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WO1989011279A1 - Nucleic acid interacting unfused heteropolycyclic compounds - Google Patents

Nucleic acid interacting unfused heteropolycyclic compounds Download PDF

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Publication number
WO1989011279A1
WO1989011279A1 PCT/US1989/002104 US8902104W WO8911279A1 WO 1989011279 A1 WO1989011279 A1 WO 1989011279A1 US 8902104 W US8902104 W US 8902104W WO 8911279 A1 WO8911279 A1 WO 8911279A1
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Prior art keywords
substituted
aromatic
general formula
bis
nhx
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PCT/US1989/002104
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French (fr)
Inventor
Raymond F. Schinazi
Lucjan Strekowski
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Georgia State University Foundation, Inc.
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Publication of WO1989011279A1 publication Critical patent/WO1989011279A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/26—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • 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
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
    • C07D213/32—Sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/02—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings
    • C07D237/06—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D237/08—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C07D237/30—Phthalazines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
    • C07D239/38—One sulfur atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/78—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the present invention relates to unfused heteropolycyclic compounds having biological activity due to their ability to interact with nucleic acids.
  • a variety of compounds have antiviral or cytotoxic activity due to interaction with DNA or RNA which interferes with translation of the genes into the encoded proteins.
  • Much research is presently being directed at compounds which interact with nucleic acids as a means for treating or preventing the disease commonly known as Acguired Immunodeficiency Syndrome, or "AIDS”.
  • HIV-1 human immunodeficiency virus
  • HIV-2 the two related but distinct retroviruses termed human immunodeficiency virus HIV-1 and HIV-2. HIV-1 is the predominant cause of AIDS in the United States. Although the genomes are only about 50% homologous at the nucleotide level, the viruses contain the same complement of genes and appear to attack and kill the same human cells by the same mechanisms. Antibodies to HIV-1 are present in over 80% of patients diagnosed as having AIDS or pre-AIDS syndrome, and have been found with high frequency in the identified risk groups.
  • AIDS is known to develop in at least 10% of the individuals infected with HIV, although this percentage is suspected to be much higher.
  • a patient is generally diagnosed as having AIDS when a previously healthy adult with an intact immune system acquires impaired T-cell immunity.
  • the impaired immunity usually appears over a period of eighteen months to three years.
  • the patient becomes susceptible to opportunistic infections, various types of cancer such as Kaposi's sarcoma, and other disorders associated with reduced functioning of the immune system.
  • Another condition associated with HIV is AIDS-related complex, or ARC. This condition is thought to lead eventually to AIDS.
  • AZT appears to be the drug of choice at this time. However, AZT exhibits toxicity in a clinical setting. See Yarchoan et al.. Lancet 575-580 (1986) and Richman, et al., N.E.J.Med. 317(4),192-197 (1987). AZT was originally synthesized by Horwitz et al., J. Pro. Chem. 29, 2076-2078, 1974. The effectiveness of AZT against HIV is described in U.S. Patent No.
  • the compounds of the present invention are derivatized unfused heteropolycyclic compounds, primarily heterotricyclic compounds.
  • diazines are derivatized by replacing halogen atoms adjacent to the diazine ring nitrogens with one of a variety of common nucleophiles such as hydroxide ion, alkoxides, mercaptides, and amines.
  • Pyridine derivatives are made by reacting 2,6-di-p-tolylpyridine with N-bromo succinimide, to form 2,6-bis[4'-(bromomethyl)phenyl]pyridine.
  • the benzylic bromine atoms are also replaced with a variety of common nucleophiles. These compounds interact with nucleic acids, thereby inhibiting translation and interfering with viral replication processes.
  • Compounds which are useful are those having a therapeutic index of greater or equal to 10, toxicity at levels of greater than or equal to 100 ⁇ M, and antiviral activity at concentrations of less than or equal to 10 ⁇ M.
  • the preferred compounds at this time are 2,6-bis[4'-[(dimethylamino)methyl]phenyl]pyridine (DH-23);
  • DH-42 bis-4,6-[4'-[[2"-(dimethylamino)ethyl]thio]phenyl] pyrimidine (LS-20); 1-methyl-4-[2'-[[4"-(naphth-2"'-yl)quinazolin-2"-yl]thio] ethyl]piperazine (M-69);
  • heteropolycyclic compounds of the present invention are produced using the method described in U.S. Serial Number 153,998 filed February 9, 1988 by Lucjan Strekowski, et al., entitled “Novel Diazines and Their Method of Preparation”.
  • Substituted halogenodiazines are used as intermediates in the synthesis of the unfused heteropolycyclic compounds containing a diazine and other aromatic moieties, such as thiophene, benzene, naphthalene, furan, pyridine or pyrrole, or a substituted aromatic. These compounds are then evaluated for antiviral activity, cytotoxicity, and toxicity in animals.
  • the method to produce the substituted halogenodiazines which are useful as intermediates in the synthesis of the novel unfused heteropolycyclic compounds, and the products thereof, is easy, efficient and results in a high yield of product.
  • the reaction consists of the addition of an organolithium reagent to a halogenodiazine with subsequent dehydrogenation of the addition product.
  • the entire reaction takes place in one reaction vessel, without isolation of the substituted halogenodihydrodiazine intermediate.
  • the reactions proceed at moderate temperature and in a short period of time, which decreases side reactions and increases yield.
  • the recovery is conducted under two-phase conditions thereby minimizing hydrolysis of the substituted halogenodiazine to a substituted hydroxydiazine.
  • the general reaction scheme in which substituted halogenodiazines are converted into unfused heteropolyaromatic compounds that may have biological application is illustrated as follows:
  • the chloro derivative was treated with a 50-fold excess of the appropriate amine.
  • the mixture was heated at 70-80 oC for one hour and the amine recovered by distillation.
  • the oily residue was treated with 5% NaOH and extracted with ether.
  • the ether solution was dried over Na 2 SO 4 , evaporated, and the product purified by flash chromatography on SiO 2 . Further purification was done via crystallization from a toluene-hexane mixture.
  • Pyridine derivatives can be made from readily available 2,6-di-p-tolylpyridine.
  • This compound is brominated with N-bromosuccinimide in a carbon tetrachloride solution under reflux conditions to give 2,6-bis[4'-(bromomethyl)phenyl]pyridine.
  • the benzylic bromine atoms in this product are readily replaced with a variety of nucleophiles such as N-methylpiperazine, dimethylamine, or 2-(dimethylamino)ethanethiolate ion to furnish final compounds.
  • Examples of the general formulas for the new classes of unfused heteropolyaromatic compounds include: A substituted phthalazine of the general formula
  • R 1 is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group and
  • R 2 is an aromatic or substituted aromatic group.
  • Aromatic groups include furanyl, thienyl, phenyl, naphthyl, and pyridyl.
  • aromatic groups can be substituted with alkoxy, alkylthio, dialkylamino, and hydrocarbon groups. If the substitution contains an -OH or -SH, the quantity and nature of the organometallic reagent should be adjusted in the preparation of these derivatives as necessary for reaction with the -OH or -SH. Examples are described in Table I.
  • R 1 is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group and
  • R 2 is any aromatic or substituted aromatic.
  • R 1 is any aromatic or substituted aromatic
  • R 2 is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group. Examples are described in Table II.
  • R is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group.
  • R 1 is any aromatic or substituted aromatic
  • R 2 is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group. Examples are described in Table IV.
  • R is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group. Examples are described in Table V.
  • R is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group.
  • Table VI An example is described in Table VI.
  • R 1 is aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic and
  • R 2 is H or alkyl. Examples are described in Table VIII.
