HEMOREGULATORY COMPOUNDS

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

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: 1 192-1 194) 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 -1- SUBSTITUTΕ SHEET (RULE 26) 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 seventy 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 suffering 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 -2- SUBST1TUTE SHEET (RULE 26) 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, Heφes and hepatitis 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 myelosuppresive agents of co-pending U.S. Application No. 07/799,465 and U.S. Patent No. 4,499,081, incorporated 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 Formula (II). This invention also constitutes a method for preventing and treating viral, fungal and bacterial infections 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 Formula (II). Detailed Description of the Invention The compounds of the invention are represented by structural Formula I wherein Rj is independently quinolinyl, 2-pyridinyl, isoquinolinyl, 2-pyrrolidonyl, pyrrolidinyl or N-methyl-2-imidazolyl; R2 is independently hydrogen, Cj_4CO2H or Cj^alkyl; R3 is hydrogen or Cj .4 alkyl; Y is (CH 2 ) n , (CH 2 ) m GsC(CH2) s , (CH 2 ) m CH=CH(CH 2 ) s , (CH 2 ) 2 -O- (CH 2 ) 2 , (CH 2 ) 2 -N(R 3 )-(CH 2 )2 or (CH 2 ) 2 -S-(CH 2 ) 2 ; n is an integer from 3 to 8; is 1 to 4; and s is 1 to 4; provided that the compound is not: N,N'-bis(picolinoyl)- 1 ,3- diaminopropane N, N'; N, N'-bis(methyl)bis(picolinoyl)-l, 3-diaminopropane; or (S), (S)-N, N'-Bis(2-pyrrolidone-5-carbonyl)-l,5-diaminopentane; or a pharmaceutically acceptable salt thereof. The invention is also a pharmaceutical composition, which comprises a compound of Formula (II): wherein Rj is independently a 4 - 10 membered mono- or bicyclic heterocydic 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_4 alkyl, F, Cl, Br, I, C1.4 alkoxy, (CH 2 )rnR5> oxo ' oxime, hydroxy, N(R4) 2 , acylamino or aminoacyl groups, 8, 9, 10 membered monocyclic ring systems being excluded; R is independently hydrogen, C \ .4 alkyl C(O)R5, C^alkyl or R 2 is benzyl which is optionally substituted by one or two C^aikyl, C1.4a.koxy, F, Cl, I, Br, OH, or N(R 4 ) 2 ; R3 is hydrogen or C 1.4 alkyl; R4 is independently hydrogen, Cj^alkyl, or benzyl; R5 is independently OR4, N(R4) 2 or SR4; Y is (CH 2 ) n , (CH 2 ) m C ≡ C(CH 2 ) s , (CH 2 )mCH=CH(CH 2 )s, (CH 2 ) 2 -O- (CH 2 )2, (CH 2 ) 2 -N(R 3 )-(CH 2 ) 2 or (CH 2 ) 2 -S-(CH 2 ) 2 ; n is an integer from 3 to 8; m is 1 to 4; and s is 1 to 4; and a pharmaceutically acceptable carrier; provided that the compound of Formula (II) is not N, N', N, N'- bis(methyl)bis(picolinoyl)-l, 3-diaminopropane or (S), (S)-N, N'-Bis(2-pyrrolidone- 5-carbonyl)- 1 ,5-diaminopentane. 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. C _4 alkyl groups may be straight or branched. The compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active form. All these compounds and diastereomers are contemplated to be within the scope of the present invention. Rl in the above Formula (II) denotes an optionally substituted pyrrolyl, isopyrrolyl, pyrazolyl, isoimidazolyl, triazolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolidinyl, piperazinyl, triazinyl, morpholinyl, indolyl, indoleninyl, isobenzazolyl, pyrindinyl, isoindazolyl, indoxazinyl, benzoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl, pyridopyridinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, indolinyl, 