NZ257477A - Bradykinin antagonist (bkan) comprising a bkan peptide joined via a bridging link to a pharmacophore and compositions thereof - Google Patents

Description

New Zealand No. 257477 International No. PCT/US93/10222 Priority Oate<s)^ 2 Complete Specificatl on FHad: 2X?.ktll.. Class: (6) /; Publication Dote: .2..6. °.0. Journal No: NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Bradykinin antagonists Name, address and nationality of applicant(s) as in international application form: CORTECH INC, of 7000 North Broadway, Denver, Colorado 80221, United States of America A U S Corryxzr^ f 47? BRADYKININ ANTAGONISTS The present: invention relates to pharmaceutically effective heterodimers comprising a bradykinin antagonist (BKAn) component covalently 5 linked to another different pharmacophore component.

Related Applications In prior application WO 92/17201 published October 15, 1992, there is described bradykinin antagonist dimers of the type: 10 X(BKAn)2 where BKAn represents a bradykinin antagonist peptide and X is a linking group which joins the two BKAn components at points intermediate to their ends. The BKAn substituents may be the same or 15 different. However, also described are certain heterodimers involving the linkage of a BKAn peptide and another peptide of different receptor activity through the linking group X, e.g. an NK, or NK: antagonist peptide or a mu-opioid receptor agonist 20 peptide. Such heterodimers are particularly useful where there is a close relationship between the activities of concern. Thus, it is known that in a number of pathophysiological^ important processes, there is an intimate interaction of inflammatory and 25 neurogenic mediators. This occurs, for example, in both pain secondary to tissue trauma (accidental and post-operative) as well a-s in asthma. In both situations, there is a complex interplay of tissue and plasma derived mediators (such as kinins acting 30 at BK2 receptors) and neuronally derived factors such as substance P (NK]_ receptors) and neurokinin A (NK2 receptors). There are also locally acting neuronal receptors of the mu-ooiate class that when PCT/IJS93/10222 2 stimulated can inhibit the release of the neurogenic peptides regardless of type (substance P, neurokinin A, neurokinin B, cholecystokinin, CGRP, etc.).

Given the interaction of these as well as other 5 inflammatory and neurogenic mediators, no one agent is likely to be universally efficacious in ameliorating the symptoms attendant to the pathophysiology. The heterodimers described in the above-noted applications are directed towards 10 addressing these problems with single agents possessing dual selectivity. Other advantages of such heterodimers will also be appreciated by those in the art.

Brief Description of the Invention 15 The present invention, in its broadest aspect, is. concerned with heterodimers obtained by linking a BKAn peptide to another pharmacophore which is not a bradykinin antagonist, i.e. which may be either a peptide as described in WO 92/17201, or a non-20 peptide, effective against a different, non- bradykinin component responsible, for example, for pain and/or the inflammatory process, or other problems related to or occurring in concert with the activity of the kinins. The resulting compounds are 25 "dual action" compounds that are capable of interacting with two receptor populations or, alternatively, with a receptor and an enzyme. This is not intended to suggest that the single molecule will engage two receptors or a receptor and an 30 enzyme simultaneously; only that the molecule is capable of interacting with either one of two receptor types or with a single class of receptors and/or an enzyme. The overall pharmacological effect of administering such a compound in an per/ US93/10222 appropriate dose, however, is at least the summation of the two types of activities. The compounds can be designed to remain intact or they can be designed to be dissociated into two separate molecules each 5 retaining its own identifiable activity.

The heterodimers of the invention can be structurally represented as follows: (Y) (X) (BKAn) where BKAn is a bradykinin antagonist peptide; X is 10 a linking group and Y is a peptide or non-peptide pharmacophore which is not a bradykinin antagonist and demonstrates activity towards a different receptor or enzyme than the BKAn component, preferably one related to pain or the inflammatory 15 process.

