HEMOREGULATORY COMPOUNDS

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.

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 9 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 duπng various stages of the hematopoietic cascade, including early pluπpotent stem cells and mature circulating effector cells The e 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 prechnical 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 morbidit 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 ol 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 Prechnical and clinical studies ol macrophage colony- stimulating factor Senun 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 yelopoietic 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 reactions I e. in bone marrow transplant surgery They may also be used to promote more rapid regeneration of bone marrow alter cytostatic chemotherapy and radiation therapy for neoplastic and viral diseases They may be of particular value where patients have seπous 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 bacteπal infectious diseases, particularly Candida and Herpes 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, 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 compπses a compound of Formula (I) and a pharmaceutically acceptable carrier This invention further constitutes a method for stimulating the myelopoietic system of an animal, including humans, which compπses administering to an animal in need thereof, an effective amount of a compound of Formula (I). 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) Detailed Description of the Invention The compounds of the invention are represented by structural Formula (I ) wherein: Ri is independently NH2, OH, SH, CN, CO 2 H or hydrogen, R2 is independently hydrogen, Cι _4 alkylC(O)R5, Cj_4 alkyl or R 2 is benzyl which is optionally substituted by one or two Cj_4 alkyl, Cj.4 alkoxy, F, C1, I, Br, OH, or N(R 4 ) 2 , R3 is independently hydrogen, C1.5 alkyl, C\_ζ alkylenehydroxy, Cj. 5alkyleneCO2H, (CH2)yN(R 4 ) 2 , (CH 2 ) m C(0)N(R 4 ) 2 , C 1 -5 alkyleneSH, CH 2 Ar. R 4 I 4 , N~ λ — CH ^ N- R. 2 ^ (CH 2 ) NH C=NR 4 (R 4 ) 2 R4 is independently hydrogen, C j _5 alkyl or benzyl, Ar is independently phenyl or indolyl optionally substituted by one or two R7 groups, Q is bιcyclo(3 3 Ojoctanyl, xylyl, benzophenonyl or 1,2,3,4- tetrahydronapthalyl, all of which are unsubstituted or substituted by one or two substituents chosen from C 1.4 alkyl, Ci _4alkoxy, halogen, mono or di C 1.4 alkyl ami no, (Cι _ 4 alkyl)2-NC(O)-, -(CH 2 ) n -R5. -(CH 2 ) n -R 6 , -(CH 2 ) n -COR 5 or -(CH 2 ) n -COR 6 , R5 is -OR 6 , -N(R 6 ) 2 , or -SR 6 ; R(3 is hydrogen, C > _4 alkyl or benzyl; R is halogen, R5 or R5; n is independently an integer from 0 to 3; m is independently an integer from 1 to 3; s is independently 0 or 1 ; and y is independently an integer from 2 to 4; provided that n is not 0 when s is 1 and further provided that the compound of Formula (I) is not: [(S),(S)]-N,N'-(.1 ,4-xylenediyl)bιs[2-acetamido-6,7,8,8a-tetrahydropyrrolo[ 1 ,2- α]pyrazine- 1 ,4(2H, 3H)-dione] ; [(/?), (Λ)]-2,2'-( 1 ,2-xylenediyl)bιs[6,7,8,8a-tetrahydropyrrolo[ 1 ,2-α]pyrazιne- l ,4(2H,3H-dione]; or [(S), (S)]-2,2'-( 1 ,3-xylenediyl)bιs[6,7,8,8a-tetrahydropyrrolo[ 1 ,2-α]pyrazιne- l ,4(2H,3H-dione]; 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, lodo, fluoro 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. Preferred compounds of Formula (I) are those wherein R j is OH or hydrogen; R3 is CH 2 OH or hydrogen and Q is xylyl or bicyclo[3.3.0]octanoyl. More preferred compounds are those wherein Ri , R2 and R3 are hydrogen and Q is xylyl. The most preferred compounds of the invention are: [3(5),3'(5),8a(5),8a'( )]-N,N-(l,4-xylenedιyl)bis[2-(acetamιdo)-3- (hydroxymethyl)-6,7,8,8a-tetrahydropyrrolo[l,2-α]pyrazιne-l,4(2H, 3H)-dione] or [(S), (5)]-2,2'-(l,2-xylenedιyl)bis[6,7,8,8a-tetrahydropyrrolo[l,2-/j]pyrazιne- l,4(2H,3H-dιone] [( l 5 r ),( )]-2,2'-(l,2-xylenediyl)bis[6,7,8,8a-tetrahydropyrrolo[l,2-c.]pyrazιne-l(2H, 3H,4H)-one] Method of Preparation Compounds of Formula (I) wherein A is tetrahydropyrrolopyrazinedione and R i , R 2 , R3 and n are defined as in Formula (I) are prepared by methods analogous to those in Scheme 1. Scheme 1 a) EDC. HOBt, ιPr 2 NEt, DMF, b) TFA, CH 2 CI 2 , c) pTSOH. toluene reflux, d) NaH, THF, e) t-butyl bromoacetate, f) TFA, CHjCI 2 , g) EDC HOBt iP^NEt, DMF, h) anhydrous HF Suitably protected amino acids ( such as 1 and 2 in Scheme 1 ) are coupled using standard solution phase peptide synthesis methods (such as EDC, HOBt, ιPr 2 NEt in DMF) giving a protected dipeptide. Removal of the protecting group on nitrogen followed by cychzation using catalytic amounts of a mild acid (such as p- toluenesulfonic acid) in a suitable solvent (such as toluene) gives the diketopφerazine (such as in Scheme 1 ). The diketopiperaz es (such as in Scheme 1 ) are alkylated with a suitably protected alkylating agent ( such as t-butyl bromoacetate) in a suitable aprotic polar solvent (such as THF) to give the N- alkylated diketopiperazine (such as 4 in Scheme 1 ). Removal of the t-butyl ester under acidic conditions (such as TFA in CH 2 C1 2 ) followed by coupling to a suitable diamine (such as 1,4-xylylene-diamine) under standard conditions (such as EDC, HOBt and iPr 2 NEt) in a suitable solvent (such as DMF) gives the protected, coupled product. Removal of the protecting groups under typical conditions (such as anhydrous HF) gives the final product (such as 5 in Scheme 1). Scheme 2 a) EDC, HOBt. iPr 2 NEt, CH CI 2 ; b) Lawesson's reagent, toluene; c) TFA, CH 2 CI 2 ; d) iPr 2 NEt, toluene; e) Ra-Ni, MeOH; f) o- dibromoxylene, NaH, THF Compounds of Formula (I) wherein A is tetrahydropyrrolopyrazinone are prepared by methods analogous to those in Scheme 2. Suitably protected amino acids (such as 1 and 2 in Scheme 2) are coupled using standard solution phase peptide synthesis methods (such as EDC, HOBt, iPr 2 NEt in CH2CI2) to give the protected dipeptide. The amide group is converted to the thioamide using conventional reagents (such as Lawesson's reagent) in a suitable solvent (such as toluene) to give the corresponding protected thioamide. Removal of the protecting group on nitrogen followed by a thermally assisted ring closure gives the cyclic thioamide (such as 3_ in Scheme 2). The thioamide is reduced using conventional reagents (such as Raney-Nickel) in a suitable polar solvent (such as MeOH) to give the tetrahydropyrrolopyrazinone (such as 4 in Scheme 2). Alkylating the sodium salt of this compound with one half an