WO1998016230A1 - Methods for reversibly inhibiting myelopoiesis in mammalian tissue - Google Patents

Methods for reversibly inhibiting myelopoiesis in mammalian tissue Download PDF

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Publication number
WO1998016230A1
WO1998016230A1 PCT/US1997/018951 US9718951W WO9816230A1 WO 1998016230 A1 WO1998016230 A1 WO 1998016230A1 US 9718951 W US9718951 W US 9718951W WO 9816230 A1 WO9816230 A1 WO 9816230A1
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alkyl
optionally substituted
aryl
crior20
compound
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PCT/US1997/018951
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French (fr)
Inventor
Daniel Horowitz
Andrew G. King
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Smithkline Beecham Corporation
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Priority to AU51475/98A priority Critical patent/AU5147598A/en
Publication of WO1998016230A1 publication Critical patent/WO1998016230A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings

Definitions

  • the present invention relates to the field of myelopoiesis in mammalian tissue, and more particularly, to the use of certain imidazoles containing compounds for reversibly inhibiting myelopoiesis in humans and animals.
  • Certain chemotherapeutic and radiation treatments for cancer and other serious illness have the undesired side effect of killing all dividing and differentiating cells. This effect is most severe on the differentiating cells of the hematopoietic system, which are essential for repopulating the bone marrow and permitting the redevelopment of a functioning immune system following such treatment.
  • bone marrow toxicity or myelosuppression is the limiting factor in the use of such treatments.
  • the acute effects of myelosuppression are the loss of neutrophils (neutropenia) and the loss of platelets (thrombocytopenia) in the blood. These conditions in part increase patient susceptibility to infections and hemorrhagic complications.
  • a variety of regulatory messengers and modifiers such as colony stimulating factors, interferons, and different types of compounds are responsible for the regulation of myelopoiesis, which is the development and growth of early progenitor cells (stem cells) of the hematopoietic system.
  • the present invention provides for a method for reversibly inhibiting myelopoiesis in mammals, mammalian tissue or other samples in vivo and ex vivo comprising administering to said animal, tissue, or sample an effective amount of a myeloprotectant compound of formula (I) below.
  • the amount of the compound is effective to reversibly inhibit the formation of myelopoietic colonies during the time the animal, tissue, or sample is exposed to myelosuppressive therapy.
  • Figure 2 demonstrates a dose response effect of (IP administered) Compounds 2 and 3 on WBC counts 9 days post 5-FU in the mouse; *P ⁇ 0.05.
  • Figure 3 demonstrates a dose response effect of (IP administered) Compounds 2 and 3 on PMN counts 9 days post 5-FU in the mouse; *P ⁇ 0.05.
  • Figure 4. demonstrates a dose response effect of (IP administered) Compounds 2 and
  • Figure 5 demonstrates a dose response effect of (IP administered) Compounds 2 and 3 on lymphocyte counts 9 days post 5-FU in the mouse; *P ⁇ 0.05.
  • Figure 6 demonstrates a dose response effect of (IP administered) Compounds 2 and
  • Figure 9 demonstrates a dose response effect of (PO administered) Compounds 2 and 3 on PMN cell counts 9 days post 5-FU in the mouse.
  • Figure 10 demonstrates a dose response effect of (PO administered) Compounds 2 and 3 on bone marrow cell counts 9 days post 5-FU in the mouse. Marrow counts obtained from pools of 3 mice/group.
  • Figure 11 demonstrates the effect of Compounds 2 and 3 and G-CSF on the kinetics of neutrophil recovery post 175 mg/kg 5-FU in mice. G-CSF (50ug kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 12 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of monocyte recovery post 175 mg/kg 5-FU in mice.
  • G-CSF 50ug/kg administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 13 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of platelet recovery post 175 mg/kg 5-FU in mice.
  • G-CSF (50ug/kg) administered daily IP animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 14 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of lymphocyte recovery post 175 mg kg 5-FU in mice.
  • G-CSF (50ug/kg) administered daily IP animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 15 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of total white blood cell recovery post 175 mg/kg 5-FU in mice.
  • mice 5-FU in mice.
  • G-CSF 50ug/kg administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days.
  • Figure 18 demonstrates the effect of Compound 3, G-CSF, and the combination of
  • Figure 19 demonstrates the effect of Compound 3, G-CSF, and the combination of
  • Figure 20 demonstrates the effect of Compound 3, G-CSF, and the combination of Compound 3 + G-CSF on the kinetics of total white blood cell recovery post
  • This invention provides for a method for reversibly inhibiting myelopoiesis in mammals, mammalian tissue, including bone marrow and other samples, in vivo and ex vivo.
  • This method entails administering to the mammal, tissue or sample an amount of a compound of formula (I) effective to reversibly inhibit the formation of myelopoietic colonies during the time the mammal, tissue or bone marrow is exposed to myelosuppressive therapy.
  • the myelopoietic colonies inhibited are CFU-C colony forming cells.
  • the myelopoietic CFU-C colony forming cells as used herein include, but are not limited to, CFU-G, CFU-M, CFU-GM, CFU-GEMM, CFU-Meg, and HPP.
  • CFU colony forming unit
  • G granulocyte
  • M macrophage or megakaryocyte
  • E erythroid colony types
  • HPP high proliferative potential colony.
  • a preferred colony forming cell is CFU-GM.
  • This method may be accomplished in vivo.
  • a mammalian subject preferably a human patient, undergoing chemotherapy or radiation may be administered the compound so that the reversible inhibition of myelopoiesis occurs in vivo.
  • Treatment with a myeloprotectant compound of this invention may be necessary throughout the time period in which the insulting agent is present within the body. Inhibition of normal hematopoietic stem cells during this period could protect normal cells while damaging only target cells such as cancer cells.
  • the compounds of this invention have demonstrated reversible inhibition, as demonstrated in the Example Section below, a more rapid rebound of hematopoietic stem cell proliferation is expected when administration of the myeloprotectant compound is ceased (concurrently with, or shortly after administration of the chemotherapeutic or radiation is ceased).
  • the end result of treatment with the compounds of this invention as myeloprotective agents is to prevent neutropenia and or accelerate neutrophil / leukocyte recovery, and prevent stem cell loss due to myelosuppression.
  • the advantage of such administration to the patient includes fewer infections due to leukopenia and the ability to use more aggressive chemotherapy regimens to kill cancer cells (dose intensification / increase dose frequency).
  • cytokine inhibitors as described in USSN 08/091,491, published as WO95/02575; WO96/21452; US Patent No.: ; USSN 08/473,396; US Patent No. 5,658,903 ; USSN 08/764,003; USSN 08/473,398; WO96/21654; WO93/14081; US Patent 5,656,644;USSN 08/095,234; US 5,656,644; WO95/03297; USSN 08/481,671; PCT/US97/00619; PCT/US97/00614; PCT/US97/00500; PCT/US 97/00529; USSN 60/013,357; USSN 60/013,358; USSN 60/013,359; WO93/14082; WO95/13067 ;WO95/31451 WO95/13067;WO95/31451 WO95/31451 WO 97/05877; WO 97/
  • a preferred group of compounds for use herein are those compounds of the formula (I):
  • Rl is 4-pyridyl, pyrimidinyl, 4-pyridazinyl, l,2,4-triazin-5-yl, quinolyl, isoquinolinyl, quinazolin-4-yl, 1-imidazolyl or 1-benzimidazolyl ring, which ring is optionally substituted independently one to three times with Y.
  • NHR a optionally substituted -4 alkyl, halogen, hydroxyl, optionally substituted C ⁇ -4 alkoxy, optionally substituted Ci-4 alkylthio, Ci-4 alkylsulfmyl, CH2OR12, amino, mono and di- -6 alkyl substituted amino, or N(R ⁇ o)C(O)R D ;
  • Y is O-R a ;
  • R4 is phenyl, naphth-1-yl or naphth-2-yl, or a heteroaryl, which is optionally substituted by one or two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-naphth-l-yl, 5-naphth-2-yl or 6-naphth-2-yl substituent, is halogen, cyano, nitro, C(Z)NR7Ri7, C(Z)ORi6, (CRioR20)vCORi2, SR5, SOR5, OR12, halo-substituted-Ci-4 alkyl, Ci-4 alkyl, ZC(Z)Ri2, NRioC(Z)Ri6, or (CRioR2 ⁇ )vNRl ⁇ R20 and which, for other positions of substitution, is halogen, cyano, C(Z)NRi3Ri4, C(Z)OR3, (CRioR20)m"COR3, S(
  • R2 is hydrogen, (CRioR20)n' OR9, heterocyclyl, heterocyclylCi-io alkyl,
  • R a is Ci-6alkyl, aryl. arylC i-6alkyl. heterocyclic, heterocyclylC i -6 alkyl, heteroaryl, or heteroarylC i-6alkyl, wherein each of these moieties may be optionally substituted;
  • Rb is hydrogen, C ⁇ . ⁇ alkyl, C3-7 cycloalkyl, aryl, arylCi- 4 alkyl, heteroaryl, heteroarylC i-4alkyl, heterocyclyl, or heterocyclylC 1 -4 alkyl;
  • R3 is heterocyclyl, heterocyclylC i-io alkyl or R ⁇ ;
  • R5 is hydrogen, Ci-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the moieties -SR5 being -SNR7R17 and -SOR5 being -SOH;
  • R6 is hydrogen, a pharmaceutically acceptable cation, Ci
  • Ci-10 alkanoyl is each independently selected from hydrogen or C 1-4 alkyl or R7 and
  • Rl7 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR15;
  • R8 is Ci-io alkyl, halo-substituted Ci-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylCi-io alkyl, heteroaryl, heteroarylC l- 10 alkyl, (CRioR20)nORl l, (CR ⁇ oR2 ⁇ )nS(O) m Ri8, (CRioR20)nNHS(O)2Rl8,
  • R9 is hydrogen, C(Z)Ri 1 or optionally substituted Ci-io alkyl, S(O)2Rl8, optionally substituted aryl or optionally substituted aryl-Ci-4 alkyl;
  • RlO and R20 is each independently selected from hydrogen or C 1-4 alkyl;
  • Rl 1 is hydrogen, Ci-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclyl
  • Ci-4 alkyl optionally substituted aryl or optionally substituted aryl-Ci-4 alkyl, or together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9;
  • Ri5 is Rio or C(Z)-Ci-4 alkyl;
  • Rl6 is Ci-4 alkyl, halo-substituted-Cl-4 alkyl, or C3.7 cycloalkyl
  • Rl8 is C i-io alkyl. C3-7 cycloalkyl, heterocyclyl, aryl, arylalkyl, heterocyclyl, heterocyclyl-Ci-ioalkyl, heteroaryl or heteroarylalkyl;
  • Rl9 is hydrogen, cyano, C 1 - 4 alkyl, C3. ⁇ cycloalkyl or aryl; or a pharmaceutically acceptable salt thereof.
  • Ri is a substituted 4-pyridyl or 4-pyrimindyl. More suitably R, is substituted by alkoxy, alkylthio, amino, methylamino, NHRa, or Y.
  • a preferred ring placement of the Ri substituent on the 4-pyridyl derivative is the 2-position, such as 2-methoxy -4-pyridyl.
  • a preferred ring placement on the 4-pyrimidinyl ring is also at the 2-position, such as in 2-methoxy-pyrimidinyl.
  • the substituent is Y, and Ra is aryl, it is preferably phenyl or naphthyl.
  • R a is aryl alkyl, it is preferably benzyl or napthylmethyl.
  • the heterocyclic portion is preferably pyrrolindinyl, piperidine, morpholino, tetrahydropyran, tetrahydrothiopyranyl, tetrahydrothipyran-sulfinyl, tetrahydrothio-pyransulfonyl, pyrrolindinyl, indole, or piperonyl.
