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Title:
HEMOREGULATORY COMPOUNDS
Document Type and Number:
WIPO Patent Application WO/1997/017958
Kind Code:
A1
Abstract:
The present invention relates to novel compounds which have hemoregulatory activities and can be used to stimulate hematopoiesis and for the treatment of viral, fungal and bacterial infectious diseases.

Inventors:
Bhatnagar
Pradip
Kumar, Heerding
Dirk
Andries, Hartmann
Michael, Hiebl
Johann, Kremminger
Peter, Rovenszky
Franz
Application Number:
PCT/US1996/018248
Publication Date:
May 22, 1997
Filing Date:
November 12, 1996
Export Citation:
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Assignee:
SMITHKLINE BEECHAM CORPORATION NYCOMED AUSTRIA GMBH BHATNAGAR
Pradip
Kumar, Heerding
Dirk
Andries, Hartmann
Michael, Hiebl
Johann, Kremminger
Peter, Rovenszky
Franz
International Classes:
A61K31/00; A61K31/395; A61K31/397; A61K31/40; A61K31/401; A61K31/4015; A61K31/44; A61P7/00; A61P31/00; A61P31/10; C07D205/04; C07D207/16; C07D207/277; C07D207/28; C07D207/34; C07D213/75; C07D213/81; C07D213/82; (IPC1-7): A61K31/40; A61K31/44; C07D401/02; C07D403/02
Foreign References:
US4499081A
Other References:
See also references of EP 0866700A1
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Claims:
CLAIMS:
1. A compound of formula (I) wherein: A1 and A2 are independently Z(CH2)jc(NR,)m; Z is a 4 10 membered mono or bicyclic heterocyclic ring system containing up to four heteroatoms N, O, S in the ring in which at least one heteroatom is N, and wherein the ring is substituted or unsubstituted by one or two dialkyl, F, Cl, Br, I, Ci _4 alkoxy, (CH2)mR4, oxo, oxime, OC 1.4 alky loxime, hydroxy, N(R3)2, acylamino or aminoacyl groups, 8, 9, 10 membered monocyclic ring systems being excluded; R and R are independently hydrogen, Cj.4alkylC(O)R4' C^alkyl or R' and R" are benzyl which is optionally substituted by one or two C 1.4 alkyl, C 1.4 alkoxy, F, Cl, I, Br, OH, or N(R3)2; k is independently an integer from 0 to 4; m is independently zero or one; Q is bicyclo[3.3.0]octanyl, xyleyl, benzophenonyl or 1,2,3,4tetrahydronapthalyl; all of which are unsubstituted or substituted by one or more C1.4a.kyl, Cj^alkoxy, halogen, mono or di C^alkylamino, (Cι_4alkyl)2NC(O), (CH2)nR2, (CH2)nR3, (CH2)nCOR2 or (CH2)nCOR3; R2 is independently OR3, NR32, SR3' R3 is independently hydrogen, Ci^alkyl or benzyl; d R4 is independently OR3, N(R3)2, SR3; an n is independently an integer from 0 to 4; or a pharmaceutically acceptable salt thereof.
2. A method of stimulating myelopoiesis in an animal in need thereof by administering a compound of Formula II wherein: A1 and A2 are independently Z(CH2)ιc(NR')m; Z is a 4 10 membered mono or bicyclic heterocyclic ring system containing up to four heteroatoms N, O, S in the ring in which at least one heteroatom is N, and wherein the ring is substituted or unsubstituted by one or two Cι_4alkyl, F, Cl, Br, I, C1.4 alkoxy, (CH2)mR4, oxo, oxime, OCι_4alkyloxime, hydroxy, N(R3)2, acylamino or aminoacyl groups, 8, 9, 10 membered monocyclic ring systems being excluded; R' and R" are independently hydrogen, C 1 _4alkylC(O)R < C ^alkyl or R' and R" are benzyl which is optionally substituted by one or two C .4a.kyl, C^alkoxy, F, Cl, I, Br, OH, or N(R3)2; k is an integer from 0 to 4; m is zero or one; Ql is a mono, bi or tricyclic aromatic or nonaromatic ring system with up to 12 carbon atoms in the ring and containing up to 4 heteroatoms which is unsubstituted or substituted by one or more Cι_4alkyl, C^alkoxy, oxo, oxime, OC1.4alkyloxi.ne, hydroxy, halogen, amino, mono or di Ci^alkylamino, (Cι_4alkyl)2NC(O), (CH2)nR2~, (CH2)nR3, (CH2)nCOR2 or (CH2)nCOR3; R2 is independently OR3, NR32, SR3; R3 is independently hydrogen, CιC4alkyl or benzyl; R4 is independently OR3, N(R3)2, SR3; and n is an integer from 0 to 4; or a pharmaceutically acceptable salt thereof.
3. A compound of Claim 1 or Claim 2 wherein Z is an optionally substituted pyrrolyl, isopyrrolyl, pyrazolyl, isoimidazolyl, triazolyl, iosxazolyl, oxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolidinyl, piperazinyl, triazinyl, moφholinyl, indolyl, indoleninyl, isobenzazolyl, pyrindinyl, ioindazolyl, indoxazinyl, benzoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl, pyridopyridinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, indolinyl, pyrrolidonyl, imidazolyl, imidazolidinyl, imidazolinyl, piperidyl, tetrazolyl, quinuclidinyl, azetidinyl, or purinyl;.
4. A compound according to Claim 1, wherein Z denotes an optionally substituted picolinoyl, 2pyrrolidonyl, quinolinyl, azetidinyl, pyrrolidinyl or pyrrolyl.
5. A compound according to Claim 2 wherein Z is optionally substituted picolinoyl, 2pyrrolidonyl, quinolinyl, azetidinyl, pyrrolidinyl or pyrrolyl; and Q' is bicyclo[3.3.0]octanyl, 1,2,3,4tetrahydronapthalyl, benzophenonyl, xylyl or phenyl.
6. A compound according to Claim 1 or Claim 2 chosen from the group consisting of : 1 ,7bis(picolinoylamino) 1 ,2,3,4tetrahydronaphthalene; N,N'bis(picolinoyl)3,7diaminobicyclo[3.3.0]octane; N,N'bis(picolinoyl) 1 ,3xylylenediamine ; N,N'bis(picolinoyl)4,4'diaminobenzophenone; N,N'bis(picolinoyl) 1 ,2phenylenediamine; or N,N'bis(picolinoyl) 1 ,3phenylenediamine.
7. A pharmaceutical composition comprising a compound according to Claim 1 and a pharmaceutically acceptable carrier.
8. A method of preventing or treating viral, fungal and bacterial infections which comprises administering to a subject in need thereof, an effective amount of a compound of Claim 2.
9. A method of preventing or treating sepsis which comprises administering to an animal in need thereof, an effective amount of a compound of Formula II of Claim 2.
Description:
HEMOREGULATORY COMPOUNDS

