EP0639075A1 - Specific inhibition of dihydrofolate reductase and compounds therefor - Google Patents

Specific inhibition of dihydrofolate reductase and compounds therefor

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Publication number
EP0639075A1
EP0639075A1 EP92900837A EP92900837A EP0639075A1 EP 0639075 A1 EP0639075 A1 EP 0639075A1 EP 92900837 A EP92900837 A EP 92900837A EP 92900837 A EP92900837 A EP 92900837A EP 0639075 A1 EP0639075 A1 EP 0639075A1
Authority
EP
European Patent Office
Prior art keywords
pyrimidine
diamino
benzyl
methoxy
bromo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP92900837A
Other languages
German (de)
French (fr)
Other versions
EP0639075A4 (en
Inventor
Ivan Kompis
Jeffrey M. Blaney
Charles K. Marlow
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chiron Corp filed Critical Chiron Corp
Publication of EP0639075A4 publication Critical patent/EP0639075A4/en
Publication of EP0639075A1 publication Critical patent/EP0639075A1/en
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • This invention relates to pharmacology and the inhibition of enzymes specific to pathogens. More particularly, the invention relates to methods for spe ⁇ cifically inhibiting the enzyme dihydrofolate reductase in fungal pathogens, and compounds therefor.
  • PCP Pneumocystis carinii pneumonia
  • trimethoprim and pyrimethamine exhibit 50% inhibition concentrations (IC 50 ) of 8 and 2,500 nM for E. coli DHFR, while IC J QS for P. carinii DHFR are 39,600 and 2,400 nM, respectively.
  • R x is 3-R 3 -4-R 4 -5-R 5 -benzyl or (N-R ⁇ - ⁇ -azabicycloI ⁇ .lloct-S-yl;
  • R. and R 2 together form where R 3 .and Rj are
  • compositions for treating a fungal infection such as P. carin ⁇
  • a fungal infection such as P. carin ⁇
  • a mammal comprising an effective amount of a compound of formula I in combination with a pharmaceutically acceptable excipient.
  • Another aspect of the invention is the use of a compound of formula I to prepare a composition for treating a fungal infection (such as P. carin ⁇ ' ) in a mam ⁇ mal comprising an effective amount of a compound of formula I in combination with a pharmaceutically acceptable excipient
  • fungal infection and "fungal pathogen” refer to the infection of a mammal with an organism of the Kingdom Fungi, for example Pneumocystis carin ⁇ . Aspergillus, Candida, Fusarium, and the like.
  • the presently preferred method of the invention is the treatment of Pneumocystis carin ⁇ using the compounds of the invention.
  • pharmaceutically acceptable refers to compounds and compo ⁇ sitions which may be administered to mammals without undue toxicity.
  • Exem ⁇ plary pharmaceutically acceptable salts include mineral acid salts such as hydro- chlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • the term "effective .amount” refers to an amount of compound sufficient to exhibit a detectable therapeutic effect.
  • the therapeutic effect may include, for example, without ⁇ mitation, inhibiting the growth of pathogens, inhibiting or pre ⁇ venting the release of toxins by pathogens, killing pathogens, and preventing the estab ⁇ shment of infection (prophylaxis).
  • the precise effective amount for a sub ⁇ ject will depend upon the subject's size and health, the nature of the pathogen, the severity of the infection, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation based on the information provided herein.
  • lower alkyl refers to saturated straight or branched-chain rad ⁇ icals consisting of carbon and hydrogen having from 1 to 6 carbon atoms, inclu ⁇ sive, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-hexyl, and the like.
  • Lower alkoxy refers to a radical of the form R-O-, where "R" is lower .alkyl as defined above. Suitable examples include methoxy, ethoxy, prop- oxy, butoxy, .and the like.
  • lower alkylthio refers to radicals of the form R-S-
  • lower alkylsulfinyl refers to groups of the form R-S(-O)-.
  • methylthio ethylthio, methylsuhlnyl, t-butylsulfinyl, and the like.
  • alkenyl refers to straight or branched-chain radicals consist- ing of carbon and hydrogen having 2-6 carbon atoms and at least one double bond between a pair of carbon atoms, such as ethenyl (vinyl), 2-propenyl, 1-methyl- ethenyl, 2-butenyl, 3-butenyl, and the like.
  • carboxy-lower alkyl refers to radicals having the form -(CH 2 ) n -COOH, where n is an integer from 1 to 6 inclusive. "Dicarboxy-lower alkyl” indicates lower allcyl chains having two COOH groups attached.
  • Aryl denotes cyc ⁇ c hydrocarbon radicals of 6-10 carbon atoms which exhibit aromatic character, for example phenyl and napthyl.
  • halo refers to fluoro, chloro, bromo, and iodo.
  • amino acid refers to any of the 20 or so commonly occurring amino acids, for example, glycine, alanine, arginine, phenylalanine, glutamic acid, va ⁇ ne, histidine, proline, ornithine, norleucine. and the like.
  • R 8 When attached as R 8 in a radical of the form -O(CH 2 ) n -COR 8 , the amino acid will preferably be attached via a peptide bond, e ⁇ by a bond between the amino group of the amino acid and the acyl car ⁇ bon of the radical.
  • coadministering means administration of a compound of the invention in combination with a second therapeutic agent.
  • the second therapeutic agent is a dihydropteroate synthase inhibitor, preferably dapsone or a sulfa drug.
  • Suitable sulfa drugs include, without limitation, sulfadiame, sulfamethoxazole, and the like.
  • Coadministration may be simultaneous, for example by administer ⁇ ing a mixture of the therapeutic agents, or may be accomplished by administration of the agents separately within a short time period.
  • the compounds of the invention are structurally related to the compound trimethoprim (2,4-diam o-5-(3,4,5-trimemoxybenzyl)pyrimidine), the synthesis for which is known in the ait. See for example U.S. Pat No. 2,909,522, which des- cribes the synthesis of trimethoprim and related compounds.
  • Compounds of for ⁇ mula I may simflarly be synthesized by those of ordinary skill in the art. Syntheses of such compounds are described in the following U.S. patents: Hitchings ___ (2,658,897); Hitchings et_al.
