WO1991008759A1

WO1991008759A1 - Alkyl substituted cyclic penicillanic acid pentapaptides

Info

Publication number: WO1991008759A1
Application number: PCT/US1990/007444
Authority: WO
Inventors: Donna L. Hammond; Donald W. Hansen; 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 invention relates to novel compounds of general formula (I) and 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, R2, R3, R4, R6 and R7¿ are independently alkyl of from one to ten carbon atoms; R5 is amino, hydroxy, alkoxy of from one to ten carbon atoms, alkyl amino or dialkylamino of from one to ten carbon atoms; and R8 is independently 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 these R8 constituents being aryl substituted thereon. The compounds and pharmaceutical compositions thereof are useful in the analgesic methods of the invention.

Description

ALKYL SU BSTITUTED CYCLIC PENICILLANIC ACID PEPTIDES

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 pentapeptide 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 agonistantagonists 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,

pentapeptide tyrosyl substituted dipenicillanic acid opioid agonists that are selective for the δ 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, R¹, R², R³,

R⁴ , R⁶ and R⁷ are independently alkyl of from

one to ten carbon atoms; R⁵ is amino, hydroxy, alkoxy of from one to ten carbon atoms, alkyl amino or dialkylamino of from one to ten carbon atoms; and R⁸ is independantly H, alkyl of from one to ten carbon atoms, carboxyl, alkoxy carbonyl of from one to ten carbonyl, alkylamino carbonyl and dialkylamino carbonyl of from one to ten carbon atoms, or any of these R⁸ 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, bisulfate, 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.

TFA = trifluoroacetic acid DIEA = diisopropylethylamine DCC = Dicyclohexylcarbodiimide HOBT = 1-hydroxybenzotriazole DMF = dimethylformamide

Boc = t-butyloxycarbonyl (a) For carboxy terminal carboxylic acid (R₅ = 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 Merrifield 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) or rats (250-300g) 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 one-way analyses of variance. Both central (intracerebroventricular [i.e.v.]) and peripheral (subcutaneous [s.c.]) routes of

administration were used to clarify site of action.

a Activity or Inactivity was determined by the significant increase in tail flick or hot plate latencies above normal latencies. Where applicable, the calculated effective dose (ED₅₀) was determined. b

Subcutaneous (s.c) administration represented in mg/kg and mtracerbroventricular (i.c.v.) administration represented in meg. 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-penicillaminyl-glycyl-L- phenylalaninyl-D-penicillamine 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^α-Boc-(S-pMeBzl)D-penicillamine

(7.96g, 22.5mmol) was dissolved in 160mL of dry,

N₂-purged dimethylforamide (DMF). To this solution was added 20g of Merrifield resin (chloromethylated

polystyrene cross linked with 1% divinylbenzene; 1.34meq Cl/gram, Lab Systems) and 5.15g (26.5mmol) CsHCO₃ and the suspension was stirred at 50ºC 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, N^α-Boc-(S- pMeBzl)D-penicillamine-Merrifield resin, was filtered, washed with 3x 75mL DMF, 3x 75mL DMF/H₂O(9 : 1), ex 75mL DMF, and 3x 75mL ethanol(ETOH) and dried under vacuum.

1.06g 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^α-Boc-2,6 dimethyl-L-tyrosyl-S-p- methylbenzyl-D-penicillaminyl-glycinyl-L-phenylalaninyl-S- p-methylbenzyl-D-penicillaminyl-resin, was prepared by stepwise addition of the protected amino acids,

N^α-Boc-L-phenylalanine, N^αBoc-glycine,

N^α-Boc-S-p-methylbenzyl-d-penicillamine, and

N^α-Boc-2,6 dimethyl-L-tyrosine using Coupling Agenda 1:

Coupling Agenda 1:

1. Wash peptide resin with methylene

chloride(CH₂Cl₂) for 2 min. (repeat 3

additional times) 2. Treat peptide resin with solution of

trifluoroacetic acid(TFA)/anisole/CH₂Cl₂

(48/2/50) for 2 min.

3. Treat peptide resin with solution of

TFA/anisole/CH₂Cl₂ (48/2/50) for 20 min.