equivalent of a suitable diha de (such as o- dibromoxylene) gives the final product (such as 5 in Scheme 2) In order to use a compound of the Formula (I) 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) 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 earners 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 mateπal 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 earners, such as lactose or sorbitol, and filling the mixture into gelatin capsules. When a liquid earner 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 earner systems may also be used Alternately pharmaceutical compositions of the compounds of this invention, or denvatives thereof, may be formulated as solutions of lyophihzed powders for parenteral administration Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier pnor 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 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) are demonstrated by the following tests Induction of Hematopoietic Synergistic Activity in Stromal Cells The munne bone marrow denved 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 munne 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 factoi (HSF) activity is measured in a munne 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 cultuied with sub optimal levels of CFU plus dilutions of test C6 4 cell 30K-E supematants from above in a standard munne 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 mynstate acetate (PMA) ( 100- 200nM) or pre-opsonized (autologous sera) live C albicans (E T = 1 10) are quantitated in a microtiter femcytochrome c reduction assay The assay is performed in the presence of 1 % gelatin/HBSS and 80uM femcytochrome c in a total volume of 200uL/well The nmoles of cytochrome c reduced/well is calculated from spectrophotometnc 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 r3( .3'(5 r ).8a(y).8a'(S)1-NN-f l .4-xylenediyl)bisl2-( ' acetamido)-3- (hydroxymethyl)-6.7.8.8a-tetrahydropyrrolo[ 1.2-αlpyrazine- 1.4(2H. 3H)-dione [ a) [3(S), 8a(S)]-2-(tert-Butoxycarbonylmethyl)-3-(benzyloxymethyl)-6,7,8,8a- tetrahydropyrrolo[ 1 ,2-α]pyrazine- 1 ,4(2H, 3H)-dione A stirred suspension of NaΗ (0.120 g, 60% dispersion in oil, 3.0 mmol) in dry TΗF ( 15 mL) under argon was cooled to 0°C. A solution of compound Pro- Ser(Bzl) diketopiperazine (0.82 g, 3.0 mmol) in anhydrous DMF ( 10 mL) was added via syringe and the mixture was stirred for 20 min. t-Butyl 2-bromoacetate (0.54 mL, 3.3 mmol) was added, the cooling bath was removed and the reaction was stined for 5 h. Water ( 10 mL) was added to quench the reaction. The mixture was diluted with Η2O (50 mL) and sat NaCl (50 mL) and then extracted with CHCI (3 x 50 mL). The combined organic layers were washed with H 2 O (2 x 50 mL), dried over MgSO 4 , filtered and concentrated in vacuo to a yellow solid. Purification by flash chromatography (2/98 MeOH/ CHCI3, silica gel) gave 1.10 g (94%) of the desired product. MS (ESI) m/z 389.2 (MH + ). b) [3(5),3 , (5),8a( ),8a'(^]-N,N'-(l,4-Xylenediyl)bis[2-(acetamιdo)-3- (benzyloxymethyl)-6,7,8,8a-tetrahydropynolo[ 1 ,2-α]pyrazine- 1 ,4(2H, 3H)-dione] To a stined solution of the compound of Example 1 (a) ( 1.1 g, 2.8 mmol) in CΗ2CI2 (10 mL) was added neat TFA ( 10 mL). After 2 h, the mixture was concentrated in vacuo to an oil, then azeotroped 3 times with toluene to remove excess acid. This product was stored under high vacuum for 1 day to dry further and then used directly in the next step. The compound obtained above was dissolved in DMF (10 mL). 