CA1074700A - Method and compositions of thiazolylphenylguanidines as antirhinovirus agents - Google Patents

Abstract

2?,166 A B S T R A C T

Compositions and method of treating rhinovirus infections, employing substituted thiazolyl phenylguanidines are described.

Description

26,166 ~7~700 This invention relates to methods for prevent-ing or treating rhinovirus infections~ In particular, this invention consists of methods of inhibition of the growth of the common cold virus (rhinovirus) with pharma-ceutical compositions containing a substituted thiazolyl phenylguanidine and a pharmaceutical carrier.
The thiazolylphenylguanidine~ which are useful -in the treatment of rhinoviru~ infections may be describ-ed by the fo}lowing formula:

... . . .. ~ _ .

R ~ NH
S NHCNH ~ ~ ~
R' wherein R is hydrogen or chloride and R' is hydrogen, fluoride, chloride, lower alkyl, caFboxyl or carbolower-~ 2 - : :
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1 alkoxy. The lower alkyl and lower alkoxy groups are those having 1 to 4 carbon atoms, Also included within the scope of the inven-tion are methods of employing the pharmaceutically ac-ceptable acid addition salts of the compounds of theabove formula. Amon~ these salts are the hydrochloride, sulfate, nitrate, hydrobromide, maleate, tartrate and benzoate. These salts are prepared in the conventional manner by reacting the base compounds with an acid to form the corresponding acid addition salt.
The active components of this invention àre prepared according to the following method:

, . . . .. . ,, _ _ NH2CHNCN NH(C2H5)2 R ~ CCH2X

(I) ~II) R ~ ~ ~

S NHCN

~ l ~NH2 .

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R~ s~ cN~3~R-(IV) -.
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wherein R and R' are as hereinbefore described.
The diethylamine salt (I) of N-cyanothiourea (R.L. McKee and J.D.
Thayer, Journal of Organic Chemistry, Volume 17, pages 1494, 1496, 1952) is treated with an ~-haloacetophenone (II) in refluxing methanol to give a 4-phenyl-2-thiazolyl-2-carbamonitrile (III) which is then reacted with ani-line or a substituted aniline derivative in refluxing ethanol to yield the 1-(4-phenyl 2-thiazolyl)-3-phenylguanidine or substituted derivative (IV).
The present invention provides a composition of matter, having antirhinoviral activity, in dosage unit form, comprising a pharmaceutical carrier and ~rom about 50 to 400 mg. of a compound selected from the group consisting of those of the formula:

-- NHaNH

R~
wherein R is selected from the group consisting of hydrogen and chloride, R' is selected from the group consisting of hydrogen, Muoride, chloride, lower alkyl, carboxyl and carboalkoxy and pharmaceutically acceptable acid addition salts thereof.
me compounds described, as well as their pharmaceutically accept-able acid addition salts, may be formulated into co~positions for use as antirhinoviral agents by methods well known to the skilled pharmaceutical

2~ chemist. When compositions are intended to be administered orally~ preferab-ly in the form of a tablet or capsule, they can be prepared in the usual manner or formulated into a sustained release preparatlon by methods well known in the art. Another preferred mode of administration is by intrana-sal application, preferably in the form of a suspension or solution which is sprayed into the nasal tissue. A further form of admlnistration can be by intramuscular or subcutaneous injection.
The tablets and capsules are prepared by known methods and may include the usual pharmace~tical excipients such as sucrose, starch~ lactose3 magnesium stearate, etc. These oral compositLons are administered in 2 to ~'`~ .

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4 doses of 50 mg. to 400 mg./dose totalling about lO0 to 1600 m~. per day.
The intranasal formulation is best adnlnistered as a 0.5-10% suspension in the form of a spray or nose drops, several times a day. The inuectable ~ormulation is adm-,nistered once at a concentra-- 4a ~ :

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1 tion of 0.5 to 2.0 mg./kg. of body wei~ht.
The compositions are preferably administered to a warm-blooded animal prior to rhinovirus infection in order to prevent or ameliorate the infection, soon after known exposure to infec~ion or upon reco~nition of symptoms in order to treat the infection and minimize its systemic effects.
A representative formulation for a tablet con taining, for example, the antirhinoviral compound 1-(4--phenylthiaæolyl-2)-3-phenylguanidine is:

Antiviral compound............ 250 mg.
Calcium carbonate............. l50 mg.
~ucrose.............~.......... 88 mg.
Starch....................... .. 20 mg.
Magnesium stearate........... ... 5 mg.
A representative formula for a capsule contain-ing the above antirhinoviral compound is:

