AU609062B2 - Amino acid derivative anticonvulsant - Google Patents

Description

A LLO V /L D 4 1W 9062a COMMVONWEALTH OF AUSTRALIA PATENTS ACT 1952 Form COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE Class Int. Class Application Number: Lodged: Complete Specification-Lodged: Accepted: Published: *Priori ty: This docunment cOntafns the a. mrendrnernts idkde tirdcr Section 49 and iscofl-(.ct o P"ifling, Related Art: Name of Applicant: TO BE COMPLETED BY APPLICANT RESEARCH CORPORATION TECHNOLOGIES, INC.

Suite 853, 25 Broadway, New York, New York, United States of America Address of Applicant: HAROLD L. KOHN DARRELL WATSON Actual Inventor: Address for Service: S/ANDERCOCK, SMITH BEAD0LE 207 Riversdale Road, Box 410) Haw~thorn, Victoria, 3122 Complete Specification for the invention entitled: AMINO ACID DERIVATIVE ANTICONVULSANT The following statement is a full description of this invention, including the best method of performing it known to me:- 5352ZY

JEL-T

1 The present invention relates to compounds having central nervous system (CNS) activity which are useful in the treatment of epilepsy and other CNS disorders. More specifically, the compounds of this invention can be characterized as protected amino acid derivatives having the following general formula:

R

)2 R-NHfC-CNH+ C-R

(I)

11 1 n l 1 O R 0 3 wherein R and R 1 independently, are hydrogen, lower alkyl, 1 lower alkenyl, lower alkynyl, aryl lower alkyl, aryl, heterocyclic, lower alkyl heterocyclic, polynuclear aromatic, or lower alkyl polynuclear aromatic, each unsubstituted or S substituted with at least one substituent; oa R 2 and R independently, are hydrogen, lower alkyl, lower alkenyl, lower alkynyl, aryl lower alkyl, aryl, heterocyclic, lower alkyl heterocyclic, polynuclear aromatic or lower alkyl polynuclear aromatic, each unsubstituted or substituted with at least one substituent, halogen or a heteroatom containing oxygen, nitrogen, sulfur or phosphorous S substituted with hydrogen, lower alkyl or aryl, said lower .,25 alkyl or aryl groups being substituted or unsubstituted; and n is 1 to 4.

i The predominant application of anticonvulsant drugs S is the control and prevention of seizures associated with Sepilepsy or related central nervous system disorders.

Epilepsy refers to many types of recurrent seizures produced by paroxysmal excessive neuronal discharges in the brain; the r, _1 -2two main generalized seizures are petit mal, which is 1 associated with myoclonic jerks, akinetic seizures, transient loss of consciousness, but without convulsion; and grand mal which manifests in a continuous series of seizures and convulsions with loss of consciousness.

The-mainstay of treatment for such disorders has been the long-term and consistent administration of anticonvulsant drugs. Most drugs in use are weak acids that, presumably, exert their action on neurons, glial cells or both of the central nervous system. The majority of these compounds are characterized by the presence of at least one amide unit and one or more benzene rings that are present as a phenyl group or part of a cyclic system.

Much attention has been focused upon the development of anticonvulsant drugs and today many such drugs are well known. For example, the hydantoins, such as phenytoin, are useful in the control of generalized seizures "0 "4 and all forms of partial seizures. The oxazolidinediones, such as trimethadione and paramethadione, are used in the treatment of nonconvulsive seizures. Phenacemide, a phenyl- 0 acetylurea, is one of the most well known anticonvulsants 0 employed today, while much attention has recently been dedicated to the investigation of the diazepines and piperazines. For example, U.S. Patent Nos. 4,002,764 and 2 4,178,378 to Allgeier, et al. disclose esterified diazepine derivatives useful in the treatment of epilepsy and other °nervous disorders. U.S. Patent No. 3,887,543 to Nakanishi, et al. describes a thieno 4 j diazepine compound also having anticonvulsant activity and other depressant S activity. U.S. Patent No. 4,209,516 to Heckendorn, et al.

relates to triazole derivatives which exhibit anticonvulsant activity and are useful in the treatment of epilepsy and 1. -3conditions of tension and agitation. U.S. Patent No.

1 4,372,974 to Fish, et al. discloses a pharmaceutical formulation containing an aliphatic amino acid compound in which the carboxylic acid and primary amine are separated by three or four units. Administration of these compounds in an acid pH range are useful in the treatment of convulsion disorders and also possess anxiolytic and sedative properties.

Unfortunately, despite the many available pharma- 1 cotherapeutic agents, a significant percentage of the S 10 population with epilepsy or related disorders are poorly o° managed. Moreover, none of the drugs presently available are capable of achieving total seizure control and most have disturbing side-effects. Clearly, current therapy has failed to "seize control" of these debilitating diseases.

The present invention relates to compounds of the following general formula: a oooo

R

12 R-NH+CCNH+ C-R (I) II I "ni 1 0 OR 3

O

o43 0 S2 wherein R and R 1 independently, are hydrogen, lower alkyl, 25 lower alkenyl, lower alkynyl, aryl lower alkyl, aryl, heterocyclic, lower alkyl heterocyclic, polynuclear aromatic or lower alkyl polynuclear aromatic, each unsubstituted or substituted with at least one substituent;

R

2 and R independently, are lower alkenyl, lower alkynyl, Heterocyclic, lower alkyl heterocyclic, polynuclear aromatic IJSasSJm-.ujm.HM.B--mi".iiiiin iin.i I i iii i 11 i ii n-^iwyt^"- -4or lower alkyl polynuclear aromatic, each unsubstituted or 1 substituted with at least one substituent, halogen or a heteroatom containing oxygen, nitrogen, sulfur or phosphorous substituted with hydrogen, lower alkyl or aryl, said lower alkyl or aryl groups being substituted or unsubstituted; and n is 1 to 4.

The present invention contemplates employing the compounds of Formula I in compositions of pharmaceutically acceptable dosage forms. Where the appropriate substituents are employed, the present invention also includes L0 pharmaceutically acceptable addition salts. Moreover, the administration of an effective amount of the present compounds, in their pharmaceutically acceptable forms or the addition salts thereof, can provide an excellent regime for the treament of epilepsy, nervous anxiety, psychosis, insomnia and other related central nervous system disorders.

The alkyl groups exemplary of the substituents are o° ~lower alkyl containing from 1 to 6 carbon atoms and may be straight chain or branched. These groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, amyl, hexyl, and the like.

.o The aryl groups of R, R 1

R

2 and R 3 are aromatic compounds containing from 6 to 10 ring carbon atoms; and include phenyl,o(- and.9 -naphthyl. Moreover, the aryl Sgroups also include organometallic compounds wherein a metal S 25 2 or metalloidal atom is sandwiched between two aromatic compounds, cyclopendienyl compounds. Ferrocene is an example of this latter class of compounds.

The aryl lower alkyl groups include, for example, benzyl, phenethyl, phenpropyl, phenisopropyl, phenbutyl, and the like, diphenylmethyl, 1,1-diphenylethyl, 1,2-diphenylethyl, and the like.

i _L rrry~L~ The lower alkenyl and lower alkynyl groups contain from 2 to 6 carbon atoms and may be straight chain or branched.

Exemplary of the unsaturated alkyl substituents, lower alkenyl and lower alkynyl, are vinyl, acetylenic, allyl, propenyl, butenyl, pentenyl, hexenyl, propynl, butynl, pentynl, hexynl, pentadienyl, and the like.

The heterocyclic substituents contemplated by the present invention are N, O or S containing rings which may be monocyclic or bicyclic or tricyclic and which may contain up to 4 heteroatoms in the rings and which may contain up to 13 ring carbon atoms and up to a total of 18 carbon atoms.

These heterocyclic substituents include heteroaromatics and saturated and partially unsaturated heterocyclic compounds such as furyl, thienyl, pyranyl, pyrrolyl, imidazoyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidyl, pyridazinyl, indolyl, thiazolyl, oxazolyl, isothiazolyl, isoxazolyl, piperidyl, pyrrolinyl, piperazinyl, quinolyl, trizaolyl, o.n. tetrazolyl, and the like.

