NO873913L - ACETYLERT CYANAMIDE MIXTURE. - Google Patents

Description

The invention was made possible in part with the help of a grant from the Veterans Administration. The state has certain rights in the invention.

The alcohol sensitizing drugs disulfiram (tetraethyl thiuram disulfide, Antabuse<®>) and carbimide (citrated calcium carbimide; Temposilw ) increase the concentration of blood acetaldehyde in the presence of ethanol by preventing the oxidation of acetaldehyde to acetic acid by aldehyde dehydrogenase (AlDH), which produces physiological effects that make alcoholics refrain from further drinking.

The absorption of calcium carbimide after oral administration is extremely rapid and causes nausea, headache and vomiting. In an attempt to reduce the rate of absorption, calcium carbimide is produced as a slow-release tablet and to prevent its breakdown into ammonia, it is produced in the citrated form (1 part drug to 2 parts citric acid).

In the gastric juice, calcium carbimide is hydrolysed to carbimide (cyanamide, I^NCN) which is quickly absorbed into the portal vein circulation system. Data from animal experiments show that the drug is rapidly absorbed, metabolised and eliminated and in light of the rapid occurrence and short duration of the calcium carbimide/ethanol reaction (CER) it is likely that absorption, metabolism and elimination are also rapid in humans. Cyanamid itself does not prevent AlDH, but must be enzymatically activated by catalase to a so far unidentified active metabolite.

Disulfiram is still widely used in the treatment of alcoholism, but there has been concern that its repeated use can induce toxicity. The use of calcium carbimide is not approved in the United States. In Canada and other countries, calcium carbimide has not been used to any great extent because of its short period of activity, lasting about 24 hours. This is due to its easy conversion in vivo to an acetylated derivative, namely acetylcyanamide (AC), which is quickly excreted in the urine. At least 94%

of the added cyanamide is eliminated within six hours via this route in rats. In the same way as cyanamide, AC is devoid of

AlDH inhibitory effect in vitro.

There is therefore still a need for drugs with an alcohol sensitizing and deterrent effect which can largely raise the level of blood acetaldehyde in the presence of ethanol for a longer period after oral administration.

SUMMARY OF THE INVENTION

The present invention is directed to a series of cyanamide derivatives with general structure RCONHCN which are produced by substitution of the amino nitrogen atom of cyanamide by lipophilic acyl and N-substituted-(-aminoacyl) groups. In vivo, the enzymatic cleavage of these acyl groups (RCO) leads to the gradual release of cyanamide. Thus, the preferred compounds according to the invention are superior to cyanamide or its salts with regard to (a) increasing the level of blood acetaldehyde after ethanol administration and/or (b) the duration of action. The present invention is therefore also directed at a method to prevent ethanol ingestion, comprising increasing the level of blood acetaldehyde in a human after ethanol ingestion by supplying an effective amount of one of the compounds according to the invention.

The present alcohol sensitizing mixture comprises compounds of the formula I:

where R is a C3-C3Q-(alkyl), aryl, or cycloalkyl group, preferably a C12-C22 alkyl or a C5-C12 cycloalkyl group, the said alkyl group comprising 0-3 double bonds. The RCO acyl group is therefore preferably obtained from a saturated or unsaturated fatty acid, such as an alkanoic, alkenoic, dienoic or trienoic acid. Such acids include lauric, oleic, palmitic, linoleic, linolenic, stearic, elastearic, palmitoleic, petro-selenic, vaccenic, erucic acids and the like. Other preferred R groups include propyl, butyl and branched alkyl groups,

such as t-butyl (from pivalic acid) or cycloalkyl groups comprising 1-4 rings, such as cyclohexyl, cyclopropylmethyl, norbornyl, cyclopentyl, adamantyl and the like. Preferred aryl groups include phenyl, benzyl, benzyloxy, tolyl and variously substituted aryl groups including heteroaryl groups.

The R group in the compounds of formula I can also be represented by the formula (R 2 )NHCH(R 1 ) where R 1 is H, benzyl or a C 1 -C 8 alkyl group and R 2 is benzoyl, (N-carbobenzyloxy) or (L-pyroglutamyl) . Thus, the RCO molecular part of the compounds of the formula I can be obtained from an (N-protected)- - amino acid or from an N- [N-protected)-( cL-amino ) acylU -(X.-amino acid. Preferably R is bezyl or i Preferred RCO groups of this type include hippuryl (CgH5CONHCH2CO), N-benzoyl-L-leucyl, (N-carbobenzyloxy)-glycyl-L-leucyl, (L-pyroglutamyl)-L-leucyl and (L-pyroglutamyl )-L-phenylalanyl RCO can also be the molecular moiety N-carbobenzyloxy-L-pyroglutamy1.

Particularly preferred acylated cyanamides of the invention due to their ability to rapidly increase blood acetaldehyde levels to alcohol-deterrent levels and/or their ability to increase blood acetaldehyde levels over an extended period of time include n-butyrylcyanamide, palmitoylcyanamide (R = C^,-- n-alkyl), stearoylcyanamide (R = C17~n-alkyl), N-benzoyl-L-leucyl-cyanamide, hippurylcyanamide, N-carbobenzyloxy-glycyl-L-leucylcyanamide and L-pyroglutamyl-L-phenylalanyl-cyanamide, and the pharmaceutically acceptable salts thereof.

The present invention is also directed to a method for getting people to refrain from consuming ethanol by increasing the amount of blood acetaldehyde in a person after ethanol ingestion by supplying an effective amount of one of the compounds according to the invention. Other compounds that can be used in this method are acetylcyanamide and propionylcyanamide. The invention can therefore also include the pharmaceutically acceptable salts of these acylated cyanamides together with a pharmaceutically acceptable carrier for administration in an effective non-toxic dosage form. Pharmaceutically acceptable amide salts may include metal salts, such as the alkali and alkaline earth metal salts, e.g.

sodium, potassium, lithium and the like.

These physiologically acceptable salts are produced by methods known in the art. Metal salts can be produced by reacting a regulated amount of metal hydroxide with the free cyanamide. Examples of metal salts that can be prepared in this way are salts containing Li, Na, K, Ca, Mg, Zn, Mn and Ba.

A less soluble metal salt can be precipitated from a solution of a more soluble salt by adding a suitable metal compound. Thus, e.g. Zn, Mg and Mn salts are prepared from the corresponding sodium salts. The metal ions for a given salt can be exchanged with hydrogen ions, other metal ions, ammonium ion, guanidinium ion and ammonium and guanidinium ions substituted with one or more organic radicals using a suitable cation exchanger.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1-4 graphically show the effectiveness of certain mixtures according to the invention with regard to increasing blood acetaldehyde (AcH) after ethanol administration in rats. Fig. 5 is a graphical representation of the effectiveness of four of the mixtures according to the invention with regard to increasing blood AcH after ethanol administration in rats over a period of

72 hours (0.5 mmol/kg i.p. dose).

Fig. 6 is a graphical representation of the effectiveness of palmitoylcyanamide and N-benzoyl-L-leucylcyanamide, administered orally, in raising blood AcH after ethanol administration in rats over a period of 72 hours.

