CA1337372C - Inhibition of bacterial ribonucleotide reductase - Google Patents

Abstract

Disclosed herein are peptides of the formula wherein R1 to R9 are designated amino acid residues; Y is hydrogen or lower alkanoyl, or Y is the hexapeptide radical W-Ile-R10-Ser-R11-Val-R12 wherein W is hydrogen or lower alkanoyl and R10, R11 and R12 are designated amino acid residues, or Y is a fragment of the hexapeptide radical wherein from one to five of the amino acid residues (i.e. Ile to Val) may be deleted serially from the amino terminus of the hexapeptide radical; and Z is hydroxy, amino, lower alkylamino or di(lower alkyl)amino.
The peptides inhibit bacterial ribonucleotide reductase and are indicated for preventing or ameliorating bacterial infections.

Description

Field of the Invention This invention relates to inhibitors of bacterial ribonucleotide reductase and to a means for preventing or ameliorating bacterial infections. More specifically, this inven-tion relates to peptide derivatives (hereinafter called "peptides") having bacterial ribonucleotide inhibiting properties, to processes for their production, to ph~ ceutical compositions of the peptides, and to their use for treating bacterial infections.

Back~round of the Invention Ribonucleotide reductase (RNR) is the enzyme respon-sible for the reductive conversion of ribonucleotides to deoxyribonucleotides. This conversion is the rate determining step in the synthesis of deoxyribonucleic acid (DNA), an essen-tial principle for the growth and replication of eucaryotic cells and procaryotic cells including virions. During the past few years, increasing attention has been given to searching for inhibitors of RNR with the aim of developing new therapeutic agents for controlling cell growth and replication. For example, B. van't Riet et al., J. Med. Chem., 22, 589 (1979) have described a series of benzohydroxamic acids which inhibited m~mmalian RNR and exhibited antineoplastic activity. P.
Gaudreau et al., J. Biol. Chem., 262, 12413 (1987) described a group of peptides which selectively inhibited herpes simplex virus RNR, noting that the peptides were important tools to study the inhibition of herpes viral replication; see also R. Friedinger et al., US patent 4,814,432, issued March 21,1989, describing a series of herpes simplex RNR inhibiting peptides. T. Spector et al., Proc. Natl. Acad. Sci. USA, 86, 1051 (1989), described a hydrazone derivative as a potent inhibitor of herpes simplex RNR and that a combination of the derivative with acyclovir produced synergistic therapy for the topical treatment of HSV-infected ~nim~l.c. W.J. Dobrogosz and S.E. Lindgren, PCI patent application W088/08452, published November 3, 1988 have reported the isolation of an antibiotic (reuterin) with RNR inhibiting properties which was active against certain bacteria, yeast, and protozoa.

Notwithst~ntlin~ the attention given the RNR inhibitors, only one such inhibitor has achieved the status as being avail-able to the physician as a therapeutic agent, namely the antineoplastic agent hydroxyurea. Hence, there is a need for RNR inhibitors with improved activity and specificity.

The present application discloses a new group of peptides which specifically inhibit bacterial RNR. This attrib-ute, together with a relative lack of toxicity, renders the peptides useful as antibacterial agents.

Summary of the Invention The peptides of this invention are represented by formula Y-Rl-R2-R3-R4-Rs-R6-R7-R8-R

S wherein R1 is Thr, Ser or Val, R2 is Asp or Glu, R3 is Asp or Glu, R4 is Leu, Ile or Val, Rs is Ser or Thr, R6 is Asn or Gln, R7 is Phe or ~-(4-halophenyl)-Ala, R8 is Gln or Asn, R9 is Leu, Ile or Phe, Y is hydrogen or lower alkanoyl, or Y is the hexapeptide radical W-Ile-R10-Ser-R11-Val-R12 wherein W is hydrogen or lower alkanoyl, R10 is Asp or Glu, R11 is Glu or Gln and R12 is Asp or Asn, or Y is a fragment of said hexapeptide radical wherein W, R10, R11 and R12 are as defined hereinabove and wherein from one to five of the amino acid residues (i.e. Ile to Val) may be deleted serially from the amino terminus of the hexapeptide radical; and Z is hydroxy, amino, lower alkylamino or di(lower alkyl)amino;
or a therapeutically acceptable salt thereof.

A preferred group of the peptides is represented by formula 1 wherein R1 to R9, inclusive, are as defined herein-above, Y is lower alkanoyl, and Z is hydroxy or amino; or a therapeutically acceptable salt thereof.

Another p,l~relled group of the peptides is represented by formula 1 wherein Rl to R9, inclusive, are as defined herein-above, Y is the hexapeptide radical or a fragment of the hexapeptide radical, as defined hereinabove, and Z is hydroxy or amino; or a thera~llically acceptable salt thereof.

A more plerelled group of the peptides is represented by formula 1 wherein Rl is Thr or Ser, R2, R3, R5, R6 and R8 are as defined hereinabove, R4 is Leu or Ile, R7 is Phe, R9 is Leu or Ile, Y is acetyl, and Z is hydroxy or amino; or a therapeuti-cally acceptable salt thereof.

Another more pler~;lled group of the peptides is repre-sented by formula 1 wherein Rl to R9, inclusive, are as defined in the last instance, Y is the aforementioned hexapeptide radical or a fragment of the hexapeptide radical wherein W is hydrogen or acetyl and Rl to Rl2, inclusive, are as defined hereinabove, and Z is hydroxy or amino; or a therapeutically acceptable salt thereof.

A most pl~felled group of the peptides is represented by formula 1 wherein Rl is Thr, R2 and R3 each independently is Asp or Glu, R4 is Leu, R5 is Ser, R6 is Asn, R7 is Phe, R8 is Gln, R9 is Leu, Y is acetyl, and Z is hydroxy; or a therapeuti-cally acceptable salt thereof.