The present heterodimers offer the possibility of.providing a wider spectrum of treatment for pain and inflammation. It is a generally held opinion that in inflammatory states, regardless of severity, 20 the likelihood that a single agent or mediator is completely responsible for all of the clinical manifestations of the syndrome being addressed is extraordinarily small.. A corollary to this is that, given the role of bradykinin in inflammatory 25 pathophysiology, any combination therapy used in the treatment of inflammatory disorders should include bradykinin antagonism as part of its overall profile of action. Broad spectrum and potent non-specific therapies (such as the use of steroids in asthma) 30 while.perhaps efficacious, carry with them the burdens of undesired and potentially serious side effects and toxicities.

In many cases, two dis.crete mediators are known to act synergistically and to account for an 3 5 overwhelming proportion of the clinically important 1 4 manifestations of the disease being treated. Such is the case, for example, with substance-P acting at NKt receptors and bradykinin acting at BK2 receptors in the contexts of asthma and post-traumatic or post-operative pain. Similarly, neutrophil elastase as one of the more important down stream effectors of inflammation and bradykinin as one of the more important initiating and sustaining inflammatory mediators also can be viewed as being synergistic in their actions.

The concept of providing homodimers of pharmaceutically active materials to improve such characteristics as metabolic stability, selectivity and receptor binding has previously been described for other systems. This prior work has included the dimerization of peptide agonists and antagonists in order to increase potency and/or duration of action. See, Caporale et al, Proc. 10th American Peptide Svmp.. Pierce Chemical Co., Rockford, IL 449-451 (1988) and Rosenblatt et al, European Patent Application No. EP 293130A2. Thus, dimerization of peptide agonists has been disclosed for enkephalins/endorphins. (Shimohig.ashi, Y., et al, BBRC. 146, 1109-1115, 1987); substance P (Higuchi, Y., et al, E.J.P.. 160, 413-416, 1989); bradykinin (Vavrek, R. and Stewart, J., J. Proc. 8th Amer.

Pept. Svmp.. 381-384, 1983); neurokinin A & B, (Kodama, H., et al, E.J.P.. 151, 317-320, 1988); insulin (Roth, R.A., et al, FEBS, 1^0, 360-364, 1984) and atrial natriuretic peptide (Chino, N., et al, BBRC. 141, 665-672, 1986). Dimerization of antagonists has been shown for parathyroid hormone (Caproale, L.H., et al, Proc. 10th Amer. Pept.

Svmp.. 449-451, 1987)). However, the literature has not disclosed heterodimers comprised of a bradykinin 7 4 77 antagonist and a different pharmacophore as contemplated herein.

The reader's attention is directed to our related New Zealand Patent Specification No. 286056 which describes and claims methods of treating pain or inflammation using a bradykinin antagonist compound covalently linked to another different pharmacophore component, and to corresponding international application PCT/US93/10222 published as WO 94/11021.

SUMMARY OF THE INVENTION Accordingly, in one aspect the present invention provides a heterodimer of the formula: (BKAn) (X) (Y) where BKAn is a bradykinin antagonist peptide; Y is a mu-opioid receptor agonist, neutrophil elastase inhibitor, cyclooxygenase inhibitor or a neurokinin receptor antagonist; and X is a linking moiety chemically joining the BKAn and Y components.

In a further aspect the present invention provides a compound comprising a pharmacophore selected from a mu-opioid receptor agonist, neutrophil elastase inhibitor, cyclooxygenase inhibitor or a neurokinin receptor antagonist and a linker group for linkage to the BKAn peptide.

In a still further aspect the present invention provides a compound comprising a BKAn peptide and a linker group.

In a still further aspect the present invention provides a pharmaceutical composition comprising a heterodimer of the invention and a pharmaceutical! acceptable carrier. (followed by page 5a) 5a 7 477 Detailed Description of the Invention Numerous bradykinin antagonist peptides are known in the art and any of these may be used for present purposes to provide the BKAn substituent of the present dimers. One of the more potent bradykinin antagonists in vitro is the peptide having the formula:' d-ARG° - Arg1 - Pro2 -Hyp3 - Gly4 - Phe5 - Ser6 - d - Phe7 - Leu8 - Arg9 See Regoli et al, Trends in Pharmacological Science, 11:156-161 (1990). This peptide is referred to herein for convenience as CP-0088.