The following abbreviations are used in the figures: CBZ = carbobenzyloxy, Gly = glycyl and pGlu = pyroglutamyl.

DETAILED DESCRIPTION OF THE INVENTION

Acylated cyanamides of the general formula I wherein R is one

<C>3<-C>30~(alkyl), aryl (e.g. phenyl) or cycloalkyl group can be readily prepared by acylation of an alkali metal cyanamide salt such as monosodium cyanamide with the acid chloride RC0Cl. The reaction can be carried out at from -5 to 30°C in an inert solvent such as tetrahydrofuran for 10-80 hours. After separation of the reaction mixture between water and an organic solvent such as ethyl acetate, the aqueous phase containing the sodium salt of the product is acidified with dilute mineral acid, extracted with an organic solvent such as chloroform,

and the solvent removed to give the crude product. The product can be purified by crystallization and/or thin-layer or column chromatography techniques. Variations of this method are indicated in the examples below.

In the case where the desired acid chloride is not commercially available, it can be prepared from the corresponding carboxylic acid by reacting the acid with thionyl chloride in the presence of triethylamine or with (COC1)2 or PCl^. See, Compendium of Organic Synthetic Methods, I.T. Harrison et al., eds., Wiley-Intersciences, Pub., New York (1971) pp. 22-24.

Acylated cyanamides of the formula I where R is R 2 CONHCH(R^)-, where R^ and R 2 are as defined above or where RCO is (N-carbobenzyloxy-L-pyroglutamyl-) can be obtained from the corresponding (N-protected)-oi- amino )-acid or from the corresponding N-(N-protected)-(oC-amino)acylj -°C-amino acid (a protected dipeptide) by reaction of the free CO 2 H largely to the corresponding activated N-hydroxysuccinimide ester by reaction with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide (DCC). The activated ester readily reacts with the cyanamide in the presence of an alkali metal hydroxide to give the protected acylated cyanamide of the formula R2CONHCH(R1)CONHCN after acidification. N-protected-cC-amino acids can also be converted to the corresponding activated N-hydroxysuccinimide esters and reacted with a second oG-amino-(benzyl)-ester to provide the dipeptide starting materials used in the present invention, (R2CONHCH(R1)CO2H ) after removal of the benzyl ester by hydrogenolysis. See L.F. Fieser et al., Reagents for Organic Synthesis, John Wiley and

Sons, Inc., New York (1967) pp. 485 and 487.

Other activated intermediates can be used, such as the mixed anhydride i-BuOCOCOR which is formed by reacting the amino acid (RC02H) with i-BuOCOCl in the presence of (N-methyl)-morpholine. Amino protecting groups are selected from those which are base stable, such as the t-butoxycarbonyl and carbobenzyloxy groups. Pyroglutamic acid is a self-protected cyclic amide of glutamic acid.

In clinical practice, the acylated cyanamides or their salts will normally be administered orally in the form of a pharmaceutical unit dosage form comprising the active ingredient in combination with a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent in a digestible capsule. A unit dose of the compound or salt can also be administered without a carrier material. Examples of pharmaceutical preparations include tablets, capsules, aqueous solutions, suspensions, liposomes and other slow-release compositions. Usually the active substance will comprise between about 0.05 and 99% by weight or between 0.1 and 95% by weight of the unit dosage form, e.g. between 0.1 and 50 percent by weight calculated on the preparations to be used for oral administration.

The amount of acylated cyanamide administered and the frequency with which it is administered to a given human patient will depend on a number of variables related to the patient's psychological profile and physical condition. For evaluation of these factors see J.E. Peachey, A Review of the Clinical Use of Disulfiram and Calcium Carbimide in Alcoholism Treatment, J. Clinical Psycho-pharmacology, 1, 368 (1981), J.F. Brien et al., Europ. J. Clin. Pharmacol., 14, 133 (1978), and Physicians' Desk Reference, Charles E. Baker, Jr., Pub., Medical Economics Co., Oradell,

NJ (34th ed., 1980) pp. 591-592. In general, although the initial unit dose of the present compounds may be similar to that given in the case of calcium cyanamide, e.g. 0.5-1.0 mg/kg, the unit dose preferably does not need to be re-administered for 24-96 h compared to 8-12 h in the case of calcium cyanamide.

The invention shall be further described with reference to

the following detailed examples.

I. Synthesis of acylated cyanamides

Example 1. Sodium acetylcyanamide ( 1).

Sodium acetylcyanamide was prepared by acetylation of sodium cyanamide (Fluka Chemical Corp., Hauppauge, N.Y.) by the method of F.N. Shirota et al., Drug Metab. Disp., _12, 337

(1984).

Example 2. Benzoylcyanamide ( 2)

Benzoyl cyanamide was prepared by adding benzyl chloride (6.33 g, 0.045 mol) in 100 mL diethyl ether to a suspension of monosodium cyanamide (5.76 g, 0.090 mol) in 100 mL ether at 4°C. The reaction was stirred overnight at room temperature and the pale yellow solid that formed was collected and dissolved in water. On acidifying the solution to pH = 1, a precipitate formed which was collected and recrystallized from ethyl acetate (EtOAc)/petroleum ether (30-60°C) to give 2 (5.89 g, 89.7% yield), m.p. . 140-142°C (reported m.p. 139-140°C). Anal. Calcd for CgH 6 H 2 O: C, 65.75; H, 4.11; N, 19,18. Found: C, 65.79; H, 4.35; N, 19.04.

Example 3. Pivaloylcyanamide ( 3)

Pivaloyl chloride (2.41 g, 2.46 mL, 0.020 mol) in 50 mL tetrahydrofuran (THF) was added dropwise to a suspension of sodium cyanamide (3.84 g, 0.060 mol) in 100 mL THF at the temperature of an ice bath with stirring . The reaction was allowed to proceed at 25°C overnight. The reaction mixture was then extracted with ethyl acetate (2 x 50 mL). The aqueous layer (pH 10.5)

was separated acidified to pH 1.5 with 10% aqueous HCl and extracted with chloroform (3 x 50 mL). The chloroform extract was dried over anhydrous sodium sulfate and filtered, and the filtrate was evaporated in vacuo to give 2.15 g (85.2% yield) of crude product. This was crystallized from ethyl acetate and petroleum ether (boiling point

range 30-60°C) to give 1.4 g of white crystalline 3_, m.p. 112-118°C TLC: Rf = 0.56 in ethyl acetate/petroleum ether/acetic acid (AcOH) (50:100:1) detected by orange color with ferricyanide spray reagent; IR (Nujol, cm"<1>) 3180 (NH), 2240 (C=N), 1730 (C=0); NMR (Silanor C, £ from TMS) 1.26 (s, (CH^C-) Anal. Calculated for CgH10N2O: C, 57.12; H, 7.99; N, 22.20. Found: C, 56.98; H, 7.93; N, 22.12.

Example 4. 1- Adamantane carbonyl cyanamide ( 1- Adamantoyl cyanamide)

( 4)

1-Adamantane carbonyl chloride (1.99 g, 0.010 mol) in 50 mL THF

was added dropwise to a suspension of sodium cyanamide (1.92

g, 0.030 mol) in 100 ml THF with stirring at ice bath temperature. The reaction was allowed to proceed at 25°C for 15 h. The reaction mixture was then extracted with ethyl acetate (100 mL).