Another most preferred group of the peptides is represented by formula 1 wherein Rl to R9, inclusive, are defined in the last instance, Y is the aforementioned 1 33737~
s hexapeptide radical or a fragment of the hexapeptide radical wherein W is hydrogen or acetyl, Rl is Asp or Glu, Rll is Glu, Rl2 is Asp or Asn, and Z is hydroxy; or a therapeutically acceptable salt thereof.

Included within the scope of this invention is an antibac-terial composition comprising an antibacterially effective amount of a peptide of formula 1, or a therapeutically acceptable salt thereof, and a pharrnaceutically or veterinarily acceptable carrier.

Also included within the scope of this invention is a method for preventing or ameliorating bacterial infections in a m~mmzl which comprises ~lmini~tering to the m~mm~l an antibacterially effective arnount of a peptide of formula 1, or a therapeutically acceptable salt thereof.

Also the invention involves a method of inhibiting the activity of bacterial ribonucleotide reductase which comprises contacting the enzyme with an amount of a peptide of formula 1 which will prevent the enzyme's capacity to catalyze the reduction of ribonucleotide diphosphates to deoxyribonucleotide diphosphates.

Processes for preparing the peptides of formula 1 are described hereinafter.

Details of the Invention General The term 'residue' with reference to an amino acid means a radical derived from the corresponding a-amino acid S by elimin~ting the hydroxyl of the carboxy group and one hydrogen of the a-amino group.

In general, the abbreviations used herein for design:~tin~;
the amino acids and the protective groups are based on recom-men~l~tions of the IUPAC-IUB Commission of Biochemical Nomenclature, see European Journal of Biochemistry, 138, 9 (1984). For instance, Val, Thr, Glu, Gln Ile, Asp, Phe, Ser, Leu and Asn represent the residues of L-valine, L-threonine, L-glutamic acid, L-glutamine, L-isoleucine, L-aspartic acid, L-phenylal~nine, L-serine, L-leucine and L-asparagine, respective-ly.

The symbol "Ac", when used herein as a prefix to a three letter symbol for an amino acid residue, denotes the N-acetyl derivative of the amino acid; for example, "AcPhe"
represents the residue of N-acetyl-L-phenyl~l~nine.

The amino acid residues possess the L-configuration, including those with prefixes such as lower aL~anoyl and acetyl.
The starting material for providing the amino acid residues, usually the corresponding Na-protected amino acids are com-mercially available or can be p~ fed by conventional methods.

The term "halo" as used herein means a halo radical selected from bromo, chloro, fluoro or iodo.

The term "lower alkanoyl" means an alkanoyl group cont~ininp: two to six carbon atoms and includes acetyl, 1-oxopropyl, 2-methyl-1-oxopropyl, 1-oxohexyl and the like.
Similarly, "lower alkanoic acid" means an alkanoic acid of two to six carbon atoms; for example, acetic acid, propionic acid and 3-methylbutyric acid.

The term "amino" as used herein means an amino radical of formula -NH2. The term "lower alkylamino" as used herein means alkylamino radicals cont~ining one to three carbon atoms and includes methylamino, ethylamino, propylamino and 1-methylethylamino. The term "di(lower alkyl)amino" means an amino radical having two lower alkyl substituents each of which contains one to three carbon atoms and includes dimethylamino, diethylamino, ethylmethylamino and the like.

The term "pharmaceutically acceptable carrier" as used herein means a non-toxic, generally inert vehicle for the active ingredient, which does not adversely affect the ingredient.

The term "veterinarily acceptable carrier" as used herein means a physiologically acceptable vehicle for a(lmini ~tering drug substances to domestic ~nim~l ~ comprising one or more non-toxic ph~rmaceutically acceptable excipients which do not react with the drug substance or reduce its effectiveness.

The term "coupling agent" as used herein means an agent capable of effecting the dehydrative coupling of an amino acid or peptide free carboxy group with a free amino group of another amino acid or peptide to form an amide bond between the reactants. The agents promote or facilitate the dehydrative coupling by activating the carboxy group. Descriptions of such 8 ~ 337372 coupling agents and activated groups are included in general textbooks of peptide chemistry; for instance, E. Schroder and K.L. Lubke, "The Peptides", Vol. 1, Academic Press, New York, N.Y., 1965, pp 2-128, and K.D. Kopple, "Peptides and Amino Acids", W.A. Benjamin, Inc., New York, N.Y., 1966, pp 33-51. Examples of coupling agents are thionyl chloride, diphenylphosphoryl azide, dicyclohexylcarbodiimide, N-hydroxysuccinimide, or l-hydroxybenzotriazole in the presence of dicyclohexylcarbodiimide. A very practical and useful coupling agent is (benzotriazol-lyloxy)tris(dimethylamino)-phosphonium hexafluorophosphate, described by B. Castro et al., Tetrahedron Letters, 1219 (1975), see also D. Hudson, J.
Org. Chem., 53, 617 (1988), either by itself or in the presence of l-hydroxybenzotriazole.

PROCESS

The peptides of formula 1 can be pre~ed by processes which incorporate therein methods commonly used in peptide synthesis such as classical solution coupling of amino acid residues and/or peptide fragments, and if desired, solid phase techniques. Such methods are described, for example, by E. Schroder and K. Lubke, cited above, in the textbook series, "The Peptides: Analysis, Synthesis, Biology", E. Gross et al., Eds., Academic Press, New York, N.Y., 1979-1987, Volumes 1 to 8, and by J.M. Stewart and J.D. Young in "Solid Phase Peptide Synthesis", 2nd ed., Pierce Chem. Co., Rockford, IL, USA, 1984.

