WO1991009051A1

WO1991009051A1 - Substituted cyclic penicillanic acid tetrapeptides

Info

Publication number: WO1991009051A1
Application number: PCT/US1990/007443
Authority: WO
Inventors: Donald Willis Hansen, Jr.; Henry I. Mosberg
Original assignee: G.D. Searle & Co.
Priority date: 1989-12-15
Filing date: 1990-12-12
Publication date: 1991-06-27

Abstract

The present invention provides novel compounds, compositions, methods of their use and methods of their manufacture. The compounds of this invention are cyclic tetrapeptide tyrosyl substituted dipenicillanic acid opioid agonists that are selective for the delta-receptor. The compounds of the invention act as neurotransmitters or neuromodulators in the central nervous system central pain-suppressant system, to induce analgesia. The present invention provides new cyclic peptide derivatives which show improved potency and bioavailability as analgesic agents by central routes of administration.

Description

SUBSTITUTED CYCLIC PENICI ANIC ACID TETRAPEPTIDES

Background of the Invention

The Government may have rights in this invention pursuant to National Institutes of Health Grant No. DA03910 awarded by the Department of Health and Human Services.

The present invention provides novel compounds, compositions, methods of their use and methods of their manufacture. Such compounds are pharmacologically useful to induce analgesia in mammals. More specifically, the compounds of the present invention are antinociceptive tetrapeptide mono and dimethyl tyrosyl penicillanic acid amides which, by acting as neurotransmitters or neuromodulators in the central nervous system central pain-suppressant system, induce analgesia.

Opioids are a group of drugs that are, to varying degrees, opium-like or morphine-like in their properties. The opioids are employed primarily as analgesics, but they have many other pharmacological effects as well, and they share some of the properties of certain naturally- occurring peptides. By 1967, researchers had concluded that the complex interactions among morphine-like drugs, morphine antagonists, and mixed morphine agonist- antagonists could best be explained by postulating the existence of more than one type of receptor for the opioids and related drugs. Subsequent research revealed that there are at least three distinct families of opioid peptides, the endorphins, the enkephalins and dynorphins, and multiple categories of opioid receptors. Although studies of the binding of opioid drugs and peptides to specific sites in brain and other organs has suggested the existence of perhaps as many as eight different types of opioid receptors, in the CNS there is reasonably firm evidence for three major categories of receptors, designated μ, K and δ.

The classical antagonist, naloxone, has been found to bind with high affinity to all opioid receptors. In 1975, Hughes and Kosterlitz described the isolation of two pentapeptides that exhibited morphine-like actions - actions that were specifically antagonized by naloxone. The same year, Goldstein and colleagues reported the presence of peptide-like substances in the pituitary gland with opioid activity. The peptide appears to act as a neurotransmitter or neuromodulator in the CNS. The natural peptide binds stereospecifically to partially purified brain opiate receptor sites, see for example, Bradberry, et al., Nature, 260,793 (1976). The natural peptide is also highly active in bioassays for opiate activity but exhibits only weak, fleeting analgesic activity when injected directly into the brain of the rat, see for example, Belluzi, et al., Nature, 260,625 (1976).

In order to attempt to overcome the lack of in vivo activity, a number of investigators have made numerous modifications to methionine enkephalin, which was the original pentapeptide reported by Hughes, et al. Among such modifications have been the synthesis and activity of short chain enkephalin-like peptides, among them tripeptide and dipeptide alkylamides by Kiso, et al., "Peptide Chemistry 1981," :65-70, Protein Research Foundation, Osaka, Japan (1982). Vavrek, et al., Peptides 2,303, 1981, disclosed analogs of enkephalin, among them the dipeptide tyrosine-D-alanine-phenylpropylamide.

The compounds of this invention are cyclic, tetrapeptide tyrosyl substituted dipenicillanic acid opioid agonists that are selective for the S receptor. The compounds of this invention have unexpected and surprisingly superior properties when compared to the non-cyclic di, tri, tetra and pentapeptides of the prior art. The present invention provides new cyclic peptide derivatives which show improved potency and bioavailability as analgesic agents by central routes of administration.

