CN1014242B

CN1014242B - Prepn. of thiadiazole compounds

Info

Publication number: CN1014242B
Application number: CN86108507A
Authority: CN
Inventors: 布伦特·杰弗里·里德; 韦恩·艾尔弗雷德·施皮策; 欧内斯特·吴群也; 小查尔斯·约翰逊·佩吉特; 威廉·贝文·布兰查德
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 1985-12-16
Filing date: 1986-12-16
Publication date: 1991-10-09
Also published as: DE3676673D1; PT83930B; KR890004197B1; IL80962A0; EG17724A; DK602786D0; GR3001371T3; HUT47092A; CN86108507A; ATE59405T1; HK2792A; SG98891G; HU198461B; IL80962A; MY101918A; ZA869439B; DK602786A; PT83930A; KR870006026A; NZ218648A

Abstract

Compounds of formula (I) wherein: R1 is hydrogen, and R2 is cyano, or -C(=S)-NH-R3; or R1 and R2 taken together are =C(NH2)2 or =C(R5)(NHR3'); R3 and R3' are hydrogen or -COOR4; R4 is C1-C10 alkyl, substituted C1-C10 alkyl, C2-C10 alkenyl, substituted C2-C10 alkenyl, phenyl, or substituted phenyl; and R5 is hydrogen or -S-R6 where R6 is C1-C4 alkyl, cyano-C1-C4 alkyl or pyridyl-C1-C4 alkyl, and salts thereof, are useful antiviral agents.

Description

Prepn. of thiadiazole compounds

The popular uneasiness that more and more causes people of the disease that causes by one or more viruses.In the past few years, the viral infection of trafficability characteristic contact and blood transfusion transmission has become the central topic that people make great efforts to study.The not really serious disease that is caused by virus also comprises common cold.

But, almost there are not several real potent antiviral agent to be developed up to nowadays.In many cases, available treatment measure only is to use antiseptic-germicide, and its effect is not antiviral, but avoids weak body to be encroached on by bacterium again.People continue research and seek potent antiviral agent with treatment with control some common disorders, as influenza and flu, and the illness that causes by various simplexviruss, and resist even more serious viral infection, as acquired immune deficiency syndrome (AIDS) (AIDS).

The present invention is related to some the 2-amino-1,3 that can effectively treat viral infection, the discovery of 4-thiadiazoles derivative.Thiadiazoles is generally known by people on document, and some is used in medical field.For example, Naik etc. are at J.lndian Chem, and Soc. has reported the antimicrobial acivity of some thiadiazolyl group thiazolidones on (LX volume: 674-678 page or leaf, July nineteen eighty-three); Grant etc. rolled up for the 10th phase at J.Med.Chem.(15: 1082-1084 page or leaf, 1972) the 2-amino-1,3 that discloses, the amino derivative that replaces of some of 4-thiadiazoles is effective depressor; United States Patent (USP) 3,772,316 have reported the 2-amino-1,3 that a class it is said the N-acidylate that is effective mycocide, 4-thiadiazole compound; Russo etc. rolled up for the 12nd phase at Farmaco.Ed.Sci.(30: 1031-1038 page or leaf, 1975) on reported a series of 1,3, the thiourea derivative of 4-thiadiazoles and their cyclization product.It is said that these compounds are antiseptic-germicides; Malinoski etc. roll up at Virology(110: 281-291 page or leaf, 1981) on narrated 2-amino-1,3, some antiviral activities of 4-thiadiazoles.

In a specific examples, the invention provides the method for a prevention, treatment and control viral infection.Say that more specifically it is a kind of at the external antiviral method that is used for protecting the substratum mammalian cell that one embodiment of the invention provide, this comprises the formula I compound that adds antiviral amount in this substratum:

In the formula:

R ¹Be hydrogen, R ²Be cyano group or-C(=S)-NH-R ³; Or

R ¹And R ²Be all together=C(NH ₂) ₂Or=C(R ⁵) (NHR ³');

R ³And R ³' be hydrogen or-COOR ⁴;

R ⁴Be C ₁-C ₁₀Alkyl, the C that tool replaces ₁-C ₁₀Alkyl, C ₂-C ₁₀Alkenyl, the C that tool replaces ₂-C ₁₀Alkenyl, the phenyl of phenyl or replacement;

R ⁵Be hydrogen or-S-R ⁶, R wherein ⁶Be C ₁-C ₄Alkyl, cyano group-C ₁-C ₄Alkyl or pyridyl-C ₁-C ₄Alkyl.

The compound of formula I also can effectively be treated the animal that is subjected to virus infection or tool susceptibility.Be used for this purpose, preferred compound is the compound of tool formula II, (III) and (IV):

In the formula: R ³Be hydrogen or-COOR ⁴, R ⁵Be hydrogen or SR ⁶, R ⁶Be C ₁-C ₄Alkyl.

The present invention also provides a series of preferred new compounds by formula (V), (VI) and (VII) regulation:

The compound of formula (V) and formula VI is special potent antiviral agent, and the compound of formula (VII) is active high in external experiment.In addition, but the present invention also comprises the salt of the hyoscine of using above-claimed cpd.

The thiadiazole compound of using in the antiviral method of the present invention is known in the document a bit.All compounds can both prepare by existing chemical technology, for example the technology by narrations such as aforesaid Naik etc. and Russo.2-thioureido-1,3 is disclosed before the aforementioned Russo etc., the 4-thiadiazoles, promptly the compound of formula III is an antiseptic-germicide, but not mentioned its antiviral activity.