  • the heterocyclic rings herein may contain unsaturation, such as in a tryptamine ring.
  • the aryl, heterocyclic and heteroaryl rings may be optionally substituted one or more times independently with halogen; Ci-4 alkyl, such as methyl, ethyl, propyl, isopropyl, or t-butyl; halosubstituted alkyl, such as CF3; hydroxy; hydroxy substituted Ci-4 alkyl; Cj-4 alkoxy, such as methoxy or ethoxy; S(O) m alkyl and S(O)m aryl (wherein m is 0, 1, or 2); C(O)OR ⁇ 1, such as C(O)Ci-4 alkyl or C(O)OH moieties; C(O)R ⁇ 1; OC(O)R c ; O-(CH2)s-O-, such as in a ketal or dioxyalkylene bridge; amino; mono- and di-Ci-6 alkylsubstituted amino; N(R ⁇ o)C(O)R D ; C(O)NR
  • R c is optionally substituted -6 alkyl, C3.7 cycloalkyl, aryl, arylCi- 4 alkyl, heteroaryl, heteroarylC i-4alkyl, heterocyclyl, or heterocyclylC 1.4 alkyl moieties.
  • the R a groups include Cj_4 alkyl, benzyl, halosubstituted benzyl, napthylmethyl, phenyl, halosubstituted phenyl, aminocarbonylphenyl, alkylphenyl, cyanophenyl.
  • alkylthiophenyl hydroxyphenyl, alkoxyphenyl, mo holinopropyl, piperonyl, piperidin-4-yl, alkyl substituted piperidine, such as 1 -methyl piperidine, or 2,2,6,6-tetramethylpiperidin-4-yl.
  • R a is aryl, arylalkyl, halosubstituted arylalkyl, halosubstituted aryl, heterocyclic alkyl, hydroxy alkyl, alkyl- 1-piperidine-carboxylate, heterocyclic, alkyl substituted heterocyclic, halosubstituted heterocyclic, or aryl substituted heterocyclic.
  • R is benzyl, halosubstituted benzyl, napthylmethyl, phenyl, halosubstituted phenyl, mo ⁇ holinopropyl, 2-hydroxy ethyl, ethyl- 1-piperidinecarboxy late, piperonyl, piperidin-4-yl, alkyl substituted piperidine, chlorotryptamine, and tetrathiohydropyranyl.
  • the alkyl chain is substituted by halogen, such as fluorine, chlorine, bromine or iodine; hydroxy, such as hydroxyethoxy; Ci-io alkoxy, such as a methoxymethoxy, S(O)m alkyl, wherein m is 0, 1 or 2; amino, mono & di- substituted amino, such as in the NR7R17 group, i.e.
  • halogen such as fluorine, chlorine, bromine or iodine
  • hydroxy such as hydroxyethoxy
  • Ci-io alkoxy such as a methoxymethoxy, S(O)m alkyl, wherein m is 0, 1 or 2
  • amino, mono & di- substituted amino such as in the NR7R17 group, i.e.
  • R2 is selected from hydrogen, Ci-io alkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylC l- 10 alkyl, (CRioR20)nNS(O)2Rl8, (CRioR20)nS(O) m Ri8, arylCi-io alkyl,
  • R2 is mo ⁇ holino propyl, piperidine, N-methylpiperidine, N-benzylpiperidine, 2,2,6,6-tetramethylpiperidine, 4-aminopiperidine, 4-amino-2,2,6,6-tetramethyl piperidine, 4-hydroxycyclohexyl, 4- methyl-4-hydroxy cyclohexyl, 4-pyrrolinindyl-cyclohexyl, 4-methyl-4- aminocyclohexyl, 4-methyl-4-acetamidocyclohexyl, 4-keto cyclohexyl, 4-oxiranyl, or 4-hydroxy-4-( 1 -propynyl)cyclohexyl.
  • R2 is hydrogen, optionally substituted heterocyclyl ring, optionally substituted heterocyclylC l- 10 alkyl, optionally substituted -io alkyl, optionally substituted C3-7cycloalkyl, optionally substituted C3-7cycloalkyl Ci-io alkyl, (CR ⁇ oR2 ⁇ )nC(Z)OR ⁇ 1 group, (CRioR20)nNRi3Rl4,
  • R2 is an optionally substituted heterocyclyl ring, and optionally substituted heterocyclylC l- 10 alkyl, optionally substituted aryl,
  • the ring is preferably a mo ⁇ holino, pyrrolidinyl, or a piperidinyl group.
  • the substituents may be directly attached to the free nitrogen, such as in the piperidinyl group or pyrrole ring, or on the ring itself.
  • the ring is a piperidine or pyrrole, more preferably piperidine.
  • the heterocyclyl ring may be optionally substituted one to four times independently by halogen; Ci-4 alkyl; aryl, such as phenyl; aryl alkyl, such as benzyl - wherein the aryl or aryl alkyl moieties themselves may be optionally substituted (as in the definition section below);
  • C(O)ORi such as the C(O)Ci-4 alkyl or C(O)OH moieties; C(O)H; C(O)C ⁇ _4 alkyl, hydroxy substituted Ci-4 alkyl, -4 alkoxy, S(O) m Ci-4 alkyl (wherein m is 0, 1, or 2), NRl ⁇ R20 (wherein Rio and R20 are independently hydrogen or Cl- 4alkyl).
  • the ring is a piperidine, the ring is attached to the imidazole at the 4-position, and the substituents are directly on the available nitrogen, i.e.
  • R2 is an optionally substituted heterocyclyl Ci-io alkyl group
  • the ring is preferably a mo ⁇ holino, pyrrolidinyl, or a piperidinyl group.
  • this alkyl moiety is from 1 to 4, more preferably 3 or 4, and most preferably 3, such as in a propyl group.
  • Preferred heterocyclic alkyl groups include but are not limited to, mo ⁇ holino ethyl, mo ⁇ holino propyl, pyrrolidinyl propyl, and piperidinyl propyl moieties.
  • the heterocyclic ring herein is also optionally substituted in a similar manner to that indicated above for the direct attachment of the heterocyclyl.
  • R2 is an optionally substituted C3-7cycloalkyl, or an optionally substituted C3-7cycloalkyl Ci-io alkyl
  • the cycloalkyl group is preferably a C4 or C6 ring, most preferably a C6 ring, which ring is optionally substituted.
  • the cycloalkyl ring may be optionally substituted one to three times independently by halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; Ci-10 alkoxy, such as methoxy or ethoxy; S(O)m alkyl, wherein m is 0, 1, or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl; S(O)m aryl; cyano, nitro, amino, mono & di- substituted amino, such as in the NR7R17 group, wherein R7 and R17 are as defined in Formula (I), or where the R7R17 may cyclize together with the nitrogen to which they are attached to form a 5 to 7 membered ring which optionally includes an additional heteroatom selected from oxygen, sulfur or NR15 (and R 15 is as defined for Formula (I)); N(R ⁇ o)C(O)X ⁇ (and Xi is Ci-4 alkyl, aryl or arylC
  • R e is a 1,3-dioxyalkylene group of the formula -O-(CH2)s-0-, wherein s is 1 to 3, preferably s is 2 yielding a 1 ,3-dioxyethylene moiety, or ketal functionality.
  • R6' is NRi9'R20' a ⁇ Y ⁇ l-6> halosubstituted alkyl 1-6; hydroxy substituted alkyl 1-6; alkenyl 2-6; aryl or heteroaryl optionally substituted by halogen, alkyl 1-6, halosubstituted alkyl 1 -6, hydroxyl, or alkoxy i- ⁇ .
  • R20 ' is H, alkyl 1.5, aryl, benzyl, heteroaryl, alkyl substituted by halogen or hydroxyl, or phenyl substituted by a member selected from the group consisting of halo, cyano, alkyl ⁇ _ 12, alkoxy 1. , halosubstituted alkyl 1-6, alkylthio, alkylsulphonyl, or alkylsulfinyl; or R19' and R20' may together with the nitrogen to which they are attached form a ring having 5 to 7 members, which members may be optionally replaced by a heteroatom selected from oxygen, sulfur or nitrogen. The ring may be saturated or contain more than one unsaturated bond.
  • R6' is NRi9'R20' and R19' and R20' are preferably hydrogen. When the R2 cycloalkyl moiety is substituted by NR7R17 group, or NR7R17
  • the substituent is preferably an amino, amino alkyl, or an optionally substituted pyrrolidinyl moiety.
  • Preferred compounds of Formula (I) include: 4-(4-Fluorophenyl)-5-(2-amino-4-pyridiminyl)- 1 -( 1 -methyl-4- piperidinyl)imidazole;
  • halogen such as fluorine, chlorine, bromine or iodine
  • hydroxy hydroxy substituted Ci-ioalkyl
  • Ci-io alkoxy such as methoxy or ethoxy
  • S(O)m alkyl wherein m is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl
  • amino, mono & di-substituted amino such as in the NR7R17 group; or where the R7R17 may together with the nitrogen to which they are attached cyclize to form a 5 to 7 membered ring which optionally includes an additional heteroatom selected from O/N/S; - io alkyl, cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl, isopropyl, t-butyl, etc.
  • halosubstituted Cl-10 alkyl such CF3
  • an optionally substituted aryl such as phenyl, or an optionally substituted arylalkyl, such as benzyl or phenethyl, wherein these aryl moieties may also be substituted one to two times by halogen; hydroxy; hydroxy substituted alkyl; Cl-10 alkoxy; S(O) m alkyl; amino, mono & di-substituted amino, such as in the NR7R17 group; alkyl, or CF3.
  • compounds of formula (I) may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration.
  • the subcutaneous and intramuscular forms of the parenteral form administration are generally preferred.
  • Appropriate dosage forms for such administration may be prepared by conventional techniques.
  • An oral administration is the most preferred route of treatment.
  • the effective amounts or dosages may be adjusted based on the amount of tissue being treated.
  • an effective in vitro concentration is in the range of about 10 ⁇ 9 M to 10 " 5 M.
  • One of skill in the art may readily determine other appropriate dosages, depending on the mode of administration, and the level of aggressiveness of therapy required in the specific circumstance.
  • One screening and identification method useful in the present invention may employ the step of screening test samples which detectably bind to a receptor for in vitro or in vivo inhibition of CFU-C colony formation in either a conventional 7 day CFU-GM assay (CFU-C Assay), well known to those skilled in the art, or in the novel, micro-screening assay as described herein.
  • CFU-C Assay 7 day CFU-GM assay
  • the micro-inhibition assay is a modification of the conventional assay and provides for an efficient and rapid screening and identification of inhibitors. The presence or amount of inhibition of CFU-C or CFU-GM colony formation can then be measured in order to identify those test samples which act as agonists.
  • PCLC pre-CFU-liquid culture
  • the assay methods may be further modified by preparing two or more additional test samples from the original test sample or samples that are determined to inhibit CFU-C colony formation activity. These additional test samples contain a lesser number of test compounds than the original test sample from which they were prepared. The steps of the assays may then be repeated as many times as desired or until the test compound or compounds which bind to the receptor preparation have been identified.
  • the Micro-Reversibility assay of this invention is performed as follows. 100,000 non-adherent murine bone marrow cells/ml were incubated for 24 hours with compound ( 5 uM, 0.5 uM, 50 nM, 5 nM) and a mixture of CSFs (mixture of IL-1, IL-3, SCF, M-CSF) in McCoys 5a liquid culture medium with 15% FBS. After 24 hours the cells were washed and resuspended in media / agar / and M-CSF and assayed according to the Micro-Inhibition assay of Example 1, above.
