WO1984002905A1

WO1984002905A1 - Antibacterial peptides

Info

Publication number: WO1984002905A1
Application number: PCT/US1984/000109
Authority: WO
Inventors: Michael A Johnston
Original assignee: University Patents Inc
Priority date: 1983-01-26
Filing date: 1984-01-26
Publication date: 1984-08-02
Also published as: EP0132441A1

Abstract

Biologically active dipeptides of the general formula (I), where R1 or R2 may be either -CH2Cl or -CH2-C=CH, and wherein the amino acid shown is of the L-configuration when R is -CH2Cl, or of either the L- or D-configurations when R is -CH2C=CH.

Description

Antibacterial Peptides

BACKGROUND OF THE INVENTION

The present invention relates to a series of new and novel biologically active dipeptides of the general formula

wherein R₁ or R₂ may be either lower alkyl of one of three carbons (preferably -CH₃), or -CH₂C≡CH, and wherein the amino acid shown is of the L-configuration when R is - CH₂Cl, or of either the L- or D-configurations when R is -CH₂C≡CH.

More specifically, the present invention relates to a series of new and novel biologically active dipeptides containing the amino acid residues B-chloroalanine and propargylglycine which have been discovered to be mechanism-based inactivators of purified microbial enzymes, more specifically the enzymes alanino racemase and cystathionine γ-synthase: The structure of Formula I is not meant to limit the size of the active compound to a dipeptide. One or more haloalanyl residues or one or more propargylglycyl residues or a number of combinations of these in any sequence may be incorporated into an oligopeptide composed of from three to ten amino acid residues (Formula II) .

wherein n is 3-10 and R¹ is a side chain of any naturally occurring amino acid.

Neuhaus and Hammes, reporting in Pharmac. Ther., 14:265 (1981), provides an excellent review of the inhibitory effect on a number of enzymes that have been found to be involved in peptidoglycan biosynthesis including alanine racemase, D-Ala:D-Ala ligase, and D-amino acid transaminase. A major effort in the design of new antibiotics has been focused, therefore, on the preparation of alanine analogs which might serve to abort the construction of an intact, functional, bacterial cell wall. Recently, a renewed interest in this series of compounds has emerged following the several discoveries which confirm that a number of sometimes antibacterial alanine analogs, notably cycloserine, O-carbamoylserine, and the B-haloalanines, are mechanism-based inactivators of at least the bacterial racemases, and perhaps also of the ligases and the transaminases found in certain bacterial species.

Mechanism-based inactivators, specifically enzyme inactivators, are substrate analogues which become activated by the ordinary catalytic mechanism of the targeted enzyme for irreversible inactivation of that enzyme. In this type of inactivation, the enzyme targeted uses some portion of its catalytic mechanism to "unmask", from an otherwise chemically unreactive group in a substrate analogue, a functionality reactive group for alkylation of the enzyme. Alkylation irreversibly denatures the protein, and since the target enzyme catalyzes its own inactivation, it is said to "commit suicide", and the substrate is commonly referred to as a "suicide substrate".

Although a great amount of effort has been expended in the development of suicide substrates, clinical utility has been compromised thus far by problems of transport and host cytotoxicity. For example, whereas β, β, β-trifluoroalanine is an exceedingly efficient inactivator of Escherichia coli alanine racemase, it is not an exceedingly effective antibiotic, most probably because it is not able to be transported into the bacteria. Similarly, O-carbamoyl-D-serine is a poor antibacterial agent because it is not readily transported by the D-alanine-gylcine permease. By contrast, β-Chloro-D-alanine is a recognized inhibitor of bacterial growth, but it is also a substrate for renal D-amino acid oxidase resulting in the formation of β-chloropyruvate; since chloropyruvate inactivates a number of mammalian enzymes, it may be expected to show a level of toxicity in human cells which presents its use as a pharmaceutical. Circumventing host toxicity is a constant concern in drug design, however, the problem of transport may be more easily remedied. In fact, selective drug delivery to and specific accumulation within the targeted pathogenic cells may reduce the incidence and severity of adventitious cytotoxicity. An approach of this type which has met with a measure of success is the incorporation of alanine analogs into peptides, designed for accumulation by several of the bacterial di- and oligopeptide translocases.