R in aforementioned formula I ²Comprise a quilt-C(=S)-NH-R ³The acyl group of definition, wherein R ³Can be another acyl moiety-COOR ⁴R ²This definition N-thiourea compound of the present invention is provided, the chemical compound lot in them has been used to one preferably in the methods of treatment.Such thiocarbamide has the structure of formula II:

R in the formula ⁴Definition as above.Many these class thiadiazoles thiocarbamides are known in the literature.For example, in Japanese Patent 71/35262, address disinfectant and weedkiller used as fungicide; And in Japanese Patent 74/7218 also used as mycocide and disinfectant.

In defined aforesaid compound, R ⁴Comprise " C ₁-C ₁₀Alkyl " and " C of replacement ₁-C ₁₀Alkyl ", these terms refer to straight chain and branched-chain alkyl, as ethyl, n-hexyl, isodecyl, 6-ethyl heptyl and the alkyl that replaces arbitrarily, as haloalkyl, hydroxyalkyl, phenylalkyl or the like." halogen " comprises fluorine, chlorine, bromine and iodine.In addition, R ⁴Can be C ₂-C ₁₀Alkenyl or substituted alkenyl base, as allyl group, 4-hexenyl, 3-chloro-5-heptenyl, 2-hydroxyl-4-iso-heptene base or the like; Can be substituted-phenyl also, as chloro-phenyl-, hydroxy phenyl, aminomethyl phenyl, cyano-phenyl or the like.Best R ⁴Group is C ₁-C ₆Alkyl and C ₂-C ₆Alkenyl.

Most of aforesaid compound is named as inferior acyl ammonia thioic acid sulfoacid (Carbamimidothioic acid) derivative of ammonia first in this article, and can pass through with 1,3 4-thiadiazoles-2-thiocarbamide and tool formula R ⁶The alkylation reactions preparation of X.R ⁶R among the X ⁶Definition the same, X is a leavings group.Suitable leavings group comprises Cl, Br, I and p-toluenesulfonic esters, methanesulfonate ester class sulfonate group.Therefore, suitable alkylating agent comprises alkyl halide, as monobromethane, or the alkyl halide that replaces, as cyanogen MB or 2-pyridylmethyl iodide.Typical this reaction can mix by 2-thiadiazoles thiocarbamide and a kind of alkylating agent with about equimolar amount, in the presence of alkali (as yellow soda ash), reacts in a kind of organic solvent (as dimethyl formamide or acetonitrile) of reactionlessness and finishes.This is reflected at about 20 ℃ to 50 ℃ reactions and the time finished in about 16 hours usually.If required, reaction solvent can under reduced pressure steam and remove.Product is easy to by the ordinary method purifying, and this is included in crystallization in ethanol, ethyl acetate and the hexane equal solvent, or carries out chromatography with silica gel etc. as solid carrier.

One group of best compound provided by the invention comprises 1,3, but the salt of 4-thiadiazoles-2-cyanamide and hyoscine thereof.This compound can be by with the inferior acyl ammonia thioester of ammonia first (Carbamimidothioic acid ester) and a kind of oxygenant, for example between reactions such as chloro peroxybenzoic acid, peracetic acid, sodium peroxide, hydrogen peroxide, chlorine and ozone make.

In addition; according to another aspect of the present invention; 1; 3; 4-thiadiazoles-2-cyanamide also can be by 2-amino-thiadiazoles derivative that has been protected, i.e. 2-imino--3-phenmethyl-1,3; 4-thiadiazoles and cyanogen bromide reaction use Lewis acid (for example aluminum chloride) debenzylation to make to remove 3 protecting group then.Debenzylating reaction is at organic solvent, as carrying out in methylene dichloride, toluene or the benzene, preferably uses at least 2 normal Lewis acids, and is better with 4～8 equivalents.Temperature requirement is not strict, and for example, debenzylation can be carried out at 0 ℃～90 ℃, preferably room temperature.

This amino prussiate also can generate when inferior acyl ammonia thioester of ammonia first and ammonia react, shown in example 8.

This amino prussiate be easy to by with organic bases or mineral alkali, but form the salt of hyoscine as reaction such as sodium-acetate, lime carbonate, sodium hydroxide.

Compound of the present invention can exist with several tautomers, and all these isomer all comprise within the scope of the invention.For example, a best compound-1,3,4-thiadiazoles-2-cyanamide can exist with following form:

Another example of the tautomer of compound can be represented by the formula among the present invention:

The all possible tautomeric form of compound all is included in this among the present invention.

Following detailed example has been illustrated the building-up process of the thiadiazole compound of using in antiviral method of the present invention.

Preparation example 1

((1,3,4-thiadiazoles-2-base is amino) sulphomethyl)-urethanum

To 19.2 gram (190 mmole) 2-amino-1,3 that stirring, in the solution of 200 milliliters of acetonitriles of 4-thiadiazoles, once add the ethoxycarbonyl isothiocyanate of 25 grams (190 mmole), reaction mixture was stirred 16 hours in 24 ℃.Filter the collecting precipitation thing,, obtain ((1,3,4-thiadiazoles-2-base is amino) the sulphomethyl)-urethanum of 35 grams (80% productive rate) with ethyl acetate washing three times and dry.

Ultimate analysis:

Molecular formula: C ₆H ₈N ₄O ₂S ₂

Theoretical value: C, 31.02; H, 3.47; N, 24.12.

Measured value: C, 31.32; H, 3.27; N, 24.40.