  • the present invention designed studies to prove whether the compounds of Formula (I) were reversible inhibitors of CFU-GM and hence myeloprotective in vivo in a high dose 5-FU model as described herein.
  • Stock solutions are diluted in PBS prior to injection.
  • the test compound concentration is adjusted so that a 0.2 ml injection delivers the appropriate dose of compound.
  • mice Female BDF1 mice were injected IP, (or the dosage may be given orally) with 10 mg/kg 1 hour prior to and 3 hours after 175 mg/kg 5-FU. Mice were sacrificed on days 3, 4, and 6 post 5- FU injection. Bone marrow cellularity and CFU-GM analysis was performed on each day. Data shown in Figure 1 demonstrate that 5-FU severely depleted the marrow compartment of CFU-GM evident on days 3 and 4. By day 6 recovery has initiated, however CFU-GM content is less than 5% of normal.
  • Example 4 A Myeloprotectant Compound Effects Stem Cells Post 5-Fluorouracil Myelosuppression
  • Compound 3 also significantly increased PMN in a dose related manner.
  • both compounds increased platelet counts in a dose responsive manner. Platelet numbers in the 5-FU model, decrease by 60% than rebound to values higher than normal on days 10 - 15. In this experiment, the 5-FU and normal platelets counts are equivalent as the 5-FU counts are beginning this rebound phase of platelet production. Both compounds resulted in higher PLT counts at day 9 indicating that PLT recovery was quicker in the CSAIDTM compound treated mice.
  • Compounds 2 and 3 significantly increased lymphocyte, monocyte, and RBC counts in a dose responsive manner. These data taken together indicate that multiple hematopoietic cell lineage's are protected by administration of compounds of Formula (I) in this murine 5-FU model.
  • mice were injected IP with compound 1 hour prior to and 3 hours after the cell cycle specific drug, 5- fluorouracil (5-FU 175mg/kg), which occurred at 0 hour.
  • 5- fluorouracil 5- fluorouracil
  • G-CSF (50 ug/kg administered daily 48 hours post 5-FU) is included as a positive control for enhanced PMN recovery in these studies. It should be noted that G-CSF is administered 1.5 hours before each bleed point. G-CSF administration under these conditions reflects PMN recovery plus additional PMN mobilization from vascular and possibly tissue sites.
  • Compounds of Formula (I) have shown efficacy as myeloprotectants in a murine high dose 5-FU model noted above.
  • Compound 3 40 mg/kg was administered IP to mice 2 hours before and 4 hours after a single injection of 175 mg/kg 5-FU G-CSF was administered daily starting 48 hours post 5-FU injection for 10 days.

Abstract

The present invention provides for the novel use of compounds of Formula (I) as myeloprotectant compounds having the biological activity of reversibly inhibiting myelopoiesis in mammalian tissue and biological samples, in vitro, ex vivo or in vivo.

Description

METHODS FOR REVERSIBLY INHIBITING MYELOPOIESIS IN MAMMALIAN TISSUE
FIELD OF THE INVENTION
The present invention relates to the field of myelopoiesis in mammalian tissue, and more particularly, to the use of certain imidazoles containing compounds for reversibly inhibiting myelopoiesis in humans and animals.
BACKGROUND OF THE INVENTION
Certain chemotherapeutic and radiation treatments for cancer and other serious illness have the undesired side effect of killing all dividing and differentiating cells. This effect is most severe on the differentiating cells of the hematopoietic system, which are essential for repopulating the bone marrow and permitting the redevelopment of a functioning immune system following such treatment. Normally, bone marrow toxicity or myelosuppression is the limiting factor in the use of such treatments. The acute effects of myelosuppression are the loss of neutrophils (neutropenia) and the loss of platelets (thrombocytopenia) in the blood. These conditions in part increase patient susceptibility to infections and hemorrhagic complications.
A variety of regulatory messengers and modifiers such as colony stimulating factors, interferons, and different types of compounds are responsible for the regulation of myelopoiesis, which is the development and growth of early progenitor cells (stem cells) of the hematopoietic system.
Compounds which have a selective inhibitory effect on the proliferation and differentiation of myelopoietic cells tend to prevent quiescent cells from entering into cell division and becoming susceptible to chemotherapeutic and radiation treatments. A number of such compounds are known, including MlPlα, HP5 monomer, TGFβ, TNF and IL- 1 (for radioprotection only) and AS 101. See, e.g., International Patent Application No. WO9313789, published July 22, 1993, and references cited therein. However, to date, there are no effective pharmaceutical compounds which protect a patient from leukopenia after treatments which create an insult to proliferating stem cells.
There remains a need for pharmaceutically acceptable compounds which are capable of serving as myeloprotectants, i.e., which can inhibit proliferation of normal hematopoietic stem cells during a period of myelosuppressive therapy.
SUMMARY OF THE INVENTION
The present invention provides for a method for reversibly inhibiting myelopoiesis in mammals, mammalian tissue or other samples in vivo and ex vivo comprising administering to said animal, tissue, or sample an effective amount of a myeloprotectant compound of formula (I) below. The amount of the compound is effective to reversibly inhibit the formation of myelopoietic colonies during the time the animal, tissue, or sample is exposed to myelosuppressive therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 demonstrates that compounds of Formula (I), Compounds 2 and 3 are myeloprotective in a high dose 5-FU in vivo model; more specifically this is the CFU-GM Kinetics Post Compounds and 5-FU administration; 4-(4- Fluorophenyl)-5-(2-phenylamino-4-pyridiminyl)- 1 -(4-piperidinyl)imidazole is Compound 2 and 4-(4-Fluorophenyl)-5-(2-N-methylamino-4-pyridiminyl)- l-(4-N-methyl-piperidinyl)imidazole is Compound 3. Figure 2 demonstrates a dose response effect of (IP administered) Compounds 2 and 3 on WBC counts 9 days post 5-FU in the mouse; *P<0.05. Figure 3 demonstrates a dose response effect of (IP administered) Compounds 2 and 3 on PMN counts 9 days post 5-FU in the mouse; *P<0.05. Figure 4. demonstrates a dose response effect of (IP administered) Compounds 2 and
3 on Platelet counts 9 days post 5-FU in the mouse; *P<0.05. Figure 5 demonstrates a dose response effect of (IP administered) Compounds 2 and 3 on lymphocyte counts 9 days post 5-FU in the mouse; *P<0.05. Figure 6 demonstrates a dose response effect of (IP administered) Compounds 2 and
3 on monocyte counts 9 days post 5-FU in the mouse; *P<0.05. Figure 7 demonstrates a dose response effect of (IP administered) Compounds 2 and
3 on RBC counts 9 days post 5-FU in the mouse; *P<0.05. Figure 8 demonstrates a dose response effect of (IP administered) Compounds 2 and
3 on bone marrow cell counts 9 days post 5-FU in the mouse. Marrow counts obtained from pools of 3 mice/group. Figure 9 demonstrates a dose response effect of (PO administered) Compounds 2 and 3 on PMN cell counts 9 days post 5-FU in the mouse. Figure 10 demonstrates a dose response effect of (PO administered) Compounds 2 and 3 on bone marrow cell counts 9 days post 5-FU in the mouse. Marrow counts obtained from pools of 3 mice/group. Figure 11 demonstrates the effect of Compounds 2 and 3 and G-CSF on the kinetics of neutrophil recovery post 175 mg/kg 5-FU in mice. G-CSF (50ug kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days. Figure 12 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of monocyte recovery post 175 mg/kg 5-FU in mice. G-CSF (50ug/kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days.
Figure 13 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of platelet recovery post 175 mg/kg 5-FU in mice. G-CSF (50ug/kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days. Figure 14 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of lymphocyte recovery post 175 mg kg 5-FU in mice. G-CSF (50ug/kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days. Figure 15 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of total white blood cell recovery post 175 mg/kg 5-FU in mice. G-CSF (50ug/kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days. Figure 16 demonstrates the effect of Compounds 2 and 3, and G-CSF on the kinetics of red blood cell recovery post 175 mg/kg 5-FU in mice. G-CSF (50ug/kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days. Figure 17 demonstrates the effect of Compound 3, G-CSF, and the combination of
Compound 3 + G-CSF on the kinetics of neutrophil recovery post 175 mg/kg
5-FU in mice. G-CSF (50ug/kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days.
Figure 18 demonstrates the effect of Compound 3, G-CSF, and the combination of
Compound 3 + G-CSF on the kinetics of platelet recovery post 175 mg/kg
5-FU in mice. G-CSF (50ug/kg) administered daily IP, animals were bleed
1.5 hours post G-CSF injection on the indicated days. Figure 19 demonstrates the effect of Compound 3, G-CSF, and the combination of
Compound 3 + G-CSF on the kinetics of monocyte recovery post 175 mg/kg
5-FU in mice. G-CSF (50ug/kg) administered daily IP, animals were bleed
1.5 hours post G-CSF injection on the indicated days. Figure 20 demonstrates the effect of Compound 3, G-CSF, and the combination of Compound 3 + G-CSF on the kinetics of total white blood cell recovery post
175 mg/kg 5-FU in mice. G-CSF (50ug/kg) administered daily IP, animals were bleed 1.5 hours post G-CSF injection on the indicated days. Figure 21 demonstrates the CFU -GM Micro-Inhibition and Reversibility Assay for
Select Compounds of Formula (I), wherein Compound 1 is 4-(4- Fluorophenyl)-5-(2-amino-4-pyridiminyl)- 1 -( 1 -methyl-4-piperidinyl)- imidazole, and Compound 2 is 4-(4-Fluorophenyl)-5-(2-phenylamino-4- pyridiminyl)- 1 -(4-piperidinyl)imidazole. DETAILED DESCRIPTION OF THE INVENTION
This invention provides for a method for reversibly inhibiting myelopoiesis in mammals, mammalian tissue, including bone marrow and other samples, in vivo and ex vivo. This method entails administering to the mammal, tissue or sample an amount of a compound of formula (I) effective to reversibly inhibit the formation of myelopoietic colonies during the time the mammal, tissue or bone marrow is exposed to myelosuppressive therapy. In this method, the myelopoietic colonies inhibited are CFU-C colony forming cells.
The myelopoietic CFU-C colony forming cells as used herein include, but are not limited to, CFU-G, CFU-M, CFU-GM, CFU-GEMM, CFU-Meg, and HPP. The terms are defined as CFU = colony forming unit, G = granulocyte, M= macrophage or megakaryocyte, E = erythroid colony types, HPP = high proliferative potential colony. A preferred colony forming cell is CFU-GM.
This method may be accomplished in vivo. In other words, a mammalian subject, preferably a human patient, undergoing chemotherapy or radiation may be administered the compound so that the reversible inhibition of myelopoiesis occurs in vivo.
Alternatively, the method may be accomplished ex vivo or in vitro. According to this embodiment of the method, the mammalian tissue, for example, bone marrow, is contacted ex vivo with an effective amount of myeloprotectant compound of this invention substantially simultaneously with exposure of the tissue to radiation or chemotherapeutic s. Inhibition of myelopoiesis is reversible when administration of the insult and the compound, which occur ex vivo, cease.
The myeloprotectant compounds, described herein, when administered prophylactically and/or therapeutically as a myelosuppressive treatment, benefit the recipient by protecting the normal hematopoietic stem cells from the damaging agent(s). Treatment with a myeloprotectant compound of this invention may be necessary throughout the time period in which the insulting agent is present within the body. Inhibition of normal hematopoietic stem cells during this period could protect normal cells while damaging only target cells such as cancer cells. Because the compounds of this invention have demonstrated reversible inhibition, as demonstrated in the Example Section below, a more rapid rebound of hematopoietic stem cell proliferation is expected when administration of the myeloprotectant compound is ceased (concurrently with, or shortly after administration of the chemotherapeutic or radiation is ceased). The end result of treatment with the compounds of this invention as myeloprotective agents is to prevent neutropenia and or accelerate neutrophil / leukocyte recovery, and prevent stem cell loss due to myelosuppression. The advantage of such administration to the patient includes fewer infections due to leukopenia and the ability to use more aggressive chemotherapy regimens to kill cancer cells (dose intensification / increase dose frequency).