  • Presently preferred compounds of the invention are: ,4-diamino-5-[3,5-dimemoxy-4-(2-hydroxyprop-2-yl)benzyl]pyrimidine; 2,4-diamino-5-(3 ,5-dimethoxy-4-N-pyrrolylbenzyl)pyrimidine; 2,4-diamino-5-(3,5-diethoxy-4-N-pyrrolylbenzyl)pyrimidine; 2,4-diar no-5-(3,5-dipropoxy-4-N-py ⁇ olylben ⁇ l)pyrimidine; 2,4-diammo-5-(3,5-dibutoxy-4-N-pyrrolylbenzyl)pyrimidine; 2,4-diammo-5-(3,5-diethoxy-4-carboethoxyber ⁇ zyl)pyrimidine; 2,4-diammo-5-(3,5-divinyl-4-vinyloxybenzyl)pyrimidine;
  • 2,2-dime yl-5-(2,4-dia ⁇ ninopyri ⁇ nidin-5-ylmethyl)-7-methoxybenz[b]dioxolane The most preferred compounds at present are 2,4-diamino-5-(3,5-diethoxy-4-N- pvrrolylbenzyl)pyrimidine and 2,4-diamino-5-[3 ⁇ -dimethoxy-4-(2-hydroxyprop-2- yl)benzyl]pyrimidine.
  • compositions of the invention for administration will generaUy include an effective amount of a compound of formula I in addition to a pharmaceutically acceptable excipient.
  • Suitable excipients include most carriers approved for oral or parenteral adininistration, including water, saline, Ringer's solution, H-ank's solution, and solutions of glucose, lactose, dextrose, ethanol, glycerol, albumin, and the like.
  • These compositions may optionally include stabilizers, antioxidants, antimicrobials, preservatives, buffering agents, surfactants, and other accessory additives.
  • a presently preferred vehicle comprises about 1 mg/mL serum albumin in phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • an effective dose of compound of formula I will range from about 10 ⁇ g/Kg to about 50 mg Kg.
  • Suitable ai ⁇ mal models include the mouse model illustrated in the Examples below. Rats and other rodents have DHFR very sim ⁇ ar to the human enzyme, and thus make suitable animal models.
  • a group of experimental animals is inoculated with 10-100 LD so s of Pneumocystis carin ⁇ , foUowed by treatment with a solution of test compound.
  • a negative con ⁇ trol group is left untreated, wh ⁇ e a positive control group is treated with a stan ⁇ dard therapy, such as trimethoprim.
  • Administration of the compounds is prefer- ably per os (e.g.. using a gavage), but may be parenteral, for example by subcu ⁇ taneous or intramuscular injection, or by inhalation of an aerosol.
  • the animals are monitored during treatment, and are sacrificed and examined after 60 days for presence of infection.
  • Buffers were prepared as follows: 4xDHFR buffe ⁇ 200 mM Tes, 300 mM BME, 4 mM EDTA, pH 7.0. +DHF buffe ⁇ 2.5 mg mL BSA, 0.25 mM NADPH, 62.5 uM dihydrofolate, 2.5x DHFR buffer. -DHF buffer: 2.5 mg/mL BSA, 0.25 mM NADPH, 2.5x DHFR buffer. Enzyme buffe ⁇ 50 mM Tes, 5 mM DTT, 1 mM EDTA, 20% glycerol, 1 mg/mL BSA, pH 7.0.
  • D ⁇ ution buffer 50 mM Tes, 5 mM DTT, 1 mM EDTA, 1 mg/mL BSA, pH 7.0.
  • PcDHFR 5 ⁇ g/mL P. carin ⁇ DHFR in enzyme buffer.
  • crude hDHFR crude recombinant human DHFR (obtained from Hoffmann-LaRoche) in enzyme buffer (9.9 mg mL total protein).
  • purified hDHFR purified recombinant human DHFR (obtained from Hoffmann- LaRoche) in enzyme buffer (3.5 mg/mL total protein).
  • Test compounds were prepared and provided by Hoffmann-L.aRoche. Stock solutions were prepared by dissolving 2-8 mg in dimethylsulfoxide (DMSO) to prepare 50 mM solutions. Compounds which did not dissolve at 50 mM were d ⁇ uted serially to 25, 16.6, or 12.5 mM.
  • DMSO dimethylsulfoxide
  • KK 2,4-dian_ ⁇ mo-5-(4,5,6-trimethoxy-2,3-dihydromd ⁇ n-l-yl)pyri ⁇ mdine; LL) 2,2-dimethyl-5-(2,4-diammopyrir din-5-ylmethyl)-7-methoxybenz[b]di- oxolane; MM) 2,4-diammcH5-(3,5-diethoxy-4-carboethoxybenzyl)pyrimidine; and NN) 2,4-diamino-5-(2,7-dimethylbenzpyrazol-5-y ethyl)pyrimidine. TABLE ⁇ :

Abstract

Compounds derived from pyrimidines having improved activity against fungi such as Pneumocystis carinii, and improved selectivity for P. carinii dihydrofolate reductase over human dihydrofolate reductase, are disclosed. P. carinii pneumonia is advantageously treated with the disclosed compounds.

Description

SPECIFIC INHIBITION OF DIHYDROFOLATE REDUCTASE AND COMPOUNDS THEREFOR
Description Technical Field This invention relates to pharmacology and the inhibition of enzymes specific to pathogens. More particularly, the invention relates to methods for spe¬ cifically inhibiting the enzyme dihydrofolate reductase in fungal pathogens, and compounds therefor.
Background of the Invention
Pneumocystis carinii pneumonia (PCP) is a significant, life-threatening infection in immunocompromised subjects, and is a leading cause of morbidity and mortality in patients presenting acquired immunodeficiency syndrome (AIDS). Since the onset of the ADDS epidemic, the incidence of PCP has risen from apprOaXimately 200 cases per year to more than 25,000 cases per year in the United States.
