 Hz), 3.37-3.2 (vbs, benzimidazole NH), 1.31 (d, 12H, J=6 Hz); MS: HRMS-ESI-POS.: calc. for C.sub.42H.sub.42FN.sub.8O.sub.2 m/z 709.3415 (M.sup.++1), found m/z 709.4542; analysis calc. for C.sub.42H.sub.41FN.sub.8O.sub.2.4HCl.1.65H.sub.2O: C, 57.14; H, 5.52; N, 12.70. Found: C, 57.35; H, 5.67; N, 12.81.
(72) By analogous methods, DB1976 and DB2115 were also prepared:
(73) ##STR00021##
(74) After synthesis DB2115 and DB2313 were dissolved as a 2.5 mM, 70 uM and 33 (mouse cells) or 66 uM (human) uM stock solutions, respectively, in sterile water and stored at 20 C.
Example 2: PU.1 Knockdown Decreases Cell Growth and Clonogenic Capacity, and Increases Apoptosis of Murine and Human AML Cells
(75) To determine whether PU.1 inhibition may be a suitable strategy in AML, we used an established model of AML driven by reduced PU.1 levels, PU.1 URE.sup./ AML, in which PU.1 expression is reduced to 20% of normal levels by disruption of an upstream enhancer (URE). Knockdown of PU.1 in PU.1 URE.sup./ AML cells by two different shRNAs led to significantly decreased cell growth and colony formation (
(76) We next investigated the effect of PU.1 knockdown on human leukemic cell lines with different PU.1 levels. MOLM13 and Kasumi-1 cell lines harbor anomalies associated with low PU.1 levels (FLT3-ITD mutation for MOLM13 and t(8; 21) for Kasumi-1), while THP1 cells have higher PU.1 levels. PU.1 decrease led to a strong growth-inhibitory effect on the growth and clonogenic capacity of MOLM13 and Kasumi-1 cells, whereas it did not have an effect on THP1 cell growth (
(77) To determine whether PU.1 inhibition has an effect on primary cells from AML patients, we seeded mononuclear cells from AML patients in semi-solid media for 2 weeks and assessed the number of colonies, the number of viable cells and the proportion of apoptotic cells. Knockdown of PU.1 significantly decreased the number of viable cells (mean decrease of 18% for shPU.1_1 and 74% for shPU.1_2) (
(78) Taken together, these data show that inhibition of PU.1 decreases cell growth and clonogenic capacity, and increases apoptosis, in murine as well as human AML, and thus provide proof-of-concept for PU.1 inhibition as a possible therapeutic strategy in AML.
Example 3
(79) To test whether these heterocyclic diamidines targeted cognate DNA binding sites for PU.1, we assessed binding to the B motif by biosensor-surface plasmon resonance (SPR). Duplex DNA harboring the B motif was immobilized on a streptavidin functionalized sensor chip with a dextran surface as described. The DNA was 5-end labeled with biotin and captured on the sensor chip. Based on the known strong AT selectivity of these heterocyclic diamidines, they are expected to bind to the AT rich B promoter sequence at the 5 side of the 5-GGAA-3 PU.1 conserved recognition sequence. Sensorgrams obtained from the compounds-DNA association and disassociation reactions were used to determine equilibrium dissociation constants. The K.sub.D values indicate strong binding by all three compounds to form a 1:1 complex, and a binding K.sup.D of 12 nM for DB2115 and 4-7 nM for DB1976 and DB2313 with the B promoter.
(80) We next tested the inhibition of PU.1 binding to the B promoter. Inhibition of the PU.1 complex with B was monitored by using the same type of sensorchip described for the compound binding experiments. Binding of PU.1 to the immobilized B DNA was monitored as with the compounds and it was found to form a 1:1 complex with a K.sub.D of 5.4 nM in good agreement with previous results. For the inhibition experiments, recombinant PU.1 was added at a concentration sufficient to occupy >95% of the DNA binding sites. The compounds inhibited PU.1 binding in a concentration dependent manner (
(81) To probe the allosteric basis of PU.1 inhibition by the three compounds more directly, we performed DNA footprinting experiments on a DNA fragment harboring the B site with or without the three selected compounds. Since DNase I activity is sensitive to local DNA structure, and both enzyme and compounds target the DNA minor groove, perturbation in the DNase I cleavage pattern would indicate an induced structural effect by the compounds. DNA fragments were saturated with compounds (1 M) and analyzed by capillary electrophoresis following limited DNase I digests. Relative to the compound-free control, the electropherograms showed significant differences in local DNase I cleavage, in one or both strands, within the AT-rich subsite occupied by the compounds (
(82) To test if the inhibition of the PU.1/DNA complex by the compounds resulted in functional inhibition of PU.1 transactivation, we tested the effects of the compounds on the expression of an EGFP reporter under the control of a M-based PU.1-dependent promoter (
(83) We modelled the interaction of the compounds with DNA by in silico docking studies. As illustrated in
Example 4
(84) To determine if our PU.1 inhibitors have an effect on PU.1.sup.low-induced AML cells, we treated the PU.1 URE.sup./ AML cell line with compounds at different concentrations. Treatment with the drugs led to a profound decrease in growth of PU.1 URE.sup./ AML cells after 48 h whereas it had only minimal or no effects on control cells with normal PU.1 levels (BaF3), even at very high doses (
(85) PU.1 inhibitors also significantly decreased colony forming capacity of PU.1 URE.sup./ AML and MOLM13 cells, but not of THP1 cells (
(86) We also explored the effect of the small molecule PU.1 inhibitors on primary human AML cells, and treated 11 samples from AML patients with PU.1 inhibitors. PU.1 inhibitors led to significant decreases in the number of viable cells (mean decrease: 81% for DB1976, 68% for DB2115, 72% for DB2313) (
(87) Taken together, treatment with PU.1 inhibitors leads to decreased cell viability, colony formation, and increased apoptosis in PU.1.sup.low-induced AML cell lines as well as in a majority of primary AML cell samples from patients.