Compounds for use herein include the cytokine inhibitors as described in USSN 08/091,491, published as WO95/02575; WO96/21452; US Patent No.: ; USSN 08/473,396; US Patent No. 5,658,903 ; USSN 08/764,003; USSN 08/473,398; WO96/21654; WO93/14081; US Patent 5,656,644;USSN 08/095,234; US 5,656,644; WO95/03297; USSN 08/481,671; PCT/US97/00619; PCT/US97/00614; PCT/US97/00500; PCT/US 97/00529; USSN 60/013,357; USSN 60/013,358; USSN 60/013,359; WO93/14082; WO95/13067 ;WO95/31451 WO95/13067;WO95/31451 WO 97/05877; WO 97/05878; WO 97/16441; WO 97/16426; and WO 97/16442. Each of these references are incorporated by reference herein in their entirety.
A preferred group of compounds for use herein are those compounds of the formula (I):
Figure imgf000008_0001
wherein:
Rl is 4-pyridyl, pyrimidinyl, 4-pyridazinyl, l,2,4-triazin-5-yl, quinolyl, isoquinolinyl, quinazolin-4-yl, 1-imidazolyl or 1-benzimidazolyl ring, which ring is optionally substituted independently one to three times with Y. NHRa, optionally substituted -4 alkyl, halogen, hydroxyl, optionally substituted C\-4 alkoxy, optionally substituted Ci-4 alkylthio, Ci-4 alkylsulfmyl, CH2OR12, amino, mono and di- -6 alkyl substituted amino, or N(Rιo)C(O)RD; Y is O-Ra;
R4 is phenyl, naphth-1-yl or naphth-2-yl, or a heteroaryl, which is optionally substituted by one or two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-naphth-l-yl, 5-naphth-2-yl or 6-naphth-2-yl substituent, is halogen, cyano, nitro, C(Z)NR7Ri7, C(Z)ORi6, (CRioR20)vCORi2, SR5, SOR5, OR12, halo-substituted-Ci-4 alkyl, Ci-4 alkyl, ZC(Z)Ri2, NRioC(Z)Ri6, or (CRioR2θ)vNRlθR20 and which, for other positions of substitution, is halogen, cyano, C(Z)NRi3Ri4, C(Z)OR3, (CRioR20)m"COR3, S(O)mR3, OR3, halo-substituted-Ci-4 alkyl, Ci-4 alkyl, (CRιoR2θ)m"NRlθC(Z)R3, NRi0S(O)m'R8, NRι0S(O)m'NR7Rl7, ZC(Z)R3 or (CRioR20)m"NRl3Rl4; v is 0, or an integer having a value of 1 or 2; n is an integer having a value of 1 to 10; n' is 0, or an integer having a value of 1 to 10; m is 0, or the integer 1 or 2; nϊ is an integer having a value of 1 or 2, m" is 0, or an integer having a value of 1 to 5;
R2 is hydrogen, (CRioR20)n' OR9, heterocyclyl, heterocyclylCi-io alkyl,
Ci.ioalkyl, halo-substituted Ci-io alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7cycloalkylCi -io alkyl, C5-7 cycloalkenyl, C5-7 cycloalkenyl Ci-ioalkyl, aryl, arylCj-io alkyl, heteroaryl, heteroarylCi-ioalkyl,
(CRlθR20)nORl l, (CRioR20)nS(O)mRi8, (CRιoR2θ)nNHS(O)2Rl8, (CRi0R20)nNRl3Rl4, (CRlθR2θ)nNO , (CRioR20)nCN, (CRioR20)n'SO Rl8, (CRioR20)nS(O)m'NRi3Rl4, (CRioR20)nC(Z)Rn, (CRlθR20)nOC(Z)Rn, (CRioR20)nC(Z)ORn, (CRioR20)nC(Z)NRi3Rl4, (CRi0R20)nC(Z)NRi 1OR9, (CRiθR2θ)nNRlθC(Z)Ri 1,
(CRi0R20)nNRl0C(Z)NRi3Rl4, (CRl0R20)nN(OR6)C(Z)NRι3Rl4, (CR ioR20)nN(OR6)C(Z)R 11 , (CR 10R20)nC(=NOR6)R 11 , (CRιoR2θ)nNRlθC(=NRi9)NRi3Rl4, (CRlθR2θ)nOC(Z)NRι3Ri4, (CR 10R20)nNR lθC(Z)NR 13R 14, (CR 10R20)nNR l θC(Z)OR 10, 5-(R 18)- 1 ,2,4- oxadizaol-3-yl or 4-(R12)-5-(Ri8Ri9)-4,5-dihydro-l,2,4-oxadiazol-3-yl; wherein the aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroaryl alkyl, heterocyclic and heterocyclic alkyl groups may be optionally substituted; Z is oxygen or sulfur;
Ra is Ci-6alkyl, aryl. arylC i-6alkyl. heterocyclic, heterocyclylC i -6 alkyl, heteroaryl, or heteroarylC i-6alkyl, wherein each of these moieties may be optionally substituted; Rb is hydrogen, C{.β alkyl, C3-7 cycloalkyl, aryl, arylCi-4 alkyl, heteroaryl, heteroarylC i-4alkyl, heterocyclyl, or heterocyclylC 1 -4 alkyl; R3 is heterocyclyl, heterocyclylC i-io alkyl or Rδ; R5 is hydrogen, Ci-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the moieties -SR5 being -SNR7R17 and -SOR5 being -SOH; R6 is hydrogen, a pharmaceutically acceptable cation, Ci-10 alkyl, C3-7 cycloalkyl, aryl, arylCi-4 alkyl, heteroaryl. heteroarylC 1.4 alkyl, heterocyclic, aroyl, or
Ci-10 alkanoyl; R7 and R17 is each independently selected from hydrogen or C 1-4 alkyl or R7 and
Rl7 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR15; R8 is Ci-io alkyl, halo-substituted Ci-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylCi-io alkyl, heteroaryl, heteroarylC l- 10 alkyl, (CRioR20)nORl l, (CRιoR2θ)nS(O)mRi8, (CRioR20)nNHS(O)2Rl8,
(CRioR20)nNRl3Rl4; wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl may be optionally substituted; R9 is hydrogen, C(Z)Ri 1 or optionally substituted Ci-io alkyl, S(O)2Rl8, optionally substituted aryl or optionally substituted aryl-Ci-4 alkyl;
RlO and R20 is each independently selected from hydrogen or C 1-4 alkyl; Rl 1 is hydrogen, Ci-10 alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclyl
Cl-ioalkyl, aryl, arylCi-io alkyl, heteroaryl or heteroarylC l- 10 alkyl; Rl2 is hydrogen or Ri ; R13 and R14 is each independently selected from hydrogen or optionally substituted
Ci-4 alkyl, optionally substituted aryl or optionally substituted aryl-Ci-4 alkyl, or together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9; Ri5 is Rio or C(Z)-Ci-4 alkyl;
Rl6 is Ci-4 alkyl, halo-substituted-Cl-4 alkyl, or C3.7 cycloalkyl; Rl8 is C i-io alkyl. C3-7 cycloalkyl, heterocyclyl, aryl, arylalkyl, heterocyclyl, heterocyclyl-Ci-ioalkyl, heteroaryl or heteroarylalkyl;
Rl9 is hydrogen, cyano, C1-4 alkyl, C3.γ cycloalkyl or aryl; or a pharmaceutically acceptable salt thereof.
Suitably Ri is a substituted 4-pyridyl or 4-pyrimindyl. More suitably R, is substituted by alkoxy, alkylthio, amino, methylamino, NHRa, or Y. A preferred ring placement of the Ri substituent on the 4-pyridyl derivative is the 2-position, such as 2-methoxy -4-pyridyl. A preferred ring placement on the 4-pyrimidinyl ring is also at the 2-position, such as in 2-methoxy-pyrimidinyl.
When the substituent is Y, and Ra is aryl, it is preferably phenyl or naphthyl. When Ra is aryl alkyl, it is preferably benzyl or napthylmethyl. When Ra is heterocyclic or heterocyclic alkyl moiety, the heterocyclic portion is preferably pyrrolindinyl, piperidine, morpholino, tetrahydropyran, tetrahydrothiopyranyl, tetrahydrothipyran-sulfinyl, tetrahydrothio-pyransulfonyl, pyrrolindinyl, indole, or piperonyl. It is noted that the heterocyclic rings herein may contain unsaturation, such as in a tryptamine ring.
The aryl, heterocyclic and heteroaryl rings may be optionally substituted one or more times independently with halogen; Ci-4 alkyl, such as methyl, ethyl, propyl, isopropyl, or t-butyl; halosubstituted alkyl, such as CF3; hydroxy; hydroxy substituted Ci-4 alkyl; Cj-4 alkoxy, such as methoxy or ethoxy; S(O)malkyl and S(O)m aryl (wherein m is 0, 1, or 2); C(O)ORι 1, such as C(O)Ci-4 alkyl or C(O)OH moieties; C(O)Rι 1; OC(O)Rc; O-(CH2)s-O-, such as in a ketal or dioxyalkylene bridge; amino; mono- and di-Ci-6 alkylsubstituted amino; N(Rιo)C(O)RD; C(O)NRioR20; cyano, nitro, or an N-heterocyclyl ring which ring has from 5 to 7 members and optionally contains an additional heteroatom selected from oxygen, sulfur or NR15; aryl, such as phenyl; an optionally substituted arylalkyl, such as benzyl or phenethyl; aryloxy, such as phenoxy; or arylalkyloxy such as benzyloxy. Rc is optionally substituted -6 alkyl, C3.7 cycloalkyl, aryl, arylCi-4 alkyl, heteroaryl, heteroarylC i-4alkyl, heterocyclyl, or heterocyclylC 1.4 alkyl moieties. Preferably, the Ra groups include Cj_4 alkyl, benzyl, halosubstituted benzyl, napthylmethyl, phenyl, halosubstituted phenyl, aminocarbonylphenyl, alkylphenyl, cyanophenyl. alkylthiophenyl, hydroxyphenyl, alkoxyphenyl, mo holinopropyl, piperonyl, piperidin-4-yl, alkyl substituted piperidine, such as 1 -methyl piperidine, or 2,2,6,6-tetramethylpiperidin-4-yl.
Preferably, when the substituent is NHRa then Ra is aryl, arylalkyl, halosubstituted arylalkyl, halosubstituted aryl, heterocyclic alkyl, hydroxy alkyl, alkyl- 1-piperidine-carboxylate, heterocyclic, alkyl substituted heterocyclic, halosubstituted heterocyclic, or aryl substituted heterocyclic. More specifically R is benzyl, halosubstituted benzyl, napthylmethyl, phenyl, halosubstituted phenyl, moφholinopropyl, 2-hydroxy ethyl, ethyl- 1-piperidinecarboxy late, piperonyl, piperidin-4-yl, alkyl substituted piperidine, chlorotryptamine, and tetrathiohydropyranyl.
