Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
  • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
  • C07C271/06—Esters of carbamic acids
  • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
  • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to a series of new and novel biologically active dipeptides of the general formula
• R 1 or R 2 may be either lower alkyl of one of three carbons (preferably -CH 3 ), or -CH 2 C ⁇ CH, and wherein the amino acid shown is of the L-configuration when R is - CH 2 Cl, or of either the L- or D-configurations when R is -CH 2 C ⁇ CH.
• 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 ⁇ -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) .
• n 3-10 and R 1 is a side chain of any naturally occurring amino acid.
• Mechanism-based inactivators are substrate analogues which become activated by the ordinary catalytic mechanism of the targeted enzyme for irreversible inactivation of that enzyme.
• 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”.
• ⁇ -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 ⁇ -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.
• 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.
• I have also developed a number of dipeptides containing propargylglycine which are directed toward the inactivation of bacterial cystathionine ⁇ -synthase, an essential enzyme in microbial methionine biosynthesis.
• 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.
• 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.
• 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 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.
• 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.
• binding agents for example, syrups, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrollidone
• fillers for
• 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.
• 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.
• effective unit dose is meant a predetermined amount sufficient to bring about the desired antibacterial effect.
• a method of producing an antibacterial effect in mammals comprising 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.
• This method has allowed the use of an unprotected ⁇ -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.
• the oxime resin method gives haloalanyl-containing peptides of exceedingly high purity and in very good yield.
• BOC tert-butoxycarbonyl
• t-Bu tert-butyl
• BuOH n-butanol
• DCC dicyclohexylcarbodiimide
• Nva norvaline
• DIEA diisopropylethylamine
• M.I.C. minimum inhibitory concentration ( ⁇ g/ml)
• ppGly propargylglycine
• TFA trifluoroacetic acid
• 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 AlCl 3 as catalyst. This afforied 10.38 g p-nitrobenzoyl polystyrene resin. The IR spectrum gave characteristic bands at 1665, 1525, 1310 cm -1 .
• 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.
• 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).
• 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-tert ⁇ butyl dicarbonate (0.62 g, 2.86 mmol) was added dropwise. The reaction mixture was stirred for 15 min.
• N-BOC-L-Propargylglycine was prepared in an identical way; yield 0.42 g, 74.3%; m.p. 95-97°C.
• 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 Na 2 CO 3 previously chilled in an ice-bath.
• N-BOC- ⁇ -Chloro-L-(and-D-)Alanine The N-BOC derivatives of ⁇ -chloro-L- and D-alanme were prepared as described for propargylglycine; yield for N-BOC- ⁇ -Cl-LAla is 1.1 g, 78.7%; m.p. 124-5°C.
• Example 6 ⁇ -Chloro-L-(and ⁇ -Chloro-D-) Alanyl t-Butyl Ester, HCl ⁇ -Chloro-L- and ⁇ -chloro-D-alanyl t-butyl ester hydrochlorides were prepared in the same way as described for D,L-propargylglycyl t-butyl ester; ⁇ -CL-L-Ala-O-t-Bu, HCl; yield 0.26 g, 32.1%; m.p. 177°C. For ⁇ -Cl-D-Ala-O-t-Bu, HCl; yield 0.26 g, 32.1%; m.p. 177°C.
• 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 CH 2 Cl 2 /H 2 O (1:1) and the aqueous phase was extracted with CH 2 Cl 2 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.
• This peptide was prepared as described in Example 9; yield 0.3 g.
• Example 11 L-Alanyl- ⁇ -Chloro-L-Alanine, TFA (Dipeptide No. 5) N-BOC-L-Alanyl-resin was prepared as outlined above for N-BOC- ⁇ -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 ⁇ -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- ⁇ -Chloro-D-Alanine, TFA (Dipeptide No. 6) ⁇ -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- ⁇ -Cl-D-Ala-O-t-Bu in TFA/anisole; yield 0.11 g.
• Example 13 ⁇ -Chloro-L-Alanyl- ⁇ -Chloro-L-Alanine, TFA (Dipeptide No. 7) An N-BOC- ⁇ -chloro-L-alanyl-resin was reacted in the usual way with ⁇ -Cl-L-Ala-O-t-Bu, HCl. After isolation and deprotection, the titled product was obtained by lyophilization; yield 0.33 g.
• 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.
• 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.
• Example 17 ⁇ -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- ⁇ -Cl-D-Ala-resin. After deprotection, 0.21 g of the titled product was obtained.
• Example 18 ⁇ -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.
• dipeptides and oligopeptides may contain any monohalo-substitution at the ⁇ -carbon (i.e. R may equal -CH 2 X, wherein X may be Br, Cl, or F) of a ⁇ -haloalanyl residue, or may bear multiple halogenation at (i. e. R may equal -CHX 2 or CX 3 ) .
• a dipeptide or oligopeptide containing a ⁇ -haolalanyl residue may also have the ⁇ -haloalanyl residue replaced by another amino acid residue such as O-acetyl-D-serinyl, ⁇ -cyano-L-alanyl, or D-cycloserinyl which is a mechanism-based inactivator of bacterial alanine racemases.
• 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 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 ⁇ g/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 10 8 colony forming units (CFU)/mL.
• CFU colony forming units
• Inocula of the other species were prepared by growing the test organisms overnight in the liquid peptide medium and diluting the cultures to approximately 1x10 7 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% CO 2 . 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.
• Table I shows that four organisms are inhibited by ⁇ -chloro-L-alanine (peptide 14) under the conditions of the test.
• the M.I.C. values are substantially greater for the free amino acid than for either peptide containing a single ⁇ -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 ⁇ -chloro-L-alanyl is potentiated by a factor of 2x10 3 when the haloalanine is incorporated into a peptide.
• 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.
• Streptococcus agalactiae, Straphylococcus aureus, and Staphylococcus epidermidis are particularly susceptible to the antibacterial action of a chloroalanyl peptide.
• 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.
• 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.
• Peptide 12 is a diastereomeric pair of D,Dand D,L-residues; consequently is, as expected, without antibacterial effect.
• 13 is enantiomerically pure and is inhibitory against ten of sixteen organisms screened, and for seven species, at ⁇ 3.12 ⁇ g/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 ⁇ -chloro-alanine and propargylglycine act synergistically.

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  • 1984-01-26 EP EP84900815A patent/EP0132441A1/de not_active Withdrawn
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