Summary of the Invention The invention relates to novel compounds of the general formula I:

and the pharmaceutically acceptable salts thereof, wherein X is H, a halogen, nitro, lower alkyl or lower alkyl substituted by halogen or nitro, aralkyl or alkaryl or substituted aralkyl or alkaryl of from one to ten carbon atoms; R 1, R2, R3 and R4 are independently H and furthermore R 1, R , R3,

R , R and R are independently alkyl of from one to ten carbon atoms; R is amino, hydroxy, alkoxy of from one to ten carbon atoms, alkyl amino or dialkyl amino of from one to ten g carbon atoms; and is independantly H, alkyl of from one to ten carbon atoms, carboxyl, alkoxy carbonyl of from one to ten carbon atoms, amino carbonyl, alkylamino carbonyl and dialkylamino carbonyl of from one to ten carbon atoms, or any of α these R constituents being aryl substituted thereon.

The compounds and pharmaceutical compositions thereof are useful in the analgesic methods of the invention.

The invention further provides dosage unit forms adapted for oral, topical or parenteral administration. Also provided for in this invention are the pharmaceutically acceptable salts of the compounds. Detailed Description of the Invention

As used herein, the expression "halogen" shall include fluorine, chlorine, bromine or iodine. The expression "alkyl" shall mean branched or straight chain carbon-carbon linkages of from one to ten carbon atoms, including one or more double or triple bonds contained therein. "Aryl" shall mean substituted or unsubstituted phenyl. The alkyl portion of "alkoxy" moieties shall be as defined above for alkyl.

The term "pharmaceutically acceptable salts" refers to non-toxic salts of the compounds of this invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid or which are prepared by reacting the free acid with a suitable base. Representative salts include the hydrochloride, hydrobromide, sulfate, biεulfate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate, clavulanate and the like salts and alkali metal salts such as sodium and potassium and alkaline earth salts such as calcium and magnesium.

As used herein, the term "analgesia" shall mean the absence of sensibility to pain, designating particularly the relief of pain without loss of consciousness.

Compounds of the invention can be prepared readily according to one of the following reaction schemes or modifications thereof using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here in greater detail. Scheme I

TFA = trifluoroacetic

DIEA = diisopropylethylamine

DCC = Dicyclohexylcarbodiimide

HOBT = l-hydroxybenzotriazole

DMF = dimethylformamide

Boc = t-butyloxycarbonyl (a) For carboxy terminal carboxylic acid (R_ = OH) in final peptide, Merrifield resin is used. Attachment to resin is via an ester formed via intermediate CS salt. For carboxy terminal carboxamide (R₅ = NH₂) in final peptide, p-methyl-benzhydrylamine (pMBHA) resin is used and linkage to resin is via amide.

(b) Complete protocol for deprotection, washing, etc. is included as a separate scheme, below.

(c) Complete protocol is included separately, below.

(d) For both Herrifield and pMBHA resins, cleavage of peptide from resin is effected by treatment with HF. In the former case an unprotected, C-terminal carboxylic acid-containing peptide is afforded; in the latter case an unprotected, C-terminal carboxamide- containing peptide results.

(e) Cyclization is achieved by treatment with K₃Fe(CN)₆ at pH=7.5 to 8.5. Solid Phase Peptide Synthesis Coupling Scheme for Chain Elongation of Resin Bound Peptide.

^a Boc Amino Acids used at 3 moles/mole vs. Resin- bound peptide. b DCC and HOBT added at 0.8 mole/mole Boc Amino Acid.

* Ninhydrin tests are run after step 6 and after step 11. A positive result after step 6 and a negative test after step 11 are required before continuation. The compounds of the present invention can be administered in such oral dosage forms as oral tablets, sublingual tablets, capsules, pills, powders, granules, elixirs, tinctures, syrups, emulsions and suspensions. Likewise, they may also be administered in intravenous, intraperitoneal, subcutaneous or intramuscular form, all using forms known to those of ordinary skill in the pharmaceutical arts. In general, the preferred form of administration is oral. An effective but non-toxic amount of the compound is employed in the induction of analgesia. The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including the type, species, age, weight, sex and medical condition of the patient. Other relevant factors are the severity of the condition to be treated, the route of administration, the renal and hepatic function of the patient, the route of administration and the particular compound employed or salt thereof. An ordinarily skilled veterinarian or physician can readily determine and prescribe an effective amount of the drug required to induce analgesia.