Among the antibacterials reported to be transported by a dipeptide permease system is D-Nva-D-Ala, which inhibits the growth of Escherichia coli K12, acts synergistically with D-cycloserine, and apparently replaces D-Ala-D-Ala as a substrate in the synthesis of the UDP-N-acetylmuramylpentapeptide. The naturally occurring compound, bacilysin, a dipeptide of L-alanine and L-anticapsin, is biologically active against a wide range of bacteria, and against at least one strain of yeast, Candida albicans. Anticapsin is a strong inhibitor of glucosamine synthase isolated from both bacilysin-sensitive and bacilysin-resistant strains of Staphylococcus aureus, however, the amino acid per se has poor activity against the whole bacterial cell.

Perhaps the most successful exploitation of the peptide-transport strategy for delivery of antibacterial amino acids has been achieved by researchers at Roche Products in England, who have synthesized a large number of di- and oligopeptides which contain a carboxy-terminal L-1-aminoethylphosphonic acid residue a recognized inhibitor of alanine racenase in vitro. The mechanism of action of phosphonoalanyl peptides appears to involve specific transport and intracellular hydrolysis to generate aminoethylphosphonate. As a result, the action of the racemase in both gram positive and gram negative bacteria is blocked; nevertheless, the phosphonoalanine has little antibacterial action alone.

It is a primary aspect of the present invention to disclose a series of mechanism-based inactivators of bacterial enzymes that are incorporated into a peptide so as to facilitate its delivery to the target cell.

In order to achieve this primary aspect of the invention, I have developed a number of β-Chlorσalanyl dipeptides which have been found to have antibacterial properties. In addition, I have also developed a number of dipeptides containing propargylglycine which are directed toward the inactivation of bacterial cystathionine γ-synthase, an essential enzyme in microbial methionine biosynthesis.

In another aspect of the invention there is provided a pharmaceutical composition comprising a compound of Formula I as has been defined or a pharmaceutically acceptable salt thereof together with any of the conventional pharmaceutically acceptable carriers or excipients.

The pharmaceutically acceptable salts of the compounds in general Formula I may be prepared by conventional reactions with equivalent amounts of organic or inorganic solutions. As exemplary, but not limiting, of pharmaceutically acceptable salts are the salts of hydrochloric, hydrobromic, sulfuric, benzenesulphonic, acetic, fumaric, oxalic, malic and citric acids, and hydroxides of potassium and sodium. The trifluoroacetyl salt of the compounds of general Formula I are used throughout this disclosure and in the tables presented herein, however, this is by no means intended to limit the present invention to only this specific non-toxic and pharmaceutically acceptable salt.

The compositions may be administered parentally in combination with conventional injectable liquid carriers such as sterile pyrogen-free water, sterile peroxide-free ethyl oleate, dehydrated alcohol or propylene glycol. Conventional pharmaceutical adjuvants for injection solutions such as stabilizing agents, solubilizing agents and buffers, for example, ethanol, complex form agents such as ethylene diamine tetraacetic acid, tartrate and citrate buffers and high-molecular weight polymers such as polyethylene oxide for viscosity regulation may be added. Such compositions may be injected intramuscularly, intraperitoneally, or intravenously. The compositions may also be formulated into orally administrable compositions containing one or more physiologically compatible carriers or excipients, and may be solid or liquid in form. These compositions may, if desired, contain conventional ingredients such as binding agents, for example, syrups, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrollidone; fillers, for example, lactose, mannitol, starch, calcium, phosphate, sorbitol or methylcellulose; lubricants, for example, magnesium stearate, high-molecular weight polymers such as polyethylene glycols, high-molecular weight fatty acids such as stearic acid or silica; disintegrants, for example, starch; acceptable wetting agents as, for example, sodium lauryl sulfate. These compositions may take any convenient form, for example, tablets, capsules, lozenges, aqueous or oily suspensions, emulsions, or dry products suitable for reconstitution with water or other liquid medium before use. The liquid oral forms of administration may, of course, contain flavors; sweeteners; preservatives, for example, methyl or propyl p-hydroxybenzoates; suspending agents, for example, sorbitol, glucose or other sugar syrup, methyl, hydroxmethyl, or carboxymethyl celluloses, or gelatin; emulsifying agents as, for example, lecithin or sorbitan monooleate; or thickening agents. Non-aqueous compositions may also be formulated which comprise edible oils as, for example, fish-liver or vegetable oils. These liquid compositions may conveniently be encapsulated in, for example, gelatin capsules in a unit dosage amount.