Preparation example 2

1,3,4-thiadiazoles-2-base thiocarbamide

10 grams ((1,3,4-thiadiazoles-2-base is amino) sulphomethyl)-urethanum (making from preparation example 1) is dissolved in 150 milliliters of 1N sodium hydroxide solutions reflux 90 minutes.Cooling and concentrating under reduced pressure.Filter the solids of collecting precipitation, it is dissolved in 20 ml waters.Add 200 milliliters of these aqueous solution of 1N hcl acidifying.Filter and collect product, and use N, the dinethylformamide recrystallization gets 5 grams 1,3,4-thiadiazoles-2-base thiocarbamide, 253 ℃ of fusing points.

Ultimate analysis:

Molecular formula: C ₃H ₄N ₄S ₂

Theoretical value: C, 22.49; H, 2.52; N, 34.97;

S，40.02。

Measured value: C, 22.73; H, 2.33; N, 34.74;

S，40.26。

Example 1

N ¹-1,3, the 4-thiadiazoles-inferior acyl ammonia of 2-base ammonia first thio-methyl ester

In 40 ℃ of heating, 4.8 grams 1,3, the mixed solution of 4-thiadiazoles-2-base thiocarbamide (obtaining from preparing 2) and 45 milliliters of 1N sodium hydroxide, 15 milliliters of ethanol and 4 milliliters of methyl iodide 10 minutes adds 50 milliliters of 1N hcl acidifyings, the reduction vaporization concentration of reaction solution then.Filter the solids and the drying of collecting precipitation, get 3.02 gram N ¹-1,3, the 4-thiadiazoles-inferior acyl ammonia of 2-base ammonia first thio-methyl ester.

Ultimate analysis: molecular formula C ₄H ₆N ₄S ₂

Theoretical value: C27.57; H3.47; N32.15;

S36.80

Measured value: C27.78; H3.55; N31.92;

S36.54

Proton nmr spectra (300MHz) (D ₆DMSO/Me ₄Si), δ: 2.42(S, 3H, S-CH ₃), the wide S of 8.90(, 2H, NH ₂), 9.08(S, 1H, H on the ring).

Field desorption(FD) mass spectrum: molecular ion peak 174.

Example 2

N ¹-1,3, the 4-thiadiazoles-inferior acyl ammonia of 2-base ammonia first thioic acid sulfoacid ethyl ester

With 3.2 gram (20 mmoles) 1,3,4-thiadiazoles-2-base thiocarbamide is dissolved in 100 milliliters of N that contain 1.6 milliliters of (20 mmole) iodoethane and 2.12 gram (20 mmole) yellow soda ash, in the dinethylformamide, stirred 24 hours in 24 ℃.Filter reaction mixture concentrates filtrate decompression, remains oily matter after boiling off solvent.Oily matter is prepared chromatography on No. 500 posts of type at Waters, with 75% ethyl acetate/hexane (volume ratio) wash-out.Collect the elutriant of suitable component, be concentrated into the dried solids that obtains, recrystallization also from ether and hexane

Fixed, get 2.08 gram N ¹-1,3, the inferior acyl ammonia of 4-thiadiazoles-2-ammonia first thioic acid sulfoacid ethyl ester.

Ultimate analysis: molecular formula C ₅H ₈N ₄S ₂

Theoretical value: C31.90; H4.28; N29.76;

S34.06

Measured value: C32.18; H4.18; N29.59;

S34.08

Proton nmr spectra (300MHz) (D ₆DMSO/Me ₄Si) δ: 1.06(t, 3H, CH ₃), 3.02(q, 2H, CH ₂), the wide S of 8.88(, 2H, NH ₂), 9.08(S, 1H, ring H).

Infrared spectra (KBr) Cm ^-1: 3259.9,3089.2,1624.2,1510.4,1416.8,1377.3,1347.4,1247.1,1209.5,718.5.

Field desorption(FD) mass spectrum: 188.

Example 3～5

The inferior acyl ammonia of following thiadiazolyl group ammonia first thioester can pass through 1,3, and 4-thiadiazoles-2-base thiocarbamide and haloalkane reaction make by operation shown in example 1 and 2.

N ¹-1,3, the 4-thiadiazoles-positive butyl ester of the 2-base inferior acyl ammonia thioic acid sulfoacid of ammonia first; Output 1.68 grams; 40 ℃ of fusing points.

Proton nmr spectra (300MHz) (D ₆DMSO/Me ₄Si) δ: 0.90(t, 3H, CH ₃), 1.38(sextet, 2H ,-CH ₂-), 1.60(quintet, 2H ,-CH ₂-), 3.04(triplet, 2H ,-CH ₂), the wide S of 8.90(, 2H, NH ₂), 9.09(S, 1H, ring H).

Field desorption(FD) mass spectrum: 216.

N ¹-1,3, the 4-thiadiazoles-inferior acyl ammonia of 2-base ammonia first thioic acid sulfoacid-2-pyridylmethyl ester hydrochloride; Output 3.72 grams.

Proton nmr spectra (300MHz) (D ₆DMSO/Me ₄Si) δ: 4.64(S, 2H, CH ₂), 7.82(t, 1H, pyridine H), 7.98(d, 1H, pyridine H), 8.40(t, H, pyridine H) and, 8.79(d, 1H, pyridine H), 9.11(S, 1H, ring H).

Field desorption(FD) mass spectrum: 251.

N ¹-1,3, the inferior acyl ammonia of 4-thiadiazoles-2-ammonia first thioic acid sulfoacid cyanomethyl ester; Output 1.04 grams.