Due to the lack of a continuous in vitro culture system, and the cumber¬ some nature of the rat model of PCP, anti-P. carinii therapy has been developed largely on the assumption that P. carinii was a species of protozoa, and thus that anti-protozoal agents were likely to be effective. The two principle therapeutic mxx ities, trimemopriin/sulfamethoxazole and pentamidine, were developed empirically. However, P. carinii has recently been suggested to belong instead to the Kingdom Fungi (J.C. Edman et_ύ., Nature (1988) 334:519-22). Trimethoprim (U.S. Pat No. 2,909,522) and pyrimethamine, and other dihydrofolate reductase (DHFR) inhibitors are known to be effective anti-bacterial, and anti-protozo.al agents due to the central role played by DHFR in the metabolic synthesis of nucleic acid precursors. Despite their efficacy when used in con- junction with a sulfonamide, trimethoprim and pyrimethamine are alone poor inhibitors of P. carinii DHFR. For example, trimethoprim and pyrimethamine exhibit 50% inhibition concentrations (IC50) of 8 and 2,500 nM for E. coli DHFR, while ICJQS for P. carinii DHFR are 39,600 and 2,400 nM, respectively. Other anti-folates have been shown to be more effective inhibitors of P. carinii DHFR, but require concomitant administration of leucovorin to prevent toxicity to the host. Allegra et al. (U.S. Pat. No. 4,694,007) suggested treatment of P. carinii and Toxoplasmosis gondii with 2,4-diaιnino-5-memyl-6-[(3,4,5-trimethoxyanilino)- methyl]quinazoline (trimetrexate), on the theory that the DHFR enzyme in this pathogens is more similar to mammalian DHFR than to prokaryotic DHFR. Prior to the ADDS epidemic, these types of agents were sufficient for treatment of the rare cases of P. carinii pneumonia. However, in the HIV-positive patient, therapy and prophylaxis with the standard anti-P. carinii agents are com¬ plicated by frequent toxic and allergic side effects. New compounds that surpass the efficacy of the known antifolates in treating PCP are desirable, especially inhibitors having greater selectivity for P. carinii DHFR relative to the host (par¬ ticularly human) DHFR than known inhibitors such as trimethoprim.
Commonly-owned copending U.S. Patent Application Serial No. 447,181, filed 7 December 1989 disclosed several DHFR inhibitors exhibiting good selectivity for P. carinii DHFR. Disclosure of the Invention
We have now found compounds which exhibit superior activity against DHFR from fungal pathogens, such as P. carinϋ, and which exhibit much higher selectivity for the fungal enzyme as compared with the mammalian (human) enzyme. These compounds are represented generally by Formula I:
Formula I:
wherein
Rx is 3-R3-4-R4-5-R5-benzyl or (N-R^-δ-azabicycloI ^.lloct-S-yl; and
Rs is H; or R. and R2 together form where R3 .and Rj are
independently selected from the group consisting of H, lower alkoxy, lower alkylthio, lower alkylsulfinyl, vinyl, carboxy-lower .alkyl, carboxy-lower .alkoxy, dicarboxy-lower alkyl, dicarboxy-lower alkoxy, aryl-lower alkoxy, arylsulfonyl- lower alkoxy, arylsulfamido-lower alkoxy, and radicals of formula -O(CH2)n-COR8, where n is an integer from 0 to 6 and R8 is an amino acid; R4 is selected from the group consisting of lower alkoxy, aryl-lower alkoxy, lower allcylthio, halo, lower alkenyl, lower alkenyloxy, and pyrrolyl; with the proviso that R3, R4, and Rj are not simultaneously methoxy; Rδ is selected from the group consisting of unsubstituted aryl and aryl substituted with one to three radicals selected from the group consisting of halo, lower alkyl, lower alkoxy, lower alkyl¬ thio, carboxy, carbamido, carboxy-lower alkyl, and carbamido-lower alkyl; and R7 is selected from the group consisting of aryl and aryl-lower alkyl, where aryl may be substituted with one to three radicals selected from the group consisting of halo, lower alkyl, lower alkoxy, lower alkylthio, carboxy, c.arb.amido, carboxy- lower alkyl, and carbamido-lower allsyl; .and lower altyl esters, amides thereof, and pharmaceutically acceptable addition salts. One aspect of the invention is a method for treating a fungal infection
(such as P. carinii) in a mammal by administering an effective amount of a compound of formula I.
Another aspect of the invention is a composition for treating a fungal infection (such as P. carinϋ) in a mammal comprising an effective amount of a compound of formula I in combination with a pharmaceutically acceptable excipient.
Another aspect of the invention is the use of a compound of formula I to prepare a composition for treating a fungal infection (such as P. carinϋ') in a mam¬ mal comprising an effective amount of a compound of formula I in combination with a pharmaceutically acceptable excipient
Modes of Carrying Out The Invention A. Definitions
The terms "fungal infection" and "fungal pathogen" refer to the infection of a mammal with an organism of the Kingdom Fungi, for example Pneumocystis carinϋ. Aspergillus, Candida, Fusarium, and the like. The presently preferred method of the invention is the treatment of Pneumocystis carinϋ using the compounds of the invention. The term "pharmaceutically acceptable" refers to compounds and compo¬ sitions which may be administered to mammals without undue toxicity. Exem¬ plary pharmaceutically acceptable salts include mineral acid salts such as hydro- chlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
The term "effective .amount" refers to an amount of compound sufficient to exhibit a detectable therapeutic effect. The therapeutic effect may include, for example, without ϋmitation, inhibiting the growth of pathogens, inhibiting or pre¬ venting the release of toxins by pathogens, killing pathogens, and preventing the estabϋshment of infection (prophylaxis). The precise effective amount for a sub¬ ject will depend upon the subject's size and health, the nature of the pathogen, the severity of the infection, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation based on the information provided herein.