Preferably, when the substituent is a substituted C 1-4 alkoxy or -4 alkylthio, the alkyl chain is substituted by halogen, such as fluorine, chlorine, bromine or iodine; hydroxy, such as hydroxyethoxy; Ci-io alkoxy, such as a methoxymethoxy, S(O)m alkyl, wherein m is 0, 1 or 2; amino, mono & di- substituted amino, such as in the NR7R17 group, i.e. tert-butylaminoethoxy; or where the R7R17 may together with the nitrogen to which they are attached cyclize to form a 5 to 7 membered ring which optionally includes an additional heteroatom selected from O/N/S; Ci-io alkyl, cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl, isopropyl, t-butyl, etc. or cyclopropyl methyl; or halosubstituted Ci-io alkyl, such as CF3. Preferably the Ri substituents are tertbutylaminoethoxy, or hydroxyethoxy. Suitably, R4 is an optionally substituted phenyl. Preferably the phenyl is substituted one or more times independently by halogen, SR5, S(O)R5, OR12, halo- substituted-Ci-4 alkyl, or Ci-4 alkyl.
Suitably, R2 is selected from hydrogen, Ci-io alkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylC l- 10 alkyl, (CRioR20)nNS(O)2Rl8, (CRioR20)nS(O)mRi8, arylCi-io alkyl,
(CRiQR20)nNRl3Rl4, optionally substituted C3-7cycloalkyl, or optionally substituted C3-7cycloalkyl -io alkyl. Preferably R2 is moφholino propyl, piperidine, N-methylpiperidine, N-benzylpiperidine, 2,2,6,6-tetramethylpiperidine, 4-aminopiperidine, 4-amino-2,2,6,6-tetramethyl piperidine, 4-hydroxycyclohexyl, 4- methyl-4-hydroxy cyclohexyl, 4-pyrrolinindyl-cyclohexyl, 4-methyl-4- aminocyclohexyl, 4-methyl-4-acetamidocyclohexyl, 4-keto cyclohexyl, 4-oxiranyl, or 4-hydroxy-4-( 1 -propynyl)cyclohexyl.
Preferably R2 is hydrogen, optionally substituted heterocyclyl ring, optionally substituted heterocyclylC l- 10 alkyl, optionally substituted -io alkyl, optionally substituted C3-7cycloalkyl, optionally substituted C3-7cycloalkyl Ci-io alkyl, (CRιoR2θ)nC(Z)ORι 1 group, (CRioR20)nNRi3Rl4,
(CRioR20)nNHS(O)2Rl8, (CRlθR2θ)nS(O)mRi8, optionally substituted aryl; optionally substituted arylCj-io alkyl, (CRioR20)nORl 1, (CRioR20)nC(Z)Rι 1, or (CRlθR20)nC (=NOR6)Rl l group.
More preferably R2 is an optionally substituted heterocyclyl ring, and optionally substituted heterocyclylC l- 10 alkyl, optionally substituted aryl,
(CRioR20)nNRl3Rl4, (CRioR20)nC(Z)ORn group, optionally substituted C3- 7cycloalkyl, or an optionally substituted C3-7cycloalkyl Ci-10 alkyl.
When R2 is an optionally substituted heterocyclyl, the ring is preferably a moφholino, pyrrolidinyl, or a piperidinyl group. When the ring is optionally substituted, the substituents may be directly attached to the free nitrogen, such as in the piperidinyl group or pyrrole ring, or on the ring itself. Preferably the ring is a piperidine or pyrrole, more preferably piperidine. The heterocyclyl ring may be optionally substituted one to four times independently by halogen; Ci-4 alkyl; aryl, such as phenyl; aryl alkyl, such as benzyl - wherein the aryl or aryl alkyl moieties themselves may be optionally substituted (as in the definition section below);
C(O)ORi 1, such as the C(O)Ci-4 alkyl or C(O)OH moieties; C(O)H; C(O)Cι_4 alkyl, hydroxy substituted Ci-4 alkyl, -4 alkoxy, S(O)mCi-4 alkyl (wherein m is 0, 1, or 2), NRlθR20 (wherein Rio and R20 are independently hydrogen or Cl- 4alkyl). Preferably if the ring is a piperidine, the ring is attached to the imidazole at the 4-position, and the substituents are directly on the available nitrogen, i.e. a l-Formyl-4-piperidine, l-benzyl-4-piperidine, l-methyl-4-piperidine, l-ethoxycarbonyl-4-piperidine. If the ring is substituted by an alkyl group and the ring is attached in the 4-position, it is preferably substituted in the 2- or 6- position or both, such as 2,2,6,6-tetramethyl-4-piperidine. Similarly, if the ring is a pyrrole, the ring is attached to the imidazole at the 3-position, and the substituents are all directly on the available nitrogen.
When R2 is an optionally substituted heterocyclyl Ci-io alkyl group, the ring is preferably a moφholino, pyrrolidinyl, or a piperidinyl group. Preferably this alkyl moiety is from 1 to 4, more preferably 3 or 4, and most preferably 3, such as in a propyl group. Preferred heterocyclic alkyl groups include but are not limited to, moφholino ethyl, moφholino propyl, pyrrolidinyl propyl, and piperidinyl propyl moieties. The heterocyclic ring herein is also optionally substituted in a similar manner to that indicated above for the direct attachment of the heterocyclyl. When R2 is an optionally substituted C3-7cycloalkyl, or an optionally substituted C3-7cycloalkyl Ci-io alkyl, the cycloalkyl group is preferably a C4 or C6 ring, most preferably a C6 ring, which ring is optionally substituted. The cycloalkyl ring may be optionally substituted one to three times independently by halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; Ci-10 alkoxy, such as methoxy or ethoxy; S(O)m alkyl, wherein m is 0, 1, or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl; S(O)m aryl; cyano, nitro, amino, mono & di- substituted amino, such as in the NR7R17 group, wherein R7 and R17 are as defined in Formula (I), or where the R7R17 may cyclize together with the nitrogen to which they are attached to form a 5 to 7 membered ring which optionally includes an additional heteroatom selected from oxygen, sulfur or NR15 (and R 15 is as defined for Formula (I)); N(Rιo)C(O)Xι (and Xi is Ci-4 alkyl, aryl or arylCi-4alkyl); N(Rio)C(O) aryl; Ci-io alkyl, such as methyl, ethyl, propyl, isopropyl, or t-butyl; optionally substituted alkyl wherein the substituents are halogen, (such as CF3), hydroxy, nitro, cyano, amino, mono & di-substituted amino, such as in the NR7R17 group, S(O)malkyl and S(O)m aryl, wherein m is 0, 1 or 2; optionally substituted alkylene, such as ethylene or propylene; optionally substituted alkyne, such as ethyne; C(O)ORι 1 (wherein Ri 1 is as defined in Formula (I)), such as the free acid or methyl ester derivative; the group Re; C(O)H; =0; =N-ORι i ; N(H)-OH (or substituted alkyl or aryl derivatives thereof on the nitrogen or the oxime moiety); N(OR )-C(O)-R6'; an optionally substituted aryl, such as phenyl; an optionally substituted arylCi-4alkyl, such as benzyl of phenethyl: an optionally substituted heterocycle or heterocyclic C i-4alkyl, and further these aryl, arylalkyl, heterocyclic, and heterocyclic alkyl moieties are optionally substituted one to two times by halogen, hydroxy, Ci-io alkoxy, S(0)m alkyl, cyano, nitro, amino, mono & di- substituted amino, such as in the NR7R17 group, an alkyl, halosubstituted alkyl.
Suitably R is hydrogen, a pharmaceutically acceptable cation, aroyl or a Ci-io alkanoyl group.
Suitably Re is a 1,3-dioxyalkylene group of the formula -O-(CH2)s-0-, wherein s is 1 to 3, preferably s is 2 yielding a 1 ,3-dioxyethylene moiety, or ketal functionality. Suitably R6' is NRi9'R20' a^Y^ l-6> halosubstituted alkyl 1-6; hydroxy substituted alkyl 1-6; alkenyl 2-6; aryl or heteroaryl optionally substituted by halogen, alkyl 1-6, halosubstituted alkyl 1 -6, hydroxyl, or alkoxy i-ζ.
Suitably R 19' is H or alkyl 1 -6.
Suitably R20' is H, alkyl 1.5, aryl, benzyl, heteroaryl, alkyl substituted by halogen or hydroxyl, or phenyl substituted by a member selected from the group consisting of halo, cyano, alkyl ι _ 12, alkoxy 1. , halosubstituted alkyl 1-6, alkylthio, alkylsulphonyl, or alkylsulfinyl; or R19' and R20' may together with the nitrogen to which they are attached form a ring having 5 to 7 members, which members may be optionally replaced by a heteroatom selected from oxygen, sulfur or nitrogen. The ring may be saturated or contain more than one unsaturated bond. Preferably R6' is NRi9'R20' and R19' and R20' are preferably hydrogen. When the R2 cycloalkyl moiety is substituted by NR7R17 group, or NR7R17
Cl-10 alkyl group, and the R7 and R17 are as defined in Formula (I), the substituent is preferably an amino, amino alkyl, or an optionally substituted pyrrolidinyl moiety.
Preferably, R^ and R^ are hydrogen, hydroxy, alkyl, substituted alkyl, optionally substituted alkyne, aryl, arylalkyl, NR7R17, and N(Rio)C(O)Rπ. Suitably, alkyl is -4 alkyl, such as methyl, ethyl, or isopropyl; NR7R17 and
NR7R17 alkyl, such as amino, methylamino, aminomethyl, aminoethyl; substituted alkyl such as in cyanomethyl, cyanoethyl, nitroethyl, pyrrolidinyl; aryl such as in phenyl; arylaikyl, such as in benzyl; optionally substituted alkyne, such as ethyne or propynyl; or together R and R^ are a keto functionality.
Preferably R2 is an optionally substituted heterocyclic, heterocylic Ci-4 alkyl, a cycloalkyl or a cycloalkyl alkyl. More preferably R2 is an optionally substituted C4 or C cycloalkyl, cyclopropyl methyl, moφholinyl butyl, moφholinyl propyl, moφholinyl ethyl, cyclohexyl substituted by methyl, phenyl, benzyl, amino, acetamide, aminomethyl, aminoethyl, cyanomethyl, cyanoethyl, hydroxy, nitroethyl, pyrrolidinyl, ethynyl, 1-propynyl, =0, O-(CH2)2O-, =NORl 1, wherein Rn is hydrogen, alkyl or aryl, NHOH, or N(OH)-C(O)-NH2; or R2 is moφholinyl propyl, aminopropyl, piperidinyl, N-benzyl-4-piperidinyl, N-methyl-4- piperidinyl, 2.2,6,6-tetramethypiperidinyl, substituted piperidine, such as 1-Formyl- 4-piperidine, or a l-ethoxycarbonyl-4-piperidine.
Preferred compounds of Formula (I) include: 4-(4-Fluorophenyl)-5-(2-amino-4-pyridiminyl)- 1 -( 1 -methyl-4- piperidinyl)imidazole;
4-(4-FIuorophenyl)-5-(2-phenylamino-4-pyridiminyl)-l-(4-piperidinyl)imidazole; 4-(4-Fluorophenyl)-5-(2-N-methylamino-4-pyridiminyl)-l-(4-N-methyl- piperidinyl)imidazole; 4-(4-Fluorophenyl)-5-(2-N-methylamino-4-pyridiminyl)- 1 -(4- piperidinyl)imidazole;or pharmaceutically acceptable salts thereof.
In all instances herein where there is an alkenyl or alkynyl moiety as a substituent group, the unsaturated linkage, i.e., the vinylene or acetylene linkage is preferably not directly attached to the nitrogen, oxygen or sulfur moieties, for instance in OR3, or for certain R2 moieties.