While CP-0088 is a convenient BKAn to use, those in the art will appreciate that other available or known bradykinin antagonist peptides can also be used for present purposes. A wide variety of such bradykinin antagonist peptides have been disclosed in the. recent patent literature and any of these can be used for present purposes. See, for example* EP-A-0334244 (Procter and Gamble) which 'discloses nona- and larger bradykinin antagonist peptides in which certain amino acid residues are modified. EP-A-0370453 (Hoechst) and WO 89/01780 and WO 89/01781 (Stewart et al) also describe bradykinin antagonist peptides. None of these patent publications appears to show dimers as contemplated herein. However, as noted, the peptides of these publications can be used in the" practice of the present invention. (followed by page 6) 6 Any linking group X may be used for present purposes to chemically or covalently link' together the BKAn and Y components provided this does not interfere with the activity of the components BKAn 5 and Y. The linking group may be inorganic (e.g. -S-) or organic and may be selected so as to hydrolyze or otherwise dissociate in order to liberate the two active components BKAn and Y in vivo.

Alternatively, the linking group may be such that 10 the heterodimer remains intact when used.

Conveniently the linking group X can include an -S- atom derived by reacting a sulfhydryl group on the BKAn peptide chain with the other pharmacophore component. This can be accomplished by reaction 15 involving a cysteine (Cys) sulfhydryl group within the peptide chain, i.e. intermediate the ends of the peptide. This may require initially modifying the starting B*C.n"peptide so that it includes a Cys group in the appropriate position in the peptide 20. chain. For example, CP-0088 may be modified by replacing the Ser in the 6-position with Cys (such modified CP-0088 being called CP-0126 hereinafter) to provide for convenient linking to the other pharmacophore through the Cys sulfhydryl. 25 CP-0126 can be structurally illustrated as follows: D-ARG-Arg-Pro-Hyp-Gly-Phe-Cys-D-Phe-Leu-Arg I SH In abbreviated fashion, the formula may be stated as: dR-R-P-J-G-F-C-DF-L-R 7 Using Cys as the position of attachment, the linking group X then includes the -S- of the cysteine sulfhydryl. This may be the entire linking group X (as in a disulfide based dimer) or only a 5 part thereof. Thus, for example, the linking group may comprise a bissuccinimidoalkane such as jbissuccinimidohexane joined at its end to the BKAn and Y components. These and other linking groups are disclosed in applicant's related application and 10 any of these may be used for present purposes.

Other linking groups X, some of which do not require or contain an -S- atom, can be derived from the six families of compounds listed below which can be generically categorized as amino acid analog linkers 15 or maleimide-based linkers. These linkers are included as examples only and are not intended to be totally inclusive of all potential linking moieties: CLASS i (Amino Acid Analogs) A) NH2 I \/«* * CH—(CH2)x C R3 co2h B) NH2 * CH— (CH2)y-CO-.H O rv- N--N^ o (CH2)—c^ R3 R, * - "D" or "L" Configuration Rj & R2 = -H. -CH3, -CHrCH3 or --Rj/R2-- = CYCLO ALIO R3 = -OH, -C02H, or -NH2 x = 1 -12 y = 1 -4 c) nh2 * ch—(ch2)y- co2h d) nh2 o ^—\ -N N- O o Ri R-, •(CH2)j C rA * CH (CH2)y JvJ jsj- Ri 1 R, c02h O (CH2)J— c r3 e) nh2 *c ft—(CH^y CO-jH O /—^ -N N- (CH2)x— C r3 R-3 o CLASS II (Maleimide.Based Linkers) R> " R.

F) II NT \ / N (CH^)X c \ O R: The amino acid analog linkers (Class I) can be directly incorporated into the peptide chain of the BKAn and then used to form esterase stable or labile heterodimers with the geminal pharmacophore 5 (component Y). Alternatively, the maleimide based linker can be reacted with the -desired pharmacophore and then conjugated to a sulfhydryl containing peptide. Finally, linkers from any of these families of compounds which contain -C02H as the R3 10 moiety can be reacted with another linker from these classes of compounds to form esterase labile (R3= -OH containing linkers) or esterase stable (R3 = -NH3 containing linkers) which can then be used to form the desired peptide/non-peptide heterodimer. and R2 can be varied so as to provide for completely unhindered or significantly hindered access to the carbonyl carbon of an ester based linking element so that the rate of in vivo hydrolysis of said ester can be controlled as desired. 2 0 Certain of the linker-modified BKAns or pharmacophores used herein to prepare the iimers of the invention are themselves novel and constitute a further aspect of the invention.