2-pyrrolidonyl, imidazolyl, imidazolidinyl, imidazolinyl, piperidyl, tetrazolyl, quinuclidinyl, azetidinyl, or purinyl; Preferred compounds of Formula (I) and Formula (IΙ)are those wherein R ] is quinolinyl, 2-pyrindinyl or 2-pyrrolidonyl; R 2 is hydrogen or Cj_4CO 2 H; Y is (CH ) n , (CH 2 ) m C=C(CH ) s or (CH 2 ) m CH=CH(CH 2 ) s ; n is an integer from 3 to 5; m is 1 or 2; and s is 1 or 2. More preferred compounds of Formula (I) and Formula (II) are those wherein Rl is 2-pyrindinyl or 2-pyrrolidonyl; R 2 is hydrogen; Y is (CH 2 ) n or (CH2) m CH=CH(CH2) s ; n is 3or 5; m is 1; and s is 1. Preferred compounds of Formula (I) are: N,N'-bis(picolinoyl)- 1 ,5-diaminopentane; (E) N,N-bis (picolinoyl)-l,4-diaminobut-2-ene; N,N'-bis(picolinoyl)- 1 ,4-diaminobutane; and (R, R)-N. N'-Bis(2-pyrrolidone-5-carbonyl)-l,3-diaminopropane; or 3, 10-bis(picolinoyl)-3,10-diaza-l,12-dodecanedioic acid. In addition to the above preferred compounds for Formula (I) the preferred compounds of Formula (II) include N, N'-bis (picolinoyl)-l,3-diaminopropane. Methods of Preparation Compounds of Formula (I) and (II) wherein R 2 is hydrogen; R3 is C j_4alkyl; and Y and Ri are defined as in Formula (I) and (II) and are prepared by methods analogous to those described in Scheme 1. Scheme 1 a) EDC, pyridine Appropriate diamines (such as 2 in Scheme 1) are bis-acylated with appropriate heterocydic acids (such as I in Scheme 1) using an activating agent (such as EDC) in a suitable polar aprotic solvent (such as pyridine) Compounds of Formula (I) and (II) wherein R2 is Cj. 4 alkyl; R3 is C 1.4 alkyl and Y, R] and n are defined as in Formula (I) and (II) are prepared by methods analogous to those described in Scheme 2. -7- SUBST1TUTΕ SHEET (RULE 26) Scheme 2 CH, CH, b, c I H,N-(CH 2 ),-NH 2 ► BOC NH-(CH 2 ),-NH-BOC ►BOC-N — (CH - — BOC < S a) (BOC) 2 )O, CH 2 C1 2 , b) NaH, THF, c) Mel; d) 4N HCl/dioxane, e) 4, EDC, pyndine Appropriate diamines (such as in Scheme 2) are bis-protected using conventional amine protecting groups (such as t-butoxycarbonyl) 2 in Scheme 2 is then bis-N- alkylated by treatment with a strong base (such as sodium hydride) in an aprotic solvent (such as THF) followed by an alkylating agent (such as methyl iodide) Removal of the protecting group of 3. in Scheme 2 under standard acidic conditions (such as 4N HCl/dioxane) followed by acylation of the resulting amine with suitable acids (such as 4 in Scheme 2) using an activating agent (such as EDC) in a suitable solvent (such as pyndine) furnishes the bis-N-alkylated product 5 in Scheme 2 Compounds of Formula (I) and (II) wherein R 2 is hydrogen; Y is (CH 2 ) - N(R3)-(CH ) 2 , R3 is hydrogen and Rj and n are defined as in Formula (I) and (II) are prepared by methods analogous to those described in Scheme 3. Scheme 3 H 2 N-(CH 2 ) 2 -N(CBZ)-(CH 2 ) 2 -Nri, a) EDC, pyridine; b) H 2 , 10% Pd C, ethanol Appropriate diamines (such as 2 in Scheme 3) are bis-acylated with appropriate heterocydic acids (such as 1 in Scheme 3) using an activating agent (such as EDC) in a suitable polar aprotic solvent (such as pyridine) to give 3 in Scheme 3. The CBZ group is then removed using hydrogen and an appropriate catalyst (such as 10% Pd/C) in a suitable solvent (such as methanol), giving 4 in Scheme 3. Compounds of Formula (I) and (II) wherein R 2 is Ci^alkyl; Y is (CH 2 ) 2 - N(R 3 )-(CH ) 2 ; R 3 is hydrogen and Rj and n are defined as in Formula (I) and (II) are prepared by methods analogous to those described in Scheme 4. Scheme 4 H 2 N-(CH 2 ) 2 -N(CBZ) — (CH 2 ) 2 -NH 2 >■ BOC-NH -(CH 2 ) 2 -N(CBZ) — (CH 2 ) 2 ~NH-BOC J_ b, c CH ^ CH T BOC-N -(CH 2 ) 2 -N(CBZ) -(CH 2 ) 2 -N-BOC 4- 1 -L a) (BOC) 2 )O, CH C1 ; b) NaH, THF; c) Mel; d) 4N HCl/dioxane; e) 4, EDC, pyridine; f) H 2 , 10% Pd/C, ethanol Appropriate diamines (such as 1 in Scheme 4) are bis-protected using conventional amine protecting groups (such as t-butoxycarbonyl). 2 in Scheme 4 is then bis-N- alkylated by treatment with a strong base (such as sodium hydride) in an aprotic solvent (such as THF) followed by an alkylating agent (such as methyl iodide). Removal of the terminal diamino-protecting groups in 3 in Scheme 4 under standard acidic conditions (such as 4N HCl/dioxane) is followed by acylation of the resulting amine with suitable acids (such as 4 in Scheme 4) using an activating agent (such as EDC) in a suitable solvent (such as pyridine). The CBZ group is then removed using hydrogen and an appropriate catalyst (such as 10% Pd C) in a suitable solvent (such as methanol) furnishing the product 5_ in Scheme 4. Compounds of Formula (I) wherein R2 is C^alkyl; Y is (CH2) m S- S(CH2) S ; Ri , m and s are defined as in formula (I) are prepared in manners analogous to those in Scheme 5. Scheme 5 CH. H,N b, c SBn BOC-NH . BOC-N, SBn SBn a) (BOQ2O, CH2CI2; b) NaH, THF; c) Mel; d) 4N HCl/dioxane; e) 4, EDC, pyridine; 0 HF, anisole; g) air, pH 7.8 Appropriate amines (such as 1 in Scheme 5) are protected using conventional amine protecting groups (such as t-butoxycarbonyl). Compound 2 in Scheme 5 is then alkylated by treatment with a strong base (such as sodium hydride) in a polar aprotic solvent (such as THF) followed by an alkylating agent (such as methyl iodide). Removal of the nitrogen protecting group of 3_ in Scheme 5 under standard acidic conditions (such as 4N HCl/dioxane) followed by acylation of the resulting amine with a suitable acid (such as 4 in Scheme 5) in a suitable solvent (such as pyridine) furnishes the protected thiol. The benzyl group is removed using a suitable acid source (such as HF with anisole). Compound 5 in Scheme 5 is then oxidized under standard conditions (such as exposure to air at pH 7.8) to give the disulfide 6 in Scheme 5. Compounds of Formula (I) wherein R2 is Cj^alkylcarboxylic acid; R3 is C al yl; Y is (CH 2 ) n . (CH 2 ) rn C ≡ C(CH 2 )s, (CH 2 ) m CH=CH(CH s ) s , (CH 2 ) m -O- (CH 2 ) S , (CH s ) m -N(R 3 )-(CH 2 ) s , (CH s ) m -S-(CH s ) s ; and Ri , m, n and s are defined as in Formula (I) are prepared by methods analogous to those described in Scheme 6. -11- SUBST1TUTE SHEET (RULE 26) Scheme 6 BOC-NH-(CH 2 ) 6 -NH-BOC — — ► 1 2 3 a) NaH, allyl bromide; b) O3, NaOMe, MeOH; c) 4N HCl dioxane; d) picolinic acid, EDC, pyridine; e) NaOH, MeOH, water A suitably protected diamine (such as I in Scheme 6, prepared as shown in Scheme 2 is treated with a strong base (such as sodium hydride) in the presence of an alkenyl halide (such as allyl bromide) in a polar aprotic solvent (such as a mixture of DMF and THF). The bis-olefin, 2 in Scheme 6 is oxidatively cleaved with an oxidizing agent (such as ozone in NaOMe/MeOH) giving the bis-ester 3_ in Scheme 6. Removal of the terminal diamino-protecting groups in 3 in Scheme 6 under standard acidic conditions (such as 4N