Oral dosages of the compounds of the present invention, when used for the indicated analgesic effects, will range between about 0.1 mg per kilogram of body weight per day (mg/kg/day) to about 1,000 mg/kg/day and preferably 10-100 mg/kg/day. Advantageously, the compounds of the present invention may be administered in a single daily dose or the total daily dosage may be administered in divided doses of 2, 3 or 4 times daily.

In the pharmaceutical compositions and methods of the present invention, the foregoing compounds described in detail above will form the active ingredients and will typically be administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of tablets or capsules, the active drug component may be combined with an oral non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form the active drug components may be combined with any oral non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In the case of oral administration and in liquid form, suitable flavoring carriers can be added such as cherry syrup and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated in the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol and various waxes. Lubricants for use in these dosage forms include boric acid, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

The compounds of this invention can also be administered by intravenous route in doses ranging from 0.01 to 10 mg/kg/day.

Furthermore, it is also contemplated that the invention can be administered in an intranasal form topically via the use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. In the case of transdermal skin patch administration, daily dosage is continuous via the transdermal delivery system rather than divided, as in an oral delivery system.

The compounds of this invention exhibit analgesic properties useful in the treatment of pain. The test procedures employed to measure this activity of the compounds of the present invention are described below.

Examples 1 and 2 Analgesia Assay

Male Charles River albino mice (20-30g) were used.

The heat induced tail flick (TF) response is a reflex reaction mediated at the level of the spinal cord. The hind paw lick (HP), however, is a more complex behavior requiring integration at higher centers in the brain. When used together, the TF and HP tests provide two different methods of concurrently measuring analgesia. Compounds active in one test are not necessarily active in the other.

Morphine and codeine are active in both tests. In contrast, aspirin and Zomax show little activity in these tests. Opiate compounds having clinical efficacy as analgesics increase tail flick and/or hot plate latencies. However, these tests are not sufficiently sensitive or of the appropriate design to demonstrate the analgesic activity of NSAID's. To determine the tail flick latency (TFL) , the time required for reflex removal of the blackened tail from a high intensity beam of light is measured. Following TFL determination, the animal is placed on a 55 degree C hot plate. The time the animal spends on the plate before either licking a hind paw or jumping is defined as the hot plate latency (HPL) . The cut-off latencies established to prevent tissue damage are 12 sec (mouse) or 14 sec (rat) for the TF and 40 sec for the HP. Latencies are measured before drug administration (baseline) and again at set intervals after dosing. The significance of any increase in TFL or HPL is determined using analyses of variance.

Intracerebroventricular (ICV) administration of the product of Example 3 increased TFL and HPL in a dose-dependent manner. The calculated ED₅₀ for TF and HP tests were 0.57 meg and 0.54 meg, respectively.

The following non-limiting examples further illustrate details for the preparation of the compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds. All temperatures are degrees Celsius unless otherwise noted. Unless otherwise noted, IR and NMR spectra were consistent with the assigned structure.

Example 3 2,6-Dimethyl-L-tyrosyl-D-cysteinyl-L-phenyl alaninyl-D-penici11amine cyclic (2-5) disulfide

N^α-Boc-(S-pMeBzl)D-penicillamine was attached to the solid phase resin support via an ester linkage using a modification of the procedure of Gisin (Helv. Chim. Acta, 56 1476 (1973)): N^αBoc-(S-pMeBzl)D-penicillamine (7.96g, 22.5mmol) was dissolved in 160mL of dry, N--purged dimethylforamide (DMF) . To this solution was added 20g of Merrifield resin (chloromethylated polystyrene cross linked with 1% divinylbenzene; 1.34 meq Cl/gram, Lab Systems) and 5.15g (26.5mmol) CsHCO. and the suspension was stirred at 50^βC under anhydrous conditions for 72hr. Progress of the reaction was followed by disappearance of N^α-Boc-(S-pMeBzl)D- penicillamine assessed by analytical HPLC which indicated >99% completion at 72 hr. The product, if-Boc- (S-pMeBzl)D-penicillamine-Merrifield resin, was filtered, washed with 3X 75mL DMF, 3X 75mL DMF/H₂0(9:1), 3x 75 mL DMF, and 3x 75mL ethanol(ETOH) and dried under vacuum.