As used herein, "optionally substituted", unless specifically defined, shall mean such groups as halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; hydroxy substituted Ci-ioalkyl; Ci-io alkoxy, such as methoxy or ethoxy; S(O)m alkyl, wherein m is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl; amino, mono & di-substituted amino, such as in the NR7R17 group; or where the R7R17 may together with the nitrogen to which they are attached cyclize to form a 5 to 7 membered ring which optionally includes an additional heteroatom selected from O/N/S; - io alkyl, cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl, isopropyl, t-butyl, etc. or cyclopropyl methyl; halosubstituted Cl-10 alkyl, such CF3; an optionally substituted aryl, such as phenyl, or an optionally substituted arylalkyl, such as benzyl or phenethyl, wherein these aryl moieties may also be substituted one to two times by halogen; hydroxy; hydroxy substituted alkyl; Cl-10 alkoxy; S(O)m alkyl; amino, mono & di-substituted amino, such as in the NR7R17 group; alkyl, or CF3. Suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methane sulphonic acid, ethane sulphonic acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid and mandelic acid. In addition, pharmaceutically acceptable salts of compounds of Formula (I) may also be formed with a pharmaceutically acceptable cation, for instance, if a substituent group comprises a carboxy moiety. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations.
The following terms, as used herein, refer to:
• "halo" or "halogens", include the halogens: chloro, fluoro, bromo and iodo.
• "Ci-ioalkyl" or "alkyl" - both straight and branched chain radicals of 1 to 10 carbon atoms, unless the chain length is otherwise limited, including, but not limited to, methyl, ethyl, rc-propyl, wσ-propyl, -butyl, sec-butyl, wσ-butyl, tert- butyl, «-pentyl and the like.
• The term "cycloalkyl" is used herein to mean cyclic radicals, preferably of 3 to 8 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the like. • The term "cycloalkenyl" is used herein to mean cyclic radicals, preferably of 5 to 8 carbons, which have at least one bond including but not limited to cyclopentenyl, cyclohexenyl, and the like.
• The term "alkenyl" is used herein at all occurrences to mean straight or branched chain radical of 2- 10 carbon atoms, unless the chain length is limited thereto, including, but not limited to ethenyl, 1-propenyl, 2-propenyl, 2-methyl-l- propenyl, 1-butenyl, 2-butenyl and the like.
• "aryl" - phenyl and naphthyl;
• "heteroaryl" (on its own or in any combination, such as "heteroaryloxy", or "heteroaryl alkyl") - a 5-10 membered aromatic ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O or S, such as, but not limited, to pyrrole, pyrazole, furan, thiophene, quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, thiazole, thiadiazole, triazole, imidazole, or benzimidazole. • "heterocyclic" (on its own or in any combination, such as
"heterocyclylalkyl") - a saturated or partially unsaturated 4-10 membered ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O, or S; such as, but not limited to, pyrrolidine, piperidine, piperazine, moφholine, tetrahydro pyran, or imidazolidine. • The term "aralkyl" or "heteroarylalkyl" or "heterocyclicalkyl" is used herein to mean Ci-4 alkyl as defined above attached to an aryl, heteroaryl or heterocyclic moiety as also defined herein unless otherwise indicated.
• "sulfinyl" - the oxide S (O) of the corresponding sulfide, the term "thio" refers to the sulfide, and the term "sulfonyl" refers to the fully oxidized S(O)2 moiety.
For the puφoses herein the "core" 4-pyrimidinyl moiety for Ri or R2 is referred to as the formula:
Figure imgf000018_0001
It is recognized that the compounds of the present invention may exist as stereoisomers, regioisomers, or diastereiomers. These compounds may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All of these compounds are included within the scope of the present invention.
As noted previously, methods of making these compounds can be found in their respective patent applications as noted above.
Compounds of Formula (I) are capable of inhibiting proinflammatory cytokines, such as IL-1, IL-6, IL-8 and TNF. A member of the MAP kinase family, alternatively termed CSBP, p38, or
RK, has been identified independently by several laboratories under different names. Activation of this novel protein kinase via dual phosphorylation has been observed in different cell systems upon stimulation by a wide spectrum of stimuli, such as physicochemical stress and treatment with lipopolysaccharide or proinflammatory cytokines such as interleukin- 1 and tumor necrosis factor. The cytokine biosynthesis inhibitors of Formula (I) have been determined to be potent and selective inhibitors of CSBP/p38/RK kinase activity. Patent Application USSN 08/123175 Lee et al., filed September 1993, USSN; Lee et al., PCT 94/10529 filed 16 September 1994 and Lee et al., Nature 300, n(72), 739-746 (Dec. 1994) whose disclosures are incoφorated by reference herein in its entirety describes the above noted method for screening drugs to identify compounds which interact with and bind to the cytokine specific binding protein (hereinafter CSBP).
In order to use a compound of Formula (I) or a pharmaceutically acceptable salt thereof in therapy, it will normally be Formulated into a pharmaceutical composition in accordance with standard pharmaceutical practice. This invention, therefore, also relates to a pharmaceutical composition comprising an effective, non- toxic amount of a compound of Formula (I) and a pharmaceutically acceptable carrier or diluent.
Compounds of Formula (I), pharmaceutically acceptable salts thereof and pharmaceutical compositions incoφorating such may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation. The compounds of Formula (I) may be administered in conventional dosage forms prepared by combining a compound of Formula (I) with standard pharmaceutical carriers according to conventional procedures. The compounds of Formula (I) may also be administered in conventional dosages in combination with a known, second therapeutically active compound. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the Formulation and not deleterious to the recipient thereof.
The pharmaceutical carrier employed may be, for example, either a solid or liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
A wide variety of pharmaceutical forms can be employed. Thus, if a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge. The amount of solid carrier will vary widely but preferably will be from about 25mg. to about lg. When a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension. It is recognized that compounds of Formula (I) can be administered by inhalation, orally, bucally, etc. and as such the previously noted patents which are incoφorated by reference herein should be conferred.
It is expected that compounds of formula (I) may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of the parenteral form administration are generally preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques. An oral administration is the most preferred route of treatment.
As used throughout the description of the methods for inhibiting myelopoiesis, the term "effective amount" refers to that amount of the compound of formula (I) which evokes a reversible inhibition of proliferating myelopoietic cells. In general, in order to exert an inhibitory effect, the compounds of the invention may be administered to human or other mammalian patients by injection in the dose range of about 0.5 mg to about 1000 mg. As one example, a desirable dosage range is from about 5 to about 1000 mg. The daily oral dosage regimen will be from about 0.1 to about 100 mg/kg of total body weight, preferably from about 0.2 to 50 mg/kg. If administered by infusion or similar means, the daily parenteral dosage regimen will be from about 0.1 to about 100 mg/kg of total body weight, preferably from about 0.2 to about 50 mg/kg. In principle, it is desirable to produce a concentration of the compound of about 10"9 M to about 10"5 M in the extracellular fluid of the patient, preferably 10"^ M.
For adaptation of the method for ex vivo or in vitro use of these compounds, e.g., for administration to bone marrow outside of the body of the patient donor, the effective amounts or dosages may be adjusted based on the amount of tissue being treated. Preferably, an effective in vitro concentration is in the range of about 10~9 M to 10"5 M. One of skill in the art may readily determine other appropriate dosages, depending on the mode of administration, and the level of aggressiveness of therapy required in the specific circumstance.
Another aspect of the present invention is a method for identifying or screening for compounds which inhibit CFU-C formation of myeloid progenitors, which method comprises the steps of:
(a) bringing together a test sample containing one or more test compounds and hematopoietic growth factors with a preparation comprising a receptor on myeloid progenitor cells capable of detectably and reversibly interacting with a compound of Formula (I) as defined herein, in soft agar; (b) incubating said test sample and said preparation under conditions which would permit the detection of inhibition of CFU-C colony growth; and
(c) determining the degree of inhibition caused by said test sample by measuring MTT conversion by extracting said preparation and measuring optical density at 570 nm with a reference filter of 750 nm on an ELIS A reader.
This method further comprises a step of comparing the degree of inhibition caused by said test sample with that caused by said compound. Preferably the receptor preparation comprises isolated myeloid progenitor cells. This method allows, preferably, for the preparation to be from bone marrow cells in soft agar.
BIOLOGICAL EXAMPLES SECTION
The following examples illustrate the preparation and uses of myeloprotectant compounds of this invention. These examples are illustrative only, and do not limit the scope of the present invention.
A. Micro-Inhibition Assay
One screening and identification method useful in the present invention may employ the step of screening test samples which detectably bind to a receptor for in vitro or in vivo inhibition of CFU-C colony formation in either a conventional 7 day CFU-GM assay (CFU-C Assay), well known to those skilled in the art, or in the novel, micro-screening assay as described herein. The micro-inhibition assay is a modification of the conventional assay and provides for an efficient and rapid screening and identification of inhibitors. The presence or amount of inhibition of CFU-C or CFU-GM colony formation can then be measured in order to identify those test samples which act as agonists. For example, in one such embodiment, an assay for the screening or identification of other myeloprotectant compounds capable of reversible inhibition includes the following steps. First, to measure the occurrence of, and degree of, inhibition, a test sample containing one or more test compounds and selected hematopoietic growth factors is contacted with bone marrow cells in soft agar (a semi-solid matrix) in a 96 well tissue culture plate. The hematopoietic growth factor may be any of the known factors. Desirable factors specifically include M-CSF, IL-1, IL-3, stem cell factor (SCF), and/or a combination thereof.
The plate is then incubated under conditions which would permit the detection of inhibition of CFU-C CFU-C or CFU-GM colony growth. Such conditions include incubation at about 37°C humidified atmosphere of about 7% CO2 for about 4 to 7 days. The degree of inhibition caused by the test sample is detected by measuring the conversion of the mitochondrial metabolism of (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), also known as MTT, to an insoluble formazan crystal which can be dissolved in sodium dodecyl sulfate (SDS). Optical density is measured at 570 nm with a reference filter of 750 nm on an ELIS A reader. The results of the assay with the peptide are compared to those of a control, i.e., bone marrow cells with growth factors and no peptide, as well as marrow cells only. Increased OD means increased MTT conversion indicative of increased cell metabolism or growth.
B. Micro-Reversibility Assay
An alternative assay method, called a pre-CFU-liquid culture (PCLC) assay, enables the determination of whether the test compound demonstrates reversibility of inhibition. In the PCLC assay or the novel micro reversibility assay as described below, the test sample is first contacted with bone marrow cells in a liquid medium with or without a test compound, in this instance a compound of formula (I), in a 96 well tissue culture plate for 1 to 4 days. Marrow cells are washed with medium three times and recultured in either a conventional CFU-C assay or the above noted Micro-Inhibition assay. The degree of inhibition caused by the test sample is again detected by measuring MTT conversion, as an index of CFU-C proliferation, and measuring optical density at 570 nm with a reference filter of 750 nm on an ELISA reader, using the same controls, as described above.
The assay methods may be further modified by preparing two or more additional test samples from the original test sample or samples that are determined to inhibit CFU-C colony formation activity. These additional test samples contain a lesser number of test compounds than the original test sample from which they were prepared. The steps of the assays may then be repeated as many times as desired or until the test compound or compounds which bind to the receptor preparation have been identified.
Example 1 - Myeloprotectant Compound Demonstrates Bone Marrow CFU-C Inhibitory Activity in vitro in a Micro-Inhibition Assay
Dilutions of compound stock solution (DMSO) are made in phosphate buffered saline (PBS) with 0.01% bovine serum albumin. Different concentrations (5 uM, 0.5 uM, 50 nM, 5 nM) of Compound were added to wells in a maximum volume of 25 microliters. Non adherent bone marrow cells (4 x 10^ cells/ 200 ul) were added in agar with 250 Units of M-CSF. These plates were grown in 37°C incubators in a humidified atmosphere of 7% CO, for 5 days. All Micro-Inhibition plates were incubated with MTT for several hours and solubilized with SDS overnight. Plates were read at 570 nm with a reference of 750 nm. All values compared to wells containing media / agar/ and cells with no M-CSF growth factor.