  • R 1 and R 2 are -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group and R 3 is aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic groups.
  • Table IX An example is described in Table IX.
  • R 1 and R 2 are aromatic or substituted aromatic groups and R 3 is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group.
  • R 3 is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group.
  • R 1 and R 2 are an aromatic or substituted aromatic group
  • R 3 is H or an alkyl group. Examples are described in Tables XI and XII. Screening for Antiviral Activity and Toxicity.
  • antiviral activity refers to the ability of a composition to inhibit the growth of HIV.
  • the claimed composition also exhibits antiviral activity towards other retroviruses.
  • the ability of the present compositions to inhibit HIV may be measured by various experimental techniques. One such technique involves the inhibition of viral replication in human peripheral blood mononuclear cells. The amount of virus produced is determined by measuring the virus-coded reverse transcriptase (an enzyme found in retroviruses).
  • PBM Human Peripheral Blood Mononuclear
  • PHA phytohemagglutinin
  • HIV-1 (strain LAV-1) was obtained from the Center for Disease Control, Atlanta, and propagated in PHA-stimulated human PBM cells using RPMI 1640 medium as above without PHA and supplemented with 7% interleuken-2 (Advanced Biotechnologies, Inc., Silver Spring, MD), 7 ⁇ g/ml DEAE-dextran (Pharmacia, Uppsala, Sweden), and 370 U/ml anti-human leukocyte (alpha) interferon (ICN, Lisle, IL). Virus was obtained from cell-free culture supernatant and stored in aliquots at -70°C until used.
  • Uninfected PHA-stimulated human PBM cells were uniformly distributed among 25 cm 2 flasks to give a 5 ml suspension containing about 2 ⁇ 10 6 cells/ml. Suitable dilutions of HIV were added to infect the cultures.
  • the mean reverse transcriptase (RT) activity of the inocula was 50,000 dpm/ml which was equivalent to about 100 TCID 50 , determined as described in AIDS Res.Human Retro.3.71-85 (1987).
  • the drugs at twice their final concentrations in 5 ml of RPMI 1640 medium, supplemented as described above, were added to the cultures.
  • Uninfected and untreated PBM cells were grown in parallel as controls. The cultures were maintained in a humidified 5% CO 2 -95% air incubator at 37oC for five days after infection at which point all cultures were sampled for supernatant RT activity. Preliminary studies had indicated that maximum RT levels are obtained at that time.
  • RT activity assay Six ml of supernatant from each culture was clarified from cells at 300 x g for 10 minutes. Virus particles were then pelleted from 5 ml samples at 40,000 rpm for 30 minutes using a Beckman 70.1 Ti rotor and suspended in 200 ⁇ l of virus disrupting buffer (50 mM Tris-HCl, pH 7.8, 800 mM NaCl, 20% glycerol, 0.5 mM PMSF, and 0.5% Triton X-100).
  • virus disrupting buffer 50 mM Tris-HCl, pH 7.8, 800 mM NaCl, 20% glycerol, 0.5 mM PMSF, and 0.5% Triton X-100.
  • the RT assay was performed by a modification of the method of Spira, et al, in J. Clin.Microbiol. 25,97-99 (1987) in 96-well microtiter plates.
  • the radioactive cocktail (180 ⁇ l) which contained 50 mM Tris-HCl pH 7.8, 9 mM MgCl 2 , 5 mM dithiothreitol 4.7 ⁇ g/ml Poly (rA) n ⁇ (dT) 12-18 , 140 ⁇ M dATP and 0.22 ⁇ M[ 3 H]dTTP (specific activity 78.0 Ci/mmol, equivalent to 17,300 cpm/pmol; NEN Research Products, Boston, MA) was added to each well.
  • the sample (20 ⁇ l) was added to the reaction mixture and incubated at 37°C for two hours.
  • the reaction was terminated by the addition of 100 ⁇ l 10% trichloroacetic acid (TCA) containing 0.45 mM sodium pyrophosphate.
  • TCA trichloroacetic acid
  • the acid insoluble nucleic acid which precipitated was collected on glass filters using a Skatron semi-automatic harvester (setting 9) .
  • the filters were washed with 5% TCA and 70% ethanol, dried, and placed in scintillation vials.
  • Four ml of scintillation fluid (Econofluor, NEN Research Products, Boston MA) was added and the amount of radioactivity in each sample determined using a
  • the drugs were evaluated for their potential toxic effects on uninfected PHA-stimulated human PBM cells. Flasks were seeded so that the final cell concentration was 3 ⁇ 10 5 cells/ml. The cells were cultured with and without drug for 6 days at which time aliquots were counted for cell viability.
  • EC 50 is the median effective concentration of the compound as determined from the reverse transcriptase assay. Although these compounds exhibit reduced toxicity to normal cells, administration of a high concentration, a dosage which would result in a blood serum concentration of approximately 100 ⁇ M or higher, of such a drug would nevertheless produce some adverse side effects. Thus, compositions having a high concentration of the active ingredient are not considered to be therapeutically effective.
  • the therapeutic index of a compound is determined by dividing the inhibitory or lethal dose for 50% of the population (IC 50 or LD S0 ) by the effective dose for 50% of the population (EC 50 ).
  • the EC 50 for a variety of compounds according to the present invention are shown in Tables I - XVII. Compounds which are toxic at 10 ⁇ M are marked with an + . Compounds which are toxic at 100 ⁇ M are marked with an *. Compounds which are toxic at an excess of 100 ⁇ M are marked with >*.
  • R 1 is aromatic or substituted aromatic groups
  • R 2 is -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group
  • R 3 is H or an alkyl. Examples are described in Table XIII below.
  • R 1 and R 2 are -NHX, -NX 2 , -OX, or -SX, where X is H or an organic group, and R 3 and R 4 is H or an alkyl. Examples are described in Table XIV.
  • R 1 and R 2 are aromatic, substituted aromatic, heteroaromatic, or substituted heteroaromatic.
  • An example is described in Table XVI.
  • Most of the compounds included in the present invention are in the form of free amines or hydrobromide salts.
  • One compound was prepared as a hydrochloride. All basic compounds may be prepared in the form of salts. Non-limiting examples are hydrochlorides, hydrobromides, hydroiodides, sulfates, phosphates, and quaternary alkylammonium derivatives. Animal Toxicity Studies.
  • mice Compounds having low cytotoxicity and good anti-HIV activity are screened in mice for systemic toxicity.
  • the following specific compounds were injected into mice under the following conditions: 4-[3'-[[2"-(dimethylamino)ethyl]thio] phenyl]-2-[[2"'- (dimethylamino)ethyl]thio ⁇ pyrimidine (DH-19), 4,6-bis[4'-[[2''-(dimethylamino)ethyl]thio]phenyl]-5-methylpyrimidine (LS-22);
  • the least toxic compounds in BALB/C mice were M-116 and LS-22.
  • M-69 produced some toxicity at 60 mg/kg per day, given once a day for 10 days. Less toxicity is present in vivo than in vitro. Toxicity at concentrations of greater than 100 ⁇ M in cell culture is generally considered to be acceptable
  • Toxicity at concentrations of greater than 100 ⁇ M in cell culture is generally considered to be acceptable
  • Even chimpanzees are not considered to be good models since HIV apparently does not replicate well in non-human primates.
  • the data obtained from in vitro testing and in vivo toxicity studies is considered to be of sufficient predictive value that the Food and Drug Administration accepts them as the basis for an Investigational New Drug application for testing in humans infected with HIV.
  • the compounds of the present invention can be delivered using a variety of pharmaceutical vehicles and modes of delivery.