The component Y of the present heterodimers may 25 be any peptide or non-peptide pharmacophore, other than a bradykinin antagonist, which demonstrates activity towards.a different (non-bradykinin) component related to pain and/or the inflammatory process so as to provide dual action compounds that 3 0 are capable of interacting independently with two different receptor populations or a receptor and an enzyme. Thus, for example, the component Y may be a non-peptide mu-opioid receptor agonist, e.g. morphine or one of its derivatives such as oxycodone 3 5 or oxymorpnone.

Indomethacin is a useful choice for che Y component when cyclooxygenase inhibition fCOI) is desired. However, any of the ocher conventional non-steroidal anti-inflammatory agents, such as 5 aspirin, ibup'rofen, naproxyn or the like can be. used. In this case, the BKAn/COI heterodimer may need to be hydrolyzed in order to obtain in vivo COI activity as cyclooxygenase is generally considered to be an intra-cellular enzyme.

Where neutrophil elastase inhibition is required, this may be an active ester, e.g. 2-phenyl-alkanoate ester. There may also be used a heteroaryl alkanoate esterase inhibitor. A preferred neutrophil elastase inhibitor for use 15 herein as component Y is identified below as CE-1218. This is believed to be a new compound and constitutes a further feature of the present invention.

Other types of elastase inhibitors which may 20 also be used as component Y, include fluoromethvl ketones, phosphonates, benzoxazoles, beta-lactams, etc.

As noted earlier,, component Y may comprise a peptide or non-peptide inhibitor having a desired 25 activity other than bradykinin antagonist activity, however, the Y component is preferably selected to provide activity against receptors or enzymes which have a common or close relationship to the activity of bradykinin, e.g. the treatment of pain or 3 0 inflammation. The rationale for using combinations of a BKAn with a mu-opioid receptor agonist, neutrophil elastase inhibitor, cyclooxygenase inhibitor or NKj or NK2 receptor antagonist in various conditions is discussed below for purposes 35 of illustration. 11 BKAn/mu-opioid receptor agonists C-Fiber afferents are known to mediate both the sensation of pain as well as the neurogenic component of inflammation. These afferent neurons 5 release a variety of neuropeptides in response to specific and non-specific stimuli in both the central nervous system (CNS) as well as in the peripherally innervated tissues. Some of these neuropeptides include: substance-P, neurokinin A, 10 neurokinin B, calcitonin gene related peptide (CGRP), cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP), and neuropeptide Y, among other neurotransmitters. To add to this complexity, different C-fibers appear to contain different 15 amounts and/or ratios of these neuropeptides depending on the tissue innervated. All of these peptides have been shown to play contributory roles in the various neurogenic processes that have been implicated in numerous diseases and clinical 20 syndromes. In fact, specific antagonists to these peptides are being developed as potential therapeutics by a variety of pharmaceutical companies.and independent research laboratories.

One apparently common feature among this 25 otherwise diverse group of neurons is that they all have mu-opioid receptors that modulate the release of these neuropeptides. Both the endogenous enkephalins as well as other exogenously administered small molecular weight compounds such 3 0 as morphine, oxymorphone, fentanyl and their derivatives will inhibit the release of the neuropeptides from peripheral C-fibers by acting as mu-opioid receptor agonists locally (at terminal mu-opioid receptors in the periphery) and in the CNS. 3 5 This inhibition is independent of both the 12 constellation of peptides contained in the specific C-fiber as well as the stimulus causing their release.