Field of the Invention The present invention relates to novel compounds which have hemoregulatory activities and can be used to stimulate hematopoiesis and for the treatment of viral, fungal and bacterial infectious diseases.

Background of the Invention The hematopoietic system is a life-long cell renewal process whereby a defined stem cell population gives rise to a larger population of mature, differentiated blood cells (Dexter TM. Stem cells in normal growth and disease. Br Med J 1987; 195:1192-1194) of at least nine different cell lineages (erythrocytes, platelets, eosinophils, basophils, neutrophils, monocytes/macrophages, osteoclasts, and lymphocytes) (Metcalf D. The Molecular Control of Blood Cells. 1988; Harvard University Press, Cambridge, MA). Stem cells are also ultimately responsible for regenerating bone marrow following treatment with cytotoxic agents or following bone marrow transplantation.

The major dose-limiting toxicities of most standard anti-neoplastic drugs are related to bone marrow suppression, which if severe and prolonged, can give rise to life-threatening infectious and hemorrhagic complications. Myelosuppression is predictable and has been reported to be dose-limiting in greater than 50% of single-agent Phase I trials cytotoxic compounds (Merrouche Y, Catimel G, Clavel M. Hematopoietic growth factors and chemoprotectants; should we move toward a two-step process for phase I clinical trials in oncology? Ann Oncol 1993; 4:471-474). The risk of infection is directly related to the degree of myelosuppression as measured by the severity and duration of neutropenia (Brody GP, Buckley M, Sathe YS, Freireich EJ. Quantitative relationship between circulating leukocytes and infections with acute leukemia. Ann In Med 1965; 64:328-334).

The control of hematopoiesis involves the interplay of a variety of cytokines and growth factors during various stages of the hematopoietic cascade,

including early pluripotent stem cells and mature circulating effector cells. These regulatory molecules include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), and a variety of interleukins which have overlapping, additive and synergistic actions which play major roles in host defense. Mechanistically, this is accomplished by enhancing the production of granulocytes and macrophages, as well as by the activation of effector cell functions (Moore MAS. Hemopoietic growth factor interactions: in vitro and in vivo preclinical evaluation. Cancer Surveys 1990; 9:7-80). These coordinated activities support optimal host defences which are necessary for fighting bacterial, viral and fungal infections.

Strategies to prevent or reduce the severity of neutropenia and myelotoxicity include the use of hematopoietic growth factors and/or other hematopoietic cytokines. Such treatments are becoming common practice, in that they offer the potential of increased doses of cytotoxic agents that may improve the therapeutic efficacy of antineoplastic agents, and reduce the morbidity associated with their use (Steward WP. Granulocyte and granulocyte-macrophage colony stimulating factors, Lancet 1993; 342: 153-157). Clinical studies have demonstrated the G-, GM- and/or M-CSF may reduce the duration of neutropenia, accelerate myeloid recovery, and reduce neutropenia-associated infections and other infectious complications in patients with malignancies who are receiving cytotoxic chemotherapy or in high infectious-risk patients following bone marrow transplantation (Steward WP. Granulocyte and granulocyte- macrophage colony stimulating factors, Lancet 1993; 342: 153-157 and Munn DH, Cheung NKV. Preclinical and clinical studies of macrophage colony- stimulating factor. Semin Oncol 1992; 19:395-407).