A common feature of the aforementioned processes for the peptides is the protection of the labile side chain groups of the various amino acid residues with suitable protective groups which will ~lcven~ a chemical reaction from occurring at that S site until the protective group is llltim~tely removed. Usually also common is the protection of an a-amino group on an amino acid or a fragment while that entity reacts at the carboxy group, followed by the selective removal of the a-amino protec-tive group to allow subsequent reaction to take place at that location. Usually another common feature is the initial protec-tion of the C-terminal carboxyl of the amino acid residue or peptide fragment, which is to become the C-terminal function of the peptide, with a suitable protective group which will prevent a chemical reaction from occurring at that site until the protec-tive group is removed after the desired sequence of the peptide has been assembled.

Hence, the peptides of formula 1 can be plcpalcd by a process comprising the stepwise coupling, in the order of the amino acid sequence of the peptide, of the a~pl~liate amino acid residues or peptide fragments (with side chain functional groups duly protected, and with the C-termin~l carboxyl of the amino acid residue or peptide fragment, which is to become the C-terminal function of the peptide, duly protected by a C-ter-minal carboxyl protecting group), in the presence of a coupling agent, to obtain the protected peptide of formula 2 Yl-Rl3-Rl4-Rl5-Rl6-Rl7-Rl8-Rl9-R2o R2l zl 2 wherein Rl3 is Thr(VI), Ser(VI) or Val wherein Vl is a protec-tive group for the hydroxyl of Thr or Ser, Rl4 is Asp(V2) or Glu(V2) wherein v2 is a protective group for the Cd carboxyl of - lo 1337372 Asp or Glu, Rl5 is Asp(V2) or Glu(V2) wherein v2 is as defined hereinabove, R16 is Leu, Ile or Val, R17 is Ser(V1) or Thr(V1) wherein V1 is as defined hereinabove, R18 is Asn or Gln, R19 is Phe or ,~-(4-halophenyl)-Ala, R20 is Gln or Asn, R2l is Leu, Ile or Phe, yl is lower alkanoyl or the hexapeptide radical W1-Ile-RZ-Ser(V1)-R23-Val-R24 wherein V1 is defined hereinabove, W1 is an a-aminoprotective group or lower alkanoyl, RZ is Asp(V2) or Glu(V2) wherein v2 is as defined above, R23 is Glu(V2) or Gln wherein v2 is as defined above, R24 as Asp(V2) or Asn wherein v2 is as defined above, or yl is a fragment of the last-named hexapeptide radical wherein W1, RZ, R23 and R24 are as defined hereinabove and wherein from one to five of the amino acid residues (i.e. Ile to Val) may be deleted serially from the amino terminus of the last-named hexapeptide radical, and Z1 is a classical carboxyl protective group or a resin support; followed by deprotecting (including cleaving the resin support if present), and acylating and/or amidating, if required, the protected peptide of formula 2 to obtain the corresponding peptide of formula 1; and if desired, transforming the peptide of formula 1 into a therapeuti-cally acceptable salt.

The term "resin support", as used herein with reference to yl~ means the radical derived from a solid resin support of the type used in solid phase peptide synthesis. Such resin supports include the well known chloromethylated resins and benzhydrylamine resins, as well as resins which provide a spacer unit between the resin and the first amino acid building block of a peptide-resin system, so that after the peptide portion is assembled the resin can be cleaved selectively from the system.

Examples of resins with spacers incorporated therein are a-(phenyl~çet~mido)benzyl resin (PAB resin), described by E.
Giralt et al., Tetrahedron 37, 2007 (1981), and 4-(2-bromo- or 4-(2-chloro-propionyl)-phenoxyacetyl BHA resins, photolabile resins described by D. Bellof and M. Mutter, Ch~mi~, 39, 317 (1985) and by J. Gauthier, C~n~ n patent application, SN
547,394, filed September 21, 1987.

Examples of side chain protecting groups are benzyl for the protective group (Vl) for the hydroxyl of Thr or Ser; and benzyl, 2,6-dichlorobenzyl or preferably cyclohexyl for the protective group (V2) for the ~carboxyl of Asp or Glu.

Examples of C-terminal carboxyl protecting groups include the classical groups, for example, benzyloxy and 4-nitrophenoxy, and for the present processes include also a "resin support".

In an embodiment of the exclusively solid phase method, the preparation of a peptide of formula 1 in which Z is hydroxy is commenced by coupling the first amino acid relative to the carboxy terminus (the amino acid having an a-amino protective group) with PAB resin in the presence of potassium fluoride or cesium chloride to give the corresponding solid resin support having the first amino acid (with Na-protection) linked thereto. The next step is the removal of the a-amino protective group of the incorporated amino acid to give the free a-amino group. In the instance where the a-amino protective group is a t-butyloxycarbonyl, trifluoroacetic acid in methylene chloride or chlorofollll, or hydrochloric acid in dioxane, is used to effect the deprotection. The deprotection is carried out at a tempera-ture between about 0C and room It;lllpelature.

Other standard cleaving reagents and conditions for removal of specific a-amino protective groups may be used as described by E. Schroder and K. Lubke, in "The Peptides", Vol.
1, ~rademic Press, New York, N.Y., 1965, pp. 72-75. After removal of the a-amino protective group from the last men-tioned intennediate, the rem:~inin~ a-amino protected amino acids (with side chain protection when required) are coupled stepwise in the desired order to obtain the corresponding pro-tected peptide of formula 2 attached to the PAB resin. Each protected amino acid is introduced into the reaction system in one to four fold excess and the coupling is effected with a coupling agent (one to three fold excess) in a medium of methylene chloride, dimethylformamide, or mixtures of dimethylformamide and methylene chloride. In cases where incomplete coupling has occured, the coupling procedure is repeated before removal of the a-amino protective group, prior to the coupling of the next protected amino acid. The success of the coupling reaction at each stage of the synthesis is moni-tored by the ninhydrin reaction as described by E. Kaiser et al., Anal. Biochem., 34, 595 (1970).