4. Wash peptide resin with CH₂Cl₂ 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 CH₂Cl₂ 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 N^α-Boc-amino

acid dissolved in CH₂Cl₂ or DMF 2.4

equivalents of dicyclohexylcarbodiimide(DCC) dissolved in CH₂Cl₂, and 2.4 equivalents of

1-hydroxybenzotriazole(HOBT) dissolved in DMF. Allow reaction to proceed with gentle agitation for 2 hrs. 9 . Wash peptide resin with CH₂Cl₂ for 2 min.

(repeat 2 additinal times)

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

11. Wash peptide resin with CH₂Cl₂ 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 N^α-Boc amino acid.

This material, N^α-Boc-2,6 dimethyl-L-tyrosyl-S-p- methylbenzyl-D-penicill-aminyl-glycinyl-L-phenylalaninyl- S-p-methylbenzyl-D-penicillaminyl-resin, was transferred to a scintered glass funnel and dried in vacuo to yield 1.35g. 1.3g of this compound was treated with 0.65g of p-thiocresol, 0.65g of cresol, and 20mL of anhydrous hydrofluoric acid(HF) at 0ºC for 45 min to effect cleavage from the resin and removal of the N-terminal Boc as well as deprotection of the D-penicillamine sulfurs. Following evaporation of the HF, the resin was extracted with 100mL of diethylether(Et₂O) (the filtrate was discarded)

followed by extraction with 20mL 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^TM 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 CHgCN; solvent A = 0.1% TFA in H₂O). The linear disulfhydryl-containing pentapeptide, eluting in ca. 65mL at 33% solvent B, was collected, diluted with 200 mL of H₂O and the pH of the solution adjusted to 8.5 with NH₄OH. 60mL of 0.01M

K₃Fe(CN)₆ in water was added to the solution and the reaction was allowed to proceed with stirring for 2 hr. An additional 30mL of 0.01M K₃Fe(CN)₆ solution was

added and the reaction was allowed to continue for an additional 1 hr. Analytical HPLC. showed that the

oxidation reaction to the cyclic dissulfide containing pentapeptide was essentially complete. The mixture was acidified to pH 4 , stirred for 20 min. with 10mL (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. The resulting solution was purified by HPLC on a Vydac 218TP^TM 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 54mg 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 673 by analysis via fast atom bombardment(FAB) mass spectrometry.

Example 4

2,6-Dimethyl-L-tyrosyl-D-penicillaminyl-glycyl-L- phenylalaninyl-L-penicillamine cyclic (2-5) disulfide

The title product was synthesized from N^α-Boc- (S-pMeBzl)L-penicillamine by the method of Example 3.

This compound was found to be >99% pure by analytical HPLC and have the appropriate molecular weight of 673 by analysis via FAB mass spectrometry.

Example 5

2,6-Dimethyl-L-tyrosyl-D-penicillaminyl-glycyl-L- (p-fluoro) phenylalaninyl-L-penicillamine cyclic (2-5) disulfide

The title compound was prepared by the method of Example 3 wherein Boc-L-(p-fluoro)phenylalanine replaced

Boc-L-phenylalanine in the synthetic sequence. This pentapeptide was determined to be >99% pure by analytical HPLC and have the appropriate molecular weight of 691 by analysis via FAB mass speetrometry. Example 6

2,6-Dimethyl-L-tyrosyl-D-penicillaminyl-glycyl- (p-chloro)-L-phenylalaninyl-D-penicillamine cyclic (2-5) disulfide

The title compound is synthesized 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-cysteinyl-glycyl- (p-chloro)-L-phenylalaninyl-D-penicillamine cyclic (2-5) disulfide

The title compound is prepared by the method of Example 3 wherein Boc-(S-p-methylbenzyl)-D-cysteine and

Boc-(p-chloro)-L-phenylalanine replace

Boc-(S-p-methylbenzyl)D-penicillamine and

Boc-L-phenylalanine respectively in the synthetic sequence. Example 8

2,6-Dimethyl-L-tyrosyl-D-penicillaminyl-glycyl- (p-chloro)-D-phenylalaninyl-D-penicillamine cyclic (2-5) disulfide

The title compound is generated by the method of Example 3 wherein Boc-(p-chloro)-D-phenylalanine replaces

Boc-L-phenylalanine in the synthetic sequence.