1.22g of N^α-Boc-(S-pMeBzl)D-penicillamine-Merrifield resin was placed in the reaction vessel of a Vega Biotechnologies 250C automated solid phase peptide synthesizer and N^αBoc-2,6dimethyl-L-tyrosyl-S-p- methylbenzyl-D-cysteinyl-L-phenyl alaninyl-S-p- methylbenzyl-D-penicillaminyl-resin, was prepared by stepwise addition of the protected amino acids, N^oc-L-phenylalanine, N ^Boc-S-p- methylbenzyl-D-cysteine, and 1^-800-2,6 dimethyl-L-tyrosine using Coupling Agenda 1:

Coupling Agenda 1:

1. Wasn peptide resin with methylene chloride (CH₂C1₂) for 2 min. (repeat 3 additional times)

2. Treat peptide resin with solution of trifluoroacetic acid(TFA/anisole/CH₂Cl₂

3. Treat peptide resin with solution of TFA/anisole/CH₂Cl₂ (48/2/50) for 20 min. 4. Wash peptide resin with CH₂C1₂ for 2 min. (repeat 3 additional times

5. Treat peptide resin with solution of diisopropylethylamine(DIEA)/CH₂Cl₂ (10/90) for 3 min. (repeat 1 additional time)

6. Wash peptide resin with CH₂C1₂ for 2 min. (repeat 3 additional times)

7. Test a small portion of the resin with the ninhydrin test of Kaiser et al. (Anal. Bioch. 34, 595 (1970)). If test is positive, proceed to step 8, if negative repeat steps 3-7.

8. Add 3 equivalents of appropriate l^-Boc-amino acid dissolved in CH₂C1₂ or DMF 2.4 equivalents of dicyclohexylcarbodiimide(DCC) dissolved in CH₂C1₂, and 2.4 equivalents of l-hydroxybenzotriazόle(HOBT) dissolved in DMF. Allow reaction to proceed with gentle agitation for 2 hrs.

9. Wash peptide resin with CH₂C1₂ for 2 min. (repeat

2 additional times)

10. Wash peptide resin with EtOH for 2 min. (repeat 2 additional times)

11. Wash peptide resin with CH₂C1₂ for 23 min. (repeat

3 additional times) 12. Test a small portion of the resin with the ninhydrin test of Kaiser et al. (Anal. Bioch, 34, 595 (1970). If test is positive, repeat steps 8-12. If test is negative repeat Agenda for next if'- oc amino acid.

This material, l^-Boc^,6dimethyl-L-tyrosyl-S-p- methylbenzyl-D-cysteinyl-L-phenylalaninyl-S-p-methylbenzyl- D-penicillaminyl-resin was transferred to a scintered glass funnel and dried in vacuo. 1.07g of this compound was treated with 0.53g of p-thiocresol, 0.53g of cresol, and lOmL of anhydrous hydrofluoric acid(HF) at 0°C for 45 min to effect cleavage from the resin and removal of the N-terminal Boc as well as deprotection of the D-penicillamine and D-cysteine sulfurs. Following evaporation of the HF, the resin was extracted with l50mL of diethylether(Et₂O) (the filtrate was discarded) followed by extraction with 15mL of a mixture of DMF and 80% acetic acid (90/10). This latter extract was diluted with 200 mL of a solution of 0.1% TFA in H₂O and was purified on a Vydac 218TP™ reverse phase HPLC column (2.2cm X 25cm) using a linear gradient of 10-50% solvent B in solvent A (solvent B = 0.1% TFA in CH-CN; solvent A = 0.1% TFA in H₂O) . The linear disulfhydryl-containing tetrapeptide eluting at 35% solvent B, was collected and lyophilized to yield 125mg of partially pure peptide. A 60mg sample of this linear disulfhydryl-containing tetrapeptide was diluted with 500 mL of H₂O and the pH of the solution adjusted to 8.5 with NH₄OH. 41mL of 0.01M K₃Fe(CN)₆ in water was added to the solution and the reaction was allowed to proceed with stirring for 2.5 hr. Analytical HPLC showed that the oxidation reaction to the cyclic dissulfide containing tetrapeptide, eluting at 32% solvent B, was essentially complete. The mixture was acidified to pH 4, stirred for 20 min with lOmL (settled volume) of anion exchange resin (AG 3x4A, Cl form), and filtered. The filter was washed with 20mL of a mixture of DMF and 80% acetic acid (90/10) and the wash added to the filtrate which was then lyophilized. The resulting dry crude peptide was dissolved in solvent A and purified by HPLC on a Vydac 218TP^TH reversed phase HPLC column (2.2cm X 25cm) using a linear gradient of 10-50% solvent B in Solvent A and lyophilized. This procedure yielded 11.6mg of the title product, which was determined to be >99% pure by analytical HPLC and which was found to have the appropriate molecular weight of 589 by analysis via fast atom bombardment mass spectrometry. Example 4 2-Methyl-L-tyrosyl-D-cysteinyl-L-phenyl alaninyl-D-penicillamine cyclic (2-5) disulfide