°20 The polynuclear aromatic substituents contemplated o .n 20 t herein are polyaromatic compounds containing up to 4 fused rings and containing up to 18 ring carbon atoms, for example, naphthyl, anthracenyl, phenanthrenyl, azulenyl, and the like.

The heteroatom containing substituents include, for example, methoxy, ethoxy, phenoxy, thiomethoxy, thioethoxy, °a 25 thiophenoxy, methylamino, ethylamino, anilino, dimethylamino, o o trimethylamino, fluoro, chloro, bromo, iodo, and the like.

The aryl groups such as phenyl, ferrocenyl, and the like, the alkyl groups, the aryl lower alkyl groups, the 't'3 lower alkenyl group, the lower alkynyl groups and heterocyclic, lower alkyl heterocyclic, polynuclear aromatic, Sand lower alkyl polynuclear aromatic may carry one or more -i i -6substituents which can be characterized as either electron 1 withdrawing groups such as halo, including bromo, fluoro, chloro, iodo, and the like, nitro, acyl, carboxyl, carboalkoxy, carboxamide, cyano, sulfonyl, sulfoxide, heterocyclic, guanidine, quaternary ammonium, and the like; or as electron donating groups such as hydroxy, alkoxy including methoxy, ethoxy, and the like, alkyl, amino, substituted amino, phenoxy, substituted phenoxy, thiol, sulfide, disulfide, and the like. One skilled in the art will appreciate that the aforesaid substituents may have electron donating or electron withdrawing properties under different chemical conditions. Moreover, the present invention contemplates any combination of substituents selected from the above-defined groups.

Preferred compounds of the present invention have the following general formula: 12 CH NHC-CNHC-R 20 0 R 0 j ou. wherein R is H or lower alkyl, R 2 and R 3 are as defined above and A is one to three substituents selected from the above-defined groups.

The alkyl groups of R 1 can be unsubstituted or o substituted with one or more substituents which can be characterized as either electron withdrawing groups or electron donating groups as defined above.

The alkyl groups of R 2 and R 3 including the alkyl portion of the aryl alkyl, or the alkyl heterocyclic and alkyl polynuclear aromatic groups, or the alkyl or aryl d" groups of the heteroatom containing substituents, as well as W i i i i :IU-e i I-.

-7the alkenyl, alkynyl, aryl, heterocyclic and polynuclear aromatic groups of R 2 and R 3 may also be unsubstituted or substituted with one or more substituents which can be characterized as either electron withdrawing groups or electron donating groups as defined above.

The preferred compounds of the present invention are those where n is 1 but di-, tri- and tetra-peptides are acceptable.

The compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. Depending upon the substituents, the present compounds may form addition salts as well. All of these forms are contemplated to be within the scope of this invention including mixtures of the stereoisomeric forms.

The following three schemes of preparation are generally exemplary of the process which can be employed for the preparation of the present complex: Scheme I yo o u« R OR a 2 12 HOOC-C-NH SOC1 excess R-NH-C-C-NH 1 2 2 T 2

R

3 MeOH RNH 2

R

3 3 2 0 0 OR 25 U 11 1 2 i R C-O-C-R RHN-C- -NH-C-R 1

R

3

O

4-44~mm4n4rw~ Scheme II

R

HOOC- C-NH 1 2 R 3 0 0 17 1 R 0 1 2 11 tIUUOG-C-NH-CR, 1 0 11 CiCOEt Et 3N O R 0 112 11 RNH- C -C -NHCR 1 RN

H

2 0Q0R 0 It it 2 11 EtOCOC-C-NH-CR 1 444.4.

4 4 444, 44 44 44 44' 44 44' ~O #44144 44 4444 20 .44 4. 4444# C0 2 EtOH Scheme III 0 11 R 1CNH2 00 1111I R2CCOH 0 OHO 11 1 11 R iCNH-C-COH 1 R 4

OH/H+

4 A 4 4n 0 OR j R 1

CNH-T--CNHR

RH2 R3H BF YOEt 2 0 RO0 R. CNH-C-CNHR 11 RNH 2 R1 CNH- T-CO 4 *I P1-u: -9- More specifically, these compounds can be prepared by art-recognized procedures from known compounds or readily preparable intermediates. For instance, compounds of Formula I can be prepared by reacting amines of Formula II with an acylating derivative of a carboxylic acid of Formula III under amide forming conditions: H OR R 11 12H ,2 R C-C-NH I n-l II 2 3 0 R3

II

0 R I

III

wherein R, R 1

R

2

R

3 are as defined hereinabove.

Alternatively, the compound of Formula I can be prepared by reacting an amine of Formula IV with an acylating derivative of a carboxylic acid of Formula V under amide forming conditions: orsc, o ra..

3 I' a 4 2 44r 4 S a 1 4 S R H OR 0 13 H 2H l| RNH +HOOC C R1> IV R 2

R

3 n-i V wherein R, R 1

R

2 and R 3 are as defined hereinabove.

Another useful method for preparing a compound of Formula I involves simple substitution reactions.

An exemplary procedure is as follows: 0 LO It H II R C-[N-C-C-NHR R L >I 1 n VI R VII wherein R, R 1

R

2

R

4 and n have the aforesaid meanings and

R

3 is defined heretofore except it is not aryl, heteroaromatic or polynuclear aromatic and L and L' are independently a good leaving group, such as halides, i tosylates, mesoylates, brosylates, benzyloxy and the like.

In this procedure the amine of Formula VI is reacted with a compound of Formula VII under substitution conditions. The reaction may take place in the presence of an acid, such as inorganic acid, hydrochloric acid, sulfuric acid or Lewis acid, such as boron trifluoride and the like or in the presence of a base, such as triethylamine.

However, when R 3 is heteroaromatic, aryl or polynuclear aromatic, L is hydrogen. In the procedure under these circumstances, the reaction should take place in the presence of an acid ca'.ilyst, such as an inorganic acid, hydrochloric acid or a Lewis acid, such as borontrifluoride.

The amide forming conditions referred to herein involve the use of ,-iown derivatives of the described acids, such as the acylhalides, R-C-X, 0 wherein X is Cl, Br, and the like), anhydrides 0 0 II II R1-C-O-C-R

I

mixed anhydrides, lower alkyl esters, °o carbodiimides, carbonyldiimidazoles, and the like. It is opreferred that the acylating derivative used is the S0 11 1 S anhydride, Ri-C-0-C-R 1 g As in any organic reaction, solvents can be employed such as methanol, ethanol, propanol, acetone, tetrahydrofuran, dioxane, dimethylformamide, dichloromethane, chloroform, and the like. The reaction is normally effected at or near room temperature, although temperatures from 0 C Sup to the reflux temperature of the reaction mixture can be employed.

a a hi~iiLL i -11- As a further convenience, the amide forming reaction can be effected in the presence of a base, such as tertiary organic amine, triethylamine, pyridine, picolines and the like, particularly where hydrogen halide is formed by the amide forming reaction, acyl halide and the amine of Formula II. Of course, in those reactions where hydrogen halide is produced, any of the commonly used hydrogen halide acceptors can also be used.

The exact mineral acid or Lewis acid employed in the reaction will vary depending on the given transformation, the temperature required for the conversion and the sensitivity of the reagent toward the acid in the reaction employed.

The various substituents on the present new compounds, as defined in R, R 1

R

2 and R 3 can be present in the starting compounds, added to any one of the intermediates or added after formation of the final products by the known methods of substitution or conversion reactions.

For example, the nitro groups can be added to the aromatic ring by nitration and the nitro group converted to other o20 o..O groups, such as amino by reduction, and halo by diazotization o of the amino group and replacement of the diazo group.

Alkanoyl groups can be substituted onto the aryl groups by 1 Friedel-Crafts acylation. The acyl groups can be then transformed to the corresponding alkyl groups by various methods, including the Wolff-Kishner reduction and Clemmenson reduction. Amino groups can be alkylated to form mono, dialkylamino and trialkylamino groups; and mercapto and hydroxy groups can be alkylated to form corresponding thioethers or ethers, respectively. Primary alcohols can be oxidized by oxidizing agents known in the art to form carboxylic acids or aldehydes, and secondary alcohols can be 4 CL-Lj I. F Z -I Ii -12oxidized to form ketones. Thus, substitution or alteration 1 reactions can be employed to provide a variety of substituents throughout the molecule of the starting material, intermediates, or the final product.