Synthetic peptides have been reported to induce the synthesis and release of hematopoietic mediators, including m-CSF from bone marrow stromal elements (see U.S. Patent Application 08/001,905). We have now found certain novel non-peptide compounds which have a stimulative effect on myelopoietic cells. They are useful in stimulating myelopoiesis in patients sufferieng from reduced myelopoietic activity, including

bone marrow damage, agranulocytosis and aplastic anemia including patients having depressed bone marrow function due to immunosuppressive treatment to suppress tissue reactions i.e. in bone marrow transplant surgery. They may also be used to promote more rapid regeneration of bone marrow after cytostatic chemotherapy and radiation therapy for neoplastic and viral diseases. They may be of particular value where patients have serious infections due to a lack of immune response following bone marrow failure. They are also useful in the treatment and prevention of viral, fungal and bacterial disease.

Summary of the Invention This invention comprises compounds, hereinafter represented as Formula (I), which have hemoregulatory activities and can be used to stimulate hematopoiesis and in the prevention and treatment of bacterial, viral and fungal diseases.

These compounds are useful in the restoration of leukocytes in patients with lowered cell counts resulting from a variety of clinical situations, such as surgical induced myelosuppression, AIDS, ARDS, congenital myelodysplacis, bone marrow and organ transplants; in the protection of patients with leukopenia from infection; in the treatment of severely burned patients and in the amelioration of the myelosuppression observed with some cell-cycle specific antiviral agents and in the treatment of infections in patients who have had bone marrow transplants, especially those with graft versus host disease, in the treatment of tuberculosis and in the treatment of fevers of unknown origin in humans and animals. The compounds are also useful in the treatment and prevention of viral, fungal and bacterial infectious diseases, particularly Candida and Heφes in both immunosuppressed and "normal" subjects. They are useful in the treatment of sepsis caused by gram negative and gram positive organisms. These compounds may also be used in combination with the myelosuppressive agents of co-pending U.S. Application No. 07/799,465 and U.S. Patent No. 4,499,081, incoφorated by reference herein, to provide alternating peaks of high and low activity in the bone marrow cells, thus

augmenting the natural circadian rhythm of hematopoiesis. In this way, cytostatic therapy can be given at periods of low bone marrow activity, thus reducing the risk of bone marrow damage, while regeneration will be promoted by the succeeding peak of activity. This invention is also a pharmaceutical composition, which comprises a compound of Formula (II) and a pharmaceutically acceptable carrier.

This invention further constitutes a method for stimulating the myelopoietic system of an animal, including humans, which comprises administering to an animal in need thereof, an effective amount of a compound of Formula (I) or (II).

This invention also constitutes a method for preventing and treating viral, fungal and bacterial infections including sepsis, in immunosuppressed and normal animals, including humans, which comprises administering to an animal in need thereof, an effective amount of a compound of Formula (I) or (II).

Detailed Description of the Invention

The compounds of the invention are represented by structural Formula (I)

wherein:

A 1 and A 2 are independently Z-(CH2) -( R') m -; Z is a 4 - 10 membered mono- or bicyclic heterocyclic ring system containing up to four heteroatoms N, O, S in the ring in which at least one heteroatom is N, and wherein the ring is substituted or unsubstituted by one or two Cj^alkyl, F, Cl, Br, I, Cι_4 alkoxy, (CH2) m R , oxo, oxime, O-C|_4alkyloxime, hydroxy, N(R3)2, acylamino or aminoacyl groups, 8, 9, 10 membered monocyclic ring systems being excluded;

R' and R" are independently hydrogen, Cι_4alkylC(O)R 4 - C^alkyl or R' and R" are benzyl which is optionally substituted by one or two Cj^alkyl, C j ^alkoxy, F, Cl, I, Br, OH, or N(R 3 ) 2 ; k is independently an integer from 0 to 4; m is independently zero or one;

Q is bicyclo[3.3.0]octanyl, xyleyl, benzophenonyl or 1,2,3,4-tetrahydronapthalyl; all of which are unsubstituted or substituted by one or more C^alkyl, C^alkoxy, halogen, mono or di C ^alkylamino, (C alkyl) -NC(O)-, -(CH 2 ) n -R 2 , -(CH 2 ) n -R 3 , -(CH 2 ) n -COR 2 or -(CH 2 ) n -COR 3 ; R 2 is independently -OR 3 , -NR 3 2, -SR 3 i

R 3 is independently hydrogen, or benzyl;

R 4 is independently -OR 3 , -N(R 3 )2, - SR 3 ; and n is independently an integer from 0 to 4; or a pharmaceutically acceptable salt thereof.

The invention is also a method of stimulating myelopoiesis in an animal, including humans, in need thereof by administering an effective amount of a compound of Formula (II)

wherein:

A 1 and A 2 are independently Z-(CH2)k-(NR') m -; Z is a 4 - 10 membered mono- or bicyclic heterocyclic ring system containing up to four heteroatoms N, O, S in the ring in which at least one heteroatom is N, and wherein the ring is substituted or unsubstituted by one or two C^alkyl, F, Cl, Br, I, C j _4 alkoxy, (CH2) R 4 , oxo, oxime, O-Cj^alkyloxime, hydroxy, N(R 3 )2, acylamino or aminoacyl groups, 8, 9, 10 membered monocyclic ring systems being excluded;

R and R" are independently hydrogen, Cι _4alkylC(O)R 4 > Cι_4alkyl or R' and R" are benzyl which is optionally substituted by one or two C^alkyl, C-^alkoxy, F, Cl, I, Br, OH, or N(R ) 2 ; k is an integer from 0 to 4; m is zero or one;