The separated aqueous portion (pH 10.5) was acidified to pH 1.5 with 10% HCl and extracted with ethyl acetate (3 x 60 mL). The ethyl acetate extract was dried over anhydrous sodium sulfate and filtered, and the filtrate was evaporated in vacuo to dryness. The resulting white solid residue was triturated in a minimal amount of distilled water, filtered and air dried to give 1.74 g (85.2% yield) of crude 4_ as a white solid. This was recrystallized from ethyl acetate and petroleum ether to give 0.55

g (lot 1) of white crystalline powder. The filtrate was concentrated when further crystallization took place to give 0.49

g (lot 2) of additional product, giving an overall yield of 50.9% mp 168-170°C TLC: Rf = 0.58 in EtoAc/petroleum ether/AcOH (50:100:1) detected by UV quenching (faint) and red-orange color with ferricyanide spray reagent; IR (Nujol, cm 1 ), 3210 (NH), 2230 (C=N), 1710 (C=O); NMR (Silanor C5" from TMS) 1.62-2.2 (m, aliphatic CHs). Anal. Calcd for C12H16N2O: C, 70.56; H, 7.90; N, 13.71. Found: C, 70.59, H, 8.04, N, 13.66.

Example 5. n-Butyrylcyanamide (5)

n-Butyryl chloride (3.2 g, 3.12 mL, 0.030 mol) in 50 mL recently

distilled dry THF was added dropwise to a suspension of sodium cyanamide (5.76 g, 0.090 mol) in 100 mL freshly distilled THF at ice bath temperature. The reaction was allowed to proceed at room temperature for 24 hours, and the mixture was then evaporated to dryness in vacuo. The resulting yellow solid residue was dissolved in distilled water (50 mL) and the alkaline solution was adjusted to pH 6.5 with 10% HCl. The mixture was then extracted with ethyl acetate (2 x 50 mL). The aqueous portion (pH 7.8) was acidified to pH 1.5 with 10% HCl and extracted with methylene chloride (3 x 30 mL). The organic layer was separated, dried over anhydrous sodium sulfate and evaporated in vacuo to give 3.33 g (99.0% yield) of the crude product as a pale yellow liquid. A portion of the crude product _5 (2.00 g, 0.0178 mol) was applied to a column of dry silica gel (22 x 2.5 cm, 230-400 mesh, EM reagent) in EtOAc/petroleum ether (1:3) and eluted with the same solvent by flash chromatography at 103 kPa. A total of 32 x 20 ml fractions were collected. The fractions containing the desired compound were combined and evaporated in vacuo to give 1.77 g (overall yield 87.6%) of pure 5^ as a clear, colorless liquid. This compound breaks down when allowed to stand even at 5°C after longer periods. TLC: Rf = 0.6 in EtOAC/petroleum ether/AcOH (50:100:1) detected by UV quenching and orange color with ferricyanide spray reagent; IR (pure, cm<-1>) 3100-3250 (NH), 2860-2960 (alkyl), 2260 (C<=>N), 1725 (C=0); NMR (Silanor C,<5from TMS) 8.2-8.5 (wide, NH), 2.3-2.6 (t, -CH2-CH2-C=0), 1.5-1.9 ( sextet, CH3~CH2-CH2~), 0.8-1.1 (t, CH3~CH2-); CI-MS (NH3: positive) m/z (rel. intensity) 147 (12.6, Qm+1) + 2m^\ ) , 130 (100 , 0 ) Qm+1) +NH.^105 (5, 2) Qm+1) + 2NH3-H2NCN]; CI-MS (NH3: negative) m/z (rel. intensity) 111 (100.0) [m-1 ](68 (2.0) Qm-1 ) -CH^H^H^

41(16.7) Qm-1)-CH3CH2CH=C=oJ.

Example 6. Palmitoylcyanamide ( 6)

A solution of palmitoyl chloride (5.5 g, 0.02 mol) in 50 mL freshly distilled dry THF was added dropwise to a suspension of sodium cyanamide (3.84 g, 0.060 mol) in 150 mL freshly distilled dry THF at ice bath temperature. After the addition, the ice bath was removed and the reaction was allowed to proceed at 25°C for 43 h. The solids that formed were filtered to give 8.48 g of a waxy solid residue. The residue was ground to a fine powder and stirred in 150 ml of distilled water at ice bath temperature. The resulting soap-like suspension (pH 11) was acidified

to pH 2 with 10% HC1, then filtered and air dried to

give 5.4 g (96.3% yield) of crude 6 as a white solid. This was dissolved in hot THF and the mixture filtered. The filtrate was concentrated in vacuo until the solution became slightly cloudy. This material was crystallized from THF/petroleum ether for

to give 3.6 g (lot 1) of white powder. The filtrate was concentrated and crystallized in the same manner to give a further 0.6 g (lot 2) of white powder (74.8% overall yield) m.p. 6 2-65°C TLC: Rf= 0.68 in ethyl acetate/petroleum ether/glacial acetic acid (10:20:1); IR (Nujol, cm-1) 3230 (NH), 2250 (C<=>N), 1740 (C=0); NMR (Silanor C,^ from TMS) 1.3-1.6 (t, -CH2-CH2"C=0), 1-1.8 (m, -(CH_2)13-), 0.8-1 (t, CH3"CH2-); Anal. Calculated for C17H32<N>2<0:>C, 72.81; H, 11.50; N, 9.99. Found: C, 72.69; H, 11.65; N, 9.68.

Example 7. Stearoylcyanamide ( 7)

Stearoyl chloride (3.03 g, 0.010 mol) in 50 mL freshly distilled dry THF was added dropwise to a suspension of sodium cyanamide (1.92 g, 0.030 mol) in 100 mL freshly distilled dry THF

with vigorous stirring. The reaction was allowed to proceed at 25°C for 63 hours. The solids that formed were collected by filtration and the solid cake was suspended in 200 ml of distilled water. The resulting soapy suspension (pH 10.5) was acidified to pH 1.5 with 10% HCl, filtered and air dried to give 2.85 g (92.4% yield) of crude 1_. This was dissolved in THF and the solution decolorized with activated charcoal. The filtrate was concentrated until the solution became cloudy. Addition of petroleum ether gave 2.29 g (lot 1) of white, waxy ]_. The filtrate was concentrated and petroleum ether added to give 0.28 g (lot 2) of additional product, giving an overall yield of 83.3%, m.p. 74-75°C TLC: Rf = 0.69 in EtOAc/petroleum ether/AcOH (50:100:1) detected by UV quenching (weak) and no color reaction with ferricyanide spray reagent; IR (Nujol, cm 1 ) 3210 (NH), 2250 (C<S>N), 1735 (C=0); NMR (Silanor C, 6 from TMS) 2.3-2.6

(t, - CH2-CH2-C=0), 1.0-1.8 (melted s, (CH2)14), 0.8-0.9 (t, CH3-CH~); Anal. Calculated for C19H36N2<0:>C, 73.97; H, 11.76;

N, 9.08. Found: C, 73.88, H, 11.50, N, 9.09.