In the above reactions, if the substituents themselves are reactive, then the substituents can themselves be protected according to the techniques known in the art. A variety of protecting groups known in the art may be employed. Examples of many of these possible groups may be found in "Protective Groups in Organic Synthesis," by T.W.

Green, John Wiley Sons, 1981.

The present compounds obviously exist in stereoisomeric forms and the products obtained thus can be mixtures of the isomers, which can be resolved. Alternatively, by selection of specific isomers as starting compounds or synthetic intermediates, the preferred stereoisomer can be produced.

The active ingredients of the therapeutic compositions and the compounds of the present invention exhibit excellent anticonvulsant activity when administered °'2O o° ~in amounts ranging from about 10 mg to about 100 mg per kilogram cf body weight per day. A preferred dosage regimen for optimum results would be from about 20 mg to about 50 mg per kilogram of body weight per day, and such dosage units are employed that a total of from about 1.0 gram to about grams of the active compound for a subject of about 70 kg of body weight are administered in a 24-hour period. This dosage regimen may be adjusted to provide the optimum therapeutic response and is preferably administered one to 0 three times a day in dosages of about 600 mg per 3o administration. For example, several divided doses may be 44 44 S administered daily or the dose may be proportionally reduced 0 4

I.:

-13as indicated by the exigencies of the therapeutic situation.

A decided practical advantage is that the active compound may be administered in an convenient manner such as by the oral, intraveneous (where water soluble), intramuscular or subcutaneous routes.

The active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of Lhe diet. For oral i0 therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.

The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 5 and 1000 mg of active compound.

0 The tablets, troches, pills, capsules and the like 2 may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a 'ubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry e 4 4 flavoring. When the dosage unit form is a capsule, it may 444,44 S -14contain, in addition to materials of the above type, a liquid 1 carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit.

For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically 10 pure and substantially non-toxic in the amounts employed. In I0 addition, the active compound may be incorporated into sustained-release preparations and formulations.

The active compound may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) °20 0° or dispersions and sterile powders for the extemporaneous O preparation of sterile injectable solutions or dispersions.

In all cases the form must be sterile and must be fluid to o the extent that easy syringability exists. It must be stable OO under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as *arqal k 4 .i E~ u lecithin, by the maintenance of the required particle size in 1 the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the 10 compositions of agents delaying absorption, for example, i0 aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated S above. In the case of sterile powders for the preparation of a o°20 sterile injectable solutions, the preferred methods of mo~ preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any So additional desired ingredient from previously sterilefiltered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and 0 absorption delaying agents, and the like. The use of such o° media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media oo or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

i i- -16- SSupplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect 10 in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active material and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

The principal active ingredient is compounded for 1 convenient and effective administration in effective amounts 2 0 with a suitable pharmaceutically acceptable carrier in dosage 0j form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts U ranging from about 5 to about 1000 mg, with from about 250 to about 750 mg being preferred. Expressed in proportions, the active compound is generally present in from about 10 to about 750 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

For a better understanding of the present invention together with other and further objects, reference is made to the following description and example.

1: -17- EXAMPLE I 1 General Methods. Melting points were determined with a Thomas-Hoover melting point apparatus and are uncorrected. Infrared spectra (IR) were run on a Beckman IR-4250 and Perkin-Elmer 1330 spectrophotometer and -1 calibrated against the 1601-cm band of polystyrene.

Absorption values are expressed in wave numbers (cm 1 Proton nuclear magnetic resonance H NMR) spectra were recorded on Varian Associates Models T-60 and FT-80A, and Nicolet NT-300 NMR spectrometers. Carbon nuclear magnetic resonance C NMR) spectra were run on a Varian Associates Models FT-80A and Nicolet NT-300 instrument. Chemical shifts are in parts per million 6 values) relative to Me 4 Si, and coupling constants (J values) are in hertz. Mass spectral data were obtained at an ionizing voltage of 70 eV on a Hewlett-Packard 5930 gas chromatograph-mass spectrometer and a Bell-Howell 21-491 spectrometer. High-resolution (El mode) mass spectra were performed by Drs. James Hudson and John Chinn at the Department of Chemistry, University of Texas at a o Austin, on a CEC21-110B double-focusing magnetic-sector I spectrometer at 70 eV. Elemental analyses were obtained at Spang Microanalytical Laboratories, Eagle Harbor, MI.

a S° The solvents and reactants were of the best 2a B commercial grade available and were used without further purification unless noted. All anhydrous reactions were run under nitrogen, and all glassware was dried before use. In particular, acetonitrile and triethylamine were distilled from CaH 2 while dichloromethane was distilled from P205.

i Acetic anhydride, benzaldehyde and ethyl chloroformate were fractionally distilled.

4 4 0 i t rr1i -18- 1Preparation of N-Acetyl--D,L-alanine-N'-benzylamide.

Acetic anhydride (2.20 g, 0.022 mol) was slowly added to a methylene chloride solution (30 mL) of D,L-alanine-N-benzylamide (3.80 g, 0.021 mol) and allowed to stir at room temperature (3 h) The mixture was then successively washed with H 2 0 (15 mL), 1% aqueous NaQE (15 mL) and H 2 0 (15 mL) dried (Na 2 so 4 and concentrated in vacuo.

The residue was recrystallized from CH 2Cl 2 Yield: 2.50 g mp 139-141'C.

Hi NMR (DMSO-d 6 1.22 (d,J =7.1 Hz, 3H), 1.84 3H), 4.04-4.50 (in, 3H), 7.26 5H), 8.11 (br d,J =7.3 Hz, 1H) 8.42 (br t,J 6 Hz, 1H) l3 C NMR (DMSO-d 6 18.2, 22.4, 41.9, 48.2, 126.5, 126.9, 128.1, 139.4, 168.9, 172.4 ppm. IR (CHC 3 3440, 3300, 3005, 1660, 1515 cm Mass spectrum (CI. mode), m/e: 221 mol wt 220.1208 (Calculated for C 12

H

16

N

2 0 2 220.1212).

I0 t -19- Preparation of N-Acetyl-D- and L-amino acid-N-benzylamides.

1 General Procedure. The D- or L-amino acid amide (11 mmol) was dissolved in dichloromethane (15 mL) and then acetic anhydride (1.23 g, 1.40 mL, 12 mmol) was added dropwise. The solution was stirred at room temperature (18 h) and then concentrated to dryness. The residue was recrystallized from chloroform/hexane.

f i o 0 3 aa«3 N-Acetyl-D-alanine-N' -benzylamide.

Yield: 1.36 g mp 139-141 0

C.

1<23= +36.2 (c 2.5, MeOH).

1 0 H NMR (80 MHz, DMSO-d 6 d1.25 7.1 Hz, 3H) 1.86 (s, 3H), 4.10-4.50 (in, 1H), 4.30 (d,j 6.0 Hz, 2H), 7.26 5H), 8.09 (d,J =7.3 Hz, 1H), 8.40 (t,J 6.0 Hz, 1H).

13NMR (80 MHz, DMSO-d 6 18.3, 22.5, 42.0, 48.4, 126.6, 127.0 (2C) 128.2 (2C) 139.4, 169.2, 172.5 ppm.

LR (KBr): 3290, 1635 (br) 1540, 1455, 700, 695 cm Mass spectrum, m/e (relative Th;_tensity): 221 (30) 114 106 91 87 (100), 77 72 (2 65 Elemental analysis ~Calculated for C 12H 16N 20 2 65.42% C, 7.34% H; 12.72% N.

Found 65.31% C; 7.28% H; 12.63% N.

~4 4 3044 -21- 1N-Acetyl-L-alanile-N' -benzylamide.

Yield: 1.11 g mp 139-142 0

C.

Ic< 2 -35.3 (c 2.5, MeOH).

H NMR (80 MHz, DMSO-d 6 '1.23 (d,j 7.2 Hz, 3H), 1.86(s 3H), 4.26-4.35 (mn, 1H), 4.29 (d,J 5.8 Hz, 2H1), 7.22-7.33 5H) 8.10 (d,j 7.4 Hz, 1H) 8.42 (t,J= 5.8 Hz, 1H).