HCl/dioxane) is followed by acylation of the resulting amine with suitable acids (such as picolinic acid) using an activating agent (such as EDC) in a suitable solvent (such as pyridine). The methyl esters are saponified under basic aqueous conditions (such as IN NaOH in MeOH) giving the product 5_ in Scheme 6. Compounds of Formula (I) wherein R 2 is C ι-4alkylcarboxyhc acid, Y is (CH 2 ) m -S-S-(CH 2 )s, Rj , m and s are defined as in Formula (I) are prepared by methods analogous to those described in Scheme 7 Scheme7 a) NaH, DMF, b) methyl bromoacetate, c) 4N HCl dioxane, d) picolinic acid, EDC, pyndine, e)PH3P, f) air, pH 7.8, g) NaOH, MeOH, water A suitably protected aminothiol (such as 1 in Scheme 7) is treated with a strong base (such as sodium hydride) in the presence of a halo-ester (such as methyl bromoacetate) in a polar aprotic solvent (such as DMF) Removal of the terminal amino-protecting groups in 2 in Scheme 7 under standard acidic conditions (such as 4N Hcl dioxane) is followed by acylation of the resulting amine with suitable acids (such as picolinic acid) using an activating agent (such as EDC) in a suitable solvent (such as pyndine) The sulfur protecting group is removed under standard conditions (such as tnphenylphosphine) and the resulting thiol is oxidized by standard means (such as exposure to air at pH 7.8) to give a disulfide 4 in Scheme 7 Basic hydrolysis (using IN NaOH, MeOH for example) gives the diacid 5 in Scheme 7 Scheme 8 BOC-NH-(CH,) N(CBZMCH 2 ) NH-BOC — ► BOC-N-(CH 2 ) 2 -N-(CBZMCH 2 > 2 -N- BOC Λ 2> a) NaH, DMF; b) allyl bromide; c) O3, NaOMe, MeOH; d) 4N HCl/dioxane; e) picolinic acid, EDC, pyridine; f) H 2 , 50 psi, 10 % Pd/C, MeOH; g) IN NaOH, MeOH Compounds of Formula (I) wherein R2 is C 1.4 alkyl carboxylic acid; Y is (CH 2 ) m -N(R3)-(CH 2 ) s ; R3 is hydrogen and Rj, m and s are defined as in Formula (I) are prepared by methods analogous to those described in Scheme 8. Appropriately protected diamines (such as I in Scheme 8, prepared as shown in Scheme 4) are bis-N-alkylated by treatment with a strong base (such as sodium hydride) in an aprotic solvent (such as THF) followed by a halo-alkene (such as allyl bromide). The bis-olefin 2 in Scheme 8 is oxidatively cleaved with an oxidizing agent (such as ozone in NaOMe/MeOH) giving the bis-ester 3_ in Scheme 8. Removal of the terminal diamino-protecting groups in 3 in Scheme 8 under standard acidic conditions (such as 4N HCl/dioxane) is followed by acylation of the resulting amine with suitable aromatic acids (such as picolinic acid) using an activating agent (such as EDC) in a suitable solvent (such as pyridine). The central amino-protecting group is cleaved under standard reductive conditions (such as H 2 in MeOH) in the presence of a suitable catalyst (such as 10 % Pd/C) to give the amino ester 4 in Scheme 8. The methyl esters are saponified under basis aqueous conditions (such as IN NaOH) giving the product 5 in Scheme 8. 