Example 8. N- Carbobenzoxycyanamide, sodium salt ( 8)

To a stirred solution of cyanamide (6.3 g, 0.15 mol) in 60

ml of distilled water, carbobenzoxychloride (8.5g, 0.050 mol) and 10% NaOH (40 ml, 0.10 mol) were added dropwise via separate separatory funnels at ice bath temperature. The reaction was allowed to proceed at this temperature for 3 hours. The reaction mixture (pH 10.1) was extracted with ethyl acetate (2 x 50 ml) and diethyl ether (2 x 50 ml) then the aqueous layer was acidified with 10% HCl to pH 1.5 and extracted with diethyl ether (4 x 50 ml) . The ether extract was dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuo to give a pale pink clear liquid. The liquid was dissolved in 50 ml of methanol at ice bath temperature. The methanolic solution (pH 1.8) was titrated with 5% methanolic NaOH to pH 5.9, the solution filtered and the filtrate evaporated in vacuo to near dryness to give a pale yellow liquid. Diethyl ether (100 mL) was added to the liquid with periodic shaking. After some scraping with a glass rod, a white firm cake was formed. The collected white solid was washed with diethyl ether to give 5.17 g (52.2% yield) of pure 8 as a white powder, m.p. 216-217°C TLC: Rf =0.56 in CH 2 Cl 2 /CH 3 OH (5:1); detected by UV quenching and orange color with ferricyanide spray reagent; IR (Nujol, cm<-1>) 3100-3040 (CgH5), 2150 (-N=C=N-Na), 1640 (-O2C-N), NMR (Silanor D20/DSS, J" from DSS), 7.38 (s, CgH5), 5.03 (s, C^-CH^-O-) . Anal. Calculated for CgHg-N2O2 • Na: C, 54.55, H, 3.56, N, 14 , 14. Found: C, 54.57, H, 3.79, N, 14.26.

Example 9. N-Carbobenzoxyglycine-N-hydroxysuccinimide ester

Acetonitrile (100 mL) was added to a reaction vessel containing N-carbobenzoxyglycine (4.18 g, 0.020 mol), dicyclohexylcarbodiimide (4.13 g, 0.020 mol), and N-hydroxysuccinimide (2.3 g, 0.020 mol). was stirred in 100 ml of acetonitrile at ice bath temperature for 5 hours. The reaction mixture was filtered for

to remove 4.12 g of white crystalline dicyclohexylurea (DCU)

as a byproduct. The filtrate was evaporated in vacuo to give a semi-solid residue which was recrystallized from chloroform/petroleum ether (boiling point range: 60-70°C) to give 4.99 g (81.5% yield) of white crystalline N-carbobenzoxyglycine-N- hydroxysuccinimide ester m.p. 111°C. IR (Nujol, cm"<1>) 3300 (NH), 3020

and 3060 (CgH5), 1820, 1780, 1740 and 1690 (C=0). Anal. Calculated for C14<H>14<N>2°6<:><c>' 54.90, H, 4.61, N, 9.15. Found: C, 54.15, H, 4.63, N, 8.75.

Example 10. N-Carbobenzoxyglycylcyanamide (9)

A solution of N-carbobenzoxyglycine-N-hydroxysuccinimide ester (1.53 g, 5.0 mol) in 50 mL THF and 10% NaOH (4 mL, 0.4 g, 0.010 mol) was added dropwise simultaneously via separate channels to a reaction vessel containing cyanamide (0.63 g, 0.015 mol) in 100 ml of distilled water with stirring at ice bath temperature. The reaction was allowed to proceed overnight at room temperature. The reaction mixture (pH 9.5) was extracted with ethyl acetate (2 x 50 mL). The ether was dried over anhydrous sodium sulfate and filtered, and the filtrate was evaporated in vacuo to give 1.0 g (86% yield) of crude product as a white solid residue. It was recrystallized from THF/petroleum ether (bop range: 60-70°C) to give 0.5 g (43% yield) of white crystalline 9, m.p. 198°C Dec. IR (Nujol, cm<-1>) 3300, 3100 (NH),

2250 (CN), 1725 and 1680 (C=0). Anal. Calculated for C^H^<NgOg:>C, 56.65, H, 4.75, N, 18.02. Found: C, 56.77, H, 4.53, N, 17.95 .

Example 11. N-Benzoylglycylcyanamide (Hippurylcyanamide) (10)

Hippuric acid (3.58 g, 0.020 mol) DCC (4.13 g, 0.020 mol) and N-hydroxysuccinimide (2.3 g, 0.020 mol) were stirred in 100 mL acetonitrile at ice bath temperature for 2 h. The reaction mixture was filtered to remove most of the by-product DCU. The filtrate was evaporated in vacuo to dryness, the residue was redissolved in THF and the mixture was filtered to remove any residual DCU. The filtrate (70 ml) was added dropwise to a solution of sodium cyanamide (3.84 g, 0.060 mol) in 100 ml of distilled water with vigorous stirring at ice bath temperature. The reaction was allowed to proceed at room temperature overnight. The reaction mixture (pH 11) was extracted with ethyl acetate (2 x 60 mL) and the separated aqueous layer was acidified to pH 1.5 with 10% HCl. The resulting solid suspension was filtered to give 2.28 g (56.1% yield) of white crystalline 10. It was recrystallized from THF/acetonitrile/petroleum ether to give 0.78 g (lot 1) of white crystalline product. The filtrate was concentrated to give 0.9 g (lot 2) of additional crystals. Temp. Dec. >155°C (turns to brown oil at 160°C); TLC: Rf = 0.36 in EtOAc/AcOH (100:1), detected by UV quenching and orange color with ferricyanide spray reagent, IR (Nujol, cm ) 3400 (NH), 3080 (CgH5), 2220 (C= N), 1710 and 1620 (C=0), NMR (Silanor C and DMSO (1:1), J from TMS) 8.2-8.4 (wide, NH), 7.3-8.0 (m , C,HC-), 4,1-— — D —D

4.2 (d, -NH-CH_2-CO). Anal. Calculated for C 2 H 2 N 2 : C, 59.11, H, 4.46, N, 20.68. Found: C, 59.26, H, 4.46, N, 20.84 (lot 1) and C, 59.03, H, 4.20, N, 20.80 (lot 2).

Example 12. N-Benzoyl-L-leucine

Benzoyl chloride (4.22 g, 0.030 mol) and 10% NaOH (12 mL, 0.030 mol) with added separately with vigorous stirring to a solution of L-leucine (3.94 g, 0.030 mol) in 150 mL of distilled water at ice bath temperature . The reaction was allowed to proceed at this temperature until the reaction mixture became clear (30 min); It was then extracted with ethyl acetate (2 x 100 mL). The aqueous layer was separated, acidified to pH 2 with 10% HCl and extracted with ethyl acetate (3 x 50 mL). The ethyl acetate extract was dried over anhydrous sodium sulfate. After filtration, the filtrate was evaporated in vacuo to give a quantitative amount of N-benzoyl-L-leucine as a semi-solid which was used directly in the following step.