1,4- Xylylenediamine (0.14 g, 1.0 mmol), iPr 2 NEt (1.75 mL 10.0 mmol), HOBt (0.31 g, 2.3 mmol) and BOP reagent (1.02 g, 2.3 mmol) were added sequentially and the reaction was stirred at room temperature for 18 h. The reaction mixture was added to a rapidly-stirred mixture of EtOAc (100 mL), IN HCI (100 mL), and sat NaCl ( 100 mL). After stirring for 1 h, the phases were separated and the aqueous layer was extracted with fresh EtOAc (2 x 50 mL). The combined organic layers were washed with H2O (3 x 50 mL), dried over MgSU 4 , filtered and concentrated in vacuo to a yellow oil. Purification by flash chromatography (20/80 MeOH/EtOAc, silica gel) gave 234 mg (31%) of the desired product as a white solid. MS (ESI) m/z 765.4 (MH + ). c) [3( ),3'(5),8a(->),8a'(5)]-N,N , -(l,4-Xylenediyl)bis[2-(acetamido)-3- (hydroxymethyl)-6,7,8,8a-tetrahydropynolo[ 1 ,2-α]pyrazine- 1 ,4(2H, 3H)-dione] The compound of Example 1 (b) (26 mg, 45 μmol) was dissolved in p-cresol (0.5 mL) in a 50-mL Teflon ΗF reaction vessel fitted with a magnetic stirring bar. The vessel was chilled to -78°C and evacuated by water aspirator. Anhydrous ΗF (ca. 5 mL) was condensed into the mixture. The reaction mixture was warmed to 0°C and stirred for 45 min. The ΗF was then carefully removed under vacuum. The residue was taken up in Et2θ (25 mL) and was extracted with 0.1 % TFA in Η 2 O (v/v, 4 x 25 mL). The combined aqueous extracts were washed with Et 2 θ (3 x 25 mL) and then concentrated in vacuo to ca 10 mL. This solution was lyophilised to a white powder. Purification by HPLC [CH3CΝ H2O (0.1 % TFA, Hamilton PRP-1] gave 6.3 mg (24%) of the title compound as a white powder. MS (ESI) m/z 585.2 (MH+). EXAMPLE 2 r< , ). f5ι1-2.2 , -f l.2-xylenediynbisr6.7.8.8a-tetrahydropyπOlor i.2-αlpyrazine- 1.4(2H.3//-dione1 To a stirred suspension of NaH (88 mg, 60% dispersion in oil, 2.2 mmol) in anhydrous DMF (5 mL) under argon was added Pro-Gly diketopiperazine (0.34 g, 2.2 mmol) in one portion (slight amount of foaming occuned). After 20 min, a, α'- dibromo- o-xylene (0.26 g, 1.0 mmol) was added and the reaction was stirred for 4 h at room temperature. Water (2 mL) was added to quench the reaction. The mixture was diluted with H2O (10 mL) and sat NaCl (50 mL), then extracted with CHCI3 (4 x 50 mL). The combined organic layers were dried over MgSO 4 , filtered and concentrated in vacuo to a clear oil. Purification by flash chromatography (2/98 MeOH/ CHCI3, silica gel) afforded 289 mg (70%) of the title compound as a glassy solid. MS (ESI) m z 411.2 (MH + ). EXAMPLE 3 \(S).(S) 2.2'-( 1.2-xylenediynbisf 6.7.8.8a-tetrahydropyrτolof 1.2- lpyrazine- If 2H. 3H.4H)-onel a) N-(t-Butoxycarbonylaminothioacetyl)proline methyl ester To Boc-Gly-Pro-OMe (2.78 g, 9.72 mmol) in toluene (200 mL) was added Lawesson's reagent (2.36 g, 5.83 mmol). The mixture was heated to reflux for 2 h, then allowed to cool to RT and concentrated under vacuum to give a yellow residue. Flash chromatography (30 % EtOAc/hexanes, silica gel) gave 2.33 g (76 %) of the desired compound as a yellow oil. This was homogeneous by TLC and Η NMR analysis