Q s a mono-, bi- or tricyclic aromatic or non-aromatic ring system with up to 12 carbon atoms in the ring and containing up to 4 heteroatoms which is unsubstituted or substituted by one or more Cj^alkyl, Cι _4alkoxy, oxo, oxime, O-Cμ4alkyloxime, hydroxy, halogen, amino, mono or di Cι_4alkylamino, (Cι_4alkyl)2-NC(O)-, -(CH2) n -R 2~ > -(CH 2 ) n -R 3 , -(CH 2 ) n -COR 2 or -(CH 2 ) n -COR 3 ; R 2 is independently -OR 3 , -NR 3 2, -SR 3 « R 3 is independently hydrogen, C*[-C4-alkyl or benzyl; R 4 is independently -OR 3 , -N(R 3 ) 2 , - SR 3 ; and n is an integer from 0 to 4; or a pharmaceutically acceptable salt thereof.

This invention is also a pharmaceutical composition, which comprises a compound of Formula (I) and a pharmaceutically acceptable carrier.

Alkyl groups may be straight or branched. Halogen may be chloro, iodo, fluro or bromo.

The compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All the compounds and diastereomers are contemplated to be within the scope of the present invention.

Z in the above Formulas (I) and (II) denotes an optionally substituted pyrrolyl, isopyrrolyl, pyrazolyl, isoimidazolyl, triazolyl, iosxazolyl, oxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolidinyl, piperazinyl, triazinyl, moφholinyl, indolyl, indoleninyl, isobenzazolyl, pyrindinyl, ioindazolyl, indoxazinyl, benzoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl, pyridopyridinyl,

tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, indolinyl, pyrrolidonyl, imidazolyl, imidazolidinyl, imidazolinyl, piperidyl, tetrazolyl, quinuclidinyl, azetidinyl, or purinyl.

Q' may be, for example, bicyclo[3.3.0]octanyl, bicyclo[4.4.0]decanyl, bicyclo[4.3.0]nonanyl, bicyclo[2.2. ljheptanyl, bicyclo[3.3. ljnonanyl, bicyclo[2.2.2]octanyl, tetrahydronapthalyl, xylyl and hexahydroindacyl

Preferred compounds of Formula (I) are those wherein Z is optionally substituted picolinoyl, 2-pyrrolidonyl, quinolinyl, azetidinyl, pyrrolidinyl or pyrrolyl.

Preferred compounds of Formula (L ) are those wherein Z is optionally substimted picolinoyl, 2-pyrrolidonyl, quinolinyl, azetidinyl, pyrrolidinyl or pyrrolyl; and Q' is bicyclo[3.3.0]octanyl, 1,2,3,4-tetrahydronapthalyl, benzophenonyl, xylyl or phenyl.

Preferred compounds are:

1 ,7-bis(picolinoylamino)- 1 ,2,3,4-tetrahydronaphthalene; N,N'-bis(picolinoyl)-3,7-diaminobicyclo[3.3.0]octane; N,N'-bis(picolinoyl)- 1 ,3-xylylenediamine;

N,N'-bis(picolinoyl)-4,4'-diaminobenzophenone; N,N'-bis(picolinoyl)- 1,2-phenylenediamine; or N,N'-bis(picolinoyl)- 1 ,3-phenylenediamine.

Methods of Preparation

Compounds of Formula (I) or (II) can be prepared by condensing two molar equivalents of an approporiately activated carboxylic acid derivatives of Formula (III).

were A s defined as in Formula (I) and (II), with molar equivalent of a suitable diamine of Formula (IV), where Q, R' and R" are defined as in Formula (I) and (II) (Q may also be Q**), in a suitable organic solvent (such as dimethyl formamide).

R " (IV)

Activated carboxylic acid derivatives can be obtained by reacting Formula (III) with a suitable activating agent (such as BOP reagent) in the presence of a suitable base

(such as di-isopropylethyl amine) in a suitable organic solvent (such as dimethylformamide) .

Alternatively, compounds of Formula (I) and (II) can be obtained by reacting two molar equivalents of the acid chloride derived from Formula (UI), obtained by reacting Formula (ID) with excess thionyl chloride, with one molar equivalent of a diamine of Formula (IV) in the presence of a suitable base (such as di-isopropylethyl amine) in a suitable organic solvent (such as THF) to give compounds of formula (I) or(II).

In order to use a compound of the Formula (I) and (II) or a pharmaceutically acceptable salt thereof for the treatment of humans and other mammals it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.

According to a still further feature of the present invention there are provided pharmaceutical compositions comprising as active ingredient one or more compounds of Formula (I) and (II) as herein before defined or physiologically compatible salts thereof, in association with a pharmaceutical carrier or excipient. The compositions according to the invention may be

presented for example, in a form suitable for oral, nasal, parenteral or rectal administration.

As used herein, the term "pharmaceutical" includes veterinary applications of the invention. These compounds may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline and water. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such a glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. Capsules containing one or several active ingredients may be produced, for example, by mixing the active ingredients with inert carriers, such as lactose or sorbitol, and filling the mixture into gelatin capsules. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule. Organ specific carrier systems may also be used.

Alternately pharmaceutical compositions of the compounds of this invention, or derivatives thereof, may be formulated as solutions of lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration and contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as

polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

For rectal administration, a pulverized powder of the compounds of this invention may be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository. The pulverized powders may also be compounded with an oily preparation, gel, cream or emulsion, buffered or unbuffered, and administered through a transdermal patch.

Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers either with an aerosol propellant or provided with means for manual compression.

Dosage units containing the compounds of this invention preferably contain .05-50 mg, for example .05-5 mg of the compound of formula (I) or (II) or salt thereof.

According to a still further feature of the present invention there is provided a method of stimulation of myelopoiesis which comprises administering an effective amount of a pharmaceutical composition as hereinbefore defined to a subject.

No unacceptable toxicological effects are expected when compounds of the invention are administered in accordance with the present invention. The biological activity of the compounds of Formula (I) and (II) are demonstrated by the following tests.

Induction of Hematopoietic Synergistic Activity in Stromal Cells

The murine bone marrow derived stromal cell line, C6.4 is grown in 12 well plates in RPMI 1640 with 10% FBS. Upon reaching confluence, the C6.4 cells are washed and the media exchanged with fresh RPMI 1640 without FBS. Confluent cell layers of murine C6.4 cells are treated with compound. Cell-free supernatants are collected 18 hours later. Supernatants are fractionated with a Centricon-30 molecular weight cut-off membrane. C6.4 cell hematopoietic synergistic factor (HSF) activity is measured in a murine CFU-C assay.

CFU-C Assav

Bone marrow cells are obtained from C57B1/6 female mice and suspended in RPMI 1640 with 10% FBS. Bone marrow cells (7.5E+4 cells/mL) are cultured with sub optimal levels of CFU plus dilutions of test C6.4 cell 30K-E supernatants from above in a standard murine soft agar CFU-C assay. Cell aggregates >50 cells are counted as colonies. The number of agar colonies counted is proportional to the amount of HSF present within the C6.4 bone marrow stromal line supernatant.

Effector Cell Function Assay

Female C57B 1 mice are administered test compound IP or PO daily for 8 days. Resident peritoneal exudate cells (PEC) utilized ex vivo from treated or untreated mice are harvested with cold calcium and magnesium-free DPBS supplemented with heparin and antibiotics within 2-4 hours following the last injection. Adherent PEM populations are prepared by incubating standardized PEC suspensions in microtiter dishes for 2 hours at 37 °C (5% CO2) and removing nonadherent cells by washing the wells with warm buffer.

The superoxide dismutase-inhibitable (SOD) superoxide released by effector cells in response to a in vitro stimulation by phorbol myristate acetate (PMA) (100- 200nM) or pre-opsonized (autologous sera) live C. albicans (E:T = 1: 10) are quantitated in a microtiter ferricytochrome c reduction assay. The assay is performed in the presence of 1% gelatinHBSS and 80uM ferricytochrome c in a total volume of 200uL/well. The nmoles of cytochrome c reduced/well is calculated from spectrophotometric readings (550 nm) taken following a 1 hour incubation at 37 °C (5% CO2). The amount of SOD-inhibitable cytochrome c reduced is determined by the inclusion of wells containing SOD (200 U/well). Baseline superoxide release is determined in the absence of stimuli. Experimental data are expressed as a percentage of the control group.

The following examples are illustrative and are not limiting of the compounds of this invention.

Example 1

Preparation of 1.7-bis(picolinoylamino)- 1.2.3.4-tetrahvdronaphthalene

a) 7-Nitro-l-tetralone oxime

7-Nitro-l-tetralone (2.00 g, 10.4 mmol) was dissolved in ethanol (20 mL) and was added to NH4OH (1.64 mL, 16.6 mmol) and NaOAc (1.71 g, 20.8 mmol) in H 2 O (30 mL). After 2 h at 65 °C, the solvent was removed under reduced pressure and the residue was titurated with H2O. The precipitate was removed by filtration and dried under vacuum to give 1.99 g (93 %) of the desired material as a white solid. MS (ES-) m/z 205 [M-H]-

b) 1 ,7-Diamino- 1 ,2,3,4-tetrahydronaphthalene

A Parr shaker was charged with the compound of Example 1(a) (1.99 g, 9.70 mmol) and 10 % Pd C (250 mg) in ethanol (60 mL). The vessel was sealed and pressurized with H2 at 50 psi for 3.5 hr. The catalyst was removed by filtering through celite. The filtrate was poured into IN NaOH and extracted with CH2CI2. The combined organic extracts were evaporated at reduced pressure to give 950 mg (60 %) of the desired product. MS (ES+) m/z 163 [M+H] +

c) l,7-Bis(picolinoylamino)-l,2,3,4-tetrahydronaphthalene The compound of Example 1(c) (0.95 g, 5.8 mmol) was dissolved in DMF

(10 mL) and was treated at O°C with picolinic acid (2.14 g, 17.4 mmol), Bop reagent (7.70 g, 17.4 mmol), HOBt (2.35 g, 17.4 mmol) and iPr2EtN (10.1 mL, 58.0 mmol). After 20 h at RT, the solvent was evaporated under high vacuum. The residue was dissolved in CHCI3 and washed with IN HCl. Removal of solvent under reduced pressure gave crude product which was purified by flash chromatography (silica gel, 40% EtOAc in hexane) to give 430 mg of the desired

material. This material was further recrystallized from CHCI3: EtOAc: Hexane (3: 1 : 1) to give 230 mg of pure material. MS (ES+) m/z 373 [M+H]+.