Example 13. N-Benzoyl-L-leucylcyanamide (11)

DCC (2.41 g, 0.0117 mol) and N-hydroxysuccinimide (1.35 g, 0.0117 mol) were added to a solution of N-benzoyl-L-leucine (2.75 g, 0.0117 mol ) in 100 ml of acetonitrile at ice bath temperature. The reaction was allowed to proceed at this temperature for 2 hours. The reaction mixture was filtered to remove most of the DCU and the filtrate was evaporated in vacuo to dryness. The resulting semi-solid residue was redissolved in THF and the mixture filtered to remove any residual DCU. The filtrate was evaporated in vacuo to give crude N-benzoyl-L-leucine-N-hydroxysuccinimide ester as a foamy residue. IR (pure, cm 1) 3340 (NH), 3060 (CgH5), 1820, 1790, 1740 and 1650 (C=0), NMR (Silanor C, cf from TMS) 7.3-7.9 (m, CgH5 ), 5.1-5.3 (m, -NH-CH-C=0), 2.8 (s, cycl. -CH2-CH2"), 1.6-2.2 (m, CH-CH_2 -), 0.9-1.1 (d, (CH_3)3CH). This was dissolved in 100 ml of THF and added dropwise to a solution of sodium cyanamide (1.50 g, 0.0234 mol) in 100 ml of distilled water at ice bath temperature. The reaction was allowed to proceed at room temperature for 24 h. The reaction mixture was then concentrated in vacuo to half its original volume and filtered. The filtrate (pH 9) was extracted with ethyl acetate (2 x 50 mL). The aqueous layer was separated, acidified to pH 1.5 with 10% HCl and extracted with methylene chloride (3 x 50 mL). The methylene chloride extract was dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuo to give 2.80 g (92.4% yield ) of crude product as a sticky white solid This was triturated with diethyl ether and collected to give 1.30 g (42.9% yield) of white t powdered 11, m.p. 136-137°C. [<1D23+ 0.7° (c1.0, CH3OH), TLC: Rf = 0.93 in EtOAc/petroleum ether/THF/CH2Cl2/AcOH (50:50:10:50:4) and 0.24 in EtOAc/ petroleum ether/AcOH (50:100:1) detected by UV quenching and orange color with ferricyanide spray reagent; IR (Nujol, cm"<1>) 3300, 3100 (NH), 2230 (C=N), 1750 and 1630 (C=0); NMR (Silanor-DMSO, S from TMS) 7.4-8.0 (d, CgHg-), 4.2-4.6 (m, -NH-CH-C=0), 1.4-1.9 (m, CH-CH_2-), 0.8-1.1 (d, (CH3)2CH-). Anal. Calculated for C14H17<N>3<0>2<:>C, 64.85, H 6.61, N, 16.20. Found C, 64.62, H, 6.73, N, 15.93.

Example 14. N-Cbz-glycyl-L-leucylcyanamide (12)

N-Cbz-glycyl-L-leucine (4.84 g, 0.015 mol) was allowed to react with DCC (3.09 g, 0.015 mol) and N-hydroxysuccinimide (1.73 g, 0.015 mol) in 50 mL THF at ice bath temperature for 2.5 hours. The reaction mixture was filtered and the light brown filtrate (50 mL) was added to a solution of sodium cyanamide (2.88 g, 0.045 mol) in 30 mL of distilled water with stirring at ice bath temperature. The reaction was allowed to proceed at room temperature

for 24 hours. The reaction mixture was concentrated in vacuo to half its original volume and filtered. The filtrate (pH 9) was acidified to pH 2.5 with 10% HCl and extracted with methylene chloride (4 x 50 mL). The combined methylene chloride extract was dried over anhydrous sodium sulfate, filtered and the filtrate was evaporated in vacuo to give 4.86 g (93.5% yield) of crude 10 as a thick semi-solid residue. This residue was dissolved in methylene chloride and the solution washed with 5% citric acid. The organic layer was separated over anhydrous sodium sulfate and decolorized with activated carbon. After removal of the carbon by filtration, the filtrate was evaporated in vacuo to give 4.10 g (78.9% yield) of white glassy 10, m.p. 50-55°C. HD<23->34.0° (c 1.0 MeOH), TLC: Rf = 0.54 in CH2Cl2/MeOH/AcOH (90:10:5), detected by UV quenching and orange color with ferricyanide - spray reagent; IR

(Nujol, cm"<1>) 3300 (NH), 3060 (CgH5), 2260 (C=N) 1650-1730 (C=0); NMR (Silanor C^ from TMS) 7.35 (s, CgH^ -), 5.7-6.1 (NH) 5.1 (s, -0-CH2), 4.3-4.7 (m, -NH-CH-CO), 3.8-4.0 (melted s, - NH-CH_2-CO), 1.4-1.8 (m, -CH2-CH=), 0.8-1.0 (melted d, (CH3CH-). Anal. Calculated for ci7H22 <N>4°4: C'58'95'H'6'40'N'16.17 Found: C, 58.90, H, 6.55, N, 15.93.

Example 15. N-Cbz-L-pyroglutamic acid-N-hydroxysuccinimide ester

DCC (2.27 g, 0.011 mol) was added to a solution of Cbz-L-pyroglutamic acid (2.63 g, 0.010 mol) and N -hydroxysuccinimide (1.15 g, 0.010 mol) in 50 mL of THF at 0° C and the mixture was stirred at room temperature for 15 hours. The reaction mixture was filtered and the filtrate was evaporated to give 3.50 g (97.1% yield) of crude product as a thick liquid which flocculated upon addition of petroleum ether. The precipitate was crystallized from isopropyl alcohol to give 2.15 g (lot 1) of white crystals. The filtrate was concentrated and crystallized in the same manner for

to give 0.71 g (lot 2) which gave a total yield of 79.4%. Temp. 132-134°C (reported mp 131-133°C); TLC: R 1 f = 0.77 µm

in EtOAc/AcOH (100:1) detected by UV quenching, IR (Nujol, cm )