As a result, one important class of compounds 5 considered to have a particularly good profile of activities for the treatment of conditions that are produced by combined humoral and neurogenic processes are BKAn/mu-opioid receptor agonist heterodimers. These compounds would be expected to 10 attenuate or block both the humoral component of the inflammatory process as represented by the kinins as well as the neurogenic aspects of inflammation produced by the release of the neuropeptides. In addition, one of the limiting aspects of the use of 15 existing mu opioid agonists is their propensity to produce sedation, confusion, and a depressed respiratory drive, not to mention their potential for the development oi addiction and/or tolerance in the patients being treated with these agents. These 20 . undesirable aspects of mu-opioid receptor agonists are due to their ability to easily penetrate the CNS. BKAn/mu-opoid receptor agonist heterodimers, however, should not penetrate the CNS due to the highly cationic nature of the BKAn. Consequently, 25 mu-opoid receptor agonist activity should be limited to the periphery and should result in a substantially reduced side effect/toxicity profile for these types of compounds.

BKAn/Neutrophil Elastase Inhibitor (NEI) 30 As previously mentioned the control of both systemic and local inflammatory responses may require interventions in more than one inflammatory pathway. In particular, the ability to block the activity of a primary mediator responsible for the 13 initiation and maintenance of the inflammatory-process (such as bradykinin) and a primary' final pathway effector responsible for actual tissue degradation and injury (such as neutrophil elastase) 5 may be a key to "single drug therapy" of sepsis or other severe inflammatory conditions requiring parenteral therapy or for the treatment of. inflammatory dermatologic or dental/periodontal conditions.

Heterodimers containing combined BKAn/NEI activities can be designed to remain intact or to be dissociable as both targets (bradykinin receptors and neutrophil elastase) are extracellular in nature. However, should dissociation of the two 15 active pharmacophores be desired, linking moieties tethering the two active components of the heterodimer can be designed to be hydrolyzed by, for example, plasma hydrolases. These types of dissociable or hydrolyzable heterodimers are 20 discussed herein.

BKAn/Cvclooxvcrenase Inhibitor (COI) A large proportion of the biological activity of bradykinin is interwoven with the generation of prostaglandins. For example, much of the 25 hyperalgesia associated with inflammatory pain appears to be dependent on the generation of certain prostaglandins both by the injured tissues and by the C-fibers themselves. In the latter case, bradykinin and substance-P appear to be the primary 30 stimuli for these "second messengers". The local generation of prostaglandins by the injured tissues is bradykinin independent. .This interaction of peptide pro-inflammatory mediators and prostaglandins occurs in other settings as well and PCI7US93/10222 14 can also be considered a target, for dual action compounds. Heterodimers containing combined BKAn/COI activities may need to undergo in vivo dissociation of the respective pharmacophores as 5 cyclooxygenase is an intracellular enzyme and functional bradykinin receptors are limited to the external plasma membrane.

BKAn/NK.-Receptor Antagonist (NKlAn) Bradykinin and substance-P are known to act synergistically in the initiation and maintenance of the inflammatory and neurogenic components of both asthma and a variety of painful conditions. In both of these situations, bradykinin is one of the more potent, if not the most potent, agents capable of stimulating C-fiber sensory afferents that mediate peripheral pain and/or the sensation of cough and dyspnea in asthma. These neurons, regardless of the primary stimulus, will release substance-P which amplifies and augments the activity of bradykinin 2 0 and other stimuli at the sensory nerve endings where these stimuli are acting. This "one/two punch" of initial stimulus followed by local amplification is well documented and has significant implications for the success or failure of any single intervention.

By targeting both components of these processes with a single compound, it is possible to provide a dually-specific agent which is superior than monospecific agents; used alone and both easier and cheaper to use than combination therapies. 3 0 BKAn/NK7 Antagonist (NK2An) Bradykinin's ability to produce acute bronchial smooth muscle constriction is at least partially dependent on the release of neurokinin A by the same FCT/US93/10222 C-fibers chat release substance-P. Neurokinin A exerts ics effect via NK2 recepcors on the bronchial smooth muscle. However, more than just bradykinin can release neurokinin A from these neurons and, as 5 a result, a dually-specific antagonist with combined BK2/NK2 antagonist activity should provide better overall amelioration of bronchoconstriction in the asthmatic patient than any other single agent.