The preceling protected peptide of formula 2 thereafter is simultaneously cleaved from the resin and deprotected by treatment with liquid hydrogen fluoride to give the correspond-ing peptide of formula 1 in which Z is hydroxy.

When it is desired to pl~pal~ the C-terminal primary amide of formula 1 (Z = NH2), the peptide can be pl~ared by the solid phase method using a benzhydrylamine resin and incol~oral~ng into the process the cleavage of the resulting S resin-bound peptide and any required deprotection according to known procedures such as described by Stewart and Young, supra.

Alternatively, a convenient and practical method for pr~illg the prece(lin~ C-terminal primary amide, as well as the corresponding secondary and tertiary amides (i.e. peptides of formula 1 wherein Z is lower alkylamino or di(lower alkyl)amino, respectively, involves the solid phase method with a photolabile resin serving as the resin support. For instance, the stepwise coupling of the appropriate amino acid residues to 4-(2-chloropropionyl)phenoxyacetyl BHA resin, noted above, gives the protected peptide of formula 2 in which Zl is 4-(2-o~ypropionyl)phenoxyacetyl BHA-resin. Subsequent photolysis of a suspension or solution of the latter peptide-resin (350 nm, 0C, 6 to 24 hours) gives the corresponding protected peptide of formula 2 in which Z' is hydroxy. Coupling of the latter protected peptide with benzylamine or the ~pro~liate lower alkylamine, e.g. methylamine or ethylamine, or the a~r~liate di(lower alkyl)amine, e.g. dimethylamine or ethylmethylamine, yields the respective protected peptide of formula 2 in which Zl is benzylamino, lower alkylamino or di(lower alkyl)amino.
Deprotection of the latter protected peptide, for example with hydrofluoric acid, provides the corresponding C-terminal pri-mary, secondary or tertiary amide of formula 1.

The terminal amino acylated derivatives of the peptides of formula 1, e.g. peptides of formula 1 wherein Y is lower alkanoyl or Y is the hexapeptide radical or fra~ment thereof wherein W is lower alkanoyl, are obtained from the correspond-ing free N-termin~l amino peptide (with side chain protection) by treatment with an ayy~pliate acylating agent under suitable conditions; for instance, by treatment with the apyl~liate acid chloride or acid anhydride in the presence of a strong organic base, e.g. l-oxobutylchloride with diisopropylethylamine or N-methylmorpholine, or by treatment with a molar equivalent of the apyroyniate lower alkanoic acid in the presence of a cou-pling agent; preferably (benzotriazol- 1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, alone or in combination with l-hydroxybenzotriazole; followed by conventional deprotection.

The peptide of formula 1 of this invention can be ob-tained in the form of therapeutically acceptable salts.

In the instance where a particular peptide has a residue which functions as a base, examples of such salts are those with organic acids, e.g. acetic, lactic, succinic, benzoic, salicylic, methanesulfonic or p-toluenesulfonic acid, as well as a polymeric acids such as tannic acid or carboxymethylcellulose, and also salts with inorganic acids such as hydrohalic acids, e.g. hydrochloric acid, or sulfuric acid, or phosphoric acid. If desired, a particular acid addition salt is converted into another acid addition salt, such as a non-toxic, pharmaceutically accep-table salt, by treatment with the ~yyuoyliate ion exhange resin in the manner described by R.A. Boissonnas et al., Helv. Chim.
Acta, 43, 1849 (1960).

-In the instance where a particular peptide has one or more free carboxy groups, example of such salts are those with the sodium, potassium or calcium cations, or with strong or-ganic bases, for example, triethylamine of N-methylmorpholine.

In general, the therapeutically acceptable salts of the peptides of formula 1 are biologically fully equivalent to the peptides themselves.

BIOLOGICAL ASPECTS

The RNR inhibiting and antibacterial properties of the peptides of formula 1, or a therapeutically acceptable salt thereof, can be demonstrated by biochemical and biological procedures.

As exemplified hereinafter, the RNR inhibitory effect of the peptides of formula 1 on bacterial RNR can be demonstrat-ed in the "Inhibition of Bacterial Ribonucleotide Reductase Assay", the procedure of which is based on similar assays reported by E.A. Cohen et al., J. Gen. Virol., 66, 733 (1985) and by H.L. Elford et al., Adv. Enz. Reg., 19, 151 (1981).

Noteworthy, is the finding that when the latter assay is repeated with m~mm~ n RNR's, including human RNR, a selective inhibition of bacterial RNR is shown.

The ability of the peptides of formula 1 to selectively inhibit bacterial RNR renders the peptides useful as agents for treating pathogenic infections.

- 1 33737~

In the laboratory, the antibacterial effect of the peptides can be demonstrated in tests with pathogenic bacteria in culture.
Minimum inhibitor,v concentration is used as the evaluation parameter. The methods are described in various publications;
S for example, F. Kavanagh in "Industrial Microbiology", B.M.
Miller and W. Litsky (eds.), McGraw-Hill, New York, N.Y., 1976, pp 13-46.

When the peptides of this invention, or their therapeuti-cally acceptable salts, are employed as agents for combatting disease states associated with bacterial infection, they are ad-ministered topically or systemically to warm-blooded ~nim~
e.g. hllm~ni, dogs, horses, in combination with ph~rm~ceutical acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the peptide, chosen route of ~tlmini~tration and standard biological practice. For topical application, the peptides may be formulated in the form of solutions, creams, or lotions in pharmaceutically acceptable vehicles containing 1.0 to 10 per cent, preferably 2 to 5 per cent of the agent, and may be atlmini~tered topically to the infected area of the body.