Example 2

Preparation of N. N'-bis(picolinoyl)-3.7-diaminobicyclof3.3.01octane

a) 3,7-Diaminobicyclo[3.3.0]octane

To bicyclo[3.3.0]octane-3,7-dione (0.25 g, 1.81 mmol) in dry MeOH (3 mL) at 0 °C was added hydroxylamine hydrochloride (0.28 g, 4.30 mmol) and NaOAc (0.65 g, 7.92 mmol). After lh, the solvent was removed under vacuum. Triturate the resulting residue with a minimum of ice-cold water to give a white precipitate. Collect and dry the precipitate under vacuum to give 0.16 g of the crude oxime.

A Parr shaker vessel was charged with the crude oxime and activated Raney nickel (0.50 g) in IN methanolic NH3 (20 mL). The reaction vessel was pressurized with H2 at 50 psi for 18 h. The catalyst was removed by filtration through celite and the filtrate was evaporated under reuced pressure to give 0.11 g of the diamine as a pale yellow residue. This material was used without further purification. MS (ES+) m/z 141.0 [M+H}+.

b) N, N'-Bis(picolinoyl)-3,7-diaminobicyclo[3.3.0]octane

The crude material from Example 2(a), picolinic acid (0.67 g, 5.44 mmol) and EDC (1.39 g, 7.25 mmol) were dissolved in pyridine (10 mL). After 24 h at RT, the solvent was removed under vacuum. The residue was dissolved in a minimum amount ifMeOH and applied to a flash column (silica gel). The product was eluted with EtOAc to give 0.21 g of a pale yellow oil. This was further purified by flash chromatography (10% EtOAc/hexanes, silica gel) to give 0.14 g of desired product as a white solid. MS (ES+) m z 351.2 [M+H] + .

Example 3

Preparation of N.N'-bis(picolinoyl)-l ,3-xylylenediamine

To a stirred solution of 1,3-xylylenediamine (132 uL, 1.00 mmol) in DMF (10 mL) was sequentially added /Pr2NEt (1.75 mL, 10.0 mmol), HOBt (405 mg, 3.00 mmol), picolinic acid (370 mg, 3.00 mmol) and BOP reagent (1.33 g, 3.00 mmol). After 18 h at room temperature, the reaction mixture was added to a rapidly-stirred mixture of EtOAc (50 mL), H2O (50 mL), and sat'd NaCl (50 mL). After stirring for ca lh, the phases were separated and the aqueous layer was extracted with EtOAc (50 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo to a brown oil. Purification by flash chromatography (silica gel, EtOAc) afforded 359 mg of the desired product as a white solid. MS (ES+) m/z 347.2 [M+H] + .

Example 4

Preparation of N.N'-bis(picolinoyl)-4.4'-diaminobenzophenone

To a stirred solution of 4,4'-diaminobenzophenone (637 mg, 3.00 mmol) in DMF (30 mL) was added sequentially /Pr 2 NEt (5.30 mL, 30.4 mmol), HOBt (1.23 g, 9.10 mmol), picolinic acid (1.14g, 9.00 mmol) and BOP reagent (3.98 g, 9.0 mmol). After 17 h at room temperature, the reaction mixture was concentrated in vacuo to ca. 20 mL and added to a rapidly-stirred mixture of EtOAc (100 mL), H2O (100 mL), and sat'd NaCl (100 mL). After stirring for ca lh, the phases were separated and the aqueous layer was extracted with EtOAc (2 x 100 mL). The combined organic layers were washed with H2O (100 mL), dried over MgSO- * ., filtered, and concentrated in vacuo to a brown solid. Purification by flash chromatography (silica gel, 1% MeOH/CHC ) afforded 303 mg (24%) of the desired product as a yellow solid. MS (ES+) m/z [M+H] + .

Example 5

Preparation of N. N'-bis(picolinoylV3.7-diaminobicyclof3.3.01octane

a) 3 ,7-Dihydroxybicyclo [3.3.0] octane

To a suspension of LAH (0.08 g, 2.11 mmol) in Et2θ (5 mL) was added bicyclo[3.3.0]octane-3,7-dione (0.14 g, 1.01 mmol). The reaction attained reflux during the addition. The reaction was allowed to stir at ambient temperatures for an additional 1.5h following the addition. The reaction mixture was cooled to 0 °C and quenched by the sequential addition of NaF (0.35 g, 8.33 mmol) and water (0.11 mL, 6.11 mmol). The mixture was rapidly stirred at 0 °C for 1 h and then filtered through celite. The filtrate was concentrated under vacuum to give 0.14 g of a clear oil. Flash chromatogφahy (EtOAc, silica gel) gave 0.10 g (70 %) of the desired material as a clear oil. MS (ES+) m z 143.0 [M+H] + .