Proton nmr spectra (300MHz) (D ₆DMSO/Me ₄Si) δ: 4.14(S, 2H, CH ₂), the wide S of 9.09(, 2H, NH ₂), 9.15(S, 1H, ring H).

Field desorption(FD) mass spectrum: 199.

Preparation example 3

((1,3,4-thiadiazoles-2-base is amino) sulphomethyl)-phenyl carbamate

3.8 gram (50 mmole) ammonium thiocyanate is dissolved in 100 milliliters of acetonitrile solutions that contain 7.8 gram (50 mmole) phenyl chloroformates, stirs one hour at 24 ℃.Reaction mixture is filtered into containing 6.88 gram (50 mmole) 2-amino-1,3, in the 75 milliliters of acetonitriles of 4-thiadiazoles hydrochloride and the suspension of 25 ml propylene oxides.Stir to filter then in two hours at 24 ℃ and obtain solid sediment, warp

Be decided to be that ((1,3,4-thiadiazoles-2-base is amino) sulphomethyl phenyl carbamate weighs 6.42 grams.

Field desorption(FD) mass spectrum: 280.

Preparation example 4～6

By the general operation of above-mentioned preparation example 3, make following product:

((1,3,4-thiadiazoles-2-base is amino) sulphomethyl) just own ester of carboxylamine.

Ultimate analysis: molecular formula: C ₁₀H ₁₆N ₄O ₂S ₂

Theoretical value: C, 41.65; H, 5.59; N, 19.43

S，22.24。

Measured value: C, 41.64; H, 5.31; N, 19.28

S，22.46。

((1,3,4-thiadiazoles-2-base is amino) sulphomethyl)-Urethylane

Field desorption(FD) mass spectrum: 218.

((1,3,4-thiadiazoles-2-base is amino) sulphomethyl)-carboxylamine ethene ester

Field desorption(FD) mass spectrum: 230.

Preparation example 7

((methylthio group) (1,3,4-thiadiazoles-2-base is amino)-methyl)-urethanum

6.96 gram (30 mmole) ((1,3,4-thiadiazoles-2-base is amino) sulphomethyl)-urethanum (by preparation example 1 described making) mixes with 45 milliliters of 1N sodium hydroxide solutions that contain 9.0 gram (4 milliliters) methyl iodide and 15 ml methanol, heated 10 minutes in 40 ℃.Reaction mixture is cooled to 5 ℃ of after-filtration, and filter cake washes with water and at air drying, gets 4.72 grams ((methylthio group) (1,3,4-thiadiazoles-2-base is amino) methyl)-urethanum.

Ultimate analysis: molecular formula: C ₇H ₁₀N ₄O ₂S ₂

Theoretical value: C, 34.13; H, 4.09; N, 22.75

S，26.04。

Measured value: C, 34.41; H, 3.84; N, 22.91;

S，25.94。

Preparation example 8

((ethylmercapto group) (1,3,4-thiadiazoles-2-base is amino) methyl)-urethanum

((1,3,4-thiadiazoles-2-base is amino) sulphomethyl)-carboxylamine (6.96 gram) and the operant response of 4 milliliters of iodoethane according to preparation example 7, make 1.83 gram ((ethylmercapto groups) (1,3,4-thiadiazoles-2-base is amino)-methyl)-urethanum.

Field desorption(FD) mass spectrum: 260.

Example 6

1,3,4-thiadiazoles-2-cyanamide

3.48 gram (20 mmole) N ¹-1,3, the inferior acyl ammonia of the amino first of 4-thiadiazoles-2-thio-methyl ester (by the preparation of example 1 method) is dissolved in 200 milliliters of methylene dichloride that contain chloro peroxybenzoic acid between 4 grams (20 mmole equivalents are by 85% purity meter), stirs 2 hours in 24 ℃.Filter this mixture, throw out stirred 2 hours with 40 ml waters.The solid collected by filtration thing, drying gets 1.7 grams 1,3,4-thiadiazoles-2-cyanamide.

Field desorption(FD) mass spectrum: 126.

Ultimate analysis: molecular formula: C ₃H ₂N ₄S

Theoretical value: C, 28.57; H, 1.60; N, 44.42;

S，25.42。

Measured value: C, 28.95; H, 1.80; N, 43.60;

S，24.87。

Example 7

1,3,4-thiadiazoles-2-cyanamide sodium salt

To being dissolved with 1.38 grams 1,3, add 100 milliliters of ethanol in 10.5 milliliters of 1N sodium hydroxide solutions of 4-thiadiazoles-2-cyanamide.Filter this mixture, the solvent of removing in the filtrate obtains oily matter.This oily matter obtains crystallization by the slow steaming of reducing pressure except that methyl alcohol from 20 ml methanol and 100 milliliters of Virahols, make 1,3,980 milligrams of the sodium salts of 4-thiadiazoles-2-cyanamide.

Proton nmr spectra (300mHz) (D DMSO/Me Si) δ 8.68(s, 1H, ring H).Field desorption(FD) mass spectrum: 126.

Ultimate analysis: molecular formula: C ₃HN ₄SNa

Theoretical value: C, 24.33; H, 0.68; N, 37.83

S，21.65。

Measured value: C, 24.59; H, 0.87; N, 38.06;

S，21.90。

Example 8

1,3,4-thiadiazoles-2-guanidine

5.0 gram N ¹-1,3, the 4-thiadiazoles-2-base inferior acyl ammonia thio-methyl ester of amino first (making from example 1) mixes with 50 milliliters of ammonia and 100 milliliters of ethanol, in a capping still in 140 ℃ of reacting by heating 14 hours.Carry out thin-layer chromatography (TLC) (developping agent: 66%CHCL with silica-gel plate ₃, 26%, MeOH, 8% HOAc) demonstrate two new points, one and 1,3, the chromatography point place same position of 4-thiadiazoles-2-cyanamide (example 6).Boil off solvent, crude product stirs and removes by filter insoluble cyanamide with 200 milliliters of 1N hydrochloric acid.The evaporation salt acid solution is to doing, and residue is by reverse phase HPLC (HPLC) purifying, and the methanol wash-out with 90% gets 1.5 grams 1,3,4-thiadiazoles-2-guanidine.