For systemic a~lmini~tration, the peptides of formula 1 are a-lmini.ctered by either hl~lavenous, subcutaneous or intra-muscular injection, in compositions with pharmaceutically acceptable vehicles or carriers. For a-lmini~tration by injection, it is ~refelled to use the peptides in solution in a sterile aqueous vehicle which may also contain other solutes such as buffer or preservatives as well as sufficient quantities of phar-maceutically acceptable salts or of glucose to make the solution isotonic.

17 l 337372 Examples of suitable excipients or carriers are found in standard ph~rm~ eutical texts, e.g. in "Remin~ton's Ph:~rm~ceuti-cal Sciences", 1 6th ed, Mack Publishing Company, Easton, Penn., USA, 1980.

The dosage of the peptides will vary with the form of a~lmini ~tration and the particular compound chosen. Further-more, it will vary with the particular host under treatment.
Generally, treatment is initiated with small dosages substantially less than ~illlulll dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. In general, the peptides of this invention are most desirably atlmini~tered at a concentration level that will generally afford effective results, i.e. antibacterial effects, without call~ing any harmful or deleteri-ous side effects.

When used systemically as an antibacterial agent, the peptide of formula 1 is aclmini~tered at a dose of 100 mcg to 1000 mcg per kilogram of body weight per day, although the aforementioned variations will occur. However, a dosage level that is in the range of from about 100 mcg to 500 mcg per kilogram of body weight per day is most desirably employed in order to achieve effective results.

In addition, as an antibacterial agent, the peptides of formula 1 can be used for cleaning and disinfecting laboratory equipment, surgical instruments, locker rooms, or shower rooms of sensitive bacteria organisms. For such purposes it is pre-ferred to use 0.1-10% solutions of the peptide in a lower alkanol, preferably methanol, and to dilute the solution with 10-100 volumes of water c~ ,~t~ ing 0.001-0.1% of a non-ionic surface-active agent, for example, polysorbate 80 U.S.P., imme-diately before applying the pre~ara~ion to the objects to be cleaned and disinfected.

Without further elaboration, it is believed that one skilled in the art can, using the prece~ling description, utilize the inven-tion to its fullest extent, the invention encompassing a peptide, or a functional derivative thereof, for use as a antibacterial agent, capable of inhibiting the activity of bacterial RNR, the peptide having an a~nino acid sequence of Thr-Asp-Asp-Leu-Ser-Asn-Phe-Gln-Leu. The following specific embodiments are, therefore, to be construed as not limitative of the rem~incler of the disclosure.

The following examples illustrate further this invention.
Solution per~ellLages or ratios express volume to volume rela-tionship, unless stated otherwise. Abbreviations used in the examples include Boc: t-butyloxycarbonyl; BOP: (benzotriazol-1-yloxy)tris(dimethylamino)-phosphonium hexafluorophosphate;
Bzl: benzyl; CH2Cl2: methylene chloride; Chxl: cyclohexyl; 2,6-DiClBzl: 2,6-dichlorobenzyl; DCC: N,N1-dicyclohexylcar-bo~liimi~le; DMF: dimethylformamide; Et20: diethyl ether;
EtOH: ethanol; HF: hydrofluoric acid; HOBT: 1-hydroxyben-zotriazole; MeOH: methanol; TFA: trifluoroacetic acid.

Example 1 Preparation of Boc-Leu-CH~-PAB resin Boc-Leu-OH (12.9 g, 56 mmol) and potassium fluoride (7.31 g, 126 mmol) were added to a mechanically stirred sus-pension of a-(4-chloromethylphenylacetamido)benzyl copoly(styrene 1% divinylbenzene) resin (25 g, 14 mmol, described by Giralt et al., supra) in DMF (1 1). The mixture was stirred at 70 C for 24 h, and then allowed to cool to ambient telllpel~ture. The solid was collected by filtration, washed successively with 100 ml portions of DMF, DMF-H2O
(1:1), H2O-dioxane (1:1), dioxane, MeOH, CH2Cl2 and EtOH, and dried under reduced pressure to give 25.3 g of the title compound. The leucine content of the product was 0.40 mmoVg as determined by deprotection of an aliquot and picric acid titration according to the method of B.F. Gisin, Anal.
Chim. Acta, 58, 248 (1972).

Example 2 Preparation of the N-acetyl nonapeptide of the formula:

Ac-Thr-Asp-Asp-Leu-Ser-Asn-Phe-Gln-Leu-OH

The title compound was synthesized by a modification of the solid-phase method of R.B. Merrifield, J. Amer. Chem.
Soc., 85, 2149 (1963). Applying the method, the corresponding protected nonapeptide-resin having the correct sequence of amino acid residues was assembled by stepwise addition of the amino acids residues to Boc-Leu-CH2-PAB resin, i.e. the title compound of Example 1. The following protocol was used:
(a) Boc-deprotection: 30% TFA in CH2Cl2 (2 times, firstly for S S min then for 25 min); (b) wash: CH2Cl2 (3 times for 2 min each); (c) wash: iso~a~lol (2 min); (d) neutralization: 5%
diisopropylethylamine in CH2C12 (2 times for 2 min each); (e) amino acid coupling: achieved by the method of D. Hudson, J.
Org. Chem., 53, 617 (1988) using the a~plo~liate protected amino acid (2.1 molar equivalents per mmol of the Boc-Leu-CH2-PAB resin) and BOP-HOBT (2.2 and 1.1 molar equiv-alents, respectively, per mmol of the Boc-Leu-CH2-PAB resin) in the presence of N-methylmorpholine (6-8 molar equivalents providing pH 8 for the reaction mixture) in CH2Cl2 or DMF;
the reaction time for coupling varied from 3 to 5 h; and (f) wash: CH2Cl2 or DMF (2 times for 2 min each). The Gln and Asn residues were coupled in DMF after activation of the cor-responding Boc-amino acid with DCC-HOBT and removal by filtration of the N,N1-dicyclohexylurea formed during the activa-tion process.