13NMR (80 MHz, DMSO-d 6 18.3, 22.6, 42.0, 48.4, 126.7, 127.0 128.3 139.5, 169.2, 172.6 ppm.

IR (KBr) 3290, 1635 1545, 1450, 700, 695 cm Mass spectrum, m/e (relative intensity) 221 (40) 114 106 (80) 106 (80) 91 (75) 87 (100) 77 72 analysis Calculated for 0 12H16 N2 02 65.42% C; 7.34t 11; 12.72% N.

Found 65.58% C; 7.32% H; 12.43% N.

00000 0 00 00 0 L~ I I r r

F

-22- Preparation of N-Acetyl-D,L-phenylglycine-N'-methylamide.

1 Acetic anhydride (2.90 g, 28 mmol) was added dropwise to D,L-phenylglycine-N-methylamid (3.4 g, 20 mmol) and allowed to stir at room temperature (1.5 During this time, a copious white precipitate formed. This material was collected by filtration, dried in vacuo and recrystallized from absolute alcohol.

Yield: 2.00 g mp 232-235 0 C (dec).

1H NMR (DMSO-d 6 1.89 3H), 2.58 (d,J 4.6 Hz, 3H), 5.42 (d,J 8.1 Hz, 1H), 7.35 5H), 8.18 (br q,J 4.2 Hz, 1H), 8.47 (d,J 8.1 Hz, 1H).

13C NMR (DMSO-d 6 22.4, 25.5, 56.3, 127.1, 127.3, 128.1, 139.0, 168.9, 170.3 ppm.

-1 IR (KBr): 3310, 1645 cm.

Mass spectrum (CI mode), m/e: 207 Elemental analysis Calculated for C 1

HI

4

N

2 0 2 64.06% C; 6.86% H; 13.58% N.

11 14 2 2 Found 63.79% C; 6.66% H; 13.27% N.

.o 94 4 4 3 i_ I -23- 000044 o 4 0000 0 00 O 0 4 *000 0 004* 4*00 0 a 0 00 00 a 004 Preparation of N-Acetylglycine-N-benzylamide.

The D,L-amino acid amide (11 mmol) was dissolved in dichioromethane (l5mL) and then acetic anhydride (.1.23 g, 1.40 mL, 12 mmol) was added dropwise. The solution was stirred at room temperature (4-6 h) and then concentrated to dryness. The residu'. was recrystallized from chloroform! hexane.

Yield: 1.84 g mp 140-1421C.

-0 HNMR (DMSO-d 6 3H), 3.74 (d,J 5.3 Hz, 2H), 4.30 (d,J 5.1 Hz, 2H), 7.27 5H), 8.37 (br s, 1H), 8.75 (br s, 1H).

13NMR (DMSO-d 6 22.5, 42.0, 42.5, 126.6, 127.1 (2C), 128.1 139.3, 169.0, 169.6 ppm.

IR (KBr): 3060, 1655, 1640, 1560, 1545, 1450, 1300, 740, 710 -5 cm Mass spectrum, m/e (relative intensity) 206 147 (12), 106 (100) 91 (75) 73 Elemental analysis 20 ~Calculated for C 11

H

14

N

2 0 2 64.05% C; 6.86% H; 13.58% N.

Found 64.03% C; 6.79% H; 13.61% N.

0000 0 0 0000 0000 0 00 00 0 00 0 0 0 4 0 04 000004 0 S 4444 4441 14 9 7, r I -i I' I i i- i. I -24- Preparation of N-Acetyl-D,L-valine-N-benzylamide.

The D,L-amino acid amide (11 mmol) was dissolved in dichloromethane (15mL) and then acetic anhydride (1.23 g, 1.40 mL, 12 mmol) was added dropwise. The solution was stirred at room temperature (4-6 h) and then concentrated to dryness. The residue was recrystallized from chloroform/ hexane.

Yield: 2.35 g mp 192-193 0

C.

1 H NMR (DMSO-d 6 0.83 (d,J 6.7 Hz, 6H), 1.87 3H), 1.73-2.09 1H), 4.11 (d,J 8.8 Hz, 1H), 4.27 (d,J o 4 5.8 Hz, 2H), 7.26 5H), 7.89 (d,J 8.8 Hz, 1H) 8.84 4.44 (t,J 5.8 Hz, 1H).

o 1C NMR (DMSO-d 6 18.1, 19.2, 22.4, 30.2, 41.9, 57.8, 126.6, 127.1 128.1 139.4, 169.2, 171.1 ppm.

15 -1 IR (KBr): 1625, 1540, 1535, 1450, 1380, 1290, 750, 695 cm Mass spectrum, m/e (relative intensity): 142 114 (43), 106 91 72 (100).

Elemental analysis -oo Calculated for C 14 H 20N202 67.70% C; 8.13% H; 11.28% N.

o 0 Found 67.58% C; 8.05% H; 11.10% N.

14' a I I1 Lb C rsi~up3lrr*- I~ll suP---r Preparation of N-Acetyl-D,L-phenylglycine-N'-benzylamide.

The D,L-amino acid amide (11 mmol) was dissolved in dichloromethane (15mL) and then acetic anhydride (1.23 g, 1.40 mL, 12 mmol) was added dropwise. The solution was stirred at room temperature (4-6 h) and then concentrated to dryness. The residue was recrystallized from chloroform/ hexane.

Yield: 2.05 g mp 202-203 0

C.

SH NMR (DMSO-d) f1.91 3H), 4.27 (d,J 5.6 Hz, 2H), 6 5.50 (d,J 7.9 Hz, 1H), 7.21 5H), 7.36 1 8.38-8.86 2H).

1C NMR (DMSO-d 6 22.3, 42.0, 56.3, 126.6 127.0, 127.1 127.4 128.1 138.9, 139.0, 168.9, 169.9 ppm.

-1 IR (KBr): 3020, 1635, 1580, 1540, 1450, 1265, 745, 690 cm Mass spectrum, m/e (relative intensity): 283 264 (21), 149 (100), 131 118 106 91 79 77 65 51 (37).

Elemental analysis 0 20 Calculated for C 17

H

8 N202 72.31% C; 6.44% H; 9.92% N.

Found 72.49% C; 6.47% H; 9.89% N.

0 o o 1 -26- 1 Preparation of N-Acetyl-D- and L-phenylglycine-N-benzylamide.

General Procedure. The chiral Boc-protected phenylglycine-N-benzylamide was dissolved in trifluoroacetic acid (0.04 M) and was stirred at room temperature (30 min), during which tie 9as evolved. The solution was concentrated in vacuo and the residue was redissolved in enough methanol to form a solution of 0.2 M. Methanesulfonic acid (1 equiv) was added dropwise and stirred for 5 min. After concentrating the solution in vacuo, the residue was 10 -repeatedly dissolved in methanol and the solvent was removed (3 times). The residue was then dried under vacuum (18 h), f' leaving a yellow oil.

Without further purification, the phenylglycine-Nbenzylamide methanesulfonate was dissolved in tetrahydrofuran (0.2 M) and then was cooled in an ice bath. Triethylamine (2 equiv) was added dropwise, followed by acetyl chloride (1 equiv). The ice bath was removed and stirring was continued at room temperature (18 The solution was concentrated in vacuo and the residue was recrystallized from e 1:1 95% ethanol/water.

a a 4o a 4 i i. i-J.

-L I -27- N-Acetyl-D-phenylglycine-N-benzylamide.

The reaction was run on an 11.9 mnol scale.

Yield: 2.97 g rnp 219-221'C.

-103.0 (c EtOH).

1 DH,2) H 8.56SOd191(,3H,42 (d,J 5.50 (Md 7. 22., 42, 564-.4 126., 127, (2C) 7.8 189 1391).0, =55 z 1) 1-3 CR 3260, 12,2, 40, 130, 720., 6970 Ca2 c late (2 o C 1 2 7 1 2 2 C) 7 2.32% C; C) 6. 3 8; .9 2% N3 .0 Found9 72.04% C;622p; .8%N

A-

IR 04-: 3 6 6 0 5 5 1 5 ,1 7 2 ,6 0c Massetu e(eaieinest)40 4 9) 10200 ,91 (2 ,8 43 ,7 1 Elmna aayi A1 204 j .1 's~ii r~ -28- N-Acetyl-L-phenylglycine-N-benzylamide.