  • the preferred dosage is that which results in a plasma concentration of 10 ⁇ M.
  • the compound should show a half-life close to one hour.
  • Humans suffering from diseases caused by HIV can be treated by administering to the patient a pharmaceutically effective amount of the compound in the presence of a pharmaceutically acceptable carrier or diluent.
  • a preferred carrier/diluent for oral administration is water, especially sterilized water. If administered intravenously, preferred carrier/diluents are physiological saline or phosphate buffered saline (PBS).
  • the compounds according to the present invention are included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to exert a therapeutically useful inhibitory effect on HIV in vivo without exhibiting adverse toxic effects on the patient treated.
  • HIV inhibitory amount is meant an amount of active ingredient sufficient to exert an HIV inhibitory effect as measured by, for example, an assay such as the ones described herein.
  • the active materials can also be mixed with other active materials which do not impair the desired action and/or supplement the desired action.
  • the active materials according to the present invention can be administered by any route, for example,, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.
  • a preferred mode of administration of the compounds of this invention is oral.
  • Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets.
  • the aforesaid compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.
  • These preparations should produce a serum concentration of active ingredient of from about 0.2 to 40 ⁇ M.
  • a preferred concentration range is from 0.2 to 20 ⁇ M and most preferably about 1 to 10 ⁇ M.
  • the concentration of active ingredient in the drug composition itself will depend on bioavailability of the drug and other factors known to those of skill in the art.
  • dosage values will also vary with the specific severity of the disease condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted to the individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compositions. It is to be further understood that the concentration ranges set forth herein are exemplary only and they do not limit the scope or practice of the invention.
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
  • the tablets, pills, capsules, troches and the like may contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose or saccharin or flavoring agent such as peppermint, methyl salicylate, or orange flavoring may be added.
  • a liquid carrier such as a fatty oil.
  • dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings.
  • tablets or pills may be coated with sugar, shellac, or other enteric coating agents.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
  • the solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraphens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl paraphens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylenediaminetetraacetic acid
  • buffers such
  • compositions of the present invention are prepared as formulations with pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers Preferred are those carriers that will protect the active compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as polyanhydrides, polyglycolic acid, collagen, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions are also preferred as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 (the pertinent portions of which are incorporated herein by reference). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container.
  • appropriate lipid(s) such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol

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Abstract

The compounds of the present invention are derivatized unfused heteropolycyclic compounds, primarily heterotricyclic compounds, formed by replacing the halogen atoms adjacent to diazine ring nitrogens or the benzylic halogenin 2,6-bis[4-(bromomethyl)phenyl]pyridine with one of a variety of common nucleophiles such as the hydroxide ion, alkoxides, mercaptides, and amines. These compounds interact with nucleic acids, thereby inhibiting translation and interfering with viral replication processes, such as the replication of HIV, the virus believed to cause AIDS. The preferred compounds at this time are 2,6-bis[4'-[(dimethylamino)methyl]phenyl]pyridine (DH-23); 4,6-bis[4'-[[2''-(dimethylamino)ethyl]thio]phenyl]-5-methylpyrimidine (LS-22); N-[2''-(dimethylamino)ethyl]-4-(benzo[b]thiophen-2'-yl)quinazolin-2-amine (M-116); N,N-bis(2''''-hydroxyethyl)-2-[[4'-(benzo[b]thiopen-2''-yl)-6'-(thien-2'''-yl)pyrimidin-2'-yl]oxy]ethylamine (DH-42); bis-4,6-[4'-[[2''-(dimethylamino)ethyl]thio]phenyl]pyrimidine (LS-20); 1-methyl-4-[2'-[[4''-(naphth-2'''-yl) quinazolin-2''-yl]thio]ethyl]piperazine (M-69); N,N-bis-(2'-hydroxyethyl)-4,6-bis(thien-2''-yl)pyrimidin-2-amine (M-103); N,N-bis(2'-hydroxyethyl)-2-[[4'',6''-bis(thien-2'''-yl)pyrimidine-2''yl]oxy]ethylamine (M-105); and 4-[3'-[[2''-(dimethylamino)ethyl]thio]phenyl]-2-[[2'''-(dimethylamino)ethyl]thio] pyrimidine (DH-19). Advantages of these compounds include low cost and ease of manufacture.

Description

Nucleic Acid Interacting
Unfused Heteropolycyclic Compounds
Background of the Invention
This application is a continuation-in-part of U.S. Serial Number 153,998 filed February 9, 1988 by Lucjan Strekowski, et al., entitled "Novel Diazines and Their Method of Preparation".
The present invention relates to unfused heteropolycyclic compounds having biological activity due to their ability to interact with nucleic acids. A variety of compounds have antiviral or cytotoxic activity due to interaction with DNA or RNA which interferes with translation of the genes into the encoded proteins. Much research is presently being directed at compounds which interact with nucleic acids as a means for treating or preventing the disease commonly known as Acguired Immunodeficiency Syndrome, or "AIDS".
AIDS was recognized as early as 1979. The Centers for Disease Control (CDC) declared AIDS a new epidemic in 1982. AIDS is generally accepted at this time to be a consequence of infection with the two related but distinct retroviruses termed human immunodeficiency virus HIV-1 and HIV-2. HIV-1 is the predominant cause of AIDS in the United States. Although the genomes are only about 50% homologous at the nucleotide level, the viruses contain the same complement of genes and appear to attack and kill the same human cells by the same mechanisms. Antibodies to HIV-1 are present in over 80% of patients diagnosed as having AIDS or pre-AIDS syndrome, and have been found with high frequency in the identified risk groups.
There is considerable difficulty in diagnosing the risk of developing AIDS. AIDS is known to develop in at least 10% of the individuals infected with HIV, although this percentage is suspected to be much higher. A patient is generally diagnosed as having AIDS when a previously healthy adult with an intact immune system acquires impaired T-cell immunity. The impaired immunity usually appears over a period of eighteen months to three years. As a result of this impaired immunity, the patient becomes susceptible to opportunistic infections, various types of cancer such as Kaposi's sarcoma, and other disorders associated with reduced functioning of the immune system. Another condition associated with HIV is AIDS-related complex, or ARC. This condition is thought to lead eventually to AIDS. No treatment capable of preventing or reversing the immunodeficiency of AIDS or ARC is currently available. All patients with opportunistic infections and approximately half of all patients with Kaposi's sarcoma have died within two years of diagnosis. Attempts at reviving the immune systems in patients with AIDS have been unsuccessful.
A number of compounds have demonstrated antiviral activity against this virus in cell culture, including HPA-23, interferons, ribavirin, phosphonoformate, ansamycin, suramin, imuthiol, penicillamine, rifabutin, AL-721, 3'-azido-3'-deoxythymidine (AZT), and other 2',3'-dideoxynucleosides. An example of the latter are 2',3'-dideoxy-5-substituted uridines, described in U.S. Patent No. 4,681,933 issued July 21, 1987 to Chu and Schinazi.
AZT appears to be the drug of choice at this time. However, AZT exhibits toxicity in a clinical setting. See Yarchoan et al.. Lancet 575-580 (1986) and Richman, et al., N.E.J.Med. 317(4),192-197 (1987). AZT was originally synthesized by Horwitz et al., J. Pro. Chem. 29, 2076-2078, 1974. The effectiveness of AZT against HIV is described in U.S. Patent No.