The Boc group gave Na protection for all amino acids.
Side chain protection was as follows: Bzl for Thr and Ser, and Chxl for Asp. After each coupling, the completeness of the re-action was checked by the ninhydrin test, E. Kaiser et al., Anal.
Biochem., 34, 595 (1970). The N-termin~l acetylation was ac-complished by coupling the free N-terminal amino protected peptide-resin with a molar equivalent of acetic acid using the BOP-HOBT method, or with acetic anhydride in the presence of diisopropylethylamine in CH2C12 or DMF.

On completion of the peptide sequence, the protected nonapeptide-resin was collected on a filter, washed with CH2Cl2 and EtOH and dried under reduced pressure over phosphorous pentoxide for 24 h to give the corresponding protected nonapeptide-resin (i.e. peptide-resin). The nonapeptide was cleaved from the peptide-resin by using HF (Sml per g of peptide-resin) in the presence of distilled anisole (1 ml per g of peptide-resin) and ethanedithiol (0.2 ml per g of peptide-resin).
The mixture was m~int~ined at -20 C for 40 min and then at 0-5 C for 40 min, with vigorous stirring. After evaporation of HF, the residue was ~ urdted with Et2O. The mixture was fil-tered through diatomaceous earth. After washing with Et2O, the filter cake was dried under reduced pressure. The residual solid was washed with several portions of 10% aqueous acetic acid, and then with 0.1M aqueous NH40H (total volume: 40 ml per g of the peptide-resin). All the aqueous filtrates were mixed at 0C (pH 6) and lyophili7~1 to afford a white solid residue.

Purification of the solid residue to greater than 95% ho-mogeneity was accomplished by reversed phase HPLC with a Waters model 600 multisolvent delivery system (Waters, Milford, MA, USA) equipped with a UV detector and using a Wh~tm~n Partisil(~3 100DS-3 C-18 column (2.2 x 50 cm2), 10 micron particle size. The elution was done with a gradient of acetonitrile in 0.1% aqueous TFA such as:
a) initial 10% acetonitrile in 0.1% aqueous TFA
for 20 min, b) followed by gradually increasing the concentra-tion of acetonitrile to 20% over a period of 20 min, followed by gradually increasing the concentration to 40% acetonitrile over a period of 50 min.

-Pure fractions, as deterrnined by analytical HPLC, were pooled and lyophilized to afford the title nonapeptide as trifluoroacetate. Analytical HPLC showed the product to be at least 95% homogeneous. Amino acid analysis: Asp + Asn, 3.08; Thr, 0.92; Ser, 0.92; Gln, 1.01; Leu, 2.03; Phe, 1.04;
FAB-MS, calcd: 1093.49, found: 1094 (M+H), etc.

Example 3 Inhibition of E. coli Ribonucleotide Reductase Assav 1) Preparation of Extracts Cont~inin~ Active RNR:

(a) Bacterial strain: E. coli B3 obtained from Dr. B.-M. Sjoberg, University of Stockholm, Stockholm, Sweden.

(b) Fermentation: Cells were produced in 80 1 batches using a standard New Brunswick Scientific Co. pilot-scale fer-menter. A two-stage fermentation process was utilized whereby an inoculum of cells was first grown in thymine-rich Davis Me-dium (3.5 1) for 16 h at 37 C to an optical density of 0.117 (~
= 640 nm). A 3.2 1 portion from the first stage was passed to 80 1 of thymine-poor Davis Medium in the fermenter and incu-bated a further 28 h at 37 C (200 rpm agitation and 1 vvm aeration). Final optical density of cells in the production bio-reactor was 0.82 at a five-fold dilution (~ = 640 nm).

Thymine Thymine Rich Poor Davis Davis Medium Medium S Sodium Citrate 0.5 g/l 0.25 g/l Potassium Phosphate Monobasic 3.0 g/l 1.5 g/l Potassium Phosphate Dibasic 7.0 g/l 3.5 g/l MgSO4 0.1 g/l 0.05 g/l (NH4)2S04 1 g/l 0.5 g/l Ethylene~ minetetracetic Acid50 ~M 25 ~M
CaCl2 5 ~lM 2.5 ',lM
FeCl3 60 ',lM 30 llM
ZnS04 0.6 ~lM 0.3 ~lM
CuS04 60 ~lM 30 ~lM
MnSO4 0.6 ~M 0.3 ,uM
CoCl2 0.75 ~M 0.375 ~lM
glucose 0.2 % 0.2 %
thymine 2.0 mg/l 0.15 mg/l DF287 antifoam (Mazer, Porritts -------- 0.1 %
Drive, Illinois, USA) (c) Preparation of cell extract cont~inin~ E. coli ribonucleotide reductase E. coli RR

The harvested fermentation media obtained above was sub-jected to microfiltration followed by high speed centrifugation.
The resulting cell pellet was processed according to the follow-ing steps. (All steps were performed at 4 C unless noted oth-erwise.) -24 l 337372 STEP

i) Storage Cells frozen at -80C until extraction ii) Extraction Buffer 50 mM tris(hydroxymethyl)aminoethane hydrochloride (Tris.-HC1, pH 7.6), 10 mM DL-dithi-S othreitol (DTT), 0.1% (w/v) Brij 58(~) (polyoxyethylene (20) cetyl ether, Atlas Chemical Industries Inc., Wilmin~ton, Delaware, USA), O.lM NaCl and 10%
(w/v) sucrose iii) Cell Disruption Cells in extraction buffer are subjected to high speed homogenization in an industrial blender in the presence of alumina abrasive (2 g of alumina abrasive per gram, wet weight, of cells) iv) Centrifugation 40,000 times gravity for 60 min; recover supernatant v) Precipitation # 1 A solution of 5% (w/v) streptomycin sulflate in 50 mM
Tris.HCl (pH 7.6) and 1 mM DTT added dropwise to supernatant to give a final concentration in the mixture of 1% (w/v) of streptomycin sulfate.