1 Beginning with 16.1 mmol N-t-Boc-L-phenylglycine- N-benzylamide.

Yield: 2.99 g mp 221-222 0

C.

[C1D +105.1 (c EtOH).

1 H NMR (DMSO-d 6 1.99 3H), 4.36 (d,J 5.6 Hz, 2H), 5.60 (d,J 8.0 Hz, 1H), 7.23-7.53 10H), 8.60 (d,J Hz, 1H), 8.83 (t,J 5.6 Hz, 1H).

13 S1C NMR (DMSO-d 6 22.4, 42.1, 56.5, 126.8, 127.1 (2C), 127.3 127.5, 128.2 139.0, 139.1, 169.1, 9 .o •170.1 ppm.

-1 IR (KBr): 3295, 1630, 1530, 1450, 1395, 720, 695 cm Mass spectrum, m/e (relative intensity): 223 203 149 106 (100), 91 86 77 (11).

S 15 Elemental analysis Calculated for C1 H1 N202 72.32% C; 6.43% H; 9.92% N.

Found 72.53% C; 6.49% H; 9.67% N.

ooo 300 -29- Preparation of N-Acetyl-D,L-alanine-N- (3-methoxy)benzylamide.

The D,L-amino acid amide (11 minol) was dissolved in dichloromethane (l5mL) and then acetic anhydride (1.23 g, 1.40 mL, 12 mmol) was added dropwise. The solution was stirred at room temperature (4-6 h) and then concentrated to dryness. The residue was recrystallized from chloroform/ hexane.

Yield: 0.47 g mp 112-1151C.

1 H NMR (DMSO-d 6 1r.

23 (d,j 7.1 Hz, 3H), 1.85 3H), e* 106 3.73 3H), 3.99-4.48 (in, 1H), 4.25 (d,J 6.1 Hz, 2H), 6.58-7.35 (mn, 4H), 8.05 (d,J 7.4 Hz, 1H), 8.35 (t,J =6.0 Hz, 1H).

13NMR (DMSO-d 6 18.1, 22.5, 41.8, 48.3, 54.9, 112.2, 15 112.3, 119.0, 129.2, 141.0, 159.3, 169.0, 172.4 ppm.

'S IR (KBr) 3270, 3065, 1625, 1580, 1450, 1260, 1150, 1095, -1 900, 775, 700, 690 cm Elemental analysis Calculated for C 13H 18N 20 362.37% C; 7.26% H; 11.19% N.

200 4 Found 62.29% C; 7.13% H; 11.08% N.

0 I I I Preparation of N-Trimethylacetyl-D, L-alanine-N'-benzylamide.

D,L-Alanine-N-benzylamide (3.56 g, 20 mmol) was dissolved in dichioromethane (25 mL) and trimethylacetic anhydride (4.10 g, 4.46 mL, 22 mmol) was added dropwise. The solution was stirred at room temperature (18 h) an-d then concentrated to dryness. The solid residue was recrystallized from benzene/petroleum ether (30-60'C).

Yield: 2.07 g mp 123-1241C.

NMR (DMSO-d 6 )5 1. 12 9H) 1. 27 J 7. 1 Hz, 3H), 4.23-4.42 111), 4.31 (d,J 5.4 Hz, 211), 7.23-7.30 (in, 5H1), 7.38 (d,J 7.4 Hz, lH) 8.26 (t,J =5.4 Hz, 1H1).

13C NMR (DMSO-d 6 18.1, 27.2 (30) 37.9, 42.0, 48.4, 126.6, 127.0 128.2 139.4, 172.5, 177.1 ppm.

IR (KBr) 3300, 1630, 1535 (br) 1455, 745, 695 cm Mass spectrum, m/e (relative intensity): 262 203 (19), 156 (18) 128 (51) 106 (31) 91 (100) 77 65 (28).

20 Elemental analysis Calculated for C015H22 N2 02 68.66% C; 8.47% H; 10.68% N.

Found 68.91% C; 8.14% H; 10.61% N.

*0 e 0*110 11 11 *0 4 0*fl~Zt~ -31- Preparation of N-Acetyl-D,L-methionine-N-benzylamide.

N-Acetyl-D,L-methionine (4.78 g, 25 mmol) was combined with acetonitrile (75 mL) and the mixture was placed into an ice/salt water bath Triethylamine (2.53 g, 3.48 mL, 25 mmol) was added dropwise, followed by ethyl chloroformate (2.71 g, 2.39 mL, 25 mmol). All additions were done slowly so that the temperature of the mixture did not rise above 0°C. The mixture was then stirred at -5°C min). Benzylamine (3.00 g, 3.06 mL, 28 mmol) in acetonitrile (5 mL) was added dropwise and the mixture was stirred at -5 C (1 h) 'and then room temperature (18 h).

The mixture was filtered and a white precipitate was collected and dried in vacuo and identified as the desired product (1H NMR and 13C NMR analyses). The filtrate was concentrated in vacuo and the residue was combined with hot tetrahydrofuran (50 mL) and cooled in the freezer (3 h), resulting in the formation of a white precipitate. The mixture was filtered and the precipitate was collected, dried in vacuo, and identified as triethylammonium hydrochloride.

.0 The latter filtrate containing tetrahydrofuran was 20 concentrated in vacuo and the resulting residue was purified by flash column chromatography (ethyl acetate). A white solid (Rf 0.50, ethyl acetate) was isolated and was identified as the desired product H NMR and 13C NMR analyses). The two solids identified as N-acetyl-D,Lmethionine-N-benzylamide were combined and recrystallized from benzene/petroleum ether (30-60 0

C).

Yield: 2.98 g mp 134-135 0

C.

1 H NMR (DMSO-d 6 :I 1.69-1.94 2H) 1.87 3H), 2.02 (s, 3H) 2.29-2.59 2H), 4.10-4.53 1H), 4.29 (d,J Hz, 2H), 7.26 5H), 8.12 (d,J 8.5 Hz, 1H), 8.47 (t,J 6.0 Hz, 1I).

-32- 13 C NMR (DMSO-d 6 14.6, 22.5, 29.7, 31.8, 42.0, 52.0, 126.6, 127.0 128.2 139.4, 169.5, 171.4 ppm.

IR (KBr): 3280, 1630, 1545, 1460, 750, 700 cm Mass spectrum, m/e (relative intensity): 280 206 (100), 164 (29) 146 (20) 106 (54) 91 (76) 77 (14) 65 (24).

Elemental analysis Calculated for C 1 4

H

2 0

N

2 0 2 S 59.96% C; 7.20% H; 9.99% N.

Found 60.02% C; 7.14% H; 9.91% N.

4 0 T. 0C w -33- SPreparation of N-Acetylalanine-N'-(3-fluoro)benzylamide.

N-Acetylalanine (3.28 g, 25 mmol) was combined with acetonitrile (100 mL) and the mixture was placed into an ice/salt bath at -5 0 C. Triethylamine (2.53 g, 3.5 mL, mmol) was added dropwise followed by the addition of ethyl chloroformate (2.71 g, 2.40 mL, 25 mmol). All additions were done slowly so that the temperature of the mixture did not rise above 0°C. The mixture was then stirred at -5 0 C for minutes. 3-Fluorobenzylamine (3.58 g, 28 mmol) and acetonitrile (5 mL) was added dropwise and was stirred at 0 C for one hour and then at room temperature for 18 hours.

The reaction became homogeneous during this time interval.

The solution was concentrated in vacuo and the residue was combined with hot tetrahydrofuran (100 mL) and cooled in the freezer for 3 hours resulting in the formation of a white precipitate. The mixture was filtered and the precipitate was collected, dried in vacuo and identified as triethylammonium hydrochloride (3.51 g, mp 253-257°C). The ,o filtrate was concentrated in vacuo and the resulting yellow solid was recrystallized from chloroform/diethyl ether.

S Yield: 3.22 g (54%) S* mp 120-121 0

C.