Proton nmr spectra (300Hz) (D ₆DMSO/Me ₄Si) the wide S of δ: 8.37(, 4H, NH ₂), 9.27(S, 1H, ring H).

Field desorption(FD) mass spectrum: 144.

Example 9

1,3,4-thiadiazoles-2-cyanamide

A.2-amino-1,3, the benzylization of 4-thiadiazoles

101 gram 2-amino-1,3, the 4-thiadiazoles mixes in 2 liters of flasks with 650 milliliters of n-propyl alcohols, 140 milliliters of bromotoluenes, and heating.When the temperature of reaction mixture reaches 88 ℃, all 2-amino-1,3, the dissolving of 4-thiadiazoles forms solution state.Reaction solution begins to reflux in 98 ℃, and move to flask in the water-bath from heating mantles and reflux with control this moment.Add 250 milliliters of n-propyl alcohols again to keep the flowability of reaction mixture.Mixture was stirred 2 hours, heat temperature of reaction in case of necessity to keep 86 ℃.Cool off reactant to 30 ℃ then.Collect product 2-(imino--3-(phenyl methyl)-1,3,4-thiadiazoles hydrogen bromide salt with n-propyl alcohol flushing, dry air, gets 190 grams (70%), 200～202 ℃ of fusing points.

B. with the reaction of cyanogen bromide

146 gram 2-imino--3-phenyl methyls-1,3 to stirring add 44 milliliters of 50%(weight in the mixture of 4-thiadiazoles hydrobromate and 1 premium on currency, 1 liter of ethyl acetate) aqueous sodium hydroxide solution, to dissolve the thiadiazoles starting raw material.Branch vibration layer in the ethyl acetate solution that stays, adds 800 milliliters of aqueous solution that contain 84 gram sodium bicarbonates again.In 25 minutes time, drip 150 milliliters of ethyl acetate solutions that are dissolved with 56.3 gram cyanogen bromides then.Mixed solution restir 30 minutes divides water-yielding stratum then and discards.Remaining ethyl acetate solution washs with 200 ml waters.And add saturated salt solution to quicken layering, divide water-yielding stratum then and discard.Ethyl acetate solution is concentrated into 1/3rd of original volume, adds 100 milliliters of toluene then, reconcentration is to 1/3rd of original volume, and then adds 100 milliliters of toluene.Repeat this concentration process several times, obtain required (3-phenmethyl-1,3,4-thiadiazoles-2(3H)-subunit) cyanamide precipitation, filter, with toluene wash and dry, output 57.7 grams (50.6%).

C. debenzylation

The aluminum chloride of the fine pulverizing of 6 equivalents (7.4 gram) is joined in the mixture of 50 milliliters of methylene dichloride of 2.0 grams (3-phenmethyl-1,3,4-thiadiazoles-2(3H)-subunit) cyanamide.Stir and add 50 milliliters of tetrahydrofuran (THF)s after 2 hours.Pour 50 milliliters of cold water then into, stirred 10 minutes, remove by filter insolubles.The sodium-chlor that add about 5 grams to quicken layering, are told the tetrahydrofuran (THF) layer to the mixture.Add 2 gram salt again in water layer, extract secondary with 25 milliliters of tetrahydrofuran (THF)s at every turn.Merge the tetrahydrofuran (THF) layer, use dried over mgso, be condensed into solids.Add 25 milliliters of methylene dichloride in this solids, collect solids and dry then, obtain 1,3 of 0.91 gram, 4-thiadiazoles-2-cyanamide (productive rate 78%), high pressure liquid chromatography (HPLC) show that purity is 155 ℃ of 96.7% fusing points).

Correspondingly, but the present invention also provides a kind of method for preparing formula I compound or its hyoscine salt.

R in the formula ¹Be hydrogen, R ²Be-CN; Or R ¹And R ²Formation=C(NH together ₂) (SR ⁶) or=C(NH ₂) ₂, R wherein ⁶Be C ₁-C ₆Alkyl, cyano group-C ₁-C ₄Alkyl, or pyridyl-C ₁-C ₄Alkyl.

This preparation method comprises:

A), with 1,3,4-thiadiazoles-2-base thiocarbamide and tool formula R ⁶The alkylation reactions of X (R wherein ⁶Definition as above, X is a leavings group), make the compound of formula I, R in the formula ¹And R ²Formation=C(NH together ₂) (SR ⁶), R ⁶Definition as above; Or

B), the compound of formula I (R in the formula ¹And R ²Formation=C(NH together ₂)-(SR ⁶), R wherein ⁶Definition as above) with a kind of oxidant reaction, obtain R in the formula I ¹Be hydrogen R ²Compound for-CN; Or

C), with R in the formula I ¹And R ²Formation=C(NH together ₂) (SR ⁶) (R ⁶Definition as above) compound and ammonia react obtain R in the formula I ¹Be hydrogen, R ²Be R in the compound of-CN and the formula I ¹And R ²Formation=C(NH together ₂) ₂Compound, separate this two compounds; Or

D), will (3-phenmethyl-1,3, the 4-thiadiazoles-2(3H)-Ji Yaji) the cyanamide debenzylation obtains 1,3,4-thiadiazoles-2-cyanamide; Or

E), with R in the formula I ¹Be hydrogen, R ²But the salt that makes hyoscine for the compound of-CN and a kind of suitable organic bases or mineral alkali reaction.