Example 5
(88) To assess on-target activity of our inhibitors in AML cells, we measured transcript levels of well-known PU.1 targets in PU.1 URE.sup./ AML cells. It has been shown that PU.1 positively regulates Csf1r, Junb, and autoregulates itself, whereas it represses E2f1. In line with this, we found a decrease in Csf1r, Junb, and PU.1 transcript expression and an increase in E2f1 expression upon treatment with DB2115, or DB2313 (
(89) To obtain insight into the genome-wide transcriptional effects following treatment of AML cells with our inhibitors, we performed gene expression analysis. We found dysregulation of 1648 transcripts (out of 34,472 total) by at least 1.2-fold after DB2313 treatment of PU.1 URE.sup./ AML cells, with 867 probe sets unregulated and 781 probe sets downregulated. We found highly significant enrichment of known genes directly downstream of PU.1 (
(90) Lastly, we compared the differentially expressed genes upon PU.1 inhibitor treatment of PU.1 URE.sup./ AML cells with publically available data in which the PU.1 regulatory transcriptional network had been identified by tamoxifen-mediated induction of PU.1 expression in engineered PU.1 null immature hematopoietic cells (PUER) (GSE13125). Of the 1,334 genes dysregulated after PU.1 inhibitor treatment of PU.1 URE.sup./ AML cells, 36% (484) overlap with previously identified canonical PU.1 targets in PUER cells (
Example 6
(91) In order to determine the effect of our PU.1 inhibitors on normal hematopoietic differentiation, we sorted immature Lin.sup.Sca1.sup.+c-Kit.sup.+ (LSK) cells from wildtype mice and studied their clonogenic potential following treatment with either DB1976, DB2115, or DB2313. After treatment with PU.1 inhibitors, total numbers of colonies were only slightly reduced; however, we saw a significant reduction of the more mature myelo-monocytic colony types (CFU-GM, CFU-G and CFU-M) (
(92) As we had observed a reduction in mature myelomonocytic colonies upon treatment with PU.1 inhibitors, but persistence or even slight increases in more immature cells (GEMM, and immature colonies), we wanted to test whether these immature cells were still functional after drug removal. We focused on the compound with the lowest IC50 and the least effect on wildtype cells (DB2313). Interestingly, the production of mature granulocytes and monocytes increased significantly by 4-fold and 22-fold, respectively, after removing the treatment in the second plating, showing that the effects of PU.1 inhibition on G/M generation are reversible (
(93) Taken together, these results indicate that treatment with our PU.1 inhibitors leads to effects on normal hematopoiesis consistent with fundamental roles of PU.1 function during hematopoiesis. These effects are reversible upon treatment discontinuation and seem to primarily affect more mature cells.
Example 7: Treatment with PU.1 Inhibitors Decreases Leukemia Progression In Vivo
(94) To assess the effect of PU.1 inhibitors on AML in vivo, we treated PU.1 URE.sup./ AML cells for 2 days in vitro and injected 210.sup.5 viable cells in sublethally irradiated recipient mice (
(95) The following compounds were prepared and tested according to the above mentioned methods:
(96) TABLE-US-00003 IC.sub.50 IC.sub.50 URE/ BAF3 Code Structure (M) (M) DB2146
(97) The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term comprising and variations thereof as used herein is used synonymously with the term including and variations thereof and are open, non-limiting terms. Although the terms comprising and including have been used herein to describe various embodiments, the terms consisting essentially of and consisting of can be used in place of comprising and including to provide for more specific embodiments of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.
REFERENCES
(98) Munde, M., Wang, S., Kumar, A., Stephens, C. E., Farahat, A. A., Boykin, D. W., Wilson, W. D., and Poon, G. M. K. (2014) Inhibition of the ETS-family transcription factor PU.1 by heterocyclic diamidines. Nucleic Acids Research. 42: 1379-90.