H NMR (L)MSO-d 6 f 1.27 (d,J 7.1 Hz, 3H), 1.90 3H), 4.233-4.41 1H), 4.33 (d,J 6.1 Hz, 2H), 7.05-7.37 (mi, 4H), 8.19 (d,J 7.1 Hz, 1H), 8.53 (t,J 6.1 Hz, 13 C NMR (DMSO-d 6 17.9, 22.4, 41.5, 48.5, 113.3 (d,J 20.4 113.5 (d,J 21.7 Hz), 122.8, 130.1 (d,J 7.9 Hz), 1i 4 4 (d,J 7.4 Hz), 162.3 (d,J 243.6 Hz), 169.6, 172. 8 ppm.

-1 IR 3280, 1645, 1545, 1450, 745, 680 cm Mb -34- Mass spectrum, m/e (relative intensity): 238 151 (22), 124 114 109 (100), 87 72 (27).

Elemental analysis Calculated 60.48% C; 6.36% H; 11.76% N.

Found 60.55% C; 6.32% H; 11.71% N.

*o a a 91 ua 0 4 4 ~ror~i*r~i~nslrp-rroI Preparation of D,L-40-Acetamido-N-benzyl-3-thiopheneacetamide D,L-o -Acetamido-3-thiopheneacetic acid (2.99 g, mmol) was combined with acetonitrile (60 mL) and the mixture was placed into an ice/salt water bath (1.51 g, 2.10 mL, 15 mmol) was added dropwise, followed by ethyl chloroformate (1.63 g, 1.43 mL, 15 mmol).

All additions were done slowly so that the temperature of the mixture did not rise above 0°C. The mixture was then stirred at -5°C (20 min). Benzylamine (1.77 g, 1.80 mL, 16.5 mmol) in acetonitrile (10 mL) was added dropwise and the mixture was stirred at -50C (1 h) and then room temperature (18 h).

The mixture was concentrated in vacuo and the residue was combined with hot tetrahydrofuran (50 mL) and cooled in the freezer (3 resulting in the formation of a white 1 precipitate. The mixture was filtered and the precipitate was collected, dried in vacuo, and identified as triethylammonium hydrochloride H NMR analysis). The filtrate was concentrated in vacuo and the resulting yellow solid was recrystallized from 1:1 95% ethanol/water.

20o Yield: 1.91 g mp 198-1990C.

1H NMR (DMSO-d 6 1.91 3H), 4.29 (d,J 5.2 Hz, 2H), 5.61 (d,J 7.9 Hz, 1H), 7.15-7.50 3H), 8.55 (d,J 7.9 Hz, 1H), 8.74 (t,J 5.2 Hz, 1H).

13C NMR (DMSO-d 6 22.3, 42.0, 52.5, 122.4, 126.1, 126.7, 127.0 128.2 139.0, 139.2, 169.0, 169.8 ppm.

IR IR (KBr): 3460, 1675, 1570, 1400, 720, 695 cm- 1 Mass spectrum, m/e (relative intensity): 288 245 155 112 (100), 91 85 65 Elemental analysis Calculated for C 5H 6N202S 62.48% C; 5.59% H; 9.71% N.

Found 62.41% C; 5.47% H; 9.55% N.

is i- r -36- Preparation of D,L-0 -Acetamido-N-benzyl-2-thiopheneacetamide 1 N-Acetyl-D,L-ethoxyglycine-N-benzylamide (6.26 g, mmol) was combined with dry ether (175 mL) and then boron trifluoride etherate (5.68 g. 5.0 mL, 40 mmol) was added dropwise, resulting in a homogeneous solution. After stirring a short time, a small amount of a yellow oil separated from the solution. Thiophene (8.41 g, 8.0 mL, 100 mmol) was then added dropwise via syringe and the reaction was stirred at room temperature (4 The mixture was cooled in an ice bath and cold aqueous saturated NaHCO 3 (200 mL) was added and the aqueous layer was extracted with ethyl acetate (2 x 100 mL). The organic washings and the original ether layer were combined, dried (Na 2

SO

4 and concentrated in vacuo. The residue was purified by flash column chromatography, using 94:6 chloroform/methanol as an eluant (Rf 0.7 94:6 chloroform/methanol), and then recrystallized from benzene.

Yield: 2.67 g mp 167-169 0

C.

1 H NMR (DMSO-d 6 1.91 3H) 4.31 (d,J 6.0 Hz, 2H), 0 .20 6 SV 5.74 (d,J 7.9 Hz, 1H), 6.99-7.44 8H), 8.64 (d,J 7.9 Hz, 1H), 8.85 (t,J 6.0 Hz, 1H).

13 C NMR (DMSO-d 6 22.4, 42.3, 52.2, 125.6, 125.8, 126.6, o 126.9, 127.3 128.3 139.0, 141.4, 169.2, 169.3 ppm.

Mass spectrum, m/e (relative intensity): 289 181 155 (100), 112 (100), 91 (100), 85 74 (24).

Elemental analysis 64 Calculated for C15H N202S 62.48% C; 5.59% H; 9.71% N.

Found 62.64% C; 5.73% H; 9.61% N.

o: 0 a 4 0 a0S cl~ L C -I -37- Preparation of D,L-0 -Acetamido-N-benzyl-2-furanacetamide.

N-Acetyl-D,L-2-(2-furyl)glycine (0.47 g, 2.56 mmol) was combined with acetonitrile (10 mL) and cooled to (ice/salt water bath). Triethylamine (0.26 g, 0.36 mL, 2.56 mmol) was then rapidly added and the mixture stirred at (3 min). Ethyl chloroformate (0.28 g, 0.25 mL, 2.56 mmol) was added dropwise beween -4 0 C and and the resulting suspension was stirred at -4°C (20 min), and then an acetonitrile solution (2 mL) of benzylamine (0.30 g, 0.31 mL, 2.82 mmol) was carefully added. During the addition of benzylamine the temperature of the solution did not go above 0°C. The mixture was stirred at -5°C (1 h) and at room temperature (18 and then concentrated in vacuo. The residue was then triturated with hot tetrahydrofuran (5 mL), cooled at -16°C (3 and the resulting white precipitate was filtered and identified as triethylamine hydrochloride 1 H NMR, 60 MHz, 1.00 (t,J 7.5 Hz, CH 3 2.82 (q,J Hz, CH 2 3.83 The filtrate was evaporated to 0o dryness in vacuo and the resulting oil purified by flash chromatography (98:2 chloroform/methanol) to give 0.09 g of the desired product as a white solid: Rf 0.30 (98:2 chloroform/methanol).

mp 178-179 0

C.

1 NMR (300 MHz, DMSO-d 6 1.90 CH 3 4.31 (d,J Hz, CH 2 5.58 (d,J 8.1 Hz, CH), 6.27-6.33 C 6.40-6.44 C4'H), 7.20-7.36 Ph), 7.60-7.64 (m,

C

5 8.57 (d,J 8.1 Hz, NH), 8.73 (t,J 6.0 Hz, NH).

S13C NMR (300 MHz, DMSO-d) 22.35 (CH 3 42.27 (CH 2 50.95 0 107.60 (C 3 110.55 (C 4 126.82 or 2C 3 127.08 (2C 2 or 2C 128.27 (C 4 139.05

(C

1 142.58 (C 151.16 (C 2 168.02 (CH 3 CO), 169.30 (NHCO) ppm.

0 a4 4 -38- IR (KBr): 3230, 1625 (br) 1525 (br) 1375 (br) 1230, 1090, 890 cm-1 Mass spectrum, in/e (relative intensity): 273 139 (100), 96 (94) 91 (51) 65 (9) Elemental analysis Calculated for C 15

H

16

N

2 0 3 66.16% C; 5.83% H; 10.29% N, Found 65.92% C; 5.83% H; 10.15% N.

4000 ~2O 000 0 0000 00 0 00 1 C

I

L.

-39- Preparation of D,L--Acetamido-N-benzyl-2-pyrroleacetamide.