The pharmaceutical compositions according to the present invention may also be administered topically as an aerosol or, formulated with conventional bases, as a cream or ointment.

A particular aspect of the present invention comprises a compound of general Formula I in an effective unit dose form. By "effective unit dose" is meant a predetermined amount sufficient to bring about the desired antibacterial effect.

And yet in a further aspect of the present invention, there is provided a method of producing an antibacterial effect in mammals, including man, which comprises the administration of an effective antibacterial amount of a compound of general Formula I or a pharmaceutically acceptable salt thereof to a mammalian host which has the bacteria to which the antibacterial action is desired.

The dosage of the compounds of general Formula I or their pharmaceutically acceotable salts will depend, of course, on the nature and severity of the bacterial infection of the mammalian host. Dosages of pharmaceutically active compounds such as those disclosed in the present invention are conventionally manufacturered in amounts sufficient to bring about the desired antibacterial effect without causing an undue burden upon the mammalian host.

Conventional methods for solution-phase peptide synthesis have been used in the preparation of compounds containing propargylglycyl residues as the single suicide substrate. For example, N-BOC-L-Ala-D,L-ppGly-O-t-Bu was readily produced by dicyclohexylcarbodiimide coupling in tetrahydrofuran. N-Butoxycarbonyl and t-butyl protecting groups were chosen for ease of. deprotection; both were cleaved in a single step using TFA in anisole, giving the deblocked peptide as its trifluoroacetate salt.

We have found the solid-phase method recently developed by DeGrado and Kaiser and reported in J. Org. Chem. 45:1295 (1980); J. Org. Chem. 47:3258 (1982), to be especially well suited for the synthesis of chloroalanyl-containing peptides. This procedure involves the DCC-coupling of an N-BOC-amino acid to a polystyrene-bound p-nitrobenzophenone oxime. The protected amino acid is subsequently removed from the support by aminolysis using the t-butyl ester of a second amino acid, a step which forms the peptide bond and regenerates the oxime resin. This method has allowed the use of an unprotected β-chloroalanine for cleavage of a resin-bound N-BOC amino acid, thus affording the introduction of a carboxy-terminal chloroalanyl residue without first having to prepare the t-butyl ester of that compound. In all cases, the oxime resin method gives haloalanyl-containing peptides of exceedingly high purity and in very good yield.

The following specific examples detailing the synthesis of the compounds according to the present invention are offered by way of further illustration of the present invention, and not by way of limitation.

(Abbreviations used in the following examples and in the disclosure of the present invention have the following meanings: BOC = tert-butoxycarbonyl; t-Bu = tert-butyl; BuOH = n-butanol; DCC = dicyclohexylcarbodiimide; Nva = norvaline; DIEA = diisopropylethylamine; M.I.C. = minimum inhibitory concentration (μg/ml); ppGly = propargylglycine; TFA = trifluoroacetic acid; and TMS = tetramethylsilane.)

Example 1 P-Nitrobenzophenone Oxime Resin The oxime resin was prepared essentially as described by DeGrado and Kaiser. 10 g. Biobeads S-Xl were reacted with nitrobenzoyl chloride (1.2 g, 5 mmol) in 1,2-dichloroethane with AlCl₃ as catalyst. This afforied 10.38 g p-nitrobenzoyl polystyrene resin. The IR spectrum gave characteristic bands at 1665, 1525, 1310 cm^-1. The nitrobenzovlated beads (4.4 g) were in turn reacted with excess hydroxylamine, HCl (4.4 g, 63.3 mmol) in 50 ml absolute ethanol and 6 ml pyridine which gave 4.5 g of the oxime resin. Strong absorbances at 3530 (oxime hydroxyl), 1525 and 1310 cm^-1 were observed in the IR spectrum; carbonyl stretching at 1665 cm^-1 (diagnostic of the unreacted nitrobenzovlated resin) is absent in the oxime product. The oxime substitution level was determined by titration with N-BOC-L-alanine on the resin and gave a value of 0.44 mmol/g resin. Elemental analysis gave 1.46% nitrogen, which corresponds to 0.52 mmol/g resin.