4,724,232 issued February 9, 1988 to Rideout, et al. In general, inhibitors of cellular processes will limit both viral replication and normal cell growth and replication. Most of the antiviral drugs that have been discovered so far cannot be prescribed for a prolonged period of time because of their toxicity. For example, idoxuridine is limited in clinical usefulness to topical application in ophthalmic solutions for treatment of herpetic keratitis because of its toxicity to normal cells. Selective toxicity of a compound, however, can be utilized in the treatment of viruses, some types of cancer and proliferative diseases.
Not only is there a strong demand for new selective antiviral agents of low toxicity, there is a need for drugs which can be administered over long periods of time which are not exorbitantly expensive. For example, major disadvantages to AZT are bone marrow toxicity and the cost of manufacturing the drug. Other nucleoside type drugs share the same high cost of manufacture. Since the AIDS virus is a latent virus, incorporating itself into the host's own cellular DNA for some indefinite period of time, carriers of the virus may be required to take drugs inhibiting replication of the virus for long periods of time to prevent an active infection from occurring. Cost of the drug, as well as toxicity, therefore becomes a significant factor in the therapy of AIDS and other HIV infections.
Since the virus has a relatively high rate of mutation, there is also a need to have alternative drugs available for the treatment of drug-resistant strains of the virus. It may ultimately become necessary to use combinations of different types of drugs to effectively treat AIDS.
It is therefore an object of the present invention to provide compounds which are useful in the treatment of diseases including AIDS, other viral infections and cancers.
It is a further object of the present invention to provide compounds which are relatively non-toxic to healthy cells.
It is another object of the present invention to provide compounds which are easier and less expensive to make than the compounds presently available for the treatment of AIDS.
Summary of the Invention
The compounds of the present invention are derivatized unfused heteropolycyclic compounds, primarily heterotricyclic compounds. For example, diazines are derivatized by replacing halogen atoms adjacent to the diazine ring nitrogens with one of a variety of common nucleophiles such as hydroxide ion, alkoxides, mercaptides, and amines. Pyridine derivatives are made by reacting 2,6-di-p-tolylpyridine with N-bromo succinimide, to form 2,6-bis[4'-(bromomethyl)phenyl]pyridine. The benzylic bromine atoms are also replaced with a variety of common nucleophiles. These compounds interact with nucleic acids, thereby inhibiting translation and interfering with viral replication processes.
Compounds which are useful are those having a therapeutic index of greater or equal to 10, toxicity at levels of greater than or equal to 100 μM, and antiviral activity at concentrations of less than or equal to 10 μM.
The preferred compounds at this time are 2,6-bis[4'-[(dimethylamino)methyl]phenyl]pyridine (DH-23);
4,6-bis[4'-[[2''-(dimethylamino)ethyl]thio]phenyl]-5-methylpyrimidine (LS-22);
N-[2"-(dimethylamino)ethyl]-4-(benzo[b]thiophen-2'-yl)quinazolin-2-amine (M-116); N,N-bis(2""-hydroxyethyl)-2-[[4'-(benzo[b]thiophen-2"-yl)-6'-(thien-2"'-yl)pyrimidin-2'-yl]oxy]ethylamine
(DH-42); bis-4,6-[4'-[[2"-(dimethylamino)ethyl]thio]phenyl] pyrimidine (LS-20); 1-methyl-4-[2'-[[4"-(naphth-2"'-yl)quinazolin-2"-yl]thio] ethyl]piperazine (M-69);
N,N-bis-(2•-hydroxyethyl)-4,6-bis(thien-2"-yl)pyrimidin-2-amine (M-103);
N,N-bis(2'-hydroxyethyl)-2-[[4",6"-bis(thien-2"'-yl)pyrimidin-2"-yl]oxy]ethylamine (M-105); and
4-[3'-[[2"-(dimethylamino)ethyl]thio]phenyl]-2-[[2"'- (dimethylamino)ethyl]thio]pyrimidine (DH-19).
The antiviral activity and low cytotoxicity of these compounds was demonstrated in human peripheral blood mononuclear (PBM) cells, comparing reverse transcriptase activity associated with virus and cell proliferation, respectively. Further testing of toxicity in animals was also conducted.
Detailed Description of the Invention
Synthesis of compounds. The heteropolycyclic compounds of the present invention are produced using the method described in U.S. Serial Number 153,998 filed February 9, 1988 by Lucjan Strekowski, et al., entitled "Novel Diazines and Their Method of Preparation". Substituted halogenodiazines are used as intermediates in the synthesis of the unfused heteropolycyclic compounds containing a diazine and other aromatic moieties, such as thiophene, benzene, naphthalene, furan, pyridine or pyrrole, or a substituted aromatic. These compounds are then evaluated for antiviral activity, cytotoxicity, and toxicity in animals.
The method to produce the substituted halogenodiazines which are useful as intermediates in the synthesis of the novel unfused heteropolycyclic compounds, and the products thereof, is easy, efficient and results in a high yield of product. The reaction consists of the addition of an organolithium reagent to a halogenodiazine with subsequent dehydrogenation of the addition product. The entire reaction takes place in one reaction vessel, without isolation of the substituted halogenodihydrodiazine intermediate. The reactions proceed at moderate temperature and in a short period of time, which decreases side reactions and increases yield. Furthermore, the recovery is conducted under two-phase conditions thereby minimizing hydrolysis of the substituted halogenodiazine to a substituted hydroxydiazine.
The general reaction scheme in which substituted halogenodiazines are converted into unfused heteropolyaromatic compounds that may have biological application is illustrated as follows: The chloro derivative was treated with a 50-fold excess of the appropriate amine. The mixture was heated at 70-80 ºC for one hour and the amine recovered by distillation. The oily residue was treated with 5% NaOH and extracted with ether. The ether solution was dried over Na2SO4, evaporated, and the product purified by flash chromatography on SiO2. Further purification was done via crystallization from a toluene-hexane mixture.
Pyridine derivatives can be made from readily available 2,6-di-p-tolylpyridine. This compound is brominated with N-bromosuccinimide in a carbon tetrachloride solution under reflux conditions to give 2,6-bis[4'-(bromomethyl)phenyl]pyridine. The benzylic bromine atoms in this product are readily replaced with a variety of nucleophiles such as N-methylpiperazine, dimethylamine, or 2-(dimethylamino)ethanethiolate ion to furnish final compounds.
Examples of the general formulas for the new classes of unfused heteropolyaromatic compounds include: A substituted phthalazine of the general formula
wherein:
Figure imgf000010_0002
R1, is -NHX, -NX2, -OX, or -SX, where X is H or an organic group and
R2 is an aromatic or substituted aromatic group.
Aromatic groups include furanyl, thienyl, phenyl, naphthyl, and pyridyl. For example, aromatic groups can be substituted with alkoxy, alkylthio, dialkylamino, and hydrocarbon groups. If the substitution contains an -OH or -SH, the quantity and nature of the organometallic reagent should be adjusted in the preparation of these derivatives as necessary for reaction with the -OH or -SH. Examples are described in Table I.
A substituted quinazoline of the general formula
Figure imgf000010_0001
wherein:
R1 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group and
R2 is any aromatic or substituted aromatic. A substituted quinazoline of the general formula
Figure imgf000011_0001
wherein:
R1 is any aromatic or substituted aromatic and
R2 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group. Examples are described in Table II.
A substituted pyrimidine of the general structure
wherein:
Figure imgf000011_0002
R is -NHX, -NX2, -OX, or -SX, where X is H or an organic group.