2-Acetamido-N-benzyl-2-ethoxyacetamide (2.00 g, mmol) was suspended in anhydrous ethyl ether (60 mL), and then boron trifluoride etherate (1.82 g, 1.57 mL, 12.8 mmol) was added in one portion and the resulting solution was stirred (15 min). The pyrrole (2.14 g, 2.22 mL, 32 mmol) was then added in one portion and the solution was stirred at room temperature (48 h) during which time a precipitate formed. Hexanes (80 mL) were then added to the suspension, and the mixture was filtered and the brown semi-solid was triturated with 95:5 chloroform/methanol (30 mL) to furnish a green solid. This material was purified by flash chromatography (95:5 chloroform/metnanol) to yield 0.94 g of the desired product as a white solid: Rf 0.29 (9( chloroform/methanol).

mp 174-175°C.

H NMR (300 MHz, CD3CN):, 1.93 CH 3 4.35 (d,J 6.0 Hz,

CH

2 5.42 (d,J 6.9 Hz, CH), 6.00-6.18 C 3

'H,

C

4 6.68-6.72 C 7.04 (d,J 6.9 Hz, NH), 2 7.17 (t,J 6.0 Hz, NH), 7.10-7.47 Ph), 9.10-9.80 (br s, NH).

3 C NMR (300 MHz, CD CN): 23.02 (CH 3 43.83 (CH 2 52.65 o O(CH), 107.57 108.85 (C 4 119.33 127.96 %on 128.01 (2C2" or 2C3"), 128.09 (2C2" or 2C 3 129.49

(C

4 140.01

(C

1 170.94

(COCH

3 171.21 (CONH) ppm.

IR (KBr): 3320, 1570 1470 1330, 1230, 950, 890, 860, 760, 710, 690, 655 cm-1 i Mass spectrum, m/e (relative intensity): 171 228 30 213 180 164 137 108 95 (100), f 30 3.0. 91 82 68 High resolution mass spectral analysis Calculated for C H17N302 271.13208.

Found 271.13144.

i .1

J-

1Preparation of D,L-2-Acetamido-N-benzyl-2-ethoxyacetamide.

An ethanolic solution (420 mL) of ethyl 2-acetamido-2-ethoxyacetate (27.92 g, 147 mmol) and benzylamine (23.70 g, 24 mL, 221 mmol) was stirred at 40-45 0

C

for 3 days. The reaction mixture was evaporated in vacuo and the residue recrystallized (1:3.5 tetrahydrofuran/hexanes (650 mL)) to yield 25.80 g of the desired product as beige crystals: Rf 0.59 (95:5 chloroform/methanol).

mp 153-155 0

C.

1H NMR (300 MHz, CDC1 3 1.20 (t,J 7.0 Hz, CH 3 2.07 (s,

CH

3 3.60-3.76 CH 2

CH

3 4.40-4.54 CH 2 NH), 5.60 (d,J 8.7 Hz, CH), 6.63 (d,J 8.7 Hz, NH), 7.00 (br s, NH), 7.26-7.36 Ph).

C NMR (300 MHz, CDC1 3 15.06 (CH 3

CH

2 23.25 (CH 3

CO),

15 43.60 (CH 2 NH), 64.51 (CH 2

CH

3 77.43 127.69 (2C2" 2 or 2C 3

C

4 128.79 (2C 2 or 2C 3 137.57 (C 1 168.13 (COCH 3 171.29 (CONH) ppm.

IR (KBr): 3260, 1630 1550 1505 1380, 1360, -1 1230, 1115, 1060, 1015, 890, 745, 690 cm 20. Mass spectrum, m/e (relative intensity): 251 163 ao i20 116 106 91 74 (100).

Elemental analysis I I: Calculated for C13H18N203 62.38%. C; 7.25% H; 11.19% N.

S r Found 62.49% C; 7.27% H; 11.24% N.

So o 1. 1. -41- Preparation of D,L-2-Acetamido-N-benzyl-2-methoxyacetamide.

To a methanolic solution (180 mL) of methyl 2-acetamido-2-methoxyacetate (8.73 g, 54 mmol) was rapidly added benzylamine (8.68 g, 8.80 mL, 81 mmol) and then the mixture was stirred at 50°C (3 days) during which time a beige precipitate appeared. The solvent was removed in vacuo and the resulting precipitate was recrystallized from tetrahydrofuran (2x) to give 7.67 g of the desired product as beige crystals: Rf 0.35 (95:5 chloroform/methanol).

mp 145-146 0

C.

1H NMR (300 MHz, CDC1 3 2.06 CH 3 CO), 3.37 4.40-4.35 CH2 5.52 (d,J 8.7 Hz, CH), 7.12 (d,J 8.7 Hz, NH), 7.20-7.40 Ph, NH).

13 C NMR (300 MHz, CDC1 3 23.03 (CH 3 CO), 43.51 (CH 2 55.84

(CH

3 78.94 127.62 (C 4 127.70 (2C 2 or 2C 3 128.70 (2C 2 or 2C 3 137.45 (C 1 166.91

(COCH

3 171.57 (CONH) ppm.

IR (KBr): 1260, 1825 1550, 1505, 1435, 1390, 1370, -1 1230, 1120, 1050, 935, 890, 690 cm Mass spectrum, m/e (relative intensity): 237 205(2), o S 177 163 146 134 121 106 (26), ,i 102 91 77 61 (100).

Elemental analysis I CIA Calculated for C 2

H

16 N203 61.00% C; 6.83% H; 11.86% N.

60.91% C; 6.85% H; 11.66% N.

ii ic r -42- 1 Pharmacology. The following compounds were tested for anticonvulsant activity using male Carworth Farms #1 mice: N-Acetyl-D ,L-alanine-N' -benzylamide N-Acetyl--D-alanine-N' -benzylamide N-Acetyl-L-alanine-N '-benzylamide N-Acetyl-D, L-phenylglycine-N'-methylamide N-Acetylglycine--N-benzylamide N-Acetyl-D, L-valine-N-benzylamide N-Acetyl-D, L-phenylglycine-N--benzylamide N-Acetyl-D-phenylglycine-N--benzylamide N-Acetyl--L-phenylglycine-N-benzylamide N-Acetyl-D, L-alanine-N- (3-methoxy) benzylamide N-Trimethylacetyl-D ,L-alanine-N-benzylamide N-Acetyl-D,L-methionine-N-benzylamide N'-Acetyl-D, L-alanine-N' -(3-fluoro) benzylamide D,L- oL-Acetamido-N-benzyl-3-thiopheneacetamide D oL-Acetamido-N-benzyl-2-thiopheleacetamfide D o(-Acetamido-N-benzyl-2- furanacetamide D,L- ix-Acetamido-N-benzyl-2-pyrroleacetamide 0 D,L-2-Acetamido-N-benzyl-2-ethoxyacetamide D ,L-2-Acetamido-N-benzyl-2-methoxyacetamide The compound was given in three dose levels (30, 100, 300 mg) and subsequently compared with phenytoin, phenobarbital, mephenytoin and phenacemide (see Table I) N-Acetyl-D,Lalanine-N'-benzylamide was tested at 600 mg/mL as well.

Seizures were then artificially induced by either electroshock or pentylenetetrazole. Maximal electroshock seizures (MES) were elicited with a 60 cycle alternating current of 50 mA intensity (5-7 times that necessary to 30 elicit minimal electroshock seizures) delivered for 0.2 sec via corneal electrodes. A drop of 0.9% saline was instilled t..

-43in the eye prior to application of the electrodes so as to 1 prevent the death of the animal. Protection in this test was defined as the abolition of the hind limb tonic extension component of the seizure. The Subcutaneous Pentylene-

R

tetrazole (Metrazol Seizure Threshold Test (sc Met) entailed the administration of 85 mg/kg of pentylenetetrazole as a 0.5% solution subcutaneously in the posterior midline.

This amount of pentylenetetrazole was expected to produce seizures in greater than 95% of mice. The animal was observed for 30 minutes. Protection was defined as the failure to observe even a threshold seizure (a single episode of clonic spasms of at least 5 sec duration). The effects of the compounds on forced and spontaneous motor activity were evaluated in mice using the Rotorod Test (Tox) or, in some cases, as indicated herein, a horizontal screen assay (HS) The animal was placed on a one-inch diameter knurled plastic rod rotating at 6 rpm after the administration of the drug.