Example 2 Propargylglycine D,L-Propargylglycine was prepared from propargylbromide and diethylformamidomalonate according to the procedures of Gershon et al., (J. Am. Chem. Soc. 76:3484 (1954)) yield 1.23 g 23%; m.p. 235°C. D,L-Propargylglycine was resolved according to the procedures of Scannell and coworkers (J. Antibiot. 24:239 (1971)). This method involves reaction of the racemic amino acid with acetic anhydride to afford N-acetyl-D,L-propargylglycine; yield 5.7 g, 83%: m.p. 137-9°C (lit. 137-9°C). The N-acetyl product (4 g, 25.8 mmol) is treated with hog kidney acylase (35 mg, 37 °C, 16 h) followed by ion exchange chromatography (Dowex 50H⁺), which separated unreacted N-acetyl-D-ppGly from L-ppGly. The latter is eluted from the column by 10% pyridine, evaporated to dryness and crystallized from water/ethanol; yield 1.3 g, 89.3%; m.p. 243-5°C(d).

Example 3 N-BOC-D,L-Proparqylglycine

N-tert-Butoxycarbonyl amino acids were prepared using the di-tert-butyl dicarbonate method of Moroder et al. (Hoppe-Seyler's J. Physiol. Chem. 357:1651 (1976)). The preparation of N-BOC-propargylglycine is illustrative. D,L-Propargylglycine (0.3 g, 2.6 mmol) was dissolved in 9 mL dioxane/water (2:1) and 2.6 mL 1N NaOH was added at 0°C. Di-tertτbutyl dicarbonate (0.62 g, 2.86 mmol) was added dropwise. The reaction mixture was stirred for 15 min. at 0°C, brought to room temperature and stirred for an additional two hours. Dioxane was then removed in vacuo, cooled to 0°C and 10 mL ethyl acetate was added. The mixture was acidified to pH 2-3 with KHSO₄ and extracted thrice to ethyl acetate. The organic phases were combined, washed with water and dried over MgSO₄. The solvent was stripped and the product crystallized from ethyl acetate/hexane; yield 0.42 g, 74.3%; m.p. 95-7°C.

N-BOC-L-Propargylglycine was prepared in an identical way; yield 0.42 g, 74.3%; m.p. 95-97°C.

Example 4 D,L-Proparqylglvcyl t-Butyl Ester, HCl.

The propargylglycyl t-butyl ester was prepared using the isobutylene method of Roeske (J. Org. Chem. 28:1251 (1963)). D ,L-Propargylglycine (1.0 g, 8.8 mmol) was added to 50 mL p-dioxane and cooled to -78°C in a 125 mL pressure bottle. Concentrated sulfuric acid (5 mL) and 50 mL liquid isobutylene were added. The mixture was shaken at room temperature for 12 hours. The reaction mixture was then poured into a 400 mL mixture of ether and saturated Na₂CO₃ previously chilled in an ice-bath. The mixture was then extracted several times with ether; the organic phases were combined, dried over MgSO₄ and evaporated to dryness. The resulting yellow oil was dissolved in 200 mL dry ethyl acetate and a white crystalline product was obtained by passing dry HCl gas through the solution: yield, 1.23 g, 67.6%; m.p. 161.5°C.

L-Propargylglycine t-butyl ester, HCl, was prepared in the same way; yield 1.3 g, 71%; m.p. 162-5°C.

Example 5 N-BOC-β-Chloro-L-(and-D-)Alanine The N-BOC derivatives of β-chloro-L- and D-alanme were prepared as described for propargylglycine; yield for N-BOC-β-Cl-LAla is 1.1 g, 78.7%; m.p. 124-5°C.

For N-BOC-β-Cl-DAla, yield 1.08 g, 77.4%; m.p. 124-5°C.