Specific examples of R are
-S-CH2-CH2-N(CH3)2; -NH-CH2-CH2-N(CH3)2; -NH-CH2-
CH2-NH2;
Figure imgf000011_0004
Figure imgf000011_0003
-NH-CH2-CH2-CH2-N(CH3)-CH2-CH2-CH2-NH2; -NH-CH2-CH2-OH; -N(CH2-CH2-OH)2; and
Figure imgf000011_0005
-O-CH2-CH2-N(CH2-CH2-OH)2. Examples are described in Table III. A substituted pyrimidine of the general formula
wherein:
Figure imgf000012_0003
R1 is any aromatic or substituted aromatic and
R2 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group. Examples are described in Table IV.
A substituted pyrimidine of the general formula
Figure imgf000012_0002
wherein:
R is -NHX, -NX2, -OX, or -SX, where X is H or an organic group. Examples are described in Table V.
A substituted pyrimidine of the general formula
Figure imgf000012_0001
wherein:
R is -NHX, -NX2, -OX, or -SX, where X is H or an organic group. An example is described in Table VI.
A substituted pyrimidine of the general formula
wherei
Figure imgf000013_0003
n R is
-NHX, -NX2, -OX, or -SX, where X is H or an organic group. Examples are described in Table VII.
A substitued pyrimidine of the general formula
wherein:
Figure imgf000013_0002
κ
R1 is aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic and
R2 is H or alkyl. Examples are described in Table VIII.
A substituted pyrimidine of the general formula
Figure imgf000013_0001
wherein:
R1 and R2 are -NHX, -NX2, -OX, or -SX, where X is H or an organic group and R3 is aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic groups. An example is described in Table IX.
A substituted pyrimidine of the general formula
Figure imgf000014_0002
wherein R1 and R2 are aromatic or substituted aromatic groups and R3 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group. An example is described in Table X.
A substituted pyrimidine of the general formula
Figure imgf000014_0001
wherein:
R1 and R2 are an aromatic or substituted aromatic group and
R3 is H or an alkyl group. Examples are described in Tables XI and XII. Screening for Antiviral Activity and Toxicity.
As used in this invention, antiviral activity refers to the ability of a composition to inhibit the growth of HIV. The claimed composition also exhibits antiviral activity towards other retroviruses. The ability of the present compositions to inhibit HIV may be measured by various experimental techniques. One such technique involves the inhibition of viral replication in human peripheral blood mononuclear cells. The amount of virus produced is determined by measuring the virus-coded reverse transcriptase (an enzyme found in retroviruses).
Methodology for Testing Antiviral Drugs Against HIV-1 in Human Peripheral Blood Mononuclear (PBM) Cells. Virus strain and cells. PBM cells from healthy HIV-1 and hepatitis B virus seronegative donors were isolated by Ficoll-Hypaque discontinuous gradient centrifugation at 1,000 x g for 30 minutes, washed twice in PBS and pelleted at 300 x g for 10 minutes. Before infection, the cells were stimulated by phytohemagglutinin (PHA) at a concentration of 16.7 μg/ml for three days in RPMI 1640 medium supplemented with 15% heat-inactivated fetal calf serum, 1.5 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 μg/ml), and 4 mM sodium bicarbonate buffer.
HIV-1 (strain LAV-1) was obtained from the Center for Disease Control, Atlanta, and propagated in PHA-stimulated human PBM cells using RPMI 1640 medium as above without PHA and supplemented with 7% interleuken-2 (Advanced Biotechnologies, Inc., Silver Spring, MD), 7 μg/ml DEAE-dextran (Pharmacia, Uppsala, Sweden), and 370 U/ml anti-human leukocyte (alpha) interferon (ICN, Lisle, IL). Virus was obtained from cell-free culture supernatant and stored in aliquots at -70°C until used.
Uninfected PHA-stimulated human PBM cells were uniformly distributed among 25 cm2 flasks to give a 5 ml suspension containing about 2 × 106 cells/ml. Suitable dilutions of HIV were added to infect the cultures. The mean reverse transcriptase (RT) activity of the inocula was 50,000 dpm/ml which was equivalent to about 100 TCID50, determined as described in AIDS Res.Human Retro.3.71-85 (1987). The drugs, at twice their final concentrations in 5 ml of RPMI 1640 medium, supplemented as described above, were added to the cultures. Uninfected and untreated PBM cells were grown in parallel as controls. The cultures were maintained in a humidified 5% CO2-95% air incubator at 37ºC for five days after infection at which point all cultures were sampled for supernatant RT activity. Preliminary studies had indicated that maximum RT levels are obtained at that time.
RT activity assay. Six ml of supernatant from each culture was clarified from cells at 300 x g for 10 minutes. Virus particles were then pelleted from 5 ml samples at 40,000 rpm for 30 minutes using a Beckman 70.1 Ti rotor and suspended in 200 μl of virus disrupting buffer (50 mM Tris-HCl, pH 7.8, 800 mM NaCl, 20% glycerol, 0.5 mM PMSF, and 0.5% Triton X-100).
The RT assay was performed by a modification of the method of Spira, et al, in J. Clin.Microbiol. 25,97-99 (1987) in 96-well microtiter plates. The radioactive cocktail (180 μl) which contained 50 mM Tris-HCl pH 7.8, 9 mM MgCl2, 5 mM dithiothreitol 4.7 μg/ml Poly (rA)n·(dT)12-18, 140 μM dATP and 0.22 μM[3H]dTTP (specific activity 78.0 Ci/mmol, equivalent to 17,300 cpm/pmol; NEN Research Products, Boston, MA) was added to each well. The sample (20 μl) was added to the reaction mixture and incubated at 37°C for two hours. The reaction was terminated by the addition of 100 μl 10% trichloroacetic acid (TCA) containing 0.45 mM sodium pyrophosphate. The acid insoluble nucleic acid which precipitated was collected on glass filters using a Skatron semi-automatic harvester (setting 9) . The filters were washed with 5% TCA and 70% ethanol, dried, and placed in scintillation vials. Four ml of scintillation fluid (Econofluor, NEN Research Products, Boston MA) was added and the amount of radioactivity in each sample determined using a
Packard Tri-Carb liquid scintillation analyzer (model 2,000CA). The results were expressed in dpm/ml of original clarified supernatant.
Cell proliferation. The drugs were evaluated for their potential toxic effects on uninfected PHA-stimulated human PBM cells. Flasks were seeded so that the final cell concentration was 3 × 105 cells/ml. The cells were cultured with and without drug for 6 days at which time aliquots were counted for cell viability.
Examples of Compounds Having Anti-viral Activity and Low Toxicity.
EC50 is the median effective concentration of the compound as determined from the reverse transcriptase assay. Although these compounds exhibit reduced toxicity to normal cells, administration of a high concentration, a dosage which would result in a blood serum concentration of approximately 100 μM or higher, of such a drug would nevertheless produce some adverse side effects. Thus, compositions having a high concentration of the active ingredient are not considered to be therapeutically effective.
The therapeutic index of a compound is determined by dividing the inhibitory or lethal dose for 50% of the population (IC50 or LDS0) by the effective dose for 50% of the population (EC50). The EC50 for a variety of compounds according to the present invention are shown in Tables I - XVII. Compounds which are toxic at 10 μM are marked with an +. Compounds which are toxic at 100 μM are marked with an *. Compounds which are toxic at an excess of 100 μM are marked with >*.