The term "lower alkyl" refers to saturated straight or branched-chain rad¬ icals consisting of carbon and hydrogen having from 1 to 6 carbon atoms, inclu¬ sive, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-hexyl, and the like. "Lower alkoxy" refers to a radical of the form R-O-, where "R" is lower .alkyl as defined above. Suitable examples include methoxy, ethoxy, prop- oxy, butoxy, .and the like. Simϋarly, "lower alkylthio" refers to radicals of the form R-S-, and "lower alkylsulfinyl" refers to groups of the form R-S(-O)-. For example, one may employ methylthio, ethylthio, methylsuhlnyl, t-butylsulfinyl, and the like. 'Lower alkenyl" refers to straight or branched-chain radicals consist- ing of carbon and hydrogen having 2-6 carbon atoms and at least one double bond between a pair of carbon atoms, such as ethenyl (vinyl), 2-propenyl, 1-methyl- ethenyl, 2-butenyl, 3-butenyl, and the like. The term "carboxy-lower alkyl" refers to radicals having the form -(CH2)n-COOH, where n is an integer from 1 to 6 inclusive. "Dicarboxy-lower alkyl" indicates lower allcyl chains having two COOH groups attached.
"Aryl" denotes cycϋc hydrocarbon radicals of 6-10 carbon atoms which exhibit aromatic character, for example phenyl and napthyl.
The term "halo" refers to fluoro, chloro, bromo, and iodo. The term "amino acid" refers to any of the 20 or so commonly occurring amino acids, for example, glycine, alanine, arginine, phenylalanine, glutamic acid, vaϋne, histidine, proline, ornithine, norleucine. and the like. When attached as R8 in a radical of the form -O(CH2)n-COR8, the amino acid will preferably be attached via a peptide bond, e^ by a bond between the amino group of the amino acid and the acyl car¬ bon of the radical.
The term "coadministering" means administration of a compound of the invention in combination with a second therapeutic agent. The second therapeutic agent is a dihydropteroate synthase inhibitor, preferably dapsone or a sulfa drug. Suitable sulfa drugs include, without limitation, sulfadiame, sulfamethoxazole, and the like. Coadministration may be simultaneous, for example by administer¬ ing a mixture of the therapeutic agents, or may be accomplished by administration of the agents separately within a short time period.
B. General Method
The compounds of the invention are structurally related to the compound trimethoprim (2,4-diam o-5-(3,4,5-trimemoxybenzyl)pyrimidine), the synthesis for which is known in the ait. See for example U.S. Pat No. 2,909,522, which des- cribes the synthesis of trimethoprim and related compounds. Compounds of for¬ mula I may simflarly be synthesized by those of ordinary skill in the art. Syntheses of such compounds are described in the following U.S. patents: Hitchings ___ (2,658,897); Hitchings et_al. (2,909,522); Hoffer (3,341,541); Roth (3,772,289); Roth _t_ύ. (3,819,629); Roth (3,822,264); Kompis e__ύ. (3,931,181); Herrling (3,980,649); Liebenow et al. (3.992,379V. Kompis (4,024,145); Rosen (4,033,962); Kompis et al. (4,039,543); Jemow et_al. (4,075,209); Perun et al. (4,087,528); Fritschi et al. (4,180,578); Kompis et al. (4,203,980); Poe et al. (4,258,045); Daluge ___ύ. (4,438,267); Dall'Asta (4,485,248); Kompis et al. (4,515.948); Swaringen et_al. (4,568,744); Roth et al. (4,587,341); Kompis et al. (4,590,270); Daluge et al. (4,603,136); Kompis et al. (4,792,557); and Seydel et_al. (4,912,112).
Presently preferred compounds of the invention .are: ,4-diamino-5-[3,5-dimemoxy-4-(2-hydroxyprop-2-yl)benzyl]pyrimidine; 2,4-diamino-5-(3 ,5-dimethoxy-4-N-pyrrolylbenzyl)pyrimidine; 2,4-diamino-5-(3,5-diethoxy-4-N-pyrrolylbenzyl)pyrimidine; 2,4-diar no-5-(3,5-dipropoxy-4-N-pyιτolylben^l)pyrimidine; 2,4-diammo-5-(3,5-dibutoxy-4-N-pyrrolylbenzyl)pyrimidine; 2,4-diammo-5-(3,5-diethoxy-4-carboethoxyberιzyl)pyrimidine; 2,4-diammo-5-(3,5-divinyl-4-vinyloxybenzyl)pyrimidine; ,4-diam cH5-[3-(4-N-acet.ammophenyl)sulfonam_inoethoxy-4,5-dimethoxybenzyl]- pyrimidine; ,4-diammcH5-[3-(4-aπ-inophenyl)sulfonaιmnoethoxy-4-methoxytenzyl]py_ ^ ,4-diarnmo-5-[3-(4-N-acetaminophenyl)sulfonaminoethoxy-4-bromo-5-methoxybenzyl]- pyrimidine; ,4-diammo-5-[3-(4-ammophenyl)su_fonaminoethoxy-4-bromo-5-methoxybenzyl]- pyrimidine; 2,4-diammo-5-(3-memoxy-4,5-dibenzyloxybenzyl)pyrimidine; 2,4-diammc 5-(4-ben^loxybenzyl)pyrirnidine; 2,4-di.amino-5-(3 ,4-dibenzyloxy-5-methoxybenzyl)pyrimidine; 2,4-diamino-5-(3,4-dimemoxy-5-benzyloxybenzyl)pyrimidine; 2,4-dian_u^o-5-[3,5-diethoxy-4-(prOpen-2-yl)benzyl]pyrimidine; 2,4-diamino-5-[3 ,5 -dimemoxy-4-(propen-2-yl)benzyl]pyrimidine; 2,4-diamincH5-(3,5-dimemoxy-4-memylthiobenzyl)pyrimidine; 