Normal mice can remain on a rod rotating at this speed S indefinitely. Neurologic toxicity was defined as the failure of the animal to remain on the rod for one minute. The MES 2 00 and sc Met Tests were conducted on single animals while four .0.0 mice were utilized for the Tox Test. The dose effect S behavior of the compounds was evaluated using the 00 above-described procedures by the administration of varying Sdose levels, treating normally eight mice at each dose.

Table I includes an evaluation of the Median Effective Dose i 4 (ED50) and the Median Toxic Dose (TD50) of representative i compounds. Mice were tested with varying doses of the anticonvulsant to define the limits of complete protection 30 (or toxicity) and no protection (or no toxicity), as well as three points in between these limits. The Median Effective Dose (ED50) was defined as the dose which produced the i 2-44- 1 desired endpoint in 50% of the animals. The Median Toxicity Dose (TD5O) was the dose which elicited evidence of minimal neurological toxicity in 50% of the animals.

N-Acetyl-D ,L-phenylglycine-N' -benzylamide, D,L-0/ -acetamido- N-benzyl-2-furanacetamide, D,L-O< -acetamido-N-benzyl-2pyrroleacetamide exhibited anticonvulsant activity at doses less than 33 mg/kg. As indicated in Table I, N-acetyl-D,Lphenylglycine-N' -benzylamide, N-acetyl-D,L-alanine-N' benzylamide and their D isomers, as well as D,L-0 -acetamido- N-benzyl-2-thiopheneacetamide, P,L-9&-acetamido-N-benzyl-2furanacetamide, D ,L-dO -acetamido-N--benzyl-2-pyrroleacetanide and D,L-2-acetamido-N-benzyl-2-ethoxyacetamide, possessed values in the MES test that compared well with phenobarbital, while their TD5Os were of similar magnitude or considerably higher. Only the ED50 (sc Met) of N-acetyl-Dalanine-N'-benzylamide was computed while the TD50 of N-acetyl-D,L-phenylglycine-N' -benzylamide, N-acetyl-Dphenylglycine-N' -benzylamide, N-acetyl-L-phenylglycine-N'- 2 benzylamide, acetamido-N-benzyl-3-thiopheneacetamide, D,L- 9- acetamido-N-benzyl-2-thiopheneacetamide, 0 D,L-O/--acetamido-N-benzyl-2-furanacetamide, D,L-O<-acetamido- N-benzyl-2-pyrroleacetamide, D,L-2-acetamido-N-benzyl-2ethoxyacetamide and D,L-2-acetamido-N-benzyl-2-methoxywere evaluated using a horizontal screen assay (HS) as opposed to the Rotorod Test.

0< TABLE I Comparative Median Effective Dosage Tox Compound TD5O_ mg/kg N-acetyl-D ,L-alanine- N' -ben zylamide N -ace ty1- D -a lanine N' -benzylamide N-acetyl-L-alanine- N' -benzylamide N-acetyl-D ,Lphenylglycine-N' benzylarnide 454 (417-501) 214 (148-262) 841 (691-594) ME S ED50 mg/kgq 77 (67-89) 55 (50-60) 548 (463-741)* sc Met ED50 mg/kg (43-67)* 0 32.1 4 0 0~300 ~2O 0 4440 4 090 N-acetyl-D-phenylglycine-N '-benzylamide I-acety 1-L-pheny 1glycine-N' -benzylamide D,L-c( -acetamido-Nbenzyl- 3-thiopheneacetamide 0 10 0-30 0 >100 30-100 40 26 .4 >300 87.80 44.80 10.33 D,L-O( -acetamido-Nbenzyl-2-thiopheneacetainide D,L-C -acetamido-Nbenzyl-2-furanac etamide 4 4 1A -46- Compar Compound D,L-OK-acetamido-Nbenzyl-2-pyrroleacet arnide D ,,-2-acetamido-Nbenzyl-2-ethoxyace tamide D, L-2-acetamido-Nbenzyl-2-methoxyace tamide phenytoin phenobarbital mephenytoin phenacemide TABLE I cont'd.

ative Median Effective Dosage Tox MES TD50 mg/kg ED50 mg/kg Sc Met ED50 mg/kg 100 112 16.10 62.01 K300 66 69 154 421 (337-549)* .ntervals .s substrate was 98 10 22 61 87 (74-100)* not effective 13 31 116 (7 1-150) 0l0~0 04 0 0 0 .00 0 00 0±0 4 0 0900 00 00 00 0 000 20 *95% confidence i #The ED50 for thi not computed I~ It I~ 4 I I 0~ Olt *4~4 0 0 4& 44 4 O *A Thus, while the invention has been described with reference to certain preferred embodiments, those skilled in the art will realize that changes and modifications may be made thereto without departing from the full and intended scope of the appended claims.

The claims form part fo the disclosure of this specification.

Claims (12)

1. An anticonvulsant composition comprising an anticonvulsant effective amount of a compound having the following general formula: 12 R-NH+C-CNH+ C-R 1 I nil 1 O R O wherein R is aryl, aryl lower alkyl, heterocyclic, lower a 10 alkyl heterocyclic, polynuclear aromatic or lower alkyl polynuclear aromatic, each unsubstituted or substituted with at least one electron withdrawing substituent or at least one electron donating substituent; be R is H or lower alkyl, unsubstituted or 1 I substituted with at least one electron withdrawing substituent or at least one electron donating substituent; R and R 3 independently. are lower alkenyl, lower alkynyl, S..heterocyclic, lower alkyl heterocyclic, polynuclear aromatic, lower alkyl polynuclear aromatic, each unsubstituted or substituted with at least one electron withdrawing substituent or at least one electron donating substituent, halogen or a heteroatom containing oxygen, nitrogen, sulfur or phosphorous substituted with hydrogen, lower alkyl or aryl, said lower alkyl or aryl groups being substituted or unsubstituted; n is 1 to 4; and a pharmaceutically acceptable carrier.

2. The composition according to Claim 1 wherein R is hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, amyl or hexyl. iu~ ~~aac 48

3. The composition in accordance with claims 1 and 2 wherein said methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, amyl or hexyl are unsubstituted or substituted with at least one electron withdrawing substituent or at least one electron donating substituent.

4. The composition in accordance with claims 1-3 wherein R is benzyl, unsubstituted or substituted with at least one electron withdrawing substituent or at least one electron donating substituent.

5. The composition in accordance with claims 1-4 wherein said electron withdrawing substituent is halo, nitro, acyl, carboxyl, carboalkoxy, carboxamide, cyano, sulfonyl, sulfoxide, heterocyclic, guanidine or quaternary ammonium.

6. The composition in accordance with claims 1-5 wherein said electron donating substituent is hydroxy, alkoxy, alkyl, amino, phenoxy, thiol, sulfide or disulfide.

7. The composition in accordance with claims 1-6 wherein said compound is in the D isomer form, L isomer form, mixtures of the D,L isomer form or D,L racemate form.

8. The composition according to claim 1 wherein the V 2U*. S compound is: N-acetyl-D,L-alanine-N'-benzylamide; N-acetyl-D-alanine-N'-benzylamide; N-acetyl-D,L-phenylglycine-N'-benzylamide; N-acetyl-D-phenylglycine-N'-benzylamide; D,L-a-acetamido-N-benzyl-3-thiopheneacetamide; D,L-a-acetamido-N-benzyl-2-thiopheneacetamide; D,L-a-acetamido-N-benzyl-2-furanacetamide; D,L-a-acetamido-N-benzyl-2-pyrroleacetamide; D,L-2-acetamido-N-benzyl-2-ethoxyacetamide; or D,L-2-acetamido-N-benzyl-2-methoxyacetamide

9. An anticonvulsant composition substantially as hereinbefore described with reference to any one of the Examples.

10. A composition as claimed in any preceding claim. wherein said compound is present as the pharmaceutically acceptable addition salt.

11. The compounds of the general formula set out in claim 1 and pharmaceutically acceptable addition salts thereof. I )mwspeO17\rescor 90 12 3 i 4 -49

12. A method of preparing the compounds of claim 11 substantially as hereinbefore described. DATED this 3 December 1990 SMITH SHELSTON BEADLE Fellows Institute of Patent Attorneys of Australia Patent Attorneys for the Applicant: RESEARCH CORPORATION TECHNOLOGIES, INC. 44 0 0 Q 0 V, mwspe017\rescor 90 12 3