Example 9

2-Methyl-L-tyrosyl-D-penicillaminyl-glycyl-L- phenylalaninyl-D-penicillamine cyclic (2-5) disulfide

The title compound is synthesized by the method of

Example 3 wherein Boc-2,6-dimethyl-L-tyrosine is replaced by Boc-2-methyl-L-tyrosine in the synthetic sequence.

Example 10

2,6-Dimethyl-(4-methyl)-L-tyrosyl-D- penicillaminyl-glycyl-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-tyrosine (the L-isomer is

synthesized by the process described for the DL-isomer in U.S. Patent #4,760,180) in the synthetic sequence.

Example 11

2,6-Dimethyl-(4-acetyl)-L-tyrosyl-D- penicillaminyl-glycyl-L-phenylalaninyl-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/ H₂O (1:2 in 2mL/mmol of product of Example 1) at room temperature. This material is then reacted with acetic anhydride.

Aqueous 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 12

2,6-Dimethyl-L-tyrosyl-D-β-methyl-D-cysteinylglycyl-L-phenylalaninyl-D-penicillamine 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-penicillamine² in the synthetic sequence.

Example 13

2,6-Dimethyl-L-tyrosyl-D-penicillaminyl-glycyl-L- phenylalaninyl-D-cysteine cyclic (2-5) disulfide

The title product is synthesized by the method of

Example 3 wherein Boc-(S-p-methylbenzyl)-D-cysteine replaces Boc-(S-p-methylbenzyl)D-penicillamine⁵ in the synthetic sequence.

Example 14

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

The title product 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 15

2,6-Dimethyl-L-tyrosyl-D-penicillaminyl-glycyl-L- phenylalaninyl-D-penicillamine methylester 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 methanol slurry.

Example 16

2 , 6-Dimethyl-L-tyrosyl-D-penicillaminyl-glycyl-L- phenylalaninyl-D-penicillamine ethyl amide cyclic ( 2-5 ) disulfide

The title product is synthesized 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 17

2 , 6-Dimethyl-(4-i-butylcarbonyl )-L-tyrosyl-D- penicillaminyl-glycyl-L-phenylalaninyl-D- penicillamine cyclic ( 2-5 ) disulfide

The product of Example 3 wherein the Merrifield resin is converted to its Boc derivative as described in

Example 11. This material is then treated with

isobutylchloroformate(2eq), and N-methylmorpholine(2eq) in CH₂Cl₂. 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 18

2,6-Dimethyl-(4-i-butylcarbamoyl)-L-tyrosyl-D- penicillaminyl-glycyl-L-phenylalaninyl-D- penicillamine cyclic (2-5) disulfide

The Boc derivative of the product of Example 3 is

synthesized as described in Example 11. This material is then treated with ethylisocyanate(2eq), and

N-methylmorpholine(2eq) in CH₂Cl₂. 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 yields the title cyclic peptide. 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 one to ten carbon atoms; R ¹, R², R³ and R⁴ are independently H and furthermore R ¹, R², R³, R⁴ , R⁶ and R⁷ are independently alkyl of from one to ten carbon atoms; and R⁵ is amino, hydroxy, alkoxy of from one to ten carbon atoms alkylamino or dialkylamino of from one to ten carbon atoms; and R⁸ is independently H, alkyl of from one to ten carbon atoms, carboxyl, alkoxy carbonyl of from one to ten carbon atoms, amino carbonyl. dialkylamino carbonyl of from one to ten carbon atoms, or any of these R⁸ 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 in 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 m 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 R⁵ is -NH₂.

9. A compound as claimed in Claim 1 in which R⁵ 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

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

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

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

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

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

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

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

34. A compoxmd as claimed in Claim 1, and which is of tne structure

35. A compoxmd as claimed in Claim 1, in which R ⁶ or R⁷ are independently methyl.

36. A compound as claimed in Claim 1, in which R⁸ is -CH₃.

37. A compoxmd as claimed in Claim 1, in which R⁸ is

-COCH₃.

38. A compoxmd as claimed in Claim 1, in which R⁸ is

-COOCH₂CHCH₃.

39. A compoxmd as claimed in Claim 1, in which R⁸ is -CONHCH₂CH₃.

40. A pharmaceutical composition comprising a

pharmaceutically acceptable non-toxic carrier and a compound as claimed in Claim 1.

41. 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.