Thiadiazole compound has shown antiviral activity in the test of standard as defined above, thereby can be used for the treatment of or prevent the common disease that is caused by multiple virus.Typical case's virus that this thiadiazole compound can effectively resist, comprise the A and the B influenzae strain virus of all tests, comprise as strains of influenza viruses such as A-Ann Arbor, A-Hong Kong, B-Great Lakes, B-Taiwan, B-Singapore, B-Brazil, A-Texas, A-Fukushina, B-Maryland.Other virus that can control according to the present invention comprise parainfluenza virus, respiratory syncytial virus, various herpes-I and II strain virus, dust can (ECHO) and vaccinia virus, Measles virus, Semliki Forest virus and the virus that causes AIDS.

The thiadiazoles antiviral agent that the present invention developed shows to have antiviral activity in vitro and in vivo, and the activity of anti-A-Ann Arbor influenzae strain virus is confirmed by data well in their body.

In a series of in vivo test,, and give the thiadiazole compound of predetermined dose with the A-Ann Arbor influenzae strain virus infection of 18 every group CD-1 mouse predetermined doses; Give in addition independent one group to take vehicle with comparing.

Test continued 10 days, noted the number of animals of every group of death every day.The animal that still survives after the 10th day is considered to the survivor, uses " survival index (Surviral lndex) " methods analyst data then, and analytic process is as follows:

Data-survival index value (the SI that obtains from control group _{X days}) calculate by every day (X) respectively, from control group (generally in X=4 that day) taking place the 1st example dead this day starts at till the 10th day.The survival index value that take place the 1st routine dead that day is defined as zero, and each day calculates with following formula up to the 10th day value later on:

SI _{X days}=(X-1) (control group is at the number of (X-1) day death)/(all control animal numbers)+SI _{(X-1) sky}

For example, be the 4th day if 1 routine control animal is taken place dead first day, and had 4 examples dead in 18 control animals, so SI at the 5th day _{The 4th day}=0, and

SI _{The 5th day}=(5-1) 3/18+0=0.66

Survival index value " SI to surviving animals _{Surviving animals}" also can use following formula by the data computation of control animal:

SI _{Surviving animals}=10 (to the 10th day dead control animal number)/(control animal sum)+SI _{The 10th day}

Then, with the index value that this cover of gained calculates, calculate the average survival value of control group and each animal subject group as follows.Average survival value " the SI of control group _{Control animal}" calculating, be by with each ^SIX days on duty with the control animal number corresponding to this sky (X) death, and will ^SISurviving animals is on duty with surviving animals number (if any), then with the product adduction, and divided by the control animal sum.The calculating of the average survival value of treatment group " SI treats animal " is with each ^SIX days on duty with the animal subject number corresponding to death in this (X) day, will ^SISurviving animals multiply by the treatment number of animals of survival, and these products of adduction are removed by the animal subject sum more then.

Then, by following formula and control animal relatively and calculate (P=largest percentage wherein:

P = \frac{SI －SI}{SI  － SI} × 100

Calculate with this formula, only equate if treat the average survival value of animal, then P=0% with the average survival value of control group; Equate with the survival index value of surviving animals if treat the average survival value of animal, then P=100%.

By laxative remedy this maximum percentage ratio of gained is converted into relative reactivity index RA then:

P RA

20% or still less=1(threshold value)

20-40%=2(has activity slightly)

The 40-60%=3(medium activity)

The 60-80%=4(activity is good)

The 80-100%=5(activity is very strong)

The table I provides the RA value of representative compound among the present invention below:

The table I

R ⁴RA

CH ₂CH ₃4.5

CH ₃5

Phenyl 5

N-hexyl 5

Vinyl 4

Table I (continuing)

R ⁵R ³RA

S-CH ₂CH ₃H 5

S-CH ₃H 5

S-CH ₃

5

S-CH ₂CH ₃

5

S-CH ₂CH ₂CH ₂CH ₃H 5

S-CH ₂-C≡N H 2.5

S-CH ₂-2-pyridyl H 2.5

In addition, in mouse influenza assay method, these compounds have also shown the activity of anti-multiple strains of influenza viruses in the good body.The table II shows, the compound of the preparation example 2 of wide dosage range in food, is fed then and given by various influenza A and the oral shown activity of B strain virus mice infected.This table has shown that the work of each test group after 10 days deposits number of animals.

The table II

Dosage (milligram/kg/day)

Strains of influenza viruses contrasts 15 30 60 120

B-Great Lake 3/36 11/18 16/18 17/18 18/18

B-Taiwan 1/36 4/18 15/18 18/18 18/18

B-Maryland 13/50 - - 19/20 20/20

B-Hong Kong 4/50 - - 10/20 16/20

(the table II continues) dosage (milligram/kg/day)

Strains of influenza viruses contrasts 15 30 60 120

A-Ann Arbor 0/50 - - 20/20 20/20

A-Texas 8/36 5/18 8/18 12/18 16/18

A-Brazil 10/28 5/15 5/16 12/18 14/18

Annotate: (one) is illustrated in this dosage level and estimates.