b) 3 ,7-Bis(methanesulfonyl)bicyclo[3.3.0]octane

To the compound of Example 5(a) (0.10 g, 0.70 mmol) in CH2CI2 (5 mL) at 0 °C was added Et3N (0.30 mL, 2.15 mmol) and methanesulfonyl chloride (0.12 mL, 1.55 mmol). The reaction was allowed to warm to RT over lh. The reaction was quenched by pouring into brine (20 mL) and extracting with EtOAc (3 x 20 mL). The organic extracts were dried over Na2SO4 and concentrated to give 0.26 g of the product as a pale yellow oil. This was used without further purification. MS (ES+) m/z 299.0 [M+H]+

c) 3,7-Di-azidobicyclo[3.3.0]octane

To the crude compound of Example 5(a) in DMF (3 mL) was added NaN3 (0.46 g, 7.17 mmol). After 24 h at RT, the reaction was quenched by pouring into 50% brine (10 mL) and extracting with EtOAc (3 x 10 mL). The combined organic extracts were dried over Na2SO4 and concentrated to give a yellow oil. Flash chromatography (5% EtOAc/hexanes, silica gel) gave 0.08 g (58% from Example 5(a)) of the desired material as a clear oil. ! H NMR (400 MHz, CDCI3) δ 3.90 (m, 2H), 2.65 (m, 2H), 2.10 - 1.35 ( , 8H).

d) 3,7-Diaminobicyclo[3.3.0]octane

To the compound of Example 5(c) (0.08 g, 0.40 mmol) in MeOH (3 mL) was added 10% Pd/C (5 mg). The reaction falsk was flushed with H2 and then

fitted with a H2 filled balloon. After stirring under H2 at RT for 4.5 h, the H2 was vented and the catalyst was removed by filtration. Removal of solvent under reduced pressure gave 0.05 g (89 %) of the desired material as a clear oil. This was used without further purification. MS (ES+) m/z 141.0 [M+H] + .

e) N, N'-Bis(picolinoyl)-3,7-diaminobicyclo[3.3.0]octane

The compound of Example 5(d) (0.05 g, 0.36 mmol). picolinic acid (0.13 g, 1.06 mmol) and EDC (0.27 g, 1.41 mmol) were combined in pyridine (5 mL). After 2 days at RT, the solvent was removed under vacuum. The residue was purified by flash chromatography (5% MeOH/EtOAc, silica gel) to give 0.05 g of a yellow oil. The product was further purified by preparative TLC (silica gel, 0.5mm thickness, 20 cm x 20 cm plate, EtOAc) to give 0.02 g (15 %) of the desired material as a white solid. MS (ES+) m/z 351.2 [M+H] + .

Example 6

Preparation of N.N'-bis(picolinoyl)-1.2-phenylenediamine

To a stirred solution of 1 ,2-phenylenediamine (111 mg, 1.00 mmol) in DMF (10 mL) was sequentially added iP- NEt (1.75 mL, 10.0 mmol), HOBt (406 mg, 3.00 mmol), picolinic acid (370 mg, 3.00 mmol), and BOP reagent (1.34 g, 3.00 mmol). After 18 h at room temperature, the reaction mixture was added to a rapidly-stirred mixture of EtOAc (50 mL), H2O (50 mL), and sat'd NaCl (50 mL). After stirring for ca lh, the phases were separated and the aqueous layer was extracted with EtOAc (50 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo to a brown oil. Purification by flash chromatography (silica gel, EtOAc) afforded 302 mg (92%) of the desired product as a white solid. MS (ES+) m/z 319.0 [M+H]+.

Example 7

Preparation of N.N'-bisψicolinoyl)-1.3-phenylenediamine

To a stirred solution of 1,3-phenylenediamine dihydrochloride (183 mg, 1.00 mmol) in DMF (10 mL) was added tPr 2 NEt (2.10 mL, 12.1 mmol), HOBt (407 mg, 3.00 mmol), picolinic acid (370 mg, 3.00 mmol), and BOP reagent (1.34 g, 3.00 mmol) in sequential fashion. After 18 h at room temperature, the reaction mixture was added to a rapidly-stirred mixture of EtOAc (50 mL), H2O (50 mL), and sat'd NaCl (50 mL). After stirring for ca lh, the phases were separated and the aqueous layer was extracted with EtOAc (50 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo to a brown oil. Purification by flash chromatography (silica gel, EtOAc) afforded 251 mg (78%) of the desired product as a white solid. MS (ES+) m/z 319.0 [M+H] + .

Example 8

Preparation of (5S). (5S)-N. N'-bis (2-pyrroIidon-5-carbonyl)-1.3-xylylenediamine

To a solution of 1 ,3-xylylenediamine in DMF is added ι ' Pr2NEt (10 molar equivalents), HOBt (3 molar equivalents), (5S)-2-pyrrolidinone carboxylic acid (3 molar equivalents) and BOP reagent (3 molar equivalents). The reaction is maintained at room temperature for 18 h and is quenched by pouring into a stirring mixture of EtOAc and 50% brine. The organic phase is seperated from the aqueous phase and the aqueous phase is further extracted with EtOAc. The combined organic layer is dried over MgSO4 and concentrated to the desired product.

Example 9

Preparation of N. N'-bis(prolyl)-1.3-xylylenediamine

a) N, N'-Bis(BOC-prolyl)-l,3-xylylenediamine To a solution of 1 ,3-xylylenediamine in DMF is added Pr2NEt (10 molar equivalents), HOBt (3 molar equivalents), BOC-proline (3 molar equivalents) and

BOP reagent (3 molar equivalents). The reaction is maintained at room temperature for 18 h and is quenched by pouring into a stirring mixture of EtOAc and 50% brine. The organic phase is seperated from the aqueous phase and the aqueous phase is further extracted with EtOAc. The combined organic layer is dried over MgSO4 and concentrated to the desired product.

b) N, N'-Bis(prolyl)-l,3-xylylenediamine

A solution of compound of Example 9(a) in CH2CI2 is treated with TFA. The reaction is stirred at room temperature for lh. The solvent is reemoved under reduced pressure to give the desired compound.