3050 (C6H5), 1820, 1785 and 1735 (C=0), NMR (Silanor C, <$* from TMS) 7.3-7.6 (m, CgHg-), 5.3-5.4 (d , -O-CHj-), 4.9-5.1 (t., 0=C-CH-N), 2.9 (s, 0=C-CH2-CH2-C=0) , 2.3 -2.8 (m, 0=C-CH_2-CH2 ;CH- ) . ;Example 16. N-Cbz-L-pyroglutamylcyanamide (13) ;Cbz-L-pyroglutamic acid-N-hydroxysuccinimide ester (1.8 g, 5.0 mmol) in 40 ml of THF was added dropwise to a solution of sodium cyanamide (0, 96 g, 0.015 mol) in 30 ml of distilled water at ice bath temperature. The reaction was allowed to proceed at this temperature for 4 hours and the reaction mixture was then extracted with ethyl acetate (2 x 50 mL). The aqueous layer (pH 11) was separated and acidified to pH 2 with 2 N HCl. The resulting white precipitate was filtered and dried to give 0.66 g (45.9% yield) of 13_ as a white powder m.p. 185-186°C (became somewhat yellow;>170°C). (oC)D<23->31.56°C (c 1.0, CH3CN), TLC: Rf = 0.5 in CH2Cl2/MeOH/AcOH (80:20:5), detected by UV quenching and gradually development of yellow color with ferricyanide spray reagent; IR (Nujol, cm"<1>), 3160 (NH), 3050 (CgHg), 2240 (C=N), 1775 and 1725 (C=0), NMR (acetone-dg,cf from TMS) 7.4 (s, CgH_5), 5.3 (s, -CH2-0-), 4.8-5.0 (m, 0=C-CH-N), 2.2-3.0 (m, 0= C-CH2-CH_2-CH) . Anal. Calcd for c14Hi3<N>3°4<:><c>' 58.53, H, 4.56, N, 14.63. Found: C, 58.33 , H, 4.73, N, 14.63.; Example 17. L- Pyroglutamic acid- N- hydroxysuccinimide ester ; DCC (4.13 g, 0.020 mol) and N-hydroxysuccinimide (2.3 g, 0.020 mol) were added to a solution of L-pyroglutamic acid (2.58g, 0.020 mol) in 50 mL THF and 5 mL dimethylformamide (DMF), at ice bath temperature. The reaction was allowed to proceed at room temperature for 48 h. The reaction mixture was then filtered to remove most of DCU and the filtrate was evaporated in vacuo to dryness. The resulting white solid residue was dissolved in 40 mL of THF, the solution was filtered and the filtrate was evaporated in vacuo to give 5.61 g of semi-solid residue. This was crystallized from hot methylene chloride to yield 2.35 g (lot 1) of white crystalline L-py roglutamic acid-N-hydroxysuc cinimide ester. Additional product 1.11 g (lot 2) was obtained from the filtrate, giving an overall yield of 76.5%. Temp. lot 1; 136-137°C, lot 2; 134-135°C; TLC: Rf = 0.4 in EtOAc/AcOH (100:1) detected by brown color development with N-chloro spray reagent. ;Example 18. L-pyroglutamyl-L-leucine benzyl ester ;L-pyroglutamic acid-N-hydroxysuccinimide ester (4.52 g, 0.020 mol) was added to a solution of L-leucine benzyl ester (4.43 g, 0.020 mol) in 60 ml of THF at room temperature and the reaction was allowed to proceed for 15 hours. The reaction mixture was evaporated in vacuo to give a pale yellow liquid. This liquid was dissolved in methylene chloride (50 mL) and washed with 10% citric acid (50 mL) followed by 1% sodium bicarbonate (50 mL). The methylene chloride layer was dried over anhydrous sodium sulfate and evaporated in vacuo to give 6.37 g (95.8% yield) of crude product as a pale yellow thick liquid. Crystallization from hot methylene chloride and petroleum ether gave 5.42 g (81.53% yield) of a white crystalline compound, m.p. 127-127°C. TLC: Rf - 0.58 in EtOAc/AcOH (100:1) detected by UV quenching, NMR (Silanor C,^ from TMS) 7.34 (s, CgH5), 5.4 (s, -O-CH ^-C^-) , 4.5-4.9 (m, -NH-CH-C=0), 3.9-4.3 (m, -NH-CH-C=0), 2.0 -2.7 (m, cycl. -NH-CH2-CH2~CH), 1.4-1.8 (m, -CH2-CH), 0.7-1.1 (melted d, -CH(CH_3 )3). Anal. Calculated for c1s<H>24N2<0>4<:><C>' 65'04'H'7'28'N'8.43. Found: C, 65.32, H, 7.51, N, 8.36. Example 19. L-pyroglutamyl-L-leucine L-pyroglutamyl-L-leucine benzyl ester (6.65 g, 0.020 mol) was hydrogenated with 9% palladium on charcoal in 100 ml methanol for 1 hour. The hydrogenation mixture was filtered through Celite and the filtrate was evaporated in vacuo to give 4.78 g of product as a white solid. This was crystallized from methanol and diethyl ether to give 4.63 g (95.6% yield) of white crystals, mp 152-154°C [reported mp. 151-152°C J; C<!D23 C-19.35° (c 1.0 MeOH); TLC: Rf = 0.57 in CHCl3/MeOH/AcOH (80:20:5) detected by brown color development with N-chloro spray reagent; NMR (methanol-d^,$ from TMS) 4.3-4.6 (m, -NH-CH-C=0), 4.1-4.3 (3, -NH-CH-C=0) , 1.9-2.6 (m, cycl. 0=C-CH2-CH2~CH), 1.5-1.8 (m, -CH2~CH), 0.7-1.1 (m, -CH(CH3)3). Anal. Calculated for cnH]_8N3°4: C'54.53, H, 7.49, N, 11.56. Found: C, 54.55, H, 7.39, N, 11.58. ;Example 20. L-pyroglutamyl-L-leucylcyanamide (14) ;L-pyroglutamyl-L-leucine (1.21 g, 5.0 mmol) was stirred with DCC (1.03 g, 5.0 mmol) and N -hydroxysuccinimide (0.58 g, 5.0 mmol) in 150 ml THF initially at ice bath temperature, then at room temperature overnight. The reaction mixture was filtered and the solvent removed in vacuo to dryness. The residue was redissolved in THF and filtered. The filtrate (70 ml) was added dropwise to a solution of sodium cyanamide (0.64 g, 0.010 mol) in 70 ml of distilled water at ice bath temperature. The reaction was allowed to proceed at this temperature for 4 hours. The reaction mixture was concentrated in vacuo to half its original volume at 25°C and filtered. The filtrate (pH 7.5) was neutralized to pH 7 with 10% HCl and passed to an AG 1x2 anion exchange resin column (15 x 2 cm, 100-200 mesh, acetate form) packed with distilled water. The column was washed with water (380 mL) until no more cyanamide was detected by spotting on TLC plates (blue-red color with ferricyanide spray reagent) then eluted with a linear pH gradient consisting of equal volumes of 0.15 N HCl in the reservoir and water in the mixing flask (1000 ml:1000 ml). A total of 45 x 20 ml fractions were collected. The fractions containing the desired product (orange color by spotting on TLC plates and sprayed with ferricyanide reagent) were combined