Research that plaque (plaque) reduces number provide quantitative evaluation vitro system to tissue culture cells in virus multiplication play the method for inhibiting inhibitor.

In this test, responsive mdck cell under 37 ℃, is grown in containing the substratum 199 of 5% inactivation foetal calf serum (FBS), penicillin (300 units per ml) and Streptomycin sulphate (300 mcg/ml) in 25 square centimeters Falcon bottle.When forming the fusion individual layer, remove substratum, in each bottle, add suitably 0.3 milliliter of viral liquid of dilution.After room temperature absorption one hour, mix agar layer with the test compound of 1% agarose of moiety, 2X substratum 199,2.5% FBS, penicillin, Streptomycin sulphate and different concns, and be coated on the cell thin that has infected.Compound is that the concentration by 10,000 mcg/ml is dissolved in the dimethyl sulfoxide (DMSO) (DMSO), then one of them aliquot arrived desired concn with nutrient agar mixture diluted.The hatching inoculation bottle shows best plaque value (2-10 millimeter) up to control bottle.In each trial jar, add the solution contain 10% formalin and 2% sodium-acetate then so that virally inactivated, and cell thin is fixed in plastic surface.After with the zone dyeing of Viola crystallina, calculate the plaque number with encircling cell.In two bottles that each concentration is same the mean deviation as a result of gained and control bottle relatively, all results with from 10% to 90% inhibiting rate of gained draw then, and calculate plaque and form and suppress 50% inhibiting rate (I ₅₀).See the table III from three kinds of results that strains of influenza viruses obtained:

The table III

The external activity of resisiting influenza virus

I ₅₀(mcg/ml)

Compd A-NWS A-Ann Arbor B-Great Lake

Preparation example 1 43＞50

Preparation example 2 3.5 3.0

Example 1 18.6 18.0

Example 2 9.8 9.5

Example 3 21.3 25.0

Example 6 0.85

Example 7 0.58 0.70 0.32

Example 8 0.04 0.3

When external application, compound can be added in the tissue culture medium (TCM), to suppress the wherein growth of virus.

The best way of using these compounds is to use in the body, and at this moment, compound can pass through non-enteron aisle, part or oral route or by sucking nasal delivery, enter in the mammalian body of suffering from virus infection or susceptible.Non-enterally administer, as by intraperitoneal administration, compound solubilized or be suspended in and contain 2% tensio-active agent particularly contains Emulphor(poly-hydroxylated lipid acid) water in.Certainly, best approach is oral.When oral, but the expansion agent of one of compound that defines among the present invention and the hyoscine of one or more standards, in the Capsules of packing into after the mixing such as starch, sucrose, lactose, lime carbonate, make each capsule contain the compound of sufficient dosage to suppress the present or growth in the future of influenza or other virus effectively.In other words, these compounds can be treated medicine as prevention or as treatment.In addition, also can be with medicine and various vehicle (as starch), the mixing such as (as stearic acid, Magnesium Stearates) of lubricant and wetting agent.With the mixture tablet forming, every contains sufficient dosage to suppress or to treat the invasion and attack of influenza or other virus effectively.Such tablet has been drawn indentation so that can be divided into half amount or 1/4th dosage during children taking.These compounds also can be taken with solution or suspension.

For implementing antiviral method of the present invention, the whole work that need do are that the thiadiazoles antiviral agent with antiviral dosage is added in the tissue culture medium (TCM) to be protected, or take for animal infective virus or susceptible.This compound also can be made into the compound preparation of easy administration with the thinner of hyoscine, for example is made into the formulation for oral, part or parenterai administration, and can be used for prevention and clinical treatment.It is 1%～95% active thiadiazoles antiviral agent that these compounds generally contain weight content.

Be used for when oral, these compounds can with common thinner and vehicle such as allotment of sucrose, starch, Microcrystalline Cellulose and gum arabic or the like, make tablet, pill then or incapsulate; Or wiring solution-forming, elixir (elixir), lozenge or the like.Externally used compound is that thiadiazoles antiviral agent and vehicle such as beeswax, lanolin and wet goods are hybridly prepared into operational formulation, as ointment, ointment, emulsifiable paste, tincture, lotion and paste or the like.

To serious virus infection, these antiviral thiadiazole compounds can be modulated into the formulation that is used for vein or intramuscular injection.Such preparation can contain about 1% to about 50% active medicine.These compounds dissolve in thinner commonly used such as isotonic saline solution or the glucose solution, and intravenous transfusion is used; Also can be dissolved in poly-hydroxy fatty alcohols, in propylene glycol or polyoxyethylene glycol, use for vein or intramuscular injection.

But the salt of hyoscine, the tool sufficient acidity that can be by the said structure formula or the compound of basicity make with reactions such as common organic acid and mineral acid, alkali example hydrochloric acid, Succinic Acid, sodium hydroxide.But the salt of these hyoscines is compared with parent compound, generally can strengthen its solvability, thereby often is more suitable for being deployed into liquid or emulsion.

Above-mentioned thiadiazoles antiviral agent all has activity in very wide dosage range.Though adopting what given dose is according to being treated or what prevent is that the infection and the severity thereof of any virus determines that route of administration can be followed the doctor's advice with relevant details.The normal dose scope is approximately from 0.1 to about 100 milligrams/kilogram, and more representative is about 0.5 to about 25 milligrams/kilogram.