and was used without further purification. b) (S)-6,7,8,8a-tetrahydroprolyl[l,2-a]pyrazine-l-one-4(2H,3H)-thione To the compound of Example 3(a) (2.22 g, 7.35 mmol) in CΗ 2 C1 2 (35 mL) at 0 °C was added TFA (35 mL). The ice bath was removed and the solution was allowed to stir for 1 h. The solvent was removed under vacuum and the residue was azeotroped from toluene (3 x 20 mL). The residue was suspended in toluene (500 mL) and ι ' Pr NEt (3.84 mL, 22.0 mmol) was added. The reaction was heated to reflux for 18 h and then allowed to cool to RT. The reaction mixture was washed with IN HCL bπne (2 x 50 mL, 1.1 v/v) and the aqueous washes were back extracted with EtOAc (100 mL) The combined organic layers were concentrated to give 0.92 g (74 %) of the desired material as a yellow solid. This material was homogeneous by TLC and ^H NMR and was used without further punfication. c) (5)-6,7,8,8a-tetrahydroprolyl[l,2-fl]pyrazιne-l(2H,3H,4H)-one To the compound of Example 3(b) (0.92 g, 5.41 mmol) in MeOΗ ( 180 mL) was added Ra-Ni (ca 9 g of a 50 % aqueous slurry). The mixture was heated to reflux for 2 h, cooled to room temperature and filtered through celite to remove the Ra-Ni. The filter cake was washed with EtOΗ and the combined filtrates were concentrated under vacuum to give 0.65 g (86 %) of the desired material as pale yellow solid. This material was homogeneous by ^Η NMR analysis and was used without further punfication d) [(S),(S)]-2,2'-( 1 ,2-xylenediyl)bis[6,7,8,8a-tetrahydropyrrolo[ 1 ,2-α]pyrazine- 1(2H, 3H,4H)-one] To the compound of Example 3(c) (0.65 g, 4.64 mmol) and o- dibromoxylene (0.55 g, 2.08 mmol) in TΗF (10 mL) was added NaΗ (0.19 g of a 60 % dispersion in oil, 4.75 mmol) portionwise. The reaction was allowed to stir at RT for 24 h and then carefully poured into IN ΗC1 (75 mL). The aqueous phase was washed with hexane (20 mL) and Et 2 O (20 mL). The combined organic phases were back extracted with IN ΗC1 (20 mL) and the combined aqueous phases were concentrated under vacuum to give a yellow residue. Reverse phase flash chromatography (step gradient, 100 % Η 2 O + 0.1 % TFA; 5 % CH 3 CN/H 2 O + 0.1 % TFA; 10 % CH3CN/H 2 O + 0.1 % TFA; YMC reverse phase silica gel) gave 0.04 g of the title compound as a white solid. MS(ES+) m/z 383.1 (MH+). EXAMPLE 4 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/In redients Per Tablet 0.5 mg 20 mg 20 mg 20 mg 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 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): wherei Ri is independently hydrogen. NH2, OH, SH, CN, CO2H or hydrogen; R2 is independently hydrogen, C1.4 alkylC(O)R5, Cj_4 alkyl or R2 is benzyl which is optionally substituted by one or two C1.4 alkyl, Cj .4 alkoxy, F, C1, I, Br, OH, or N(R 4 ) 2 ; R3 is independently hydrogen, C1.5 alkyl, C\.ζ alkylenehydroxy, C l -5 alkyleneCO2H, (CH 2 )yN(R 4 )2, (CH 2 ) m C(O)N(R 4 ) 2 , C 1.5 alkyleneSH, CFbAr, (CH a ) NH C=NR 4 ; (R 4 ) 2 R4 is independently hydrogen, C 1.5 alkyl or benzyl; Ar is independently phenyl or indolyl optionally substituted by one or two R7 groups; Q is bιcyclo[3.3.0]octanyl, xylyl, benzophenonyl or 1,2,3,4- tetrahydronapthalyl; all of which are unsubstituted or substituted by one or two substituents chosen from. C 1.4 alkyl, Cj^alkoxy, halogen, mono or di C 1 . 4 alkylamιno, (C 1 . 