and extracted with EtOAc/THF (5:1) (2 x 30 mL) and EtOAc (4 x 30 mL). The organic extract was dried over anhydrous sodium sulfate and evaporated in vacuo to dryness. The resulting solids were collected to give 1.29 g (96.9% yield) of crude product as a white powder. A portion of the product (140 mg) was dissolved in methanol and applied to 4 preparative TLC plates (Preadsorbent, Analtech, 1000 u thickness) then eluted with CHCl3/MeOH/AcOH (80:20:5). The bands corresponding to the desired product were removed, extracted with EtOAc/absolute EtOH (2:1) and the extract was then filtered. The filtrate was evaporated in vacuo to dryness, the residue triturated with diethyl ether and the product collected and dried in vacuo to give 79 mg (54.7% overall yield) of whitish powder m.p. Dec. 175°C (gradually changing to a yellow, then to a dark brown residue at 205°C) . IK1 D23~29 , 93° (c 1.0, MeOH); TLC: Rf = 0.36 in CHCl3/MeOH/AcOH (80:20:5) detected by UV quenching (weak); orange color with ferricyanide spray reagent and brown color with N-Cl spray reagent, IR (Nujol, cm "<1>) 3300 (NH), 2170 (C=N), 1670-1690 (C=0), NMR (methanol-d4, é from TMS) 4.1-4.6 (m, -NH-CH -C=0), 2.1-2.6 (m, cycl. -CH_2-CH2-), 1.4-1.8 (m, -CH_2-CH) , 0.8-1.1 (m , -CH(CH )2). ;Example 21. L- Pyroglutamyl- L- phenylalanine methyl ester ;N-Methylmorpholine (2.02 g, 0.020 mol) and isobutyl chloroformate ;(2.73 g, 0.020 mol) were added to a solution of L-pyroglutamic acid (2.58 g, 0.020 mol) in 100 mL THF/DMF (6:1) at -15° C. After a 2 minute coupling period, a mixture of L-phenyl-alanine methyl ester hydrochloride (4.31 g, 0.020 mol) (suspension); and N-methylmorpholine (2.02 g, 0.020 mol) in 50 mL DMF was added to the reaction mixture. The reaction was allowed to proceed at this temperature for 30 min and then at room temperature for an additional 1 h. The reaction mixture was then filtered to remove finely divided material and filtrate t was evaporated in vacuo to initial dryness. The resulting yellow liquid was dissolved in 100 mL of ethyl acetate and the ethyl acetate solution was washed with 5% citric acid saturated with sodium chloride (50 mL) and 5% sodium bicarbonate saturated with NaCl (50 mL). The separated organic layer was dried over anhydrous sodium sulfate and evaporated in vacuo to give quantitative amounts of a pale yellow liquid which was used directly in the following step. TLC: Rf = 0.22 in EtOAc/ AcOH (100:1) detected by UV quenching, IR (pure, cm ) 3300 (NH), 3020-3060 (CgHg), 2860-2960 (alkyl), 1650-1750 (C=0), ;NMR (Silanor C, cf from TMS) 7.25 (melted s, CgH^-), 5.5-5.7 ;and 6.6-6.8 (wide, NH's ), 4.6-5.1 (3, -NH-CH-C=0), 3.0-3.2 ;(q, -CH2-CgH5), 1.6-2.5 (m, cycle .0=C-CH2"CH2). ;Example 22. L-Pyroglutamyl-L-phenylalanine ;L-Pyroglutamyl-L-phenylalanine methyl ester (1.27 g, 4.4 mmol) in ;50 ml of methanol was stirred with 10% NaOH (1.93 mL, 0.193 g NaOH, 4.8 mmol) at room temperature for 1 h. The reaction mixture was evaporated in vacuo to dryness, the residue then dissolved in 1 distilled water and the mixture extracted with ethyl acetate (100 mL). The aqueous layer ( pH 10.5) was separated, acidified to pH 2 with 10% HCl, then saturated with sodium chloride and extracted with ethyl acetate (6 x 50 mL). The combined ethyl acetate extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated in vacuo for to give a white foam-like solid residue.This was finely divided m ed anhydrous diethyl ether, collected, and dried in vacuo to give 1.04 g (86.0% yield) of white powder which was used directly in the following step. Temp. 128-130°C; G?C]D + 13.51 (c 1.0, MeOH); TLC: Rf = 0.66 in CHCl3/MeOH/AcOH ;(80:20:5), detected by UV quenching and brown color with N-Cl spray reagent, NMR (methanol-d4, f from TMS) 7.22 (s_, CgH^-), 4.6-4.8 (m, -NH-CH- C=0), 4.0-4.2 (q_, -NH-CH-C=0), 2, 8-3.2 (m, ;-CH2-CgH5), 1.7-2.5 (m, cycl. 0=C-CH2-CH_2-CH-). ;Example 23. L-Pyroglutamyl-L-phenylalanylcyanamide ( 15 ) ;L-Pyroglutamyl-L-phenylalanine (4.10 g, 0.0148 mol) was allowed ;to react with DCC (3.37 g, 0.0163 mol ) and N-hydroxysuccinimide (1.88 g, 0.0163 mol) in 60 mL of THF at ice bath temperature for 3 hours, and then at room temperature overnight. The reaction mixture was filtered and the filtrate was added dropwise to a solution of sodium cyanamide (1.90 g, 0.0297 mol) in 60 ml of distilled water at ice bath temperature. The reaction was allowed to proceed at this temperature for 6 hours. The reaction mixture (pH 8) was then neutralized to pH 7 with 10% HCl saturated with sodium chloride and extracted with ethyl acetate (2 x 50 mL). The aqueous portion (pH 6.2) was acidified to pH 1.5 with 10% HCl and extracted with EtOAc/THF (3:1) (100 mL) and ethyl acetate (2 x 50 mL). The combined organic extract was dried over anhydrous sodium sulfate and evaporated in vacuo to give 4.20 g (94.5% yield) of crude product as an off-white solid residue. This was crystallized from abs. EtOH/diethyl ether to give 1.36 g (lot 1) of 1_5. The filtrate was concentrated and crystallized in the same manner to give 9.53 g (lot 2), giving an overall yield of 42.5%. Temp. 163-165°C, HD<23>+ 7.81° (c1.0, MeOH); TLC: Rf = 0.29 in CHCl-VMeOH/AcOH (80:20:5), detected by UV quench -1 and orange color with ferricyanide spray reagent; IR (Nujol, cm ) 3350 (NH), 3020 (CgH5), 2160 (C=N), 1650-1690 (C=0), NMR (methanol-d4,/from TMS) 7.22 (s, -CgH -^, 4.5-4.7 (3, -NH-CH, -C=0) , 4.0-4.2 (m, -NH-CH-C=0), 2.8-3, 2 (m, -CH_2-C6H5), 1.7-2.6 (m, cycl. 0=C-CH2-CH2-). Anal. Calculated for C15<H>16<N>4°3: C' 59.99, H, 5.37, N, 18.66. Found: C, 59.61, H, 5.61, N, 18.40. ;II. Evaluation of acylated cyanamides in vivo. ;Compounds _l- ijL b-*-e evaluated in rats for their ability to