At one preferably in the treatment plan, be the Mammals that these thiadiazole compounds is applied to the susceptible influenza virus, comprising horse, mouse, pig and people.When taking, these compounds also can be used for prevention for the people, particularly to the personnel of the elderly, children, nurse, doctor and other hospitals or the public health department preventing infection when existing evidence proof influenza popular approaching.These compounds also can give contact anyone of history with the influenza patient.The special benefits of therapeutic process of the present invention is: these compounds can be used for preventing or treating patient's infection, are to belong to influenza virus A strain or B strain and needn't pre-determine virus, because these compounds are all effective to these two kinds of virus strain.

Following example has illustrated some exemplary formulations of the compound allotment of using the formula I.

Example 10

The preparation of tablet

100 milligrams of the compounds of preparation example 1

200 milligrams of lactose

300 milligrams of W-Gums

50 milligrams of corn starch pastes

5 milligrams of calcium stearates

45 milligrams of Lin Suanergais

Activeconstituents, W-Gum, lactose and Lin Suanergai are mixed.In addition corn starch paste is made 10% water paste, and be added to mixing in the mixture.Add the calcium stearate mixing again, be pressed into tablet then.

Example 11

The preparation of suppository

500 milligrams of the compounds of example 1

1500 milligrams of theobroma oils

Above composition mixes at about 60 ℃, cools off in conical die then

Example 12

The preparation of oral administration mixed suspension

500 milligrams of the compounds of example 8

40 milligrams of sorbitol solution (70%N.F.)

150 milligrams of Sodium Benzoates

10 milligrams of lactose

50 milligrams of cherry flavor

100 milliliters of ethanol

Above composition is mixed so that every milliliter of syrup contains 5 milligrams activeconstituents.Take about 5～20 milliliters of this syrup every day, can protect human body to avoid virus, for example infection of influenza virus.

Example 13

The nasal cavity preparation

%(weight)

The compound 1.0 of example 8

The Antarox(non-ionic type gathers hydroxyl

The ethylization fixed oil, GAF company) 38.5

Ethanol 10.0

Freon-11 (triclosan-fluoro-methane) 25.0

Freon 12 (Refrigerant 12) 25.0

Menthol 0.5

Under about 70～80 ℃ of conditions, activeconstituents is added among the Antarox, stir this mixture, cool off this solution and use menthol and alcoholic acid mixture diluted up to becoming solution.The solution of gained is poured in the aerosol container also freezing to 0 ℃, adds freonll-11 aerosol agent matrix, with valve aerosol container is obturaged.

Claims

But 1, a kind of method for preparing the salt of formula I compound or its hyoscine,

In the formula: R ¹Be hydrogen, R ²For-CN; Or R ¹And R ²Formation=C (NH together ₂) (SR ⁶) or=C (NH ₂) ₂, R wherein ⁶Be C ₁-C ₆Alkyl, cyano group-C ₁-C ₄Alkyl, or pyridyl-C ₁-C ₄Alkyl,

This preparation method comprises:

A) with 1,3,4-thiadiazoles-2-thiocarbamide and tool structural formula are R ⁶The alkylating agent of X reacts, and makes the compound of formula I;

At R ⁶Among the X, R ⁶Definition as above, X is a leavings group;

In the formula I compound that makes, R ¹And R ²Formation=C (NH together ₂) (SR ⁶), R wherein ⁶Definition as above; Or

B) with R in the formula I ¹And R ²Formation=C (NH together ₂) (SR ⁶) (R ⁶Definition as above) compound and a kind of oxidant reaction, make R in the formula I ¹Be hydrogen, R ²Compound for-CN; Or

C) with R in the formula I ¹And R ²Formation=C (NH together ₂) (SR ⁶) (R ⁶Definition as above) compound and ammonia react, make R in the formula I ¹Be hydrogen, R ²Be R in the compound of-CN and the formula I ¹And R ²Formation=C (NH together ₂) ₂Compound, and separate this two compounds; Or

D) with (3-phenmethyl-1,3,4-thiadiazoles-2 (3H)-Ji Yaji) cyanamide debenzylation make R in the formula I ¹Be hydrogen, R ²Compound for-CN; Or

E) with R in the formula I ¹Be hydrogen, R ²Be-compound of CN and suitable organic bases or mineral alkali reaction, but make the salt of hyoscine.
2, according to the process of claim 1 wherein R in the formula I ¹And R ²Formation=C(NH together ₂) (SR ⁶), R ⁶Definition compound as claimed in claim 1 and a kind of oxidant reaction, obtain 1,3,4-thiadiazoles-2-cyanamide.
3, according to the process of claim 1 wherein with 1,3, but the salt that 4-thiadiazoles-2-cyanamide and suitable organic bases or mineral alkali reaction obtain hyoscine.
4, according to the process of claim 1 wherein with R in the formula I ¹And R ²Formation=C(NH together ₂) (SR ⁶), R ⁶Definition with the compound and the ammonia react of claim 1, make 1,3,4-thiadiazoles-2-guanidine.
5, according to the process of claim 1 wherein with 1,3 4-thiadiazoles-2-thiocarbamide and tool formula R ⁶X, R ⁶R among the X ⁶Definition with claim 1, X is a leavings group, alkylating agent reaction, make R in the formula I ¹And R ²Formation=C(NH together ₂) (SR ⁶) compound;
6, according to the process of claim 1 wherein by (3-phenmethyl-1,3, the cyanamide debenzylation of 4-thiadiazoles-2(3H)-Ji Yaji) makes R in the formula I ¹Be hydrogen, R ²Be-compound of CN.
7, according to the method for claim 6, wherein 1,3, the 4-thiadiazoles is to use the Lewis acid debenzylation.
8, according to the method for claim 7, wherein Lewis acid is an aluminum chloride.