The heterodimers■of the invention may be 10 prepared in generally the same manner as described in WO 92/17201. Ncnr..*ily this involves adding the linking group X to the BKAn component at an appropriate position along the peptide chain followed by joining the non-peptide pharmacophore to 15 the BKAn through the linking group. Alternatively, the linking group may be added to the non-peptide pharmacophore and the BKAn thereafter joined to the linker-modified pharmacophore. Representative procedures are described below although it will be 2 0 recognized that various modifications may be used.

The invention is illustrated but not limited by the following examples: Examples 1-4 ' (BKAn/mu-opioid agonist) Four different peptide/opiate heterodimers 25 '(designated CP-0477, CP-0488, CP-0494 and CP-0499) were made in order to illustrate the invention.

Three of these compounds were made using CP-0126 (dR-R-P-J-G-F-C-dF-L-R) and the fourth used CP-0347 (dR-R'-P-J-G-Thi-C-DTic-Oic-R) . Similarly, two 30 different opiates (oxycodone and oxymorphone) and Cwo different linker chemistries were used to provide the respective heterodimers as follows: 16 Examole Compound # Peptide Opiate 1 CP-0477 CP-0126 Oxycodone 2 CP-0488 CP-0126 Oxycodone 3 CP-0494 CP-0126 Oxymorphone 4 CP-0499 CP-0347 Oxymorphone The heterodimers CP-0477, CP-0488, CP-0494 and CP-0499 were prepared as detailed hereinafter with reference to the accompanying Figure 1: Preparation of Compound I: Oxycodone hydrochloride (0.182g, 0.52 mmol), acetic acid (0.475 ml, 8.3 mmol), S-benzyl cysteamine (0.174g, 1.04 mmol) and methanol (5 ml) were combined and stirred at room temperature for an hour. Sodium cyanoborohydride (95%, 0.033g, 0.52 15 mmol) was added, and the reaction stirred at room temperature for 24 h. The mixture was concentrated in vacuo. The resulting oil was dissolved in ethyl acetate and the ethyl acetate fraction was washed with saturated sodium bicarbonate solution, dried 2 0 over magnesium sulfate and evaporated in vacuo. The crude material was chromatographed on a silica column and eluted with EtOAc, EtOAc-MeOH (9:1, v/v) and EtOAcMeOH-Et3N (9:1:0.2, v/v/v) successively Compound I was isolated as an oil in 25.0% (59.0 mg) 25 yield.

Preparation of Compound II (CP-0477): I (0.059g, 0.127 mmol) was dissolved in 2 mL dry tetrahydrofuran, and was transferred to a oven dried three-necked 100 ml flask. The flask was 3 0 fitted with a dewar condensor, a nitrogen source and an ammonia inlet. Approximately 10 ml of ammonia was ■ condensed into the flask maintained at -78C. Small- 17 pieces of sodium were added until the intense blue color was maintained and then quenched after 4 0 seconds with solid ammonium chloride. The reaction mixture was allowed to warm to room temperature and 5 the ammonia boiled off through a bubbler, methanol (25 ml x 3) was added and evaporated in vacuo. The thiol isolated was dissolved in a minimum quantity of DMF (N,N-dimethyl formamide, 2 ml). Compound X (approximately 0.3 equiv) was dissolved in tris 10 buffer (0.5 M, 4.0 ml) and added to the DMF solution and then stirred for 17h. The crude mixture was purified on a reverse phase Vydac C-18 HPLC column using the gradierlt 15 - 40% CH3CN in water, 0.1% constant TFA, over 20 minutes. Retention time was 15 16.0 minutes. 26.4 mg of II was isolated, as a white powder on lyophillization.

Analysis: The mass spectra was run on a Finnigan Lasermat Mass Analyzer. calulated molecular weight--1916 observed molecular weight--1918 Amino Acid Analysis: Gly 1.02 (1), Arg 3.14 (3), Pro 1.01 (1), Leu 0.97 (1), Phe 1.92 (2) and Hyp 0.94 (1).