vi) Centrifugation 40,000 times gravity for 60 min; recover supernatant vii) Precipitation Saturated (NH4)2SO4 in Tris.HC1/DTT buffer (see step v) added slowly to supernatant to yield 60%
saturated solution; solution agitated for 60 min viii) Centrifugation 17,000 times gravity for 60 min.; recover pellet ix) Solubilization Take up pellet in minimum volume of 50 mM Tris.HC1 (pH7.6), 10 mM Dl~

x) Dialysis Dialysis carried out overnight (18h) using stan-dard dialysis tubing (nominal molecular weight cut-off limit of 12,000 to 14,000 daltons) against a 50 fold ex-cess of the solubilization buffer [50mM Tris.HC1 (pH
7.6), 10 mM DTT]

xi) Storage Frozen at -80C

2) Assa~ Protocol:

(a) Standard Reaction Mixture:

Component Amount*

HEPES Buffer (pH 7.8) 50 mM
Adenosine Triphosphate 4 mM
DTT 30 mM
MgCl2 11.5 mM
NaF 4 mM
Cytidine Diphosphate (CDP) 0.054 mM
(3H)CDP (DuPont Chem. Co. 4.2 IlCi/ ml Lachine, QC, Canada) Bacitracin 1 mM

Test Compound 1-500 ~M

* Final concentration of component in standard reaction mixture.

(b) Assay Procedure:

The activity of RNR was qu:~ntit~ted by following the conversion of radiolabeled cytidine diphosphate to radiolabeled deoxycytidine diphosphate, i.e. (3H)CDP to (3H)dCDP. The amount of cell extract utilized in the assay was that which gave a linear response between enzyme concentration and CDP con-version (ca. 100 ~g of protein per assay).

After addition of the cell extract, the assay mixture was incubated at 25C for 30 min. The reaction was stopped by immersing the vessel cont~inin~ the assay mixture in boiling water for 4 min. Nucleotides in the supernatant were then con-S verted to nucleosides by the addition of one part of a Crotalus adamenteus snake venom p~ ion [40 mg/ml of the venom in an aqueous solution of 14 mM
tris(hydroxymethyl)aminomethane (pH 8.8) and 46.5 mM
MgC12], to three parts supern~t~nt, followed by incubating the resulting mixture for 60 min at 37 C. The enzymatic reaction was stopped by immersing the vessel cont~ining the reaction mixture in boiling water for 6 min. Thereafter, the mixture is centrifuged at 10,000 rpm on a clinical centrifuge for 5 min.

A 10 ,ul aliquot of unlabelled nucleoside standards con-taining S mM each of cytidine (C) and deoxycytidine (dC) was added to the supernatant and the resulting mixture was sepa-rated by thin layer chromotography on polyethyleneimine-cellulose plates pretreated with boric acid. Elution of 5 ,ul sam-ples was accomplished using a solution of ethanol / 20 mM
aqueous ammonium formate (1:1), pH 4.7. Quantitation of radiolabel migrating as C and dC was carried out by visll~li7ing the standards under ultraviolet light and cutting out those sec-tions of the TLC plates for each of the assay lanes. The resi-dues from the sections then were extracted into a buffer of one ml of 20 mM Tris.HCl (pH 7.5)/0.7 M MgC12 by agitating the sections in the buffer for a period of 20 min. Aliquots of scintillation fluid (10 ml) were added to each extract and radiolabel was subsequently quanti-tated with a LKB-WALLAC BETA (trade mark) liquid scin~illa~ion counter sold by LKB-Produkten AB, Bromma, Sweden.
Substrate conversion was calculated as:

(3H)deoxycytidine (3H)deoxycytidine + (3H~cytidine . .

A unit of ribonucleotide reductase activity is defined as that amount which reduces one nmole of CDP/minute under the conditions described above. Activity was calculated from sub-strate conversion using the following relationship:

substrate substrate conversion - conversion X conversion factor =
(sample) (blank) activity units The conversion factor for the E. coli assay was 0.108.
Specific activity was expressed as units/mg of protein in the in-cubation rnixture. In one embodiment, the specific activity of the E.coli extract was found to be 0.2 units/mg.

The peptides of formula 1 were tested at a minimum of three concentrations. IC50's were estimated from graphs plot-ting the results for each peptide, the IC50 being the concentra-tion of the peptide in micromoles (~M) producing 50% of the S m~xim~l inhibition of the enzyme.

When the N-acetyl-nonapeptide of Example 2 having the formula Ac-Thr-Asp-Asp-Leu-Ser-Asn-Phe-Gln-Leu-OH was tested according to the assay of this example, an IC50 of 400 ~lM was determined for the compound.