Antiviral compound........... . 250 mg.
Lactose.............~......... 150 mg.
Magnesium stearate......... ..... 6 mg.
A representative formulation for an intranasal spray suspension containing the same antirhinoviral com-pound is:

Antiviral compound 5.0% w/v Sodium carboxymethyl cellulose 1.0% w/v Sodium citrate 0.2~ w/v `
Potassium biphthalate 0.13% w/v Eucalyptol 0.02~ w/v Thimerosal 0.001~ w/v Purified water...... qs to 100% w/v Other compounds within the scope o~ the gen-eral formula may be substituted in the above compositions or formulated into similar compositions.
The compounds of the present invention are active in vitro against a variety of viruses causing 1~7~7(~

1 respiratory illness such as the "common cold" or rhino~
virus.
Confluent monolayers of a continuous cell-line :
such as HeLa, HEp-2, KB or L-132, grown in plastic tis-sue culture dishes, were infected with one of the rhino-viruses, for example, types lB, 2, 5, 14 or 23 and other members of the picornavirus group, including the entero-viruses, for example, coxsackie A-ll and A-21.
Protection of the tissues to the cytopathic effects of the viruses were ascertained either by means of a plaque inhibition test in which the test compound was adsorbed onto a filter paper disc and placed on the agar used to overlay the infected cell monolayers, or by incorporation into the said agar overlay. The agar .
15 overlay medium used for this purpose was of the follow- .
ing formulation: .

Minimum Essential Medium (Eaglej containing Earle's salts (Grant Island Biological Co., : .
Grand Island, N.Y.) and to which has been ;.
added . .

Ionagar No. 2 0O4% ... .:
Diethylaminoethyl dextran 0.01% .
Magnesium chloride. 0.06%
Fetal calf serum 2.0% v/v :
The virus infected cell monolayers plus test compound ~.-:
were incubated in a humidifier atmosphere of 5% carbon dioxide at 33C. for three to five days. Th~ ability of these compounds to pro.tect tissues from destruction ~y the viruses was then evident after staining the res-idual uninfected cells with 0.5% crystal violet in 20%
ethanol. The results appear in Table I.

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1 In plaque assays, to determine the minimal inhibitory concentration (~IIC ), 1- (4-phenylthiazolyl-2)-

3-phenylquanidine was dissolved in dimethylsulfoxide or polyethylene glycol 400 or was homogenized in culture medium at 100-250 mg/ml. Two-fold, serial dilutions were prepared in culture medium or in the respective solvent and aliquots o~ each dilution were incorporated in the culture overlay medium at the desired drug con-centration. When the drug was dissolved in solvent, a final, constant concentration of dimethylsulfoxide at 0.04~ or polyethylene glycol 400 at 0.2% was present in the culture overlay medium.
To determine the MIC (that concentration of drug inhibiting virus plaque formation by 50~), plaque tests were performed on HeLa, cells infected with 100--300 plaque-forming units of rhinovirus, poliovirus or coxsackievirus. Followin~ virus adsorption, 10 ml. of agar overlay medium containing 1-(4-phenyl~2-thiazolyl)--3-phenylguanidine was added to each dish. Cultures infected with rhinovirus were incubated at 33C. for

4 to 5 days. Poliovirus and coxackievirus-infected cult-ures were incubated at 37C. for 2 to 4 days. Uninfect-ed cultures with drug and cultures with dimethyIsulfox~
ide or polyethylene glycol 400 only were included as 2S controls in the tests. At the end of the incubation period, cultures were stained with 0.1~ crystal ~iolet in 20% ethanol and the plaques were counted. The results appear in Table II.

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1 In the test for viral sensitivity, by khe fil-ter disc method, 5 mg. of 1-(4-phenylthiazolyl-2)-3--pheny]guanidine was dissolved in 1-2 drops o~ dimethyl-sulfox:Lde and 2.5 ml. of saline. Filter paper discs, when saturated wi~h the solution, absorbed about 50 mcg.
of the compound.
Sensitivity tests were performed by a modifica-tion oE the method of Herrmann, et al., Proc. Soc. Exp.
Biol. ,~ Med., 103, 625 (1960). HeLa monolayers in 150 mm plastic dishes were infected with 1.0 ml. of rhino-virus suspension known to produce destruction of 80-90%
of the cells in 4 to 5 days. Following viral adsorption, the ce]Lls were overlayed with 30 ml. of Eaglels minimum essent:ial medium containing Noble agar at 0~5%, bovine -lS fetal serum at 2.0%, diethylaminoethyl dextran at 100 mcg/ml. and magnesium chloride at 3 millimoles~ Filter discs (1/4 inch Carl Schleicher and Schuell Co., No.
740E), containing about 50 mcg. of 1-(4-phenylthiazolyl- ~ ;
2)-3-phenylguanidine, were placed on the solidified sur-face a~nd the culture plates incubated at 33C. After 4 to S days, the overlay medium was decanted and the cells stained with 0.1% crystal violet in 20% ethanol.
¦ A zone! of 10 mm or more in which at least 2/3 of the I cells were protected from viral cytopathic effects, in-i 25 dicat~!s antiviral activity. The following Table III
lists 35 rhinovirus serotypes which were inhibited by this compound.
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_hinovirus Type Strain lB s63 Norman 11 lCV15 19 6076CVl~