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Table III. Substituted pyrimidines continued,
Code & Molecular Mol. Drug Name EC50 Toxicity Formula Weight (μM)
+RW-15 C16H18N4S2.2HBr 429.3 N-[2"-(Dimethyl- 0.5 amino)ethyl]-4,
6-bis(thien-2'-yl) pyrimidin-2-amine
*M-97 C19H25N5S2 387.6 N-[3"-[(3'''- 67.7 Aminopropyl) methylamino] propyl]-4,6-bis (thien-2'-yl) pyrimidin-2-amine
*M-93 C17H18N4S2 342.5 1-Methyl-4-[4', 21 6'-bis(thien-2"- yl)pyrimidin-2'- yl]piperazine
+M-127 C14H14N4S2 302.4 N-(2"-Amino- 2.6 ethyl)-4,6-bis (thien-2'-yl) pyrimidin-2-amine
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000028_0002
Figure imgf000029_0001
Figure imgf000030_0001
The following substituted pyrimidines are representative of additional known compounds which are useful as antiviral agents.
wherein:
Figure imgf000031_0001
R1 is aromatic or substituted aromatic groups,
R2 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group, and
R3 is H or an alkyl. Examples are described in Table XIII below.
Table XIII. Substituted pyrimidines.
Code & Molecular Mol. EC50 Toxicity Formula Weight Drug Name (μM)
*LMS-2 C15H20N4S2. 2HBr 482.3 1-Methyl-4-[2'- 16.9 [[4"-(thien- 2'''-yl) pyrimidin-2"- yl]thio]ethyl] piperazine
*LMS-3 C19H22N4S3 402.6 4-(2',2"- 12.9 Bithiophen-5'- yl)-2-[[2'"-(4""- methyl-piperazino) ethyl]thio] pyrimidine
+M-59 C20H24N4S2. 2HBr 546.4 1-[2'-[[4"-(Benzo 6.5 [b]thiophen-2'" -yl)ι-5"-methyl pyrimidin-2"- yl]thio]ethyl]- 4-methylpiperazine
*DH-19 C 18H26N4S2. 2HBr 524.4 4-[3'-[[2"- 9.3 Dimethyl-amino) ethyl]thio]phenyl] -2-[[2'"- (dimethylamino) ethyl]thio] pyrimidine Substituted pyrimidines having the general formula
Figure imgf000032_0001
wherein:
R1 and R2 are -NHX, -NX2, -OX, or -SX, where X is H or an organic group, and R3 and R4 is H or an alkyl. Examples are described in Table XIV.
Table XIV. Substituted pyrimidines.
Code & Molecular Mol. EC50 Toxicity Formula Weight Drug Name (μM
*M-40 C26H36N8S3·4HBr 916.5 2,5-Bis[2'- 17.5 ·2H2O [[2"-(4"'methylpiperazino)ethyl] thio]pyrimidin- -4'-yl]thiophene
+RW-12 C20H26N6S3·2HBr 608.5 2,5-bis[2'- 1,4 [[2"-(dimethylamino)ethyl] thio]pyrimidin- 4-yl]thiophene
+RW-20 C22H30N6S3 474.7 2,5-Bis[2'-[[2"- 2.1 dimethylamino) ethyl]thio]-5'- methyl pyrimidin-4'-yl] thiophene Substituted pyrimidines having the general formula
Figure imgf000033_0001
wherein:
R1 and R2 are aromatic, substituted aromatic, heteroaromatic, or substituted heteroaromatic. An example is described in Table XVI.
Figure imgf000033_0002
Figure imgf000034_0001
Table XVII. Substituted pyridines.
Code & Molecular Mol. EC50 Toxicity Formula Weight Drug Name (μM)
+M-23 C23H27N3.2HBr 507.3 2-[4'-[[[2''- 4 (Dimethyl-aminoethyl]amino] methyl]phenyl]-6- (p-tolyl)pyridine
+M-25 C27H37N5.4HBr 755.3 2,6-Bis[4'-[[[2"- 3.7 (di-methylamino) ethyl]amino] methyl]phenyl]pyridine
*M-95 C29H37N5 455.6 2,6-Bis[4'-[(4"- 22.0 methylpiperazino) methyl]phenyl] pyridine
+DH-22 C33C45N5S2.4HBr 899.5 2,6-Bis[4'-[[[2"- 5.9 (4"'-methylpiperazino) ethyl]thio]methyl] phenyl]pyridine
*DH-23 C23H27N3.2HBr 507.3 2,6-Bis[4'- 1.6 [(dimethyl-amino) methyl]phenyl] pyridine
Most of the compounds included in the present invention are in the form of free amines or hydrobromide salts. One compound was prepared as a hydrochloride. All basic compounds may be prepared in the form of salts. Non-limiting examples are hydrochlorides, hydrobromides, hydroiodides, sulfates, phosphates, and quaternary alkylammonium derivatives. Animal Toxicity Studies.
Compounds having low cytotoxicity and good anti-HIV activity are screened in mice for systemic toxicity. The following specific compounds were injected into mice under the following conditions: 4-[3'-[[2"-(dimethylamino)ethyl]thio] phenyl]-2-[[2"'- (dimethylamino)ethyl]thio}pyrimidine (DH-19), 4,6-bis[4'-[[2''-(dimethylamino)ethyl]thio]phenyl]-5-methylpyrimidine (LS-22);
N,N-bis(2""-hydroxyethyl)-2-[[4'-(benzo[b]thiophen-2"-yl)-6'-(thien-2"'-yl)pyrimidin-2'-yl]oxy]ethylamine (DH-42); 1-methyl-4-[2'-[[4"-(naphth-2'"-yl)quinazolin-2"-yl]thio]ethyl]piperazine (M-69); N,N-bis(2•-hydroxyethyl)-2-[[4",6"-bis(thien-2"'yl)pyrimidin-2"-yl]oxy]ethylamine (M-105); N-[2"-(dimethylamino)ethyl]-4-(benzo[b]thiophen-2'-yl)quinazolin-2-amine (M-116);
N,N-bis(2'-hydroxyethyl)-4,6-bis(thien-2"-yl)pyrimidin-2-amine (M-103);
2,6-bis[4'-[(dimethylamino)methyl]phenyl]pyridine (DH-23); 4,6-bis[4'-[[2"-(dimethylamino)ethyl]thio]phenyl]pyrimidine (LS-20).
The least toxic compounds in BALB/C mice were M-116 and LS-22. M-69 produced some toxicity at 60 mg/kg per day, given once a day for 10 days. Less toxicity is present in vivo than in vitro. Toxicity at concentrations of greater than 100 μM in cell culture is generally considered to be acceptable Unfortunately, there are no good animal models to study the effectiveness of these compounds in the treatment of AIDS. Even chimpanzees are not considered to be good models since HIV apparently does not replicate well in non-human primates. However, the data obtained from in vitro testing and in vivo toxicity studies is considered to be of sufficient predictive value that the Food and Drug Administration accepts them as the basis for an Investigational New Drug application for testing in humans infected with HIV. On the basis of the data above, it is believed that these and other related compounds will be effective in the treatment or prevention of AIDS and ARC. The compounds of the present invention can be delivered using a variety of pharmaceutical vehicles and modes of delivery. The preferred dosage is that which results in a plasma concentration of 10 μM. The compound should show a half-life close to one hour. Humans suffering from diseases caused by HIV can be treated by administering to the patient a pharmaceutically effective amount of the compound in the presence of a pharmaceutically acceptable carrier or diluent. A preferred carrier/diluent for oral administration is water, especially sterilized water. If administered intravenously, preferred carrier/diluents are physiological saline or phosphate buffered saline (PBS). The compounds according to the present invention are included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to exert a therapeutically useful inhibitory effect on HIV in vivo without exhibiting adverse toxic effects on the patient treated. By "HIV inhibitory amount" is meant an amount of active ingredient sufficient to exert an HIV inhibitory effect as measured by, for example, an assay such as the ones described herein. There may also be included as part of the composition pharmaceutically compatible binding agents, and/or adjuvant materials. The active materials can also be mixed with other active materials which do not impair the desired action and/or supplement the desired action. The active materials according to the present invention can be administered by any route, for example,, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form. A preferred mode of administration of the compounds of this invention is oral. Oral compositions will generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the aforesaid compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should produce a serum concentration of active ingredient of from about 0.2 to 40 μM. A preferred concentration range is from 0.2 to 20 μM and most preferably about 1 to 10 μM. However, the concentration of active ingredient in the drug composition itself will depend on bioavailability of the drug and other factors known to those of skill in the art.