2,4-diamino-5-(3-memylsulfmyl-4-memoxy-5-memyltMobenzyl)pyrimidine; 2,4-damino-5-[3-(4,6-dicarboxyhexyloxy)-4-bromo-5-methoxybenzyl]pyrimidine; 2,4-dian-ι_nc 5-[3-(3-carboxymethylamino-3-oxopropoxy)-4-methoxybenzyl]- pyrimidine; ,4-diammo-5-(3-[3-(l,3-dicarboxypropyl)amino-3-oxopropoxy]-4-bromo-5-methoxy- benzyl)pyrimidine; ,4-diammo-5-[3,5-dimemoxy-4-((2-phenylsulfeyl)acetyl)benzyl]pyrimidine; ,4-dia.rnmo-5-[3-ammcH4-methyl-5-(N-pyrrolyl)benzyl]pyrinϋdine; ,4-diam cH5-(3,5-di-N-pyrrolyl-4-memoxyben^l)pyrimidine; ,4-diammc 5-[3,5-di-me oxy-4-(3-hydrocarboxy-l-oxopropylammo)benzyl]pyrimidine; ,4-diamino-5-[3,5-dimethoxy-(4-acetammophenylsulfon.am o)benzyl]pyriιnidme; 2,4-diamino-5-(3,5-dimemoxy-4-propylbenzyl)pyrimidine; 2,4-diammo-5-(3,5-dic orc>-4-N-pyπOlylbenzyl)pyrimidine; ,4-diamino-5-[3,5-dimethoxy-4-(2-(2-(2-methoxy)ethoxy)ethoxy)ethoxybenzyl]- pyrimidine; ,4-diaιn c 5-[3-(3-benzyloxycarbonylmemylamino-3-oxopropoxy)-4-bromo-5-methoxy- benzyl]pyrimidine; ,4-diammo-5-[3-(3-carboxymemylamino-3-oxopropoxy)-4-bromo-5-methoxybenzyl]- pyrimidine; ,4-diammch5-[3-methoxy-4-bromo-5-(4-memylaminobenzamidoethoxy)benzyl]- pyrimidine; -(2,4-diaιmrtop3πimidin-5-ylmethyl)-8-(3,5-dimethoxyphenyl)-8-
.azabicyclo[3.2.1]octane; H,3H-dihydro-5-(2,4-diaιmnopy- nidin-5-y]methyl)-6,7-dimethoxybenzofuran; -(2,4-diarmnopyrimidin-5-ylmethyl)-7-methoxy-8-bromo-l,2-benzopyran; -(2,4-d.iammopyrimidin-5-ylmemyl)-7,8-dimethoxy-l,2-benzopyran; ,4-diammch5-[3-phenyl-5-(3-memoxypropoxy)benzyl]pvrimidine; ,4-diatnino-7-(3,5-dimethoxybenzyl)pyrrolo[2,3-f]quina2Oline; ,4-diamino-5-[6-(4-methoxybutoxy)naphth-l-yl]pyrimidine; 2,4-diam o-5-(2,7-dimethylbenzpyrazol-5-y-_memyl)pyrijnidine; ,4-diam ch5-(4,5,6-lτ_jnethoxy-2,3-dihydromden-l-yl)pyrirnidine; and
2,2-dime yl-5-(2,4-diaιninopyriιnidin-5-ylmethyl)-7-methoxybenz[b]dioxolane. The most preferred compounds at present are 2,4-diamino-5-(3,5-diethoxy-4-N- pvrrolylbenzyl)pyrimidine and 2,4-diamino-5-[3^-dimethoxy-4-(2-hydroxyprop-2- yl)benzyl]pyrimidine.
Compositions of the invention for administration will generaUy include an effective amount of a compound of formula I in addition to a pharmaceutically acceptable excipient. Suitable excipients include most carriers approved for oral or parenteral adininistration, including water, saline, Ringer's solution, H-ank's solution, and solutions of glucose, lactose, dextrose, ethanol, glycerol, albumin, and the like. These compositions may optionally include stabilizers, antioxidants, antimicrobials, preservatives, buffering agents, surfactants, and other accessory additives. A presently preferred vehicle comprises about 1 mg/mL serum albumin in phosphate-buffered saline (PBS). A thorough discussion of suitable vehicles for parenteral administration may be found in E.W. Martin, "Remington's Pharma¬ ceutical Sciences" (Mack Pub. Co., current edition).
The precise dosage necessary will vary with the age, size, and condition of the subject, the nature and severity of the disorder to be treated, and the like: thus, a precise, effective amount cannot be specified in advance. However, appro¬ priate amounts may be determined by routine experimentation with animal models. In general terms, an effective dose of compound of formula I will range from about 10 μg/Kg to about 50 mg Kg. Suitable aiύmal models include the mouse model illustrated in the Examples below. Rats and other rodents have DHFR very simϋar to the human enzyme, and thus make suitable animal models. A group of experimental animals is inoculated with 10-100 LDsos of Pneumocystis carinϋ, foUowed by treatment with a solution of test compound. A negative con¬ trol group is left untreated, whϋe a positive control group is treated with a stan¬ dard therapy, such as trimethoprim. Administration of the compounds is prefer- ably per os (e.g.. using a gavage), but may be parenteral, for example by subcu¬ taneous or intramuscular injection, or by inhalation of an aerosol. The animals are monitored during treatment, and are sacrificed and examined after 60 days for presence of infection.
C. Examples
The examples presented below are provided as a further guide to the practitioner of ordinary skill in the ait, and are not to be construed as limiting the invention in anv way.
Example 1 (Demonstration of Activity) A. Materials
Buffers were prepared as follows: 4xDHFR buffeπ 200 mM Tes, 300 mM BME, 4 mM EDTA, pH 7.0. +DHF buffeπ 2.5 mg mL BSA, 0.25 mM NADPH, 62.5 uM dihydrofolate, 2.5x DHFR buffer. -DHF buffer: 2.5 mg/mL BSA, 0.25 mM NADPH, 2.5x DHFR buffer. Enzyme buffeπ 50 mM Tes, 5 mM DTT, 1 mM EDTA, 20% glycerol, 1 mg/mL BSA, pH 7.0.