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 (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) and (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 of Formula (II) as hereinbefore defined to a subject. -16- SUBSTITUTΈ SHEET (RULE 26) 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 supematants are collected 18 hours later Supematants are fractionated with a Centπcon-30 molecular weight cut-off membrane C6 4 cell hematopoietic synergistic factor (HSF) activity is measured in a murine CFU-C assay CFU-C Assay Bone marrow cells are obtained from C57B 1/6 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 supematants 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% CO 2 ) 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 % gelatin/HBSS and 80uM ferricytochrome c in a total volume of 200uL/welI. The nmoles of cytochrome c reduced/well is calculated from spectrophotometric readings (550 nm) taken following a 1 hour incubation at 37 °C (5% CO 2 ). 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 N. N'-bis(picolinoyl)-l, 3-diaminopropane Picolinic acid (3.00g, 24.4 mmol), diaminopropane (0.83 mL, 10.0 mmol), EtNιPr 2 (3.90 mL, 22.4 mmol) and EDC (4.80 g, 25.0 mmol) were combined in CH 2 CI2 (20 mL) at room temperature. After 24 h, the reaction was diluted with CH 2 C1 2 (50 mL) and washed with IN AcOH (20 mL), sat NaHCO 3 (20 mL) and brine (20 mL). The organic layer was dried over Na 2 SO4 and concentrated in vacuo to give a dark oil (1.00 g). Flash chromatography (10% MeOH/EtOAc, silica gel) gave the title compound as a pale yellow solid (0.94 g, 33 %). MS (ES+) m/z 285.0 [M+H] + . -18- SUBSTITUTΈ SHEET (RULE 26) Example 2 Preparation of N. N'-bis(picolinoyl)- 1. 4-diaminobutane In an analogous fashion to Example 1, picolinic acid (3.00 g, 24.4 mmol), diaminobutane (0.88 g, 10.0 mmol), EtN Pr 2 (3.90 mL, 22.4 mmol) and EDC (4.80 g, 25.0 mmol) in CH2CI 2 (20 mL) gave after flash chromatography the title compound as a pale yellow solid (0.66 g, 22 %). MS (ES+) m z 299.0 [M+H]+. Example 3 Preparation of N. N'-bis(picolinoyl)-l. 5-diaminopentane In an analogous fashion to Example 1, picolinic acid (1.00 g, 8.12 mmol), diaminopentane dihydrochloride (0.64 g, 3.65 mmol), EtN Pr 2 (2.80 mL, 16.1 mmol) and EDC ( 1.56 g, 8.14 mmol) in CH2CI 2 (10 mL) gave after flash chromatography the title compound as a clear oil (0.22 g, 19 %). MS (ES+) m/z 313.0 [M+H] + . Example 4 Preparation of N. N'-bis(quinaldoyP- 1. 3-diaminopropane EDC (1.10 g, 5.73 mmol) was added to a solution of quinaldic acid (0.96 g, 5.54 mmol) and 1, 3-diaminopropane (0.21 mL, 2.51 mmol) in pyridine (12 mL). After 20 h at room temperature, the bulk of the pyridine was removed in vacuo. The residue was subjected to flash chromatography (5% MeOH/EtOAc, silica gel) to give the title compound as a pale yellow foam (0.85 g, 88%). MS (ES+) m/z 385.2 [M+H] + . Example 5 (R. R)-N. N'-Bis(2-pyrrolidone-5-carbonyl)-1.3-diaminopropane a) (R, R)-N, N'-Bis(l-benzyloxycarbonyI-2-pyrrolidone-5-carbonyl)-l,3- diaminopropane (R)- 1 -Benzyloxycarbonyl-2-pyrrolidone-5-carboxylic acid (0.45 g, 1.71 mmol), 1 ,3-diaminoproρane (65 uL, 0.78 mmol), EDC (0.40 g, 2.08 mmol), HOBt (0.28 g, 2.07 mmol) and ιPr NEt (0.60 mL, 3.44 mmol) were combined in CH 2 CI 2 (10 mL). After 18h at RT, the reaction was diluted with CH 2 CI 2 (50 mL) and washed sequentially with H 2 O (20 mL), IN HCl (20mL), sat NaHCO3 ( 20 mL) and brine (20mL). The organic layer was dried over Na 2 SO4. Removal of solvent gave 0.44 g of the product as a white solid. This was used without purification in the next step. MS (ES+) m/z 565.2 [M+H]+. b) (R, ?)