Preparation of Compound III: To the mixture of oxycodone hydrochloride (1.0 g, 2.84 mmol) and ammonium acetate (2.2 g, 28.4 mmol) dissolved in methanol (10.0 ml) was added a methanolic (4.0 ml) solution of NaCNBH3 (0 13 g, 2.84 30 mmol). The resulting solution was adjusted to pH 7.0 with concentrated hydrochloric acid, stirred for • 18 17h, and acidified to pH 1.0 with concentrated hydrochloric acid. The solvent was removed in vacuo and the remaining material was dissolved in water. The aqueous layer was extracted with chloroform, 5 adjusted to pH 9.0 with 10% sodium carbonate solution, saturated with NaCl and extracted with chloroform. The chloroform layer was dried over magnesium sulfate and evaporated in vacuo. The crude oil was purified by silica gel chromatography and 10 eluted with EtOAc, EtOAc-MeOH (9:1, v/v), EtOAc- MeOH-EtjN (9:1:0.3, v/v/v) successively. Compound III was isolated as an oil in 47 0% (0.42g). yield.

Preparation of Compound IV: BOC-Glycine (0.16g, 0.91 mmol), HOBt (0.125g, 15 0.91 mmol) and 1-(3-dimethylaminopropyl) -3_-ethylcarbodiimide hydrochloride (EDC) ( 98 0%, 0.18g, 0.91 mmol) were dissolved in DMF (2.0 ml) and stirred at 0°C for an hour. The amine III (0.24g, 0.76 mmol) dissolved in DMF (3.0 ml) was added to 2 0 the reaction mixture, the reaction mixture was warmed to room temperature and stirred for 17h. DMF was removed in vacuo and the resulting material was dissolved in ethyl acetate. The ethyl acetate layer was washed with saturated sodium bicarbonate 25 solution,- brine and dried over magnesium sulfate. The organic layer was evaporated in vacuo and the crude mixture was flash chromatograhed on a silica gel column and eluted with EtOAc-MeOH-Et3N (9.5:0.5:0.3, v/v/v). Compound IV was isolated as an 3 0 oil in 82.0% (0.29g) yieid.

Preparation of Compound V: The BOC protecting group.was removed off the compound IV with TFA (5.0 ml) in methylene chloride 19 (5.0 ml). Methylene chloride was removed in vacuo and the residue was stripped with methylene chloride (20 ml x 3) and then with triethyl amine (3 ml x 3). 3-S-benzyl mercapto propionic acid (0.15g, 0.75 5 mmol), EDC (0.15g, 0.75 mmol), HOBt (0.103g, 0.75 mmol) and Et3N (0.35 ml, 2.48 mmol)were dissolved in DMF (5.0 ml) and stirred at 0°C for an hour. The solution of the amine (0.23g, 0.62 mmol) in DMF (3.0-ml) was added to the reaction mixture. The reaction 10 mixture was warmed to room temperature and stirred for 17h. DMF was evaporated in vacuo and the residue was dissolved in EtOAc. The EtOAc layer was washed with 10% NajCOj, brine, dried (over MgS04) and evaporated in vacuo. The crude material was purified 15 on a flash silica gel column and eluted with EtOAc-MeOH-Et3N (9:1:0.3, v/v/v) . Compound V was isolated as an oil in 60.0% (0.205g) yield.

Preparation of Compound VI (CP-0488): 32.0 mg (0.057 mmol) of V was deprotected using 2 0 the procedure described for II and the thiol isolated was reacted with compound X (0.073g, 0.048 mmol) in tris buffer. The crude mixture was purified using the procedure for II. Retention time of the product was 16.82 minutes. 9.5 mg (10%) of VI was 25 obtained as a white powder on lyophillization.

Mass spectral Data: calulated molecular weight 2002 observed molecular weight 2004 Amino Acid Analysis: 3 0 Gly 1.76 (2), Arg 3.19 (3), Pro 1.06 (1), Leu 0.99 (1), Phe 2.06 (2), Hyp 0.95 (1).