Dϋution buffer. 50 mM Tes, 5 mM DTT, 1 mM EDTA, 1 mg/mL BSA, pH 7.0. PcDHFR: 5 μg/mL P. carinϋ DHFR in enzyme buffer. crude hDHFR: crude recombinant human DHFR (obtained from Hoffmann-LaRoche) in enzyme buffer (9.9 mg mL total protein). purified hDHFR: purified recombinant human DHFR (obtained from Hoffmann- LaRoche) in enzyme buffer (3.5 mg/mL total protein). Test compounds were prepared and provided by Hoffmann-L.aRoche. Stock solutions were prepared by dissolving 2-8 mg in dimethylsulfoxide (DMSO) to prepare 50 mM solutions. Compounds which did not dissolve at 50 mM were dϋuted serially to 25, 16.6, or 12.5 mM. B. Assay
Eight compounds (6.7 μL each) were added to wells in one column of a Falcon® 96-weU microtiter plate, and dϋuted with 160 μL of water. The remaining weUs received 126 μL of water. The solutions in the first column were then seriaUy dϋuted (4x) into columns 2-9, with column 10 containing a control sample, and columns 11-12 containing water blanks. The final compound concentrations were 1 mM to 15 nM (after the remaining reagents were added). Using an 8-channel pipet, 100 μL of -DHF buffer were added to each weU in columns 11 .and 12 (blanks containing only water). Next, 100 μL of +DHF buffer were added to each weU in columns 1-10. PcDHFR or crude or purified rhDHFR enzyme was then added (25 μL) to each weU, and the plate contents mixed using a Titertek shaker. The plates were read on a Molecular Devices Plate Reader at an absorbance of 340 nm, -0.05 O.D. scale, for 10 min¬ utes reading every 10 seconds. The kinetic data was analyzed using Delta Soft software (Biometalϋcs), and the IC50 and Ki calculated for both human and carinii enzymes. Selectivity was calculated as Ki0l-,π,m Ki( a^), thus, higher values for selectivity indicate that the compound inhibits the P. carinϋ enzyme to a greater degree than the human enzyme. The results .are shown in Table 1 below. For purposes of comparison, trimethoprim in this assay exhibits an IC50 of 20 μM, with a selectivity of 0.1.
TABLE 1: Compounds of Formula I
R4 *5 IC50 Selectivity
Example 2
Proceeding as described in Example 1 above, the compounds ϋsted below were assayed and found to exhibit high activity .and selectivity, as set forth in Table II: A) 2,4-diammo-5-(4-benzyloxybenzyl)pyrimidine; B) 2,4-diamino-5-(3,4-dimellιoxy-5-benzyloxybenzyl)pyrimidine:
C) 2,4-diamino-5-(3 ,a4-dibenzyloxy-5-memoxybenzyl)pyrimidine;
D) 2,4-diammo-5-[3,5-dimethoxy-4-(2-hydroxyprop-2-yl)benzyl]pyri_rnidine;
E) 2,4-diamino-5-(3,5-dimethoxy-4-N-pyrrolylbenzyl)pyrimidine; F) 2,4-diaιnmo-5-(3,5-diethoxy-4-N-py]τolylbenzyl)pyrimidine;
G) 2,4-diaιnmo-5-(3,5-divmyl-4-vmyloxybenzyl)pyriιnidine;
H) 2,4-diammo-5-[3-(4-N-acetammophenyl)sulfonaminoethoxy-4,5-dimeth- oxybenzyljpyrimidine; I) 2,4-diammo-5-[3-(4-am ophenyl)sulfonaminoethoxy-4-methoxybenzyl]- pyrimidine;
J) 2,4-di.am o-5-[3-(4-N-acetan-inophenyl)su]fonaιninoethoxy-4-bromo-5- methoxybenzyljpyrimidine; K) 2,4-diaιnmcH5-[3-(4-aminophenyl)sulfonaminoethoxy-4-bromo-5-methoxy- benzyljpyrimidine; L) 2,4-diamino-5-[3,5-diethoxy-4-(propen-2-yl)benzyl]pyrimidine;
M) 2,4-diammo-5-[3,5-dimemoxy-4-(propen-2-yl)benzyl]pyrimidine; N) 2,4-diamino-5-(3 ,5-dimemoxy-4-methylttøotenzyl)pyrimidine;
O) 2,4-diammo-5-(3-memylsu]finyl-4-methoxy-5-methylthiobenzyl)- pyrimidine; P) 2,4-diamino-5-[3-(4,6-dic^boxyhexyloxy)-4-bromo-5-methoxybenzyl]- pyrimidine; Q) 2,4-diaιnino-5-[3-(3-carboxymethylamino-3-oxopropoxy)-4-methoxy- benzyljpyrimidine; R) 2,4-diamino-5-(3-[3-(l ,3-dicarboxypropyl)am o-3-oxopropoxy_|-4-bromo- 5-memoxybenzyl)pyrimidine;
S) 2,4-diamino-5-[3,5-dimethoxy-4-((2-phenylsulfonyl)acetyl)benzyl]- pyrimidine; T) 2,4-diammo-5-[3-am o-4-methyl-5-(N-pyrrolyl)benzyl]pyrimidine;
U) 2,4-diammch5-(3,5-di-N-pyrrolyl-4-memoxybenzyl)pyrimidine; V) 2,4-diamino-5-[3,5-di-memoxy-4-(3-hydrc)carboxy-l-oxopropylamino)- benzyl]pyrirnidine; W) 2,4-diammo-5-[3,5-dimemoxy-(4-acetam ophenylsu3fonamino)benzyl]- pyrimidine; X) 2,4-diammcH5-(3,5-dimemoxy-4-propylbenzyl)pyrimidine;
Y) 2,4-diamino-5-(3,5-dicMoro-4-N-pyrrolylbenzyl)py-±nidine;
Z) 2,4-diamino-5-[3,5-dimethoxy-4-(2-(2-(2-methoxy)ethoxy)ethoxy)ethoxy- benzyljpyrimidine; AA) 2,4-diam c^5-[3-(3-beiizyloxycarbonylmethylamino-3-oxopropoxy)-4- bromo-5-methoxybenzyl]pyrimidine;