The title peptide is synthesized by the method of Example 3 wherein Boc-2-methyl-L-tyrosine replaces Boc-2,6-dimethyl-L-tyrosine in the synthetic sequence.

Example 5 2,6-Dimethyl-L-tyrosyl-D-cysteinyl-L-phenylalaninyl- D-penicillamine cyclic ethylamide (2-5) disulfide

The title product is prepared by the method of Example 3 wherein the Merrifield resin is treated with excess ethyl amine before N^α-Boc-(S-pMeBzl)D-penicillamine is attached to the solid phase resin support via the amide linkage (Internat. Peptide Protein Res. 25, 1985, 414-420). The title peptide is isolated after HF cleavage from the resin, cyclization, and chromatographic purification. Example 6 2,6-Dimethyl-L-tyrosyl-D-cysteinyl-(p-chloro)-L-phenyl- alaninyl-D-penicillamine cyclic ethylamide (2-5) disulfide

The title compound is generated by the method of Example 3 wherein Boc-(p-chloro)-L-phenylalanine replaces Boc-L-phenylalanine in the synthetic sequence.

Example 7 2,6-Dimethyl-L-tyrosyl-D-penicillaminyl-L-phenyl alaninyl-D-penicillamine cyclic (2-5) disulfide

The title product is synthesized by the method of Example

2

3 wherein Boc-(S-p-methylbenzyl)-D-cysteme is replaced by Boc-(S-p-methylbenzyl)D-penicillamine in the synthetic sequence.

Example 8 2,6-Dimethyl-L-tyrosyl-D-cysteinyl-L-phenyl alaninyl-β-methyl-D-cysteine cyclic (2-5) disulfide

The title peptide is obtained by the method of Example 3 wherein Boc-(S-p-methylbenzyl)-β-methyl-D-cysteine replaces Boc-(S-p-methylbenzyl)D-penicillamine in the synthetic sequence. Both diastereomers differing only in the stereochemistry at the β carbon of β-methyl-D-cysteine position are generated and separated by chromatography.

Example 9 2 , 6-Diraethyl-( 4-benzylcarbamoyl ) L-tyrosyl-D-cysteinyl-L- phenyl alaninyl-D-penicillamine cyclic ( 2-5 ) disulfide

The product of Example 3 is converted to its Boc derivative by treatment with di-t-butyldicarbonate (l.leq) and sodium hydroxide (1.2eq) in t-butanol/ H₂0 (1:2 in 2mL/mmol of product of Example 3) at room temperature. This material is then treated with benzylisocyanate(2eq), and N-methylmorpholine(2eq) in CH₂C1₂. Aqueous 0.5 sodium bisulfate (NaHSO.) work up of this reaction yields the Boc protected title product. Exposure of this derivative to 6N hydrochloric acid(HCl) in dioxane at room temperature under an argon atmosphere provides the title compound. Example 10 2,6-Dimethyl-(4-i-butylcarbonyl)L-tyrosyl-D-cysteinyl-L- phenyl alaninyl-D-penicillaraine cyclic (2-5) disulfide

The product of Example 3 is converted to its Boc derivative as described in Example 9. This material is then treated with isobutylchloroformate(2eq), and N-methylmorpholine(2eq) in CH₂C1₂. Aqueous work up of this reaction yields the Boc protected title product. Exposure of this derivative to 6N HCl in dioxane at room temperature under an argon atmosphere provides the title cyclic peptide.