Example 10

Preparation of N. N'-bis(azetidinecarbonyl ' )-1.3-xylylenediamine

a) N, N'-Bis(BOC-azetidinecarbonyl)-l,3-xylylenediamine

To a solution of 1,3-xylylenediamine in DMF is added ι ' Pr2NEt (10 molar equivalents), HOBt (3 molar equivalents), BOC-azetidine carboxylic acid (3 molar equivalents) and BOP reagent (3 molar equivalents). The reaction is maintained at room temperature for 18 h and is quenched by pouring into a stirring mixture of EtOAc and 50% brine. The organic phase is seperated from the aqueous phase and the aqueous phase is further extracted with EtOAc. The combined organic layer is dried over MgSO4 and concentrated to the desired product.

b) N, N'-Bis(azetidinecarbonyl)-l,3-xylylenediamine A solution of compound of Example 10(a) in CH2CI2 is treated with TFA.

The reaction is stirred at room temperature for lh. The solvent is reemoved under reduced pressure to give the desired compound.

Example 1

Preparation of N. N'. N. N'-bisψicolinoyl)dimethyl-1.3-xylv.enediamine

To a solution of N, N'-dimethyl-l,3-xylylenediamine in DMF is added j ' Pr2NEt (10 molar equivalents), HOBt (3 molar equivalents), picolinic acid (3 molar equivalents) and BOP reagent (3 molar equivalents). The reaction is maintained at room temperature for 18 h and is quenched by pouring into a stirring mixture of EtOAc and 50% brine. The organic phase is seperated from the aqueous

phase and the aqueous phase is further extracted with EtOAc. The combined organic layer is dried over MgSO4 and concentrated to the desired product.

Example 12

N. N'-bisψicolinoyl)-l .2-xylylenediamine

a) α,α'-Diazido-l,2-xylene

To α,α'-dibromo-l,2-xylene (0.26 g, 1.00 mmol) in DMF (5 mL) was added NaN 3 (0.64 g, 10.0 mmol). After 5 min at RT, the reaction was quenched by poring into 50% brine (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic extracts were washed with 50 % brine (20 mL) and dried over Na.SO 4 . Removal of solvent gave 0.17 g (90% crude yield) of the desired product as a yellow oil. This material was used in the next step without further purification. Η NMR (250 Mhz, CDC1 3 ) δ 7.40 (s, 4H), 4.45 (s, 4H).

b) 1 ,2-Xylylenediamine

To the compound of Example 12 (a) (0.17 g, 0.90 mmol) in MeOH (10 mL) was added 10% Pd/C (ca. 10 mg). The reaction flask was evacuated and back- flushed with hydrogen three time. The flask was then fitted with a hydrogen filled balloon. After 3.5h, the hydrogen was vented and the reaction mixture was filtered through celite to remove the catalyst. Removal of solvent gave 0.10 g (84 % crude yield) of the desired material as a yellow oil. This material was used without further purification. Η NMR (250 Mhz, CDC1 3 ) δ 7.40 (m, 4H), 4.00 (broad s, 4H), 3.50 (broad s, 4H).

c) N, N'-Bis(picolinoyl)-l,2-xylylenediamine

To the compound of Example 12 (b) (0.10 g, 0.76 mmol) in pyridine (5 mL) was added picolinic acid (0.28 g, 2.27 mmol) and EDC (0.87 g, 4.54 mmol). After 18h, at RT, the bulk of the pyridine was removed in vacuo. Flash chromatography (10% MeOH/ EtOAc, silica gel) of the residue gave 0.22 g (84 %) of the title compound as a pale yellow solid. MS (ES+) m z 347.2 [M+H] + .

Example 13

Formulations for pharmaceutical use incoφorating compounds of the present invention can be prepared in various forms and with numerous excipients. Examples of such formulations are given below.

Tablets/Ingredients Per Tablet

1. Active ingredient 0.5 mg (Cpd of Form. I or II)

2. Corn Starch 20 mg 3. Alginic acid 20 mg

4. Sodium alginate 20 mg

5. Mg stearate 1.3 mg

Procedure for tablets: Step 1 Blend ingredients No. 1, No. 2, No. 3 and No. 4 in a suitable mixer/blender. Step 2 Add sufficient water portion-wise to the blend from Step 1 with careful mixing after each addition. Such additions of water and mixing until the mass is of a consistency to permit its converion to wet granules. Step 3 The wet mass is converted to granules by passing

it through an oscillating granulator using a

No. 8 mesh (2.38 mm) screen. Step 4 The wet granules are then dried in an oven at

140°F (60°C) until dry. Step 5 The dry granules are lubricated with ingredient No. 5.

Step 6 The lubricated granules are compressed on a suitable tablet press.

Parenteral Formulation

A pharmaceutical composition for parenteral administration is prepared by dissolving an appropriate amount of a compound of formula I or II in polyethylene glycol with heating. This solution is then diluted with water for injections Ph Eur. (to 100 ml). The solution is then sterilized by filtration through a 0.22 micron membrane filter and sealed in sterile containers.




 
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