4 alkyl) 2 -NC(O)-(CH 2 ) n -R 5 , -(CH 2 ) n -R 6 , -(CH 2 ) n -COR 5 or -(CH 2 ) n -COR6; R 5 is -OR 6 , -N(R 6 ) 2 , or -SR 6 ; Rg is hydrogen, C j _4 alkyl or benzyl; R7 is halogen, R5 or Rg; n is independently an integer from 0 to 3; m is independently an integer from 1 to 3; s is independently 0 or 1 ; and y is independently an integer from 2 to 4; provided that n is not 0 when s is 1 and further provided that the compound of Formula (I) is not: [(S),(S)]-N,N'-( 1 ,4-xylenedιyl)bιs[2-acetamιdo-6,7,8,8a-tetrahydropyrrolo[ 1 ,2- α]pyrazme-l,4(2H, 3H)-dιone], [(/?), ( ?)]-2,2'-(l,2-xylenediyl)bis[6,7,8,8a-tetrahydropyrrolo[l,2-a]pyrazine- l,4(2H,3H-dιone]; or [(S), (S)]-2,2'-( l ,3-xylenedιyl)bιs[6,7,8,8a-tetrahydropynolo[l,2-α]pyrazιne- l,4(2H,3H-dιone], or a pharmaceutically acceptable salt thereof 2. A compound of Formula I wherein R] is OΗ or Η; R3 is CΗ 2 OΗ or H; and Q is xylyl or bιcyclo[3.3 0]octanoyl. 3 A compound of Formula I wherein Ri , R 2 and R3 are hydrogen and Q is xylyl. 4 A compound of Formula I which is [3(S),3'(S),8a(5),8a'(5)]-N,N , -(l,4-xylenedιyl)bιs[2-(acetamιdo) 3- (hydroxymethyl)-6,7,8,8a-tetrahydropyrrolo[ 1 ,2-α]pyrazιne- 1 ,4( IH, 3H)-dιone] or {{S), (5)]-2,2'-(l,2-xylenedιyl)bιs[6,7,8,8a-tetrahydropyrrolo[l,2- α]pyrazιne- 1 ,4(2H,3H-dιone] [(-7),( )]-2,2'-(l,2-xylenedιyl)bιs[6,7,8,8a-tetrahydropyrrolo[l,2- α]ρyrazme-l(2H, 3H,4H)-one] 5 A method of preventing or treating viral, fungal or bacterial infections which compnses adrmnistenng to an animal in need thereof, an effective amount of a compound of claim 1 6 A method of stimulating the myelopoietic system which comprises adrmnistenng to an animal in need thereof, an effective amount to stimulate said myelopoietic system of a compound of claim 1 7 A method of preventing or treating sepsis which comprises administering to an animal in need thereof, an effective amount of a compound of claim 1 8 A process for preparing a compound of Formula (I) which compπses a) For compounds wherein A is tetrahydropyrrolopyrazmedione, coupling two equivalents of a compound of Formula (II) or two equivalents of a compound of Formula (IH) in which R j and R3 are as defined in Formula (I) or a group covertable thereto and Y is a suitable protecting group, with a suitable diarmne of Formula (IV)- HN(R 2 )-Q-(R 2 )NH (IV) in which R 2 and Q are as defined in Formula (I), with a coupling agent in a suitable solvent and removing any protecting groups, or b) For compounds where A is tetrahydropyrrolopyrazinone, alkylating a compound of Formula (V) wherein R] and R3 are as defined in Formula (I) or a group convertable thereto, with one half equivalent of a suitable diha de of Formula (VI) X-Q-X (VI) in which X is Cl, F or I and Q is defined in Formula (I), followed if necessary by I) converting R 1 and R3 , and or II) salt formauon

Inventors: BHATNAGAR PRADIP KUMAR, HEERDING DIRK ANDRIES, LOCASTRO STEPHEN MICHAEL, BHATNAGAR, PRADIP, KUMAR, HEERDING, DIRK, ANDRIES, LOCASTRO, STEPHEN, MICHAEL
Applicants: BHATNAGAR PRADIP KUMAR, HEERDING DIRK ANDRIES, LOCASTRO STEPHEN MICHAEL, SMITHKLINE BEECHAM CORP, BHATNAGAR, PRADIP, KUMAR, HEERDING, DIRK, ANDRIES, LOCASTRO, STEPHEN, MICHAEL, SMITHKLINE BEECHAM CORPORATION
Application year: 1996
Main classification: C07D487
IPC codes: A61K31, A61P7, A61P31, C07D487, C07D519