Example 11 2,6-Dimethyl-L-tyrosyl-D-cysteinyl-L-phenyl alaninyl-D-penicillamine amide cyclic (2-5) disulfide

The title tetrapeptide amide is synthesized by the method of Example 3 wherein a resin such as U.S. Biochemical Benzhydryl Amine resin is substituted for the Merrifield resin.

Example 12 2,6-Dimethyl-L-tyrosyl-D-cysteinyl-L-phenylalaninyl- D-penicillamine n-propylester cyclic (2-5) disulfide

The title product is obtained by the method of Example 3 wherein the hydrofluoric acid(HF) cleavage of the peptide from the resin prior to the cyclization is carried out in a slurry with n-propanol.

Example 13 2,6-Dimethyl-L-tyrosyl-β-methyl-D-cysteinyl-L-phenyl alaninyl-D-penicillamine cyclic (2-5) disulfide

The title peptide is produced by the method of Example 3 wherein Boc-(S-p-methylbenzyl)-β-methyl-D-cysteine replaces Boc-(S-p-methylbenzyl)-D-cysteine in the synthetic sequence. Both diastereomers differing only in the stereochemistry at the β carbon of β-methyl-D- cystein position are generated and separated by chromatography.

Example 14 2 , 6-Dimethyl-( 4-methoxy )-L-phenylalaninyl-D-cysteinyl-L- phenylalaninyl-D-penicillamine cyclic ( 2-5 ) disulfide

The title compound is prepared by the method of Example 3 wherein Boc-2,6-dimethyl-L-tyrosine is replaced by Boc-2,6 Dimethyl-(4-methyl)_^L-phenyl alanine (L-isomer is synthesized by the process described for the DL-isomer in U.S. Patent #4,760,180) in the synthetic sequence.

Example 15 2,6-Dimethyl-(4-propanyl)L-tyrosyl-D-cysteinyl-L-phenyl alaninyl-D-penicillamine ethylamide cyclic (2-5) disulfide

^ιr^c

The product of Example 5 is converted to its Boc derivative as described in Example 9. This material is then treated with propanoylchloride(leq), and N- methylmorpholine(leq) in CH₂C1₂. Aqueous 0.5N NaHSO. work up of this reaction yields the Boc protected title product. Exposure of this derivative to 6N HCl in dioxane at room temperature under an argon atmosphere provides the title cyclic peptide. Example 16 2,6-Dimethyl-L-tyrosyl-D-cysteinyl-D-(p-chloro)phenyl alaninyl-D-penicillamine cyclic (2-5) disulfide

The title compound is synthesized by the method of Example 3 wherein Boc-(p-chloro)-D-phenylalanine replaces Boc-L-phenylalanine in the synthetic sequence.

Example 17 2,6-Dimethyl-L-tyrosyl-D-cysteinyl-L-phenyl alaninyl-L-penicillamine cyclic (2-5) disulfide

The title cyclic tetrapeptide is obtained by the method of

Example 3 wherein Boc-(S-p-methylbenzyl)-L-penici11amine

4 rreeppllaacceess BBoocc--((SS--pp--mmethylbenzyl)-D-penicillamine in the synthetic sequence.

While the invention has been described and illustrated with reference to certain prepared embodiments thereof, those skilled in the art will appreciate that various changes, modifications and substitutions can be made therein without departing from the spirit and scope of the invention. For example, effective dosages other than the preferred range as set forth herein above may be applicable as a consequence of variations in the responsiveness of the mammal being treated to induce analgesia, dosage-related adverse effects, if any, and analogous considerations. Likewise, the specific pharmacological responses observed may vary according to and depending upon the particular active compound selected or whether there are present certain pharmaceutical carriers, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the results are contemplated in accordance with the objects and practices of the present invention. It is intended therefore that the invention be limited only by the scope of the claims which follow, and that such claims be interpreted as broadly as is reasonable.