BB) 2,4-diaιnino-5-[3-(3-carboxymethylamino-3-oxopropoxy)-4-bromo-5- methoxybenzyl]pyrimidine; CC) 2,4-diammo-5-[3-methoxy-4-bromo-5-(4-methylammobenz.amidoethoxy)- benzyl]pyrimidine; , DD) 3-(2,4-d. mmopyrimidin-5-ylmethyl)-8-(3,5-dimethoxyphenyl)-8-aza- bicyclo[3.2.1]octane; EE) 2H,3H-dihydrc 5-(2,4-diammopyrir din-5-ylmethyl)-6,7-dimethoxybenzo- furan; FF) 5-(2,4-diammopyrimidin-5-ylmethyl)-7-methoxy-8-bromo-1.2-benzopyran; GG) 5-(2,4-diam opyrimidin-5-ylmethyl)-7,8-dimethoxy-l,2-benzopyran; HH) 2,4-diammo-5-[3-phenyl-5-(3-methoxypropoxy)benzyl]pyrimidine; II) 2,4-diammo-7-(3,5-dimethoxybenzyl)pyrrolo[2,3-flquinazoϋne;
JJ) 2,4-diamino-5-[6-(4-methoxybutoxy)naphth-l-yl]pyrimidine;
KK) 2,4-dian_ιmo-5-(4,5,6-trimethoxy-2,3-dihydromdεn-l-yl)pyriιmdine; LL) 2,2-dimethyl-5-(2,4-diammopyrir din-5-ylmethyl)-7-methoxybenz[b]di- oxolane; MM) 2,4-diammcH5-(3,5-diethoxy-4-carboethoxybenzyl)pyrimidine; and NN) 2,4-diamino-5-(2,7-dimethylbenzpyrazol-5-y ethyl)pyrimidine. TABLEΠ:
TABLE II (continued) :

Claims

C 1 5- 17 -What is Claimed:
1. A method for treating fungal infection in a mammal, which method comprises: 5 administering to a mammal infected with a fungal pathogen an effective amount of a compound of formula I:
Rl
wherein 20 Rι is 3-R3-4-R4-5-R5-benzyl or (N-R6)-8-azabicyclo[3.2.1]oct-3-yl;
and R2 is H; or Rj and R2 together form
where R3 .and j are independently selected from the group consist¬ ing of H, lower alkoxy, lower allcylthio, lower alkylsulfinyl, vinyl, carboxy-lower alkyl, carboxy-lower alkoxy, dicarboxy-lower alkyl, dicarboxy-lower alkoxy, aryl- 25 lower alkoxy, arylsulfonyl-lower alkoxy, aiylsulfamido-lower .alkoxy, and radicals of formula -O(CH2)n-COR8, where n is an integer from 0 to 6 .and R8 is an amino acid; R4 is selected from the group consisting of lower alkoxy, aryl-lower alkoxy, lower alkylthio, halo, lower alkenyl, lower alkenyloxy, and pyrrolyl; with the proviso that R3, R4, and R5 are not simultaneously methoxy;
R^ is selected from the group consisting of unsubstituted .aryl and aryl substituted with one to three radicals selected from the group consisting of halo, lower alkyl, lower alkoxy, lower alkylthio, caΛoxy, carbamido, carboxy- lower alkyl, and carbamido-lower alkyl; and
R7 is selected from the group consisting of aryl and aryl-lower alkyl, where aryl may be substituted with one to three radicals selected from the group consisting of halo, lower alkyl, lower .aϋcoxy, lower alkylthio, carboxy, carbamido, carboxy-lower alkyl, and carbamido-lower alkyl; and lower alkyl esters, amides thereof, and pharmaceuticaϋy acceptable addition salts.
2. The method of claim 1, wherein R2 is H and Rt is
3-R3-4-R4-5-R5-benzyl.
3. The method of claim 2, wherein R3 is methoxy.
4. The method of claim 3, wherein R4 is bromo.
5. The method of claim 4, wherein Rj is
-O(CH2)2CH(COOH)(CH2)2COOH, or a pharmaceuticaUy acceptable mono- or diester thereof.
6. The method of claim 4, wherein R5 is -O(CH2)n-COR8.
7. The method of claim 6, wherein n is 2 and R8 is -NHCH(COOH)CH2CH,COOH or a mono- or di-ester thereof.
8. The method of claim 6, wherein n is 2 and R8 is -NHCH2COOH or an ester thereof.
9. The method of claim 3 wherein R5 is methoxy. 5
10. The method of claim 9, wherein R4 is 2-hydroxyprop-2-yl.
11. The method of claim 9, wherein R4 is propen-2-yl.
10 12. The method of claim 9, wherein R4 is N-pyrrolyl.
13. The method of clam 9, wherein R4 is methylthio.
14. The method of claim 3, wherein R4 is benzyloxy.
15
15. The method of claim 14, wherein R5 is benzyloxy.
16. The method of claim 3, wherein R4 is methoxy, and Rj is -OCH2CH2NHSO2(C6H6)NHCOCH3.
20
17. The method of claim 2 wherein R3 is H.
18. The method of claim 9 wherein R4 is methoxy.
25 19. The method of claim 10 wherein Rj is -O(CH2)n-COR8, where n is 2 and R8 is -NHCH2COOH or an ester thereof.
%
20. The method of claim 9, wherein R4 is benzyloxy and R^ is H.
21. The method of claim 2, where R3 and R5 are each ethoxy.
22. The method of claim 21, wherein R4 is pyrrolyl.
23. The method of claim 21, wherein R4 is propen-2-yl.
24. The method of claim 2, where R3 and Rj are each vinyl.
25. The method of claim 2, where R3 is methylthio, R4 is methoxy, and Rs is methylsulfinyl.
26. The method of claim 1, wherein R2 is H and Ri is (N-Rs)- 8-azabicyclo[3.2.1]oct-3-yl.
27. The method of claim 26, wherein Rg is selected from the group consisting of 2-naphthyl, 3,5-dimethoxyphenyl, and 4-carboethoxyphenyl.