It is to be noted that dosage values will also vary with the specific severity of the disease condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted to the individual need and the professional judgment of the person administering or supervising the administration of the aforesaid compositions. It is to be further understood that the concentration ranges set forth herein are exemplary only and they do not limit the scope or practice of the invention. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
The tablets, pills, capsules, troches and the like may contain the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, corn starch and the like; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; and a sweetening agent such as sucrose or saccharin or flavoring agent such as peppermint, methyl salicylate, or orange flavoring may be added. When the dosage unit form is a capsule, it may contain, in addition to material of the above type, a liquid carrier such as a fatty oil. Other dosage unit forms may contain other various materials which modify the physical form of the dosage unit, for example, as coatings. Thus tablets or pills may be coated with sugar, shellac, or other enteric coating agents. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
The solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraphens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.
The compositions of the present invention are prepared as formulations with pharmaceutically acceptable carriers. Preferred are those carriers that will protect the active compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as polyanhydrides, polyglycolic acid, collagen, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811 (the pertinent portions of which are incorporated herein by reference). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension. Modifications and variations of the substituted heteropolycyclic compounds having biological activity will be obvious to those skilled in the art from the foregoing detailed description of the invention. Such modifications and variations are intended to come within the scope of the present invention. We claim:

Claims

CLA IMS :
1. An antiviral composition comprising an unfused heteropolycyclic compound selected from the group consisting of:
Substituted phthalazines of the general formula
Figure imgf000042_0003
wherein:
R1 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group and
R2 is an aromatic or substituted aromatic;
Substituted quinazolines of the general formula
Figure imgf000042_0002
wherein:
R1 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group and
R2 is any aromatic or substituted aromatic;
Substituted quinazolines of the general formula
Figure imgf000042_0001
wherein:
R1 is any aromatic or substituted aromatic and R2 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group;
Substituted pyrimidines of the general formula
wherein:
Figure imgf000043_0003
R is -NHX -NX2, -OX or -SX where X is H or an organic group;
Substituted pyrimidines of the general formula
wherein:
Figure imgf000043_0002
R1 is any aromatic or substituted -aromatic and R2 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group;
Substituted pyrimidines of the general formula
wherein:
Figure imgf000043_0001
R is -NHX, -NX2, -OX, or -SX, where X is H or an organic group; Substituted pyrimidines of the general formula
wherein:
Figure imgf000044_0003
R is -NHX -NX2, -OX, or -SX, where X is H or an organic group;
Substituted pyrimidines of the general formula
wherein:
Figure imgf000044_0002
R is -NX2, -NH2, -OX, or -SX, where X is H or an organic group;
Substituted pyrimidines of the general formula
wherein:
Figure imgf000044_0001
R1 is aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic and
R2 is H or alkyl; Substituted pyrimidines of the general formula
Figure imgf000045_0002
wherein:
R1 and R2 are -NHX, -NX2, -OX, or -SX, where X is H or an organic group, and
R3 is aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic groups;
Substituted pyrimidines of the general formula
Figure imgf000045_0001
wherein: R1 and R2 are aromatic or substituted aromatic groups and R3 is -NHX, -NX2, -OX or -SX, where X is H or an organic group;
Substituted pyrimidines of the general formula
Figure imgf000045_0003
wherein:
R1 and R2 are an aromatic or substituted aromatic group and
R3 is H or an alkyl group; Substituted pyrimidines having the general formula
wherein:
Figure imgf000046_0003
R1 is aromatic or substituted aromatic groups,
R2 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group, and
R3 is H or an alkyl;
Substituted pyrimidines having the general formula
Figure imgf000046_0002
wherein:
R1 and R2 are -NHX, -NX2, -OX, or -SX, where X is H or an organic group, and
R3 and R4 is H or an alkyl;
Substituted pyrimidines having the general formula
Figure imgf000046_0001
wherein:
R1 and R2 are aromatic, substituted aromatic, heteroaromatic, or substituted heteroaromatic; Substituted pyridazines having the general formula
Figure imgf000047_0002
wherein:
R1 and R2 are aromatic, substituted aromatic, heteroaromatic, or substituted heteroaromatic; Substituted pyridines having the general formula
Figure imgf000047_0001
wherein:
R1 and R2 are H or -NHX, -NX2, -OX, or -SX, where X is H or an organic group; and salts thereof.
2. The antiviral composition of claim 1 wherein said aromatic or substituted aromatic groups are selected from the groups consisting of furanyl, thienyl, phenyl, naphthyl, pyridyl and aromatic groups substituted with alkoxy, alkylthio, dialkylamino, and hydrocarbon groups.
3. The antiviral composition of claim 1 wherein R is selected from the group consisting of -S-CH2-CH2-N(CH3)2; -NH-CH2-CH2-N(CH3)2; -NH-CH2-CH2-NH2;
Figure imgf000048_0001
Figure imgf000048_0002
-NH-CH2-CH2-CH2-N(CH3)-CH2-CH2-CH2-NH2;
Figure imgf000048_0003
-NH-CH2-CH2-OH; -N(CH2-CH2-OH)2; and
-O-CH2-CH2-N(CH2-CH2-OH)2.
4. The antiviral composition of claim 1 wherein the compound is selected from the group consisting of
2 ,6-bis[4'-[(dimethylamino)methyl]phenyl]pyridine (DH-23);
4,6-bis[4'-[[2''-(dimethylamino)ethyl]thio]phenyl]-5-methylpyrimidine (LS-22);
N-[2"-(dimethylamino)ethyl]-4-(benzo[b]thiophen-2'-yl)quinazolin-2-amine (M-116);
N,N-bis(2""-hydroxyethyl)-2-[[4'-(benzo[b]thiophen-2"-yl)-6'-(thien-2"'-yl)pyrimidin-2'-yl]oxy]ethylamine (DH-42); bis-4,6-[4'-[[2"-(dimethylamino)ethyl]thio]phenyl]pyrimidine (LS-20);
1-methyl-4-[2'-[[4"-(naphth-2"'-yl) quinazolin-2"-yl]thio]ethyl]piperazine (M-69); N,N-bis-(2'-hydroxyethyl)-4,6-bis(thien-2"-yl)pyrimidin-2-amine (M-103);
N,N-bis(2'-hydroxyethyl)-2-[[4",6"-bis(thien-2"'-yl)pyrimidin-2"yl]oxy]ethylamine (M-105); and 4-[3'-[[2''-(dimethylamino)ethyl]thio]phenyl]-2-[[2"'- (dimethylamino)ethyl]thio]pyrimidine (DH-19).
5. The antiviral composition of claim 1 further comprising a pharmaceutical vehicle.
6. The antiviral composition of claim 5 wherein said compound is in a dosage delivering an anti-Human Immunodeficiency Virus effective amount of said compound to a patient.