Example 6 β-Chloro-L-(and β-Chloro-D-) Alanyl t-Butyl Ester, HCl β-Chloro-L- and β -chloro-D-alanyl t-butyl ester hydrochlorides were prepared in the same way as described for D,L-propargylglycyl t-butyl ester; β-CL-L-Ala-O-t-Bu, HCl; yield 0.26 g, 32.1%; m.p. 177°C. For β-Cl-D-Ala-O-t-Bu, HCl; yield 0.26 g, 32.1%; m.p. 177°C.

Example 7

L-Alanyl-L-Alanine, TFA

(Peptide No. 1)

To a mixture of L-alanine t-butyl ester, HCl (0.6 g, 3.3 mmol) and Et₃N (0.49 mL, 3.5 mmol) in 15 mL THF was added N-BOC-L-alanine (0.62 g, 3.3 mmol) in 10 mL THF at 0°C with stirring. N,N'-Dicyclohexylcarbodiimide (0.76 g, 3.6 mmol) was added and the reaction stirred at 4°C for 25 hours. Dicyclohexylurea and Et₃N,HCl were removed by filtration and the filtrate was evaporated to dryness. The oily residue was dissolved in chloroform and washed with 1N HCl, 5% NaHCO₃ and water, dried over MgSO₄ and evaporated to dryness.

The crude oily product was dissolved in 4 mL cold anisole, to which 20 mL TFA was added dropwise. The mixture was then stirred for 3 hr at room temperature. The TFA/anisole mixture was pumped to dryness. The solid residue was dissolved in 20 mL CH₂Cl₂/H₂O (1:1) and the aqueous phase was extracted with CH₂Cl₂ to remove trace amounts of anisole. Lyophilization of the aqueous phase afforded 0.5 g L-Ala-L-Ala, TFA salt.

Example 8 D-Alanyl-D-Alanine ,TFA (Peptide No. 2) Peptide 2 was prepared as described above for L-Ala-L-Ala; yield 0.5 g.

Example 9 β-Chloro-L-Alanyl-L-Alanine, TFA

(Peptide No. 3)

(The synthesis of this peptide is illustrative for the preparation of all the β-Chloroalanyl-containing peptides, that is, peptides nos. 4-7, 12, and 13.)

The oxime resin (9 g) was swelled in 100 mL CH₂Cl₂. N-BOC-β-Chloro-L-alanine (0.78 g, 3.5 mmol) and DCC (0.72 g, 3.8 mmol) were added; and the mixture was shaken in a plastic screw-cap bottle for 20 hr at room temperature. The resin was then washed with CH₂Cl₂ (4x) and CH₃OH (4x) and dried; IR = 1720, 1775 cm^-1, corresponding to the carbonyl groups of N-BOC-β-Cl-L-Ala-resin.

In 45 mL Ch₂Cl₂ were mixed N-BOC-β-Cl-L-Ala-resin (4.6 g, 1.7 mmol equiv. amino acid) L-Ala-O-t-Bu,HCl (0.37 g, 2.0 mmol), DIEA (0.36 mL, 2.0 mmol) and CH₃COOH (0.12 mL, 2.0 mmol). The reaction was allowed to shake for 24 hr at room temperature, after which the resin was washed with CH₂Cl₂ (3x) and CH₃OH (3x). The filtrates were combined and evaporated to dryness. The residue was dissolved in ethyl acetate and washed with 5% citric acid (3x), 5% NaHCO₃ and water (3x). The organic phase was recovered, dried over MgSO₄ and evaporated to dryness to yield 0.44 g (oil) N-BOC-β-Cl-L-Ala-L-Ala-O-t-Bu. The oily product was dissolved in 2.5 mL of anisole and 12.5 ml TFA was added dropwise at 0°C. The reaction was then stirred for 3.5 hr at room temperature. The TFA/anisole mixture was pumped off and the residue was dissolved in 25 mL CH₂Cl₂/H₂O (1:1). The aqueous phase was washed with CH₂Cl₂, recovered and lyophilized to afford a white solid of β-Cl-L-Ala-L-Ala,TFA; yield 0.29 g, 74.7%.

Example 10 β-Chloro-D-Alanyl-D-Alanine, TFA (Dipeptide No. 4)

This peptide was prepared as described in Example 9; yield 0.3 g.