Other examples of peptides within the scope of this invention include:

H-Ser-Asp-Asp-Leu-Ser-Asn-Phe-Gln-Ile-OH

H-Thr-Asp-Asp-Ile-Thr-Asn-Phe-Gln-Ile-NH2 H-Ser-Glu-Glu-Val-Ser-Gln-NHCHCO-Gln-Leu-OH
Cl~CH~

H-Ser-Asp-Asp-Leu-Ser-Asn-Phe-Asn-Phe-OH

H-Val-Asn-Thr-Asp-Asp-Leu-Ser-Asn-Phe-Gln-Leu-OH

Claims (12)

1. A peptide of formula 1 wherein R1 is Thr, Ser or Val, R2 is Asp or Glu, R3 is Asp or Glu, R4 is Leu, Ile or Val, R5 is Ser or Thr, R6 is Asn or Gln, R7 is Phe or .beta.-(4-halophenyl)-Ala, R8 is Gln or Asn, R9 is Leu, Ile or Phe, Y is hydrogen or lower alkanoyl, or Y is the hexapeptide radical W-Ile-R10-Ser-R11-Val-R12 wherein W is hydrogen or lower alkanoyl, R10 is Asp or Glu, R11 is Glu or Gln and R12 is Asp or Asn, or Y is a fragment of said hexapeptide radical wherein W, R10, R11 and R12 are as defined hereinabove and wherein from one to five of the amino acid residues may be deleted serially from the amino terminus of the hexapeptide radical; and Z is hydroxy, amino, lower alkylamino or di(lower alkyl)amino; or a therapeutically acceptable salt thereof.

2. A peptide as recited in claim 1 wherein R1 to R9, inclusive, are as defined in claim 1, Y is lower alkanoyl and Z is hydroxy or amino, or a therapeutically acceptable salt thereof.

3. A peptide as recited in claim 1 wherein R1 to R9, inclusive, are as defined in claim 1, Y is the hexapeptide radical or a fragment of the hexapeptide radical as defined in claim 1 and Z is hydroxy or amino, or a therapeutically acceptable salt thereof.

4. A peptide as recited in claim 2 wherein R1 is Thr or Ser, R2, R3, R5, R6 and R8 are as defined in claim 2, R4 is Leu or Ile, R7 is Phe, R9 is Leu or Ile, Y is acetyl, and Z is hydroxy or amino; or a therapeutically acceptable salt thereof.

5. A peptide as recited in claim 3 wherein R1 to R9, inclusive, are as defined in claim 3, Y is the hexapeptide radical or a fragment of the hexapeptide radical wherein W is hydrogen or acetyl and R10 to R12, inclusive, are as defined in claim 3, and Z is hydroxy or amino; or a therapeutically acceptable salt thereof.

6. A peptide as recited in claim 4 wherein R1 is Thr, R2 and R3 each idependently is Asp or Glu, R4 is Leu, R5 is Ser, R6 is Asn, R7 is Phe, R8 is Gln, R9 is Leu, Y is acetyl, and Z is hydroxy; or a therapeutically acceptable salt thereof.

7. A peptide as recited in claim 5 wherein R1 to R9, inclusive, are as defined in claim 5, Y is the hexapeptide radical or a fragment of the hexapeptide radical wherein W is hydrogen or acetyl, R10 is Asp or Glu, R11 is Glu, R12 is Asp or Asn, and Z is hydroxy; or a therapeutically acceptable salt thereof.

8. A peptide as recited in claim 1 having the formula Ac-Thr-Asp-Asp-Leu-Ser-Asn-Phe-Gln-Leu-OH, or a therapeutically acceptable salt thereof.

9. A pharmaceutical composition comprising an antibacterially effective amount of a peptide of formula 1, or a therapeuti-cally acceptable salt thereof, and a pharmaceutically or vete-rinarily acceptable carrier.

10. Use of a peptide of formula 1 as defined in claim 1, or a therapeutically acceptable salt thereof, for preventing or ameliorating bacterial infections in a mammal.

11. Use of a peptide of formula 1 as defined in claim 1, or a therapeutically acceptable salt thereof, for inhibiting the activity of bacterial ribonucleotide reductase.

12. A process for preparing a peptide of formula 1 of claim 1, which comprises the stepwise coupling, in the order of the amino acid sequence of the peptide, of the appropriate amino acid residues or peptide fragments (with side chain functional groups duly protected, and with the C-terminal carboxyl of the amino acid residue or peptide fragment, which is to become the C-terminal function of the peptide, duly protected by a C-terminal carboxyl protecting group), in the presence of a coupling agent, to obtain the protected peptide of formula wherein R13 is Thr(V1), Ser(V1) or Val wherein V1 is a protective group for the hydroxyl of Thr or Ser, R14 is Asp(V2) or Glu(V2) wherein V2 is a protective group for the .omega.-carboxyl of Asp or Glu, R15 is Asp(V2) or Glu(V2) wherein V2 is as defined hereinabove, R16 is Leu, Ile or Val, R17 is Ser(V1) or Thr(V1) wherein V1 is as defined hereinabove, R18 is Asn or Gln, R19 is Phe or .beta.-(4-halophenyl)-Ala, R20 is Gln or Asn, R21 is Leu, Ile or Phe, Y1 is lower alkanoyl or the hexapeptide radical W1-Ile-R22-Ser(V1)-R23-Val-R24 wherein V1 is defined hereinabove, W1 is an .alpha.-aminoprotective group or lower alkanoyl, R22 is Asp(V2) or Glu(V2) wherein v2 is as defined above, R23 is Glu(V2) or Gln wherein V2 is as defined above, R24 as Asp(V2) or Asn wherein V2 is as defined above, or Y1 is a fragment of the last-named hexapeptide radical wherein W1, R22, R23 and R24 are as defined hereinabove and wherein from one to five of the amino acid re-sidues may be deleted serially from the amino terminus of the last-named hexapeptide radical, and Z1 is a clas-sical carboxyl protective group or a resin support; followed by deprotecting (including cleaving the resin support if present), and acylating and/or amidating, if required, the protected peptide of formula 2 to obtain the corresponding peptide of formula 1; and if desired, transforming the peptide of formula 1 into a therapeutically acceptable salt.