7. A method for treating or preventing retroviral infections, including AIDS, comprising administering an antiviral composition selected from the group of unfused heteropolycyclic derivatives consisting of:
Substituted phthalazines of the general formula
Figure imgf000049_0001
wherein: R1 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group and
R2 is an aromatic or substituted aromatic;
Substituted quinazolines of the general formula
Figure imgf000050_0003
wherein:
R1 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group and
R2 is any aromatic or substituted aromatic;
Substituted quinazolines of the general formula
Figure imgf000050_0002
wherein:
R1 is any aromatic or substituted aromatic and R2 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group;
Substituted pyrimidines of the general formula
Figure imgf000050_0001
wherein:
R is -NHX, -NX2, -OX, or -SX, where X is H or an organic group; Substituted pyrimidines of the general formula
wherein:
Figure imgf000051_0003
R1 is any aromatic or substituted aromatic and R2 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group;
Substituted pyrimidines of the general formula
whe
Figure imgf000051_0002
rein:
R is -NHX, -NX2, -OX, or -SX, where X is H or an organic group;
Substituted pyrimidines of the general formula
Figure imgf000051_0001
wherein:
R is -NHX, -NX2, -OX, or -SX, where X is H or an organic group; Substituted pyrimidines of the general formula
wherein:
Figure imgf000052_0003
R is -NX2, -NH2, -OX, or -SX, where X is H or an organic group;
Substitued pyrimidines of the general formula
wherein:
Figure imgf000052_0002
R1 is aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic and
R2 is H or alkyl;
Substituted pyrimidines of the general formula
Figure imgf000052_0001
wherein:
R1 and R2 are -NHX, -NX2, -OX, or -SX, where X is H or an organic group and
R3 is aromatic, substituted aromatic, heteroaromatic or substituted heteroaromatic groups; Substituted pyrimidines of the general formula
Figure imgf000053_0004
wherein:
Figure imgf000053_0003
R1 and R2 are aromatic or substituted aromatic groups and R3 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group;
Substituted pyrimidines of the general formula
Figure imgf000053_0002
wherein:
R1 and R2 are an aromatic or substituted aromatic group and
R3 is H or an alkyl group;
Substituted pyrimidines having the general formula
wherein:
Figure imgf000053_0001
R1 is aromatic or substituted aromatic groups,
R2 is -NHX, -NX2, -OX, or -SX, where X is H or an organic group, and
R3 is H or an alkyl; Substituted pyrimidines having the general formula
Figure imgf000054_0003
wherein:
R1 and R2 are -NHX, -NX2, -OX, or -SX, where X is H or an organic group, and
R3 and R4 is H or an alkyl;
Substituted pyrimidines having the general formula
Figure imgf000054_0002
wherein:
R1 and R2 are aromatic, substituted aromatic, heteroaromatic, or substituted heteroaromatic;
SubstituteSd pyridazines having the general formula
Figure imgf000054_0001
wherein:
R1 and R2 are aromatic, substituted aromatic, heteroaromatic, or substituted heteroaromatic; Substituted pyridines having the general formula
Figure imgf000055_0001
wherein:
R1 and R2 are H or -NHX, -NX2, -OX, or -SX, where X is H or an organic group; and salts thereof.
8. The method of claim 7 wherein said aromatic or substituted aromatic groups are selected from the group consisting of furanyl, thienyl, phenyl, naphthyl, pyridyl. and aromatic groups substituted with alkoxy, alkylthio, dialkylamino, and hydrocarbon groups.
9. The method of claim 7 wherein R is selected from the group consisting of -S-CH2-CH2-N(CH3)2; -NH-CH2-CH2-N(CH3)2; -NH-CH2-CH2-NH2;
Figure imgf000055_0002
Figure imgf000055_0003
-NH-CH2-CH2-CH2-N(CH3)-CH2-CH2-CH2-NH2;
Figure imgf000055_0004
-NH-CH2-CH2-OH; -N(CH2-CH2-OH)2; and
-O-CH2-CH2-N(CH2-CH2-OH)2.
10. The method of claim 7 wherein the compound is selected from the group consisting of 2,6-bis[4'-[(dimethylamino)methyl]phenyl]pyridine (DH-23);
4,6-bis[4'-[[2''-(dimethylamino)ethyl]thio]phenyl]-5-methylpyrimidine (LS-22);
N-[2"-(dimethylamino)ethyl]-4-(benzo[b]thiophen-2'-yl)quinazolin-2-amine (M-116);
N,N-bis(2""-hydroxyethyl)-2-[[4'-(benzo[b]thiophen-2"-yl)-6'-(thien-2"'-yl)pyrimidin-2'-yl]oxy]ethylamine
(DH-42); bis-4,6-[4'-[[2"-(dimethylamino)ethyl]thio]phenyl]pyrimidine (LS-20);
1-methyl-4-[2'-[[4"-(naphth-2"'-yl) quinazolin-2"-yl]thio]ethyl]piperazine (M-69);
N,N-bis-(2'-hydroxyethyl)-4,6-bis(thien-2"-yl)pyrimidin-2-amine (M-103);
N,N-bis(2'-hydroxyethyl)-2-[[4".,6"-bis(thien-2"'-yl)pyrimidin-2"yl]oxy]ethylamine (M-105); and
4-[3'-[[2"-(dimethylamino)ethyl]thio]phenyl]-2-[[2"'- (dimethylamino)ethyl]thio] pyrimidine (DH-19).
11. The method of claim 7 further comprising providing a pharmaceutical vehicle.
12. The method of claim 7 further comprising administering an anti-Human Immunodeficiency Virus effective amount of said compound to a patient.
13. A method for making an antiviral composition containing an unfused heteropolycyclic compound comprising: reacting a halogenodiazine with an organometallic reagent in an organic solvent; quenching the reaction with a compound selected from the group consisting of water, alcohol and aqueous acid solutions; adding a quinone to dehydrogenate the addition product; removing the hydroquinone; recovering the resulting substituted halogenodiazine.
14. The method of claim 13 wherein the organometallic compound is an organolithium compound.
15. The method of claim 14 wherein the reaction is quenched with between approximately 1 and 2 molar equivalents of water.
16. The method of claim 15 wherein the organolithium reagent is reacted with the halogenodiazine at a temperature between approximately -30°C and -15°C.
17. The method of claim 16 wherein the the addition product is dehydrogenated at approximately room 25°C for up to one hour.
18. The method of claim 13 wherein the hydroquinone is extracted with an aqueous solution having a pH greater than about 9 and the halogenodiazine is recovered from the organic layer.
19. The method of claim 13 wherein the halogen atoms adjacent to the diazine ring nitrogens of the recovered substituted halogenodiazine are reacted with a nucleophile.
20. The method of claim 19 wherein the halogen atoms are replaced by reaction with a nucleophile.
21. The method of claim 20 wherein the nucleophile is selected from the group consisting of hydroxide ion, alkoxides, mercaptides, and amines.
22. The method of claim 19 wherein the halogen atoms are replaced by reaction with a nucleophile selected from the group consisting of 2-dimethylaminoethanethiol,
N,N-dimethylethylenediamine, ethylenediamine, N-methylpiperazine, 2-(4-methylpiperazino) ethanethiol, 3,3'-diamino-N-methyldipropylamine, morpholine, ethanolamine, diethanolamine, and triethanolamine.
23. The antiviral composition of claim 1 further comprising at least one additional compound selected from the group consisting of antiviral compounds, antibiotics, antifungals, and immunostimulants.
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US11618751B1 (en) 2022-03-25 2023-04-04 Ventus Therapeutics U.S., Inc. Pyrido-[3,4-d]pyridazine amine derivatives useful as NLRP3 derivatives
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