-N,N'-Bis(2-pyrrolidone-5-carbonyl)- 1 ,3-diaminopropane To the compound of Example 5(a) (0.1 1 g, 0.20 mmol) in EtOH (10 mL) was added Pd black (ca. lOmg). The reaction vessel was flushed with hydrogen and then fitted with a hydrogen filled balloon. After 5.5h at RT, the hydrogen was vented to the atmosphere and the catalyst was removed by filtration. Removal of solvent followed by trituration of the resulting residue with CHCI3 gave 0.40 g (68 %) of the title compound as a white powder. MS (ES+) m/z 297.0 [M+H] + . Example 6 (E)-N.N-bis (picolinoyl)- 1.4-diaminobut-2-ene a) (E)-N.N-bis (phthalyl)- 1 ,4-diaminobut-2-ene A solution of (E)-l ,4-dichlorobut-2-ene (1.0 g, 8 mmol) in DMF was treated with potassium phthalimide (4.45 g, 24 mmol) at 60 °C for 72 h. The reaction mixture was evaporated under reduced pressure and the residue taken into CHCI3, washed with IN HCl (aqueous), dried over anhydrous MgSO , filtered and evaporated. Purification of the residue by flash chromatography (silica gel, 4x20 cm column, 99: 1 to 95:5, CHCl 3 :CH 3 OH) gave 3.06 g of (E) N,N-bis (phthalyl)- 1 ,4- diaminobut-2-ene. This material was used directly in the next step. b) 1 ,4-diaminobut-2-ene (E) N,N-Bis (phthalyl)- 1 ,4-diaminobut-2-ene (1.55 g, 4.48 mmol) was dissolved in ethanohbenzene (9: 1, 100 mL) and was treated with hydrazine monohydrate (1.8 mL). The resulting solution was stirred at room temperature for 5 d. The reaction was filtered and evaporated. The residue was dissolved in CHCI 3 , the insoluble filtered and the solution evaporated to give 1 12 mg of 1 ,4-diaminobut- 2-ene as an oil which was used directly in the next step. c) (E) N,N-bis (picolinoyl)-l ,4-diaminobut-2-ene Picolinic acid (Aldrich, 480 mg, 3.9 mmol) ' , HOBt (527 mg, 3.9 mmol), EDC-HCl (746 mg, 3.9 mmol) and Et 3 N (544 uL, 3.9 mmol) was added to a solution of l,4-diaminobut-2-ene (112 mg, 1.3 mmol) in DMF and the resulting mixture was stirred for 48 h. at room temperature. The reaction mixture was then evaporated, the residue dissolved in ethyl acetate, washed with IN NaOH (aqueous), NaCl(sat. aqueous), dried over anhydrous MgSO 4 , filtered and evaporated. Purification of the residue by flash chromatography (silica gel, 4x20 cm column, 80% ethyl acetate in hexane) gave 128 mg of (E) N,N-bis (picolinoyl)-l,4-diaminobut-2-ene. This material was then purified by preparative hplc (Hamilton PRP-1, 25% A(A = CH 3 CN (0.1%TFA); B = 0.1 % aqueous TFA). The fractions containing product were pooled, evaporated. The residue was taken into ethyl acetate, washed with IN NaOH (aqueous), dried over anhydrous MgSO 4 and evaporated to give 95.4 mg of (E) N,N-bis (picolinoyl)-l ,4-diaminobut-2-ene. MS (ES+) m z 297.1 [M+H]+. Example 7 Formulations for pharmaceutical use incorporating 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 ID 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.

Claims: CLAIMS: 1. A compound of formula (I) ft a wherein Rl is independently quinolinyl, 2-pyridinyl, isoquinolinyl, 2-pyrrolidonyl, pyrrolidinyl or N-methyl-2-imidazolyl; R2 is independently hydrogen, C1.4CO2H or Cj^alkyl; R3 is hydrogen or Cι_4alkyl; Y is (CH 2 ) n , (CH 2 )mC S C(CH 2 ) s , (CH 2 )mCH=CH(CH 2 )s, (CH 2 ) -O- (CH 2 ) 2 , (CH 2 ) 2 -N(R 3 )-(CH 2 ) 2 or (CH 2 ) 2 -S-(CH 2 ) 2 ; n is an integer from 3 to 8; m is 1 to 4; and s is 1 to 4; provided that the compound is not: N,N'-bis(picolinoyl)-l,3-diaminopropane N, N'; N, N'-bis(methyl)bis(picoιinoyl)-l, 3-diaminopropane; or (S), (S)-N, N'-Bis(2-pyrrolidone-5-carbonyl)-l ,5-diaminopentane; or a pharmaceutically acceptable salt thereof. 