Claims

What is claimed is:

1. A compound of the general formula

and the pharmaceutically acceptable salts thereof, wherein X is H, halogen, nitro, lower alkyl, lower alkyl substituted by halogen or nitro, aralkyl or alkaryl or substituted aralkyl or alkaryl of from

1 2 3 4 one to ten carbon atoms; R , R , R and R

1 2 are independently H and furthermore R , R ,

"5 A 7

R , R , R and R are independently alkyl of ς from one to ten carbon atoms; and R is amino, hydroxy, alkoxy of from one to ten carbon atoms, alkylamino or dialkylamino of from one to ten carbon atoms; and R is independently H, alkyl of from one to ten carbon atoms, carboxyl, alkoxy carbonyl of from one to ten carbon atoms, amino carbonyl, alkylaminocarbonyl and dialkylamino carbonyl of from one to ten carbon g atoms, or any of these constituents being aryl substituted thereon.

2. A compound as claimed in Claim 1 in which R 1 and R2

are H.

3. A compound as claimed m Claim 1 in which R 3 and R4

are H.

4. A compound as claimed in Claim 1 in which R 1 and R2

are -CH₃.

5. A compound as claimed in Claim 1 in which R 3 and R4

are -CH₃.

6. A compound as claimed in Claim 1 in which X is F.

7. A compound as claimed in Claim 1 in which X is H.

8. A compound as claimed in Claim 1 in which R is -NH₂<

9. A compound as claimed in Claim 1 in which is -OH.

10. A compound as claimed in Claim 1, and which is of the structure

11. A compound as claimed in Claim 1, and which is of the structure

12. A compound as claimed in Claim 1, and which is of the structure

13. A compound as claimed in Claim 1, and which is of the structure

14. A compound as claimed in Claim 1, and which is of the structure

15. A compound as claimed in Claim 1, and which is of the structure

LAUD

16. A compound as claimed in Claim 1, and which is of the structure

17. A compound as claimed in Claim 1, and which is of the structure

18. A compound as claimed in Claim 1, and which is of the structure

19. A compound as claimed in Claim 1, and which is of the structure

20. A compound as claimed in Claim 1, and which is of the structure

21. A compound as claimed in Claim 1, and which is of the structure

22. A compound as claimed in Claim 1, and which is of the structure

23. A compound as claimed in Claim 1, and which is of the structure

L.D.D.D

24. A compound as claimed in Claim 1, and which is of the structure

25. A compound as claimed in Claim 1, and which is of the structure

LALL

26. A compound as claimed in Claim 1, and which is of the structure

27. A compound as claimed in Claim 1, and which is of the structure

LLLL

28. A compound as claimed in Claim 1, and which is of the structure

29. A compound as claimed in Claim 1, and which is of the structure

LD,LD

30. A compound as claimed in Claim 1, and which is of the structure

31. A compound as claimed in Claim 1, and which is of the structure

LLLL

32. A compound as claimed in Claim 1, and which is of the structure

33. A compound as claimed in Claim 1, and which is of the structure

LLLD

34. A compound as claimed in Claim 1, and which is of the structure

35. A compound as claimed in Claim 1, and which is of the structure

LALL

36. A compound as claimed in Claim 1, and which is of the structure

37. A compound as claimed in Claim 1, and which is of the structure

LLLD

38. A compound as claimed in Claim 1, in which R6 or R7

are independently -CH₃.

39. A compound as claimed in Claim 1, in which R is -CH₃

Q

40. A compound as claimed in Claim 1, in which R is -CONHCH₂C_gH₆.

41. A compound as claimed in Claim 1, in which R is -COOHCH₂CH(CH₃)₂.

Q

42. A compound as claimed in Claim 1, in which R is -COCH₂CH₃.

g

43. A compound as claimed in Claim 1, in which"R is

44. A pharmaceutical composition comprising a pharmaceutically acceptable non-toxic carrier and a compound as claimed in Claim 1.

45. A method of inducing analgesia in a mammal in need thereof, comprising administering to said mammal a pharmacologically effective amount of a compound as claimed in Claim 1.