Example 11 L-Alanyl-β-Chloro-L-Alanine, TFA (Dipeptide No. 5) N-BOC-L-Alanyl-resin was prepared as outlined above for N-BOC-β-chloro-L-analyl-resin. The resin cleavage reaction (peptide bond formation) was carried out in the way usual for solid phase peptide synthesis, except that unprotected β-chloro-L-alanine,HCl (0.64 g, 4 mmol), rather than its t-butyl ester, was used. Deprotection was again accomplished with TFA in anisole to give the named dipeptide; yield 0.20 g.

Example 12 D-Alanyl-β-Chloro-D-Alanine, TFA (Dipeptide No. 6) β-Chloro-D-Ala-O-t-Bu, HCl was reacted with an N-BOC-D-alanyl-resin prepared in the ordinary way. After deprotection of the resultant N-BOC-D-Ala-β-Cl-D-Ala-O-t-Bu in TFA/anisole; yield 0.11 g.

Example 13 β-Chloro-L-Alanyl-β-Chloro-L-Alanine, TFA (Dipeptide No. 7) An N-BOC-β-chloro-L-alanyl-resin was reacted in the usual way with β-Cl-L-Ala-O-t-Bu, HCl. After isolation and deprotection, the titled product was obtained by lyophilization; yield 0.33 g.

Example 14 L-Alanyl-D,L-Propargylglycine, TFA (Dipeptide No. 8)

D,L-Propargylglycyl t-butyl ester,HCl (0.5 g, 2.4 mmol) was dissolved in 20 mL THF and chilled to 0°C; Et₃N (0.35 mL, 2.5 mmol) was added dropwise. To this mixture was added N-BOC-L-Ala (0.46 g, 2.4 mmol) and DCC (0.56 g, 2.5 mmol); and the reaction was stirred at 4°C for 23 hours. The precipitated dicyclohexylurea and Et₃N,HCl were removed by filtration and the filtrate was evaporated to dryness. The solid residue was then redissolved in CHCl₃ and washed with 1N HCl, 5% NaHCO₃ and water. The chloroform was dried over MgSO₄ and then evaporated in vacuo. A solid residue was obtained which crystallized from ethyl acetate/hexane, giving 0.32 g (39%) N-BOC-L-Ala-D,LppGly-O-t-Bu as a white powder, m.p. 85-89°C.

The protected dipeptide was then stirred with TFA/anisole, as described above, to give the desired product; yield 0.17 g, 95.8%.

Example 15 D, L-PropargyIglycyl-L-Alamine, TFA (Dipeptide No. 9) N-BOC-D,L-Propargylglycine (0.58 g, 2.7 mmol) was coupled to L-alanyl-t-butyl ester (0.50 g, 2.7 mmol) using the DCC coupling method described in Example 14. After deprotection 0.2 g D,L-ppGly-L-Ala was afforded as the TFA salt.

Example 16

D,L-Propargylglycyl-D,L-Propargylglycine, TFA;

L-Propargylglycyl-L-Propargylglycine, TFA (Dipeptides Nos. 10 and 11) Both peptides were prepared by DCC-coupling, as outlined in Example 14, using the appropriately protected amino acids. For the diastereomeric mixture (dipeptide No. 10), 0.26 g of the TFA salt was obtained.

For the enantiomerically pure dipeptide No. 11, 0.3 g of the TFA salt was obtained.

Example 17 β-Chloro-D-Alanyl-D,L-Propargylglycine, TFA (Dipeptide No. 12) This peptide was synthesized using the oxime resin method described above, wherein D,L-ppGly-O-t-Bu, HCl (0.31 g, 1.5 mmol) was used to cleave an N-BOC-β-Cl-D-Ala-resin. After deprotection, 0.21 g of the titled product was obtained.

Example 18 β-Chloro-L-Alanyl-L-Propargylglycine, TFA (Dipeptide No. 13) This peptide was prepared in a fashion identicalat described in Example 17; yield 0.25 g.