28. The method of claim 1, wherein Rx and R2 together form
29. The method of claim 28, wherein R7 is benzyl.
30. The method of claim 28 where R7 is 3,5-dimethoxyphenyl.
31. The method of claim 2 where R3 .and Rj are propoxy and R4 is N-pyrrolyl.
32. The method of claim 1 wherein said fungal pathogen is Pneumocystis carinϋ.
33. The method of claim 1, further comprising coadminstering a dihydropteroate synthase inhibitor.
34. The method of claim 33, wherein said dihydropteroate synthase inhibitor is selected from the group consisting of dapsone and sulfa drugs.
35. The method of claim 1, wherein said compound of formula I is selected from the group consisting of 2,4-di.amino-5-(4-benzyloxy- benzyl)pyrimidine; 2,4-diamino-5-(3,4-dimethoxy-5-benzyloxybenzyl)pyrimidine; 2,4-diamino-5-(3,4-dibenzyloxy-5-methoxybenzyl)pyrimidine; 2,4-diamino-5-[3,5- dimethoxy-4-(2-hydroxyprop-2-yl)benzyl]pyrimidine; 2,4-diamino-5-(3,5- dimemoxy-4-N-pyrrolylbenzyl)pyrirnidine; 2,4-diamino-5-(3 ,5-diethoxy-4-N- pyι olylbenzyl)pyrimidine; 2,4-diaamino-5-(3,5-divinyl-4-vinyloxybenzyl)- pyrimidine; 2,4-di.ammo-5-[3-(a4-N-acetammophenyl)sulfonaminoethoxy-4,5-di- memoxybenzyljpyrirnidine; 2,4-diaι nc 5-[3-(4-am ophenyl)suffonaminoethoxy- 4-memoxybenzyl]pyrimidine; 2,4-diamino-5-[3-(4-N- acetaιninophenyl)su_fonammc>ethoxy-4-bromo-5-memoxybenzyl]pyrimidine; 2,4-di- ammo-5-[3-(4-ammophenyl)sulfonaminoethoxy-4-bromo-5-methoxybenzyl]- pyrimidine; 2,4-diammo-5-[3,5-diethoxy-4-(propen-2-yl)benzyl]pyrimidine; 2,4- diaιnino-5-[3,5-dimethoxy-4-(propen-2-yl)benzyl]pyrimidine; 2,4-diamino-5-(3,5- dimethoxy-4-methylt obenzyl)pyrimidine; 2,4-diamino-5-(3-methylsul^yl-4- memoxy-5-methyllMobenzyl)pyrimidine; 2,4-diamino-5-[3-(4,6- dicarboxyhexyloxy)-4-bromo-5-memoxyberιzyl]pyrimidme; 2,4-diamino-5-[3-(3- carboxymemylammo-3-oxopropoxy)-4-memoxyberizyl]pyrimidine; 2,4-diamino-5- (3-[3-(l,3-dic^boxypropyl)amino-3-oxopropoxy]-4-bromo-5-methoxybenzyl)- pyrimidine; 2,4-diamino-5-[3 ,5-dimethoxy-4-((2-phenylsulfonyl)acetyl)benzyl]- pyrimidine; 2,-4-diaιn o-5-[3-ammo-4-memyl-5-(N-pyιrolyl)ben2tyl]pyrimidine; 2,4-dian__ino-5-(3,5-di-N-pyrrolyl-4-methoxyber_zyl)pyrinϋdm 2,4-diamino-5-[3,5- di-memoxy-4-(3-hydrocarboxy-l-oxopropylam o)benzyl]pyrimidine; 2,4-diamino- 5-[3,5-dimethoxy-(4-acetaminophenylsulfonammo)benzyl]pyrimidine; 2,4-diamino- 5-(3,5-dimethoxy-4-propylbenzyl)pyrimidine; 2,4-diammo-5-(3,5-dichloro-4-N- pyrrolylben2yl)pyrimidine; 2,4-di.amino-5-[3 ,5-dimethoxy-4-(2-(2-(2-methoxy)- ethoxy)ethoxy)ethoxybenzyl]pyrimidine; 2,4-diamino-5-[3-(3-benzyloxycarbonyl- memylammo-3-oxopropoxy)-4-bromo-5-memoxyber_zyl]pyrimidine; 2,4-diamino-5- [3-(3-cMboxymemylamino-3-oxopropoxy)-4-bromo-5-methoxybenzyl]pyrimidine; 2,4-diamino-5-[3-memoxy-4-bromo-5-(4-methylammobenzamidoethoxy)benzyl]pyr- imidine; 3-(2,4-diam opyrimidin-5-ylmethyl)-8-(3,5-dimethoxyphenyl)-8-aza- bicyclo[3.2.1]octane; 2H,3H-dihydrch5-(2,4-diaιninopyrimidin-5-ylmethyl)-6,7-di- methoxybenzofuran; 5-(2,4-dian- nopyrimidin-5-ylmethyl)-7-methoxy-8-bromo- 1 ,2- benzopyran; 5-(2,4-diaιninopyrimidm-5-ylmethyl)-7,8-drmethoxy-l ,2-benzopyran; 2,4-diaιn o-5-[3-phenyl-5-(3-metiιoxypropoxy)benzyl]pyrimidine; 2,4-diamino-7- (3,5-dimethoxybenzyl)pyrrolo[2,3-f]quinazoline; 2,4-diamino-5-[6-(4-methoxy- butoxy)naphth-l-yl]pyrimidine; 2,4-diamino-5-(4,5,6-trimethoxy-2,3-dihydroinden- l-yl)pyrimidine; 2,2-dιimemyl-5-(2,4-diaminopyrinϋdin-5-ylmethyl)-7-methoxy- benz[b]dioxolane; 2,4-diammo-5-(3,5-diethoxy-4-carboethoxybenzyl)pyrimidine; ^d 2,4-diamino-5-(2,7-dimethylbenzpyrazol-5-ylmethyl)pyrimidine.
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PT99514A (en) 1992-10-30
AU657348B2 (en) 1995-03-09
PT99514B (en) 1999-04-30
AU2202792A (en) 1994-04-28
IE913974A1 (en) 1992-05-20
CA2095518A1 (en) 1992-05-15
JPH07509215A (en) 1995-10-12
EP0639075A4 (en) 1993-09-29
WO1992008461A1 (en) 1992-05-29

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