2. A compound of Claim 1 wherein Ri is quinolinyl, 2-pyrindinyl or 2- pyrrolidonyl; R 2 is hydrogen or C^^H; Y is (CH 2 ) n , (CH 2 ) m C=C(CH 2 ) s or (CH ) m CH=CH(CH2) s ; n is an integer from 3 to 5; m is 1 or 2; and s is 1 or 2. -24- SUBSTITUTΕ SHEET (RULE 26) 3. A compound of Claim 2 wherein Ri is 2-pyrindinyl or 2- pyrrolidonyl; R 2 is hydrogen; Y is (CH 2 ) n or (CH 2 ) m CH=CH(CH 2 ) s ; n is 3or 5; m is 1 ; and s is 1. 4. A compound of Claim 1 selected from: N,N'-bis(picolinoyl)- 1 ,5-diaminopentane; (E) N,N-bis (picolinoyl)-l,4-diaminobut-2-ene; N,N'-bis(picolinoyl)- 1 ,4-diaminobutane; and (R, R)-N. N'-Bis(2-pyrrolidone-5-carbonyl)-l,3-diaminopropane; or 3, 10-bis(picolinoyl)-3, 10-diaza-l,12-dodecanedioic acid. 5. A pharmaceutical composition, which comprises a compound of Formula (II): wherein Rj is independently a 4 - 10 membered mono- or bicyclic heterocydic 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 4 alkyl, F, Cl, Br, I, C .4 alkoxy, (CH 2 ) m R5, oxo, oxime, O-Cι_4alkyloxime, hydroxy, N(R4) 2 , acylamino or aminoacyl groups, 8, 9, 10 membered monocyclic ring systems being excluded; R 2 is independently hydrogen, C1.4a.kyl C(O)R5, Cι_4alkyl or R 2 is benzyl which is optionally substituted by one or two C1.4a.kyl, C 1.4 alkoxy, F, Cl, I, Br, OH, or N(R 4 ) 2 ; R3 is hydrogen or C1.4a.kyl; R4 is independently hydrogen, C1.4a.kyl, or benzyl; R5 is independently OR4, N(R4) 2 or SR4; Y is (CH 2 ) n , (CH 2 ) m G≡C(CH 2 ) s , (CH 2 ) m CH=CH(CH 2 ) s , (CH 2 ) 2 -O- (CH 2 ) 2 , (CH 2 )2-N(R3)-(CH 2 )2 or (CH 2 )2-S-(CH 2 ) 2 ; n is an integer from 3 to 8; m is 1 to 4; and s is 1 to 4; and a pharmaceutically acceptable carrier; provided that the compound of Formula (II) is not N, N', N, N'- bis(methyl)bis(picolinoyl)-l, 3-diaminopropane or (S), (S)-N, N'-Bis(2-pyrrolidone- 5-carbonyl)- 1 ,5-diaminopentane. 6. A pharmaceutical composition of Claim 5 which is N,N'- bis(picolinoyl)- 1 ,3-diaminopropane. 7. A method of preventing or treating viral, fungal or bacterial infections which comprises administering to an animal in need thereof, an effective amount of a compound of claim 1 or claim 5. 8. A method of stimulating the myelopoietic system which comprises administering to an animal in need thereof, an effective amount to stimulate said myelopoietic system of a compound of claim 1 or claim 5. 9. A process for preparing a compound of Formula (I) which comprises: Bisacylating a diamine of Formula (III): HN(R 2 )-Y-(R2)NH Formula (III) wherein R2 and Y are as defined in Formula (I) or Y may additionally be protected by a suitable protecting group; with a compound of Formula (IV): R!-CO 2 H Formula (IV) wherein Ri is as defined in Formula (I), using a suitable activating agent in a suitable polar aprotic solvent, followed by removal of any protecting groups and if desired by salt formation. -27- SUBSTITUTΈ SHEET (RULE 26)

Inventors: BHATNAGAR PRADIP KUMAR, HARTMANN MICHAEL, HEERDING DIRK ANDRIES, HIEBL JOHANN, KREMMINGER PETER, ROVENSZKY FRANZ, BHATNAGAR, PRADIP, KUMAR, HARTMANN, MICHAEL, HEERDING, DIRK, ANDRIES, HIEBL, JOHANN, KREMMINGER, PETER, ROVENSZKY, FRANZ
Applicants: BHATNAGAR PRADIP KUMAR, HARTMANN MICHAEL, HEERDING DIRK ANDRIES, HIEBL JOHANN, KREMMINGER PETER, NYCOMED AUSTRIA GMBH, ROVENSZKY FRANZ, SMITHKLINE BEECHAM CORP, BHATNAGAR, PRADIP, KUMAR, HARTMANN, MICHAEL, HEERDING, DIRK, ANDRIES, HIEBL, JOHANN, KREMMINGER, PETER, ROVENSZKY, FRANZ, SMITHKLINE BEECHAM CORPORATION
Application year: 1996
Main classification: A61K31
IPC codes: A61K31, A61P7, A61P31, C07D207, C07D213, C07D215, C07D217, C07D233