to increase and maintain the level of blood acetaldehyde (AcH) after ethanol administration using the methodology shown in Table I.

Levels of blood AcH were measured one hour after ethanol (2 g/kg, ip)

in treated and control animals largely as described by Shirota et al. in J. Med Chem., 23, 669 (1980). The animal was paralyzed by a quick blow to the head and blood was immediately withdrawn by cardiac puncture with the chest open. Aliquots (0.2 ml) were placed in 20 ml serum bottles containing 1.0 ml of 5 mM sodium azide (to minimize the artifactual formation of AcH from ethanol) and the bottles were immediately sealed with a rubber membrane, frozen on dry ice, and kept frozen

-20°C until they were analyzed. AcH was determined in duplicate by the head-space gas chromatographic method previously described by Nagasawa et al. in Life Sei. 20 187

(1977), and quantified using a standard curve based on standards with known concentrations of AcH. The results of this investigation are shown in fig. 1-4. Cyanamid, a known potent AlDH inhibitor and positive control, increased blood AcH 150-fold over drug-free control samples at 3 hours, and more than 25-fold over control samples at 16 hours post-drug administration. Sodium acetylcyanamide, (1_) , the salt of the main urinary metabolite of cyanamide was, as expected, much weaker than cyanamide with respect to ethanol-induced increases in blood AcH, but the compound still significantly increased blood AcH at 3 hours (25-fold compared to comparison samples). Benzoyl cyanamide (2_) showed similar activity to 1_, giving rise to a somewhat greater increase in blood AcH (10 times that of the control samples) at 16 h than 1 .

n-Butyrylcyanamide (3_) with a four-carbon aliphatic acyl group was found to be a short-acting AlDH inhibitor and was even more potent than cyanamide itself. The compound increased/blood^AcH 170-fold relative to control samples at 3 hours, but the blood AcH levels were rapidly reduced to about 20-fold relative to control levels at 16 hours. Pivaloylcyanamide (4_)

and 1-adamantoylcyanamide (5), which was engineered to retard the rate of hydrolysis of the acylcyanamide bond by attaching sterically large substituents to the carbonyl group was found to be potent but short-lived.

Palmitoyl cyanamide (_6) and stearoyl cyanamide (]_), two aliphatic fatty acyl cyanamides and are almost as potent as cyanamide at 3 h and significantly higher blood AcH were still present at 16 h, namely 100-fold and 50-fold greater, respectively, than the comparison samples.

Pretreatment of rats with N-carbobenzoxycyanamide (<8) followed by ethanol raised blood AcH levels 120-fold relative to control samples at 3 hours, suggesting that the carbobenzoxy group must have been efficiently cleaved by an enzymatic process . N-Cbz-glycylcyanamide (9<_>) also raised blood AcH more than 80-fold relative to controls at 3 hours, but blood AcH was reduced to nearly control levels at 16 hours. N-Cbz-glycyl-L-leucylcyanamide (1_2) was more potent than (_9) at 3 h, but similarly to the latter, it did not maintain significantly increased levels of blood AcH at 16 h.

Hippurylcyanamide (1_0) was less potent than N-benzoyl-L-leucyl-cyanamide (11), probably because 10 is a poorer substrate for leucine aminopeptidase than 1_1 is. These compounds not only raise the levels of blood AcH at 3 hours, but also had a long-term effect with significant levels of blood AcH present at 16 hours.

N-Cbz-L-pyroglutamylcyanamide (13_) and L-pyroglutamyl-L-leucylcyanamide (14) were not nearly as potent as the other prodrugs. In contrast to _14, L-pyroglutamyl-L-phenylalanylcyanamide (1_5) was found to be a very potent inhibitor of AlDH' in vivo, giving rise to blood levels more than 110-fold higher than those of control samples at 3 h.

On the basis of the above results, the compounds 1'Z'i£ °9ii were selected for further investigations in vivo. Their duration of efficacy in increasing blood AcH was assessed over a 72 hour period according to the protocol described above.

As shown in fig. 5, the level of blood AcH by cyanamide reached a maximum within 2 hours, then the levels dropped rapidly, almost to the values of the control samples at 36 hours. When palmitoylcyanamide (6) and stearoylcyanamide (7_) were added in advance before ethanol, the levels of blood AcH reached their maximum value at about 5 hours, and then gradually declined, but the value of blood AcH produced by 6^ was significantly increased even at 48 hours. Hippurylcyanamide 1£ was not effective in maintaining high blood AcH beyond 12 hours. In contrast, N-benzoyl-L-leucylcyanamide (11) initially increased the level of blood AcH less than cyanamide at 2 h, but this compound continued to maintain high blood AcH even at 7?. hours.

When 6^ and LI were given orally, both compounds gave similar curves for increasing blood AcH, i.e. maximum AcH levels were observed at 2 hours, AcH levels rapidly decreased to a trough at 12 hours and increased then again at 24 hours (Fig. 6). Blood AcH then gradually fell back to control sample levels at approximately 36 hours.

Thus, a number of different acylated cyanamide analogs have been shown to be effective in substantially raising the levels of blood AcH in mammals in the presence of ethanol. A number of the analogs of the invention produced higher initial AcH levels and/or maintained the high AcH levels for longer periods than those produced by cyanamide itself. However, even relatively less potent or short-acting compounds may be useful clinically as alcohol deterrents if they are found to be more specifically effective than cyanamide itself and/or are properly assembled.

The invention has been described with reference to various particular and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made within the spirit and scope of the invention.

Claims (14)

1. Mixture comprising a compound of the formula: where R is (C^-C^^)-alkyl with 0-3 double bonds, aryl, cycloalkyl, benzyloxy or a group of the formula (R2)NHCH(R^)-, where R1 is H, benzyl or a (C3-Cg )-alkyl group and R 2 is benzoyl, N-carbobenzyloxy or L-pyroglutamyl and the pharmaceutically acceptable salts thereof. 2. Mixtures as stated in claim 1, characterized in that R is a C12-C22 alkyl group. 3. Mixture as stated in claim 2, characterized in that R is C17-n-alkyl, C15-n-alkyl or t-butyl. 4. Mixture as stated in claim 1, characterized in that R is benzyloxy. 5. Mixture as stated in claim 1, characterized in that R is cyclohexyl, norbornyl, cyclopentyl or adamantyl. 6. Mixture as stated in claim 1, characterized in that the group RCO is hippuryl, N-benzoyl-L-leucyl, (N-carbobenzyloxy)-glycyl-L-leucyl, (L-pyroglutamyl)-L-leucyl, (L-pyroglutamyl )-L-phenylalanyl or N-carbobenzyloxy-L-pyroglutamy1. 7. Mixture as specified in claim 1, characterized in that R is propyl. 8. Application of a compound with the formula: where R is (-C^Q)-alkyl with 0-3 double bonds, aryl, cycloalkyl, benzyloxy or a group of the formula (R^)NHCH(R^)-, where R^ is H, benzyl or a (C^ -C 6 )-alkyl group and R 2 is benzoyl, N-carbobenzyloxy or L-pyroglutamyl and the pharmaceutically acceptable salts thereof, for the preparation of a medicament for increasing the level of blood acetaldehyde in mammals. 9. Use of the compound as stated in claim 1, characterized in that R is a C12-C22<->alkyl group. 10. Use of the compound as stated in claim 8, characterized in that R is C1-n-alkyl, C1-5-n-alkyl or t-butyl. 11. Use of the compound as stated in claim 1, characterized in that R is benzyloxy. 12. Use of the compound as stated in claim 1, characterized in that R is cyclohexyl, norbornyl, cyclopentyl or adamantyl. 13. Use of the compound as stated in claim 1, characterized in that the group RCO is hippuryl, N-benzoyl-L-leucyl, (N-carbobenzyloxy)-glycyl-L-leucyl, (L-pyroglutamyl)-L-leucyl, (L -pyroglutamyl)-L-phenylalanyl or N-carbobenzyloxy-L-pyroglutamyl. 14. Use of the compound as stated in claim 1, characterized in that R is propyl.