23 512~CV24 31 140F . . .

38 CH79 :

Baylor 1 46 Baylor 2 49 8213 . . .
A2 No. 58 51 . 605CV~5 .: ..
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~11;)7~7~g l Virus Yield Reducti__ Test In a test to determine virus yield 1-(4-phenyl- :
-2-thiazolyl)-3-phenylguanidine is incorporated in Eagle~s minimum essential medium supplemented with bovine fetal serum at 2.0~. HeLa cell monolayers in 60 mm dishes were exposed to this drug in this medium for 6 hours, during and following infection with rhinovirus serotype lB at a virus multiplicity of l. Drug treated and con-trol cultures were washed 3 times and medium without drug was added for the remainin~ period of incubation.
Virus yields were determined by plaque assay o cultur fluidra harvested at 24 and 48 hours after infection.
The results of this test, showing significant inhibi-tion of rhinovirus type lB as indicated by the suppres-sion of 1-2 logs of virus yield at 24 hours in cultures treated with 0.4 to 0.8 mcg/ml~ of drug appear in Table IV.

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~t~4700 1 In addition to the above, compound 1-~4-phenyl--2-thiazolyl)-3-phenylguanidine is active against a broad spectrum of rhinoviruses and other picornaviruses, in-cluding coxsackie and poliovirus in human cell cultures.
1-(4-phenyl-2-thiazolyl)-3-phenylguanidine produces con-centration-related inhibition of virus growth as de~er-mined by inhibitory effects on virus plague formation and virus yield. The compound is equally active against rhinoviruses in three differen~ human epithelial ¢ell lines (Elela, L-132 and Detroit-6).
The following example~ describe in detail the preparation of represen~ative compounds of the invention.
' EXAMPLE 1 Preparation,of 4-phe~yl-2-~hlazolecarbamonitrile A mixture of 20 gm. (117 mmoles) of diethyl-amine N-cyanourea ~R.L. McKee and J.D. ~hayerj J. Organic Chem. 17, 1494 (1953)3, 18.2 gm. (117 mmoles) of phen-acyl chloride and 90 ml. o~ methanol is heated to reflux - for 15 minutes, during which time everythring dissolves and a new solid separates. The mixture is cooled and filtered yielding 19.8 gm. of the product, melting point . 177-179C. tdark green melt).
EXAMPLE_2 Preparation o~ 4-(4-chlorophenyl)-2-thiazolecarbamonitrile The procedure of Example 1 is repeated employ-ing 4-chlorophenacyl chloride in place of phenacyl ohlor ide. The product had a melting poin~ of 173-176C.
(dark green melt).

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Preparation oE l-t4-phenyl-2-thiazol ~ e A ~olution of 19.8 gm. (100 mmoles) of 4-phenyl--2-thiazolecc!rbamonitrile and 10 ml. (10.2 gm., 110 mmoles) of anili~e in 350 ml. of ethanol is heated to reflux for 8 hours and then cooled overnight to give 23 gm. of product, mel~ing point 179 180C. This prod-uct is recry~tallized from 550 ml. of ethanol to give 19.5 g. of produc~, melting point 180-181C.
EXAMP~E 4 Preparation of 1-(4-phenyl-2-thiazolyl)-3-(3-chlorophenyl)-_ guanidine A solution of 1.6 gm. (8 mmoles) of 4-phenyl--2-thiazolecarbamonitrile and 0.9 ml. (1.1 gm., 8.6 mmoles) of m-chloroaniline in 30 ml, of ethanol is heat-ed to reflux for 30 hours and then cooled overnight to give 0.7 gm~ of product. This pro~uct is recrystallized from 5 ml. of ethanol to give 0.45 gm. of p~oduct, melt-ing point 134-135C.