The results of this assay demonstrate that compounds of Formula (I) inhibit murine bone marrow CFU-C growth. Bovine serum albumin (0.01%) represents background M-CSF stimulated CFU-C growth detected in this assay format. TGFβ- 1 (25 ng/ml) was included as a positive control for CFU-C growth inhibition.
Compounds of Formula (I) which inhibited murine bone marrow CHU-C growth are: 1 -(3'-N-Moφholino- 1 ,-propyl)-4-(4'-fluorophenyl)-5-(4'-pyridyl)imidazole
5-(2- Amino-4-pyrimidinyl)-4-(4- fluorophenyl)- 1 -( 1 -methy l-4-piperidinyl)imidazole 5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-l-(2,2,6,6-tetramethyl-4- piperidinyl)imidazole 5-[4-(2-N-Methylamino)pyrimidinyl]-4-(4-fluorophenyl)-l-(4-N- methylpiperidine)imidazole 5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)- l-(trans-4- hydroxycyclohexyl)imidazole 5-(2-Methylamino-4-pyrimidinyl)-4-(4-fluorophenyl)-l-(4-piperidinyl)imidazole 4-(4-Fluorophenyl)-5-(4-pyridyl)- 1 -(4-piperidinyl)imidazole 5-(2-Pheny lamino-4-pyrimidinyl)-4-(4-fluorophenyl)- 1 -(4-piperidinyl)imidazole Trans- l-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-[(2-methoxy)pyrimidin-4- yljimidazole 5-(2-Phenoxy-4-pyrimidinyl)-4-(4-fluorophenyl)-l-(4-piperidinyl)imidazole 5-[4-(2-Hydroxy)pyrimidinyl]-4-(4-fluorophenyl)-l-(4-ketocyclohexyl)imidazole 1 -(4-piperidinyl)-4-(4-fluorophenyl)-5-(2-phenoxy-4-pyridinyl)imidazole l-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-isopropoxy-4-pyrimidinyl) imidazole l-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)imidazole l-(4-Piperidinyl)-[2-3H]-4-(4-fluorophenyl)-5- (2-methoxy-4-pyrimidinyl) imidazole Trans-5-[4-(2-Phenoxy)pyrimidinyl]-4-(4-fluorophenyl)-l-(4- hydroxycyclohexyl)imidazole l-(4-Piperidinyl)-4-(4-fluorophenyl)-5-(2-anilino-4-pyridinyl)imidazole 1 -(Piperidin-4-yl)-4-(4-fluoropheny l)-5 - [2-(4-methoxyphenoxy )pyrimidin-4- yl]imidazole l-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-fluorophenoxy)pyrimidin-4- yl] imidazole l-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-aminocarbonylphenoxy)pyrimidin-4- yl]imidazole l-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-ethylphenoxy)pyrimidin-4- yljimidazole l-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-benzyloxyphenoxy)pyrimidin-4- yljimidazole l-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-cyanophenoxy)pyrimidin-4- yljimidazole l-(Piperidin-4-yl)-4-(4-fluorophenyl)-5-[2-(4-hydroxyphenoxy)pyrimidin-4- yljimidazole Example 2 - Micro-Reversibility Assay - In Vitro Reversibility of CFU-C Inhibitory Activity
The Micro-Reversibility assay of this invention is performed as follows. 100,000 non-adherent murine bone marrow cells/ml were incubated for 24 hours with compound ( 5 uM, 0.5 uM, 50 nM, 5 nM) and a mixture of CSFs (mixture of IL-1, IL-3, SCF, M-CSF) in McCoys 5a liquid culture medium with 15% FBS. After 24 hours the cells were washed and resuspended in media / agar / and M-CSF and assayed according to the Micro-Inhibition assay of Example 1, above.
The results of this assay demonstrate that incubation of murine bone marrow cells with BSA buffer and CSF for 24 hours in liquid medium prior to washing and replating in a Micro-Inhibition assay results in measurable CFU-C growth as measured by MTT conversion. However, cells cultured for 24 hours in CSF and compound in liquid media, followed by washing and replating resulted in slightly greater CFU-C growth as measured by MTT conversion. There was no difference in the number of cells recovered from both sets of cultures. Marrow cells cultured with TGFb did not grow as well as BSA buffer of compound treated cells indicating an incomplete reversal of initial inhibitory effects of this concentration of TGFβ. These data indicate the inhibition of CFU-C formation by compound is completely reversible at these doses and that the inhibition of CFU-C growth does not represent cytotoxicity. In the two graphs shown below as Figure 21, Compound 1 is 4-(4-Fluorophenyl)-5-(2-amino-4-pyridiminyl)- 1 -( 1 -methy 1-4- piperidinyl)imidazole, and Compound 2 is 4-(4-Fluorophenyl)-5-(2-phenylamino-4- pyridiminyl)- 1 -(4-piperidinyl)imidazole.
Example 3 - In Vivo Inhibitory Activity of a Myeloprotectant Compound on CFU-C Formation
The present invention designed studies to prove whether the compounds of Formula (I) were reversible inhibitors of CFU-GM and hence myeloprotective in vivo in a high dose 5-FU model as described herein. Stock solutions are diluted in PBS prior to injection. The test compound concentration is adjusted so that a 0.2 ml injection delivers the appropriate dose of compound.
To assess the in vivo inhibition of CFU-C formation, Female BDF1 mice were injected IP, (or the dosage may be given orally) with 10 mg/kg 1 hour prior to and 3 hours after 175 mg/kg 5-FU. Mice were sacrificed on days 3, 4, and 6 post 5- FU injection. Bone marrow cellularity and CFU-GM analysis was performed on each day. Data shown in Figure 1 demonstrate that 5-FU severely depleted the marrow compartment of CFU-GM evident on days 3 and 4. By day 6 recovery has initiated, however CFU-GM content is less than 5% of normal. Mice injected with either 4-(4-Fluorophenyl)-5-(2-pheny lamino-4-pyridiminyl)- 1 -(4- piperidinyl)imidazole as Compound 2 or 4-(4-Fluorophenyl)-5-(2-N-methylamino- 4-pyridiminyl)-l-(4-N-methyl-piperidinyl)imidazoleas Compound 3. Both Compound 2 and Compound 3 had significantly greater numbers of CFU-GM in the femur at each day tested. On day 4 the CFU-GM content of the marrow was approximately 50% of normal values. This experiment was repeated twice more at day 3 resulting in the same conclusions. Administration of either Compound 2 or at these doses protected a proportion of CFU-GM from 5-FU toxicity. Similar to Compounds 2 and 3 and oral dosage of Compound 4, 4-(4-Fluorophenyl)-5-(2-N- methylamino-4-pyridiminyl)-l-(4-piperidinyl)imidazole at levels of 50 mg/kg resulted in the same protection.
Example 4 - A Myeloprotectant Compound Effects Stem Cells Post 5-Fluorouracil Myelosuppression
One of the utilities of a myeloprotectant agent is for prevention of neutrophil nadir or a more rapid recovery of neutrophils post chemotherapeutic insult. Administration of either Compound 2 or 3 resulted in significant increases in mature blood cell elements and bone marrow cellularity on day 9 post 5-FU. While the pattern / kinetics of activity for these two compounds were not identical, the specific cell lineages were more effected by one compound and not the other, however both compounds were effective as myeloprotectants. Shown in Figure 2, below Compound 3, in a dose responsive manner increased total white blood cell (WBC) count over 5-FU. In Figure 3, Compound 2 in a dose responsive manner, increased neutrophil counts to levels equivalent to normal non-5-FU treated mice. Compound 3, also significantly increased PMN in a dose related manner. In Figure 4, both compounds increased platelet counts in a dose responsive manner. Platelet numbers in the 5-FU model, decrease by 60% than rebound to values higher than normal on days 10 - 15. In this experiment, the 5-FU and normal platelets counts are equivalent as the 5-FU counts are beginning this rebound phase of platelet production. Both compounds resulted in higher PLT counts at day 9 indicating that PLT recovery was quicker in the CSAID™ compound treated mice. In figures 5, 6 and 7 Compounds 2 and 3 significantly increased lymphocyte, monocyte, and RBC counts in a dose responsive manner. These data taken together indicate that multiple hematopoietic cell lineage's are protected by administration of compounds of Formula (I) in this murine 5-FU model. This pattern of mature blood cell recovery is unprecedented. No known agent / drug (i.e. G-CSF) increases every cell lineage in chemotherapy models of myelosuppression. In addition to mature blood cell elements, bone marrow cellularity was also increased in a dose responsive manner (Figure 8) indicating myeloprotection and enhanced cell recovery within the marrow. This study (Figure 8) was repeated with Compounds 2 and 3 administered orally. Figures 9 and 10, show only the PMN and marrow cellularity data. Both compounds appear to be more effective post oral administration than IP.
In order to determine whether a compound could increase PMN could increase mature blood cell counts the following experiment was done. In a preliminary experiment compounds of Formula (I), Compounds 2 and 3 increased PMN counts on day 9 post 5-FU. Therefore, day 9 post 5-FU was chosen as an indicator time because mature blood cell counts are still significantly suppressed in 5-FU treated mice at this time. In addition, from previous experiments it was known that G-CSF administered every day post 5-FU begins to increase PMN counts starting around day 9 - 10. Based on these results a dose response analysis of Compounds 2 and 3 were performed utilizing the Day 9 5-FU myelosuppression model shown herein. Similar to Example 3 above, mice were injected IP with compound 1 hour prior to and 3 hours after the cell cycle specific drug, 5- fluorouracil (5-FU 175mg/kg), which occurred at 0 hour. The timing of the compound injections in the figures 2 to 10 were made wherein the compound was injected IP 2 hours prior and 4 hours after the myelosuppresive dose was administered.
Example 5: Evaluation of Compounds of Formula (Is) and G-CSF in a murine 5-FU myelosuppression model As shown above compounds of Formula (I) are effective reversible inhibitors of CFU-GM growth in vitro and in vivo. Examples 1 to 4, above demonstrate that compounds of Formula (I) in experiments in which a high dose of 5-FU (175 mg/kg) is are myeloprotectants for normal hematopoietic stem cells. The following experiments demonstrate the myeloprotective effects of compounds of Formula (I), in particular Compounds 2 and 3, in a kinetic model of 5-FU induced myelosuppression.
G-CSF (50 ug/kg administered daily 48 hours post 5-FU) is included as a positive control for enhanced PMN recovery in these studies. It should be noted that G-CSF is administered 1.5 hours before each bleed point. G-CSF administration under these conditions reflects PMN recovery plus additional PMN mobilization from vascular and possibly tissue sites.
Administration of 5-FU resulted in significant neutropenia lasting 8 days. All mature cell elements were depleted by 5-FU, however the extent and duration vary with each cell lineage. Administration of either Compound 2 or 3 (IP, 40 mg/kg; 2 hours pre and 4 hours post 5-FU) resulted in enhanced mature blood cell recovery across multiple lineage's. The most dramatic effects were on PMN, monocytes, and platelet recoveries. Data shown in Figures 1 1 through 16.
Administration of either Compound 2 or 3, 2 hours before and 4 hours after high dose 5-FU significantly enhance overall mature blood cell recovery. The data from the above Figures can be summarized as the following table of hematology values. The lower the value the faster the cell lineage has recovered. Compounds 2 or 3 were effective myeloprotectants with activity better or equal to that of therapeutic G-CSF.