In addition to the specific examples described, other structures are also encompassed within the broad terms of the present invention. For example, dipeptides and oligopeptides may contain any monohalo-substitution at the β-carbon (i.e. R may equal -CH₂X, wherein X may be Br, Cl, or F) of a β-haloalanyl residue, or may bear multiple halogenation at (i. e. R may equal -CHX₂ or CX₃) . A dipeptide or oligopeptide containing a β-haolalanyl residue may also have the β-haloalanyl residue replaced by another amino acid residue such as O-acetyl-D-serinyl, β-cyano-L-alanyl, or D-cycloserinyl which is a mechanism-based inactivator of bacterial alanine racemases. Furthermore, peptides according to the present invention which contain a propargylglycinyl residue may have this residue replaced by an amino acid residue which is a mechanism-based inactivator of bacterial cystathionine synthases such as 2-amino-3-halobutanoic acid.

The activity of this series of dipeptides was tested in a variety of organisms obtained either from the American Type Culture Collection, or as fresh clinical isolates from the Clinical Microbiology Laboratory of the University of Chicago Hospitals and Clinics.

The minimum inhibitory concentration (M.I.C.) of each peptide for each strain was determined on a defined peptide susceptibility medium,. Hemin (25 ug/mL), nicotinamide adenine dinucleotide (25 μg/mL), and Isovitalex (1%) were added to the medium to support growth of Hemophilus influenzae; Streptococcus agalactiae; and Streptococcus pyoqenes. Inocula of these three species were prepared by picking colonies of each after overnight growth on a chocolate or blood agar plate and resuspending the cells into the liquid peptide medium to a concentration of 10⁸ colony forming units (CFU)/mL.

Inocula of the other species were prepared by growing the test organisms overnight in the liquid peptide medium and diluting the cultures to approximately 1x10⁷ CFU/mL. The inocula were applied to plates containing peptides in serial two-fold dilutions, and the plates were incubated overnight at 37°C. The plates with Hemophilus influenzae; Streptococcus agalactiae, and Streptococcus pyoqenes were incubated in the presence of 7% CO₂. The M.I.C. was defined as the lowest concentration of peptide which allowed growth of fewer than ten colonies after 16-18 hr incubation. The results of the M.I.C. evaluations may be found in Table I.

The data contained within Table I shows that peptides containing a single β-chloro-L-alanyl residue (peptides 3 and 5), show substantial antibacterial activity against three gram-positive cocci: Streptococcus agalactiae; Staphylococcus aureus; and Staphylococcus epidermidis. In the very best cases, that is peptide 5 tested against Staphylococcus aureus and Staphylococcus epidermidis, the minimum inhibitory concentration corresponds to approximately 0.06 μM peptide. Biological activity is not appreciably affected by the position of the chloroalanyl residue in the peptide; it may be either amino- or carboxyterminal.

Table I shows that four organisms are inhibited by β-chloro-L-alanine (peptide 14) under the conditions of the test. The M.I.C. values, however, are substantially greater for the free amino acid than for either peptide containing a single β-chloro-L-alanyl residue. Attention is drawn, for example, to the action of peptide 5 on Staphylococcus epidermidis, in which case the antibacterial action of β-chloro-L-alanyl is potentiated by a factor of 2x10³ when the haloalanine is incorporated into a peptide. In the "worst case", the enhancement of activity is only eight-fold, as observed for the action of peptide 3 on Streptococcus agalactiae. Since the dipeptides 3 and 5 have, on a molar basis, only one-half as much of the "active component" as does the free chloroalanine, peptide 3 is actually 16-fold more active than the control peptide 14 against Streptococcus agalactiae, and peptide 5 effectively enhances the activity of chloroalanine against Staphylococcus epidermidis by a factor of 4,000. Twelve of the sixteen organisms surveyed are inhibited by peptide 7 at concentrations less than 100 yg/ml, and seven at ≤ 3.12 ug/ml. Streptococcus agalactiae, Straphylococcus aureus, and Staphylococcus epidermidis are particularly susceptible to the antibacterial action of a chloroalanyl peptide. On the other hand, Streptococcus pyrogenes, Streptococcus faecalis, Escherichia coli, and Hemophilus influenzae are susceptible to the action of peptide 7, and yet not inhibited by the monohaloalanyl containing peptides 3 and 5.