Preparation of 1-(4-phenyl-2-thia201yl)-3-(4-carbomethoxy-phen~l)guanidlne _ A solution of 2.0 gm. (10 mmoles) of 4-phenyl--2-thiazolecarbamonitrile and 1.65 gm. (11 mmoles) o~
methyl 4-aminobenzoate in 40 ml. of ethanol is hPated to reflux for 30 hours and then cooled overniyht to give 0.75 gm. of product. This produat is recrystallized from lS ml. of ethanol to give 0.6 gm. of product, melt~
ing point 189-191C.

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Preparation of 1-(4-phenyl-2-thiazolyl)-3-(4-fluorophenyl)-a'uanidine A solution of 2.0 gm. (10 mmoles) of 4-phenyl--2-thiazolecarbamonitrile and 1.24 gm. (10.5 mmoles) of 4-fluoroaniline in 25 ml. of ethanol is heated to reflux for 7 hours and then cooled overnight to give 1.4 gm. of product. This product is recrystallized from 8 ml. of ethanol yielding 1.0 gm. of product, melting point 156-158C.

Preparation of 1-(4~phenyl-2-thiazolyl)-3-(4-carboxy-~henvl)auani~ine ~ . _ .

A solution of 10.0 gm. (50 mmoles) of 4-phenyl--2-thiazolecarbamonitrile and 7O25 gm. (52.5 mmoles) of ~-aminobenzoic acid in 125 ml. of ethanol is heated to reflux for 30 hours during which time a solid appears.
The mixture is filterad while hot, to give 5.2 gm. of product. This product is recrystallized from 30 ml.
of acetic acid and then dried overnight in an Abderhalden pistol at 110C., to give 5.1 gm. of product; melting point 245-247C.

Preparation of 1-(4-phenyl-2~thiazolyl~-3-(~-tolyl)guani-` ~ dine A solution of 10.0 gm. (50 mmoles) of 4-phenyl--2-thiazolecarbamonitrile and 6.0 gm. (55 moles) of ~-toluidine in 150 ml. of ethanol is heated to reflux for 7 hours and then cooled over~ight to glve llo 5 gm.
of product. Tbis product is recrystal1ized from 100 ~07~L7(:~

1 ml. of ethanol to give 9.3 gm. of product, melting point 152-153C.

Preparation of 1-[4-(4-chlorophenyl)thiazolyl-2]-3-(p--tolyl)guanidihe __ A solution of 2.4 gm. (10 mmoles) of 4-(4-chlor-ophenyl)-2-thiazolecarbamonitrile and 1.2 g~. (11 mmoles) of p-toluidine in 30 ml. of e~hanol is heated to reflux for 8 houxs and then cooled overnight to give 2.7 gm.
of product. Thi~ product is recrystallized from 50 ml.
of ethanol and then from 20 ml. of toluene yielding 1.9 gm. of product, melting poin~ 175~177C. ;
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Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition of matter, having antirhinoviral activity, in dosage unit form, comprising a pharmaceutical carrier and from about 50 to 400 mg.
of a compound selected from the group consisting of those of the formula:

wherein R is selected from the group consisting of hydrogen and chloride, R' is selected from the group consisting of hydrogen, fluoride, chloride, lower alkyl, carboxyl and carboalkoxy and pharmaceutically acceptable acid addition salts thereof.

2. The composition in accordance with claim 1 wherein the antirhino-viral compound is 1-(4-phenyl-2-thiazolyl)-3-phenylguanidine.

3. The composition in accordance with claim 1 wherein the antirhino-viral compound is 1-(4-phenyl-2-thiazolyl)-3-(3-chlorophenyl)-guanidine.

4. The composition in accordance with claim 1 wherein the antirhino-viral compound is 1-(4-phenyl-2-thiazolyl)-3-(4-carbomethoxy) phenylguanidine.

5. The composition in accordance with claim 1 wherein the antirhinoviral compound is 1-(4-phenyl-2-thiazolyl)-3-(4-fluorophenyl) guanidine.

6. The composition in accordance with claim 1 wherein the antirhino-viral compound is 1-(4-phenyl-2-thiazolyl)-3-(4-carboxyphenyl) guanidine.

7. The composition in accordance with claim 1 wherein the antirhino-viral compound is 1-(4-phenyl-2-thiazolyl)-3-(p-tolyl) guanidine.

8. The composition in accordance with claim 1 wherein the antirhino-viral compound is 1-[4-(4-chlorophenyl)-2-thiazolyl]-3-(p-tolyl) guanidine.