TABLE 2. Summary Table of Key Hematology End Points in the Murine 5-FU Myeloprotection Model. Values extrapolated from the kinetic study graphs.
Figure imgf000030_0001
Example 6: Evaluation of Compound of Formula (I) alone or in combination with therapeutic G-CSF in a high dose 5-FU model
Compounds of Formula (I) have shown efficacy as myeloprotectants in a murine high dose 5-FU model noted above. In this experiment, Compound 3 (40 mg/kg) was administered IP to mice 2 hours before and 4 hours after a single injection of 175 mg/kg 5-FU G-CSF was administered daily starting 48 hours post 5-FU injection for 10 days.
As shown in earlier experiments, IP administration of Compound 3 is effective as a myeloprotectant, and the data demonstrates that a trend towards rapid recovery were seen in every cell lineage. G-CSF alone resulted in enhanced recovery of PMN and monocyte populations as well as total WBC's. The combination of a compound of Formula (I) and pre-treatment with G-CSF therapy resulted in dramatic increases in multiple cell lineage recovery rates. The most dramatic increase in recovery rate was the PMN population with evidence over several days of complete protection of neutropenia in a subpopulation of treated mice. These data are shown in Figures 17 to 20 herein.
Consequently another aspect of the present invention is the novel administration of a compound of Formula (I) along with G-CSF for the protection of multiple lineage cell lines and their enhanced recovery.
Overall mature blood cell recovery and is summarized as the following table of hematology values. The lower the value the faster the cell lineage has recovered. Compound 3 + therapeutic G-CSF [QDxlO] were effective myeloprotectants with activity better or equal to that of G-CSF alone.
Figure imgf000031_0001
All publications, including but not limited to patents and patent applications, cited in this specification are herein incoφorated by reference as if each individual publication were specifically and individually indicated to be incoφorated by reference herein as though fully set forth.
The above description fully discloses the invention including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration, it is believed that one skilled in the are can, using the preceding description, utilize the present invention to its fullest extent. Therefore the Examples herein are to be construed as merely illustrative and not a limitation of the scope of the present invention in any way. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.

Claims

What is claimed is:
1. A method for reversibly inhibiting myelopoiesis in mammalian tissue in vivo and ex vivo comprising administering to said tissue an amount effective to reversibly inhibit the formation of myelopoietic colonies during the time said tissue is exposed to myelosuppressive therapy of a compound of the formula:
Figure imgf000033_0001
wherein:
Rl is 4-pyridyl, pyrimidinyl, 4-pyridazinyl, l,2,4-triazin-5-yl, quinolyl, isoquinolinyl, quinazolin-4-yl, 1-imidazolyl or 1-benzimidazolyl ring, which ring is optionally substituted independently one to three times with Y, NHRa, optionally substituted Ci-4 alkyl, halogen, hydroxyl, optionally substituted
Ci-4 alkoxy, optionally substituted Ci .4 alkylthio, Ci-4 alkylsulfinyl, CH2OR12, amino, mono and di- Ci-6 alkyl substituted amino, or N(Rio)C(O)Rb; Y is O-Ra; R4 is phenyl, naphth-1-yl or naphth-2-yl, or a heteroaryl, which is optionally substituted by one or two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-naphth-l-yl, 5-naphth-2-yl or 6-naphth-2-yl substituent, is halogen, cyano, nitro, C(Z)NR7Ri7, C(Z)ORi6, (CRioR20)vCORi2, SR5, SOR5, OR12, halo-substituted-Ci-4 alkyl, Ci-4 alkyl, ZC(Z)Ri2, NRiθC(Z)Rl6, or (CRioR2θ)vNRlθR20 and which, for other positions of substitution, is halogen, cyano, C(Z)NRi3Ri4, C(Z)OR3, (CRioR20)m"COR3, S(O)mR3, OR3, halo-substituted-Ci-4 alkyl, Ci-4 alkyl, (CRioR20)m"NRioC(Z)R3, NRioS(O)m'R8, NRιoS(O)m'NR7Rl7, ZC(Z)R3 or (CRioR20)m"NRi3Rl4; v is 0, or an integer having a value of 1 or 2; n is an integer having a value of 1 to 10; n' is 0, or an integer having a value of 1 to 10; m is 0, or the integer 1 or 2; nϊ is an integer having a value of 1 or 2, m" is 0, or an integer having a value of 1 to 5; R2 is hydrogen, (CRioR20)n OR9, heterocyclyl, heterocyclylC i- 10 alkyl,
Ci-ioalkyl, halo-substituted Cl- 10 alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C3-7cycloalkylCi-lO alkyl, C5-7 cycloalkenyl, C5-7 cycloalkenyl Ci-ioalkyl, aryl, arylCi-io alkyl, heteroaryl, heteroarylC i-ioalkyl, (CRioR20)nORl l, (CRιoR2θ)nS(O)mRι8, (CRioR20)nNHS(O) Rl8, (CRιoR20)nNRl3Rl4, (CRlθR2θ)nNθ2, (CRioR20)nCN,
(CRioR20)n'SO2Rl8, (CRioR20)nS(O)m'NRi3Rl4, (CRlθR20)nC(Z)Rn, (CRi0R20)nOC(Z)Rn, (CRioR20)nC(Z)ORn, (CRiθR20)nC(Z)NRι3Rl4, (CRl0R20)nC(Z)NRι ιOR9, (CRi0R20)nNRl0C(Z)Rn,
(CRi0R20)nNRl0C(Z)NRi3Rl4, (CRl0R20)nN(OR6)C(Z)NRι3Rl4, (CRioR20)nN(OR6)C(Z)Ri 1, (CRι0R20)nC(=NOR6)Rl 1,
(CRi0R20)nNRl0C(=NRi9)NRi3Rl4, (CRl0R20)nOC(Z)NRι3Rl4,
(CRlθR2θ)nNRlθC(Z)NRi3Rl4, (CRlθR2θ)nNRlθC(Z)ORιo, 5-(Rι8)-l,2,4- oxadizaol-3-yl or 4-(R 12)-5-(R 1 sR i9)-4,5-dihydro- 1 ,2,4-oxadiazol-3-yl; wherein the aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroaryl alkyl, heterocyclic and heterocyclic alkyl groups may be optionally substituted;
Z is oxygen or sulfur; Ra is Ci-6alkyl, aryl, arylCi-6alkyl, heterocyclic, heterocyclylC 1 -6 alkyl, heteroaryl, or heteroarylC i-6alkyl, wherein each of these moieties may be optionally substituted; Rb is hydrogen, -6 alkyl, C3_7 cycloalkyl, aryl, arylCj-4 alkyl, heteroaryl, heteroarylC i-4alkyl, heterocyclyl, or heterocyclylC 1.4 alkyl; R3 is heterocyclyl, heterocyclylC l- 10 alkyl or Rs; R5 is hydrogen, -4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the moieties -SR5 being -SNR7R17 and -SOR5 being -SOH; R6 is hydrogen, a pharmaceutically acceptable cation, Ci-io alkyl, Ci-η cycloalkyl, aryl, arylCi-4 alkyl, heteroaryl, heteroarylC 1.4 alkyl, heterocyclic, aroyl, or
Ci-io alkanoyl; R7 and R17 is each independently selected from hydrogen or Ci-4 alkyl or R7 and
Rl7 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR15; R8 is Ci-io alkyl, halo-substituted Ci-io alkyl, C2-10 alkenyl, C2-10 alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, arylCj-io alkyl, heteroaryl, heteroarylC 1-10 alkyl, (CRioR20)nORl l, (CRioR2θ)nS(0)mRi8, (CRioR20)nNHS(O)2Rl8, (CRioR20)nNP>l3Rl4; wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl may be optionally substituted;
R9 is hydrogen, C(Z)Rι 1 or optionally substituted Cl-10 alkyl, S(O)2Rl8, optionally substituted aryl or optionally substituted aryl- -4 alkyl;
RlO and R20 is each independently selected from hydrogen or Ci-4 alkyl;
Rl l is hydrogen, Ci-io alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclyl Ci-ioalkyl, aryl, arylCj-io alkyl, heteroaryl or heteroarylC i- 10 alkyl;
Rl2 is hydrogen or R16;
Rl3 and R14 is each independently selected from hydrogen or optionally substituted Ci-4 alkyl, optionally substituted aryl or optionally substituted aryl- Ci-4 alkyl, or together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 members which ring optionally contains an additional heteroatom selected from oxygen, sulfur or NR9;
Rl5 is Rio or C(Z)-Ci-4 alkyl;
Rl6 is Ci-4 alkyl, halo-substituted-Ci-4 alkyl, or C3-7 cycloalkyl;
Rl8 is Ci-io alkyl, C3-7 cycloalkyl, heterocyclyl, aryl, arylalkyl, heterocyclyl, heterocyclyl-C 1 - 1 Oalky 1, heteroaryl or heteroary lalkyl ;
Rl9 is hydrogen, cyano, C1.4 alkyl, 03.7 cycloalkyl or aryl; or a pharmaceutically acceptable salt thereof.
2. The method according to claim 1 wherein said myelopoietic colonies are CFU-C colony forming cells.
3. The method according to claim 2 wherein said CFU-C colonies are CFU-GM colony forming cells.
4. The method according to claim 1 wherein reversible inhibition occurs in vivo.
5. The method according to claim 1 wherein the compound is administered to mammalian tissue ex vivo and wherein said reversible inhibition occurs ex vivo.
6. The method according to claim 1 for reversibly inhibiting myelopoiesis in mammalian tissue in vivo and ex vivo comprising administering to said tissue an effective amount of a compound of Formula (I).
7. The method according to anyone one of Claims 1 to 6 wherein the compound, or a pharmaceutically acceptable salt thereof is:
4-(4-Fluorophenyl)-5-(2-amino-4-pyridiminyl)- 1 -( 1 -methyl-4- piperidinyl)imidazole,
4-(4-Fluorophenyl)-5-(2-phenylamino-4-pyridiminyl)-l-(4-piperidinyl)imidazole; 4-(4-Fluorophenyl)-5-(2-N-methylamino-4-pyridiminyl)-l-(4-N-methyl- piperidinyl)imidazole; or
4-(4-Fluoropheny l)-5-(2-N-methylamino-4-pyridiminyl)- 1 -(4- piperidiny l)imidazole .
8. The method according to Claim 1 wherein the compound is administered in an oral dosage form.
9. The method according to Claim 1 wherein the compound is administered in a parenteral dosage form.
10. A method for identifying or screening for compounds which inhibit
CFU-C formation of myeloid progenitors which comprises the steps of:
(a) bringing together a test sample containing one or more test compounds and hematopoietic growth factors with a preparation comprising a receptor on myeloid progenitor cells capable of detectably and reversibly interacting with a compound of Formula (I), according to Claim 1 , in soft agar; (b) incubating said test sample and said preparation under conditions which would permit the detection of inhibition of CFU-C colony growth; and
(c) determining the degree of inhibition caused by said test sample by measuring MTT conversion by extracting said preparation and measuring optical density at 570 nm with a reference filter of 750 nm on an ELIS A reader.
11. The method according to claim 10 comprising a further step of comparing the degree of inhibition caused by said test sample with that caused by a compound of Formula (I) as defined in Claim 1.
12. The method according to claim 10 wherein said receptor preparation comprises isolated myeloid progenitor cells.
13. The method according to claim 10 wherein said preparation is bone marrow cells in soft agar.
14. The method according to claim 10 wherein said test sample comprises one or more known compounds.
PCT/US1997/018951 1996-10-17 1997-10-17 Methods for reversibly inhibiting myelopoiesis in mammalian tissue WO1998016230A1 (en)

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