Peptides containing a single propargylglycyl residue, that is peptides 8 and 9, are active only against Straphylococcus epidermidis. Introduction of a second D,L-propargylglycyl residue such as in peptide 10, improves biological activity somewhat against Streptococcus agalactiae and Staphylococcus aureus. Note, however, that the enantiomerically pure peptide 11 has a M.I.C. against Staphylococcus epidermidis which is four-fold lower than that of peptide 10 which consists of two pairs of diastereomers. The enhancement of the antimicrobial action of peptide 11 over that of peptide 10 corresponds precisely to an effective four-fold increase in the concentration of the L,L-diastereomer in peptide 11 as compared to peptide 10. An identical pair of results obtains for the action of peptide 10 on Staphylococcus aureus when compared with peptide 11; these findings draw particular attention to the relationship between the biological activity of the peptide and the sterero stereochemical configuration of its component amino acids.

Compounds 12 and 13 which contain both chloroalanyl and propargylglycyl residues, were designed specifically for multienzyme targeting in vitro. Peptide 12 is a diastereomeric pair of D,Dand D,L-residues; consequently is, as expected, without antibacterial effect. By contrast, 13 is enantiomerically pure and is inhibitory against ten of sixteen organisms screened, and for seven species, at ≤ 3.12 μg/mL. The spectrum and degree of activity for this compound are similar to those observed for peptide 7. Distinct patterns of sensitivity are discerned from the data which may suggest multiple sites of action for peptide 13 in vivo; for example, this peptide inhibits the growth of a number of organisms which are resistant to peptides (specifically 3 and 5) containing only a single chloroalanyl residue. Shigella, moreover, is apparently four-fold more sensitive to 13 than to 7 and 11, the peptides containing two units each of haloalanine and propargylglycine, respectively. If, in fact, peptide 13 is actually cleaved to its component amino acids in situ, it would appear that β-chloro-alanine and propargylglycine act synergistically.

The physical depiction of the peptides according to the present invention are shown in Table II.

TABLE II. PHYSICAL DATA FOR SYNTHETIC PEPTIDES

The omission of the hyphen in abbreviations of amino acid residues (e.g., DAla) conforms with suggestions cited in Biochemistry 5:2485 (1966). Thus, while I have illustrated and described the preferred embodiment of my invention, it is to be understood that this invention is capable of variation and modification, and I therefore do not wish to be limited to the precise terms set forth, but desire to avail myself of such changes and alterations which may be made for adapting the invention to various usages and conditions. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview, of the following claims.

Having thus described my invention and the manner and process of making and using it, in such full, clear, concise, and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same:

Claims

I CLAIM :

1. A compound of the general formula

wherein R₁ or R₂ may be either lower alkyl of one to three carbons, -CH₂Cl, or -CH₂C≡CH with the proviso that the amino acid is of the L-configuration and the pharmaceutically acceptable salts thereof.

2. A compound according to Claim 1 wherein lower alkyl is -CH₃.

3. A compound according to Claim 2 wherein R₁ is -CH₃.

4. The compound according to Claim 3 wherein R₂ is -CH₂Cl and which is L-Ala-β-Cl-L-Ala.

5. The compound according to Claim 3 wherein R₂ is -CH₂-C≡CH and which is L-Ala,L-ppGly.

6. A compound according to Claim 2 wherein R₂ is -CH₃.

7. The compound according to Claim 6 wherein R₁ is -CH₂Cl and which is β-Cl-L-Ala-L-Ala.

8. The compound according to Claim 6 wherein R₁ is -CH₂C=CH and which is L-ppGly-L-Ala.

9. A compound according to Claim 1 wherein R₂ is -CH₂C≡CH.

10. The compound according to Claim 9 wherein R₁ is -CH₂C≡CH and which is L-ppGly=L-ppGly.

11. The compound according to Claim 9 wherein R₁ is -CH₂Cl and which is β-Cl-L-Ala-L-ppGly.

12. The compound according to Claim 1 wherein R₁ and R₂ are -CH₂Cl and which is β-Cl-L-Ala-β-ClL-Ala.

13. A compound according to Claim 1 wherein R, is CH₂CH₂CH₃ and R₂ is CH₂CL and which is L-Nva-BC₁-L-Ala.

14. A pharmaceutical composition containing a biologically active component formula

wherein R₁ or R₂ may be either lower alkyl of one to three carbons, -CH₂Cl, or -CH₂C=CH with the proviso that the amino acid is of the L-configuration and the pharmaceutically acceptable salts thereof.