Preparation of Compound VII: Oxymorphone hydrochloride (0.56g, 1.66 mmol) , S-benzyl cysteamine (0.69g, 4.15 mmol), acetic acid (1.52 ml, 26.5 mmol) and sodium cyanoborohyaride 5 (O.llg, 1.66 mmol) were used according to the procedure for I. The composition of the third eluant, EtOAc-MeOH-Et3N, used in the purification of the crude mixture was 9:1:0.3, v/v/v. On purification, 0.213g of compound VII (29.0%) was 10 isolated as an oil.

Preparation of Compound VIII (CP-04 94): VII (0.063g, 0.14 mmol) was deprotected following the procedure for II. The thiol was then treated with X (0.335g, 0.152 mmol) in tris buffer.-15 The crude mixture was purified on a reverse phase Vydac C-18 .HPLC column using the gradient 15 - 70% CHjCN in water, 0.1% constant TFA over 35 minutes. VIII had a retention time of 15.0 minutes. 119.0 rag (45.0%) of VII was isolated as a white powder on 2 0 lyophillization.

Mass Spectral Data: calculated molecular weight 1902 observed molecular weight 1904 Amino Acid Analysis: Gly 0.81 (1), Arg 3.12 (3), Pro 1.07 (1), Leu 0.99 (1), Phe 2.04 (2), Hyp 0.98 (1).

Preparation of Compound IX (CP-0499): VII (0.009g, 0.02 mmol) was deprotected following the procedure for II and the thiol was 30 then reacted with XI (0.026g, 0.016 mmol) in tris buffer. Crude mixture was purified on a Vydac C-18 21 reverse phase HPLC using the gradient 15 - 70% CH3CN in water, 0.1% constant TFA, flow rate of 8.0 ml/min over 40 minutes. Retention time of IX was 14.22 minutes. IX (6.4 mg, 20.0%) was isolated as white 5 powder on lyophillization.

Mass Spectral Data; Calculatedmolecularweight 1957 Observed molecular weight 1958 In Vitro Testing The BKAn/mu-opioid receptor agonist heterodimers were evaluated in vitro using the rat uterus (3K2-receptor activity) and the electrically stimulated guinea pig ileum (mu-opiate receptor activity) assays. These assays are well known in 15 the art. The results obtained are shown in Table I: TABLE I Compound pA2-Rat Uterus CP-0126 7.1 CP-0347 9.5 oxycodone inactive oxymorphone inactive CP-0477 7.9 CP-0488 8.2 CP-0494 8.4 CP-0499 8.9 IC,0 Guinea Pig Ileum (nmolar) inactive inactive inactive 21.7 inactive inactive 24.0 17.0 It should be noted that neither oxycodone nor the heterodimers derived from oxycodone (CP-0477 and CP-0488) showed any activity in the in vitro guinea pig ileum assay of mu-opiate receptor agonist activity. This is probably due to the fact that for 22 complete activity, oxycodone apparently needs to be demethylated in vivo. As a result, oxycodone and oxycodone-based compounds would not be expected to show activity in an assay wherein the appropriate 5 demethylating enzymes were missing.

More important, however, are the data regarding the activity of the BKAn component of these heterodimers on the rat uterus and the data regarding the activity of the oxymorphone containing 10 compounds. As can be seen from the data outlined in Table I, full BKAn activity was retained in all of these heterodimers and in those compounds utilizing oxymorphone as the opiate, full mu-opiate receptor agonist activity was also retained. From these 15 data, it is evident that BKAn/mu-opioid receptor agonist heterodimers can interact with their respective receptor populations in in vitro systems.

In Vivo Testing In order to test the activity of these 20 compounds in vivo, a model of inflammatory and neurogenic pain was used. This model measures the behavioral responses of mice injected in the hind limb foot pad with 50 ul of formalin. The data from these studies are summarized in Figures 2, 3 and 4, 25 Control mice (open circles) show a characteristic bi-phasic response to the injected formalin wherein there is a short lasting initial response followed by a quiescent period which is then followed by a sustained period of hind limb licking. The licking 3 0 behavior is interpreted to mean that the limb is irritated and painful. The greater the time spent licking, the more painful the stimulus.

Oxymorphone (Fig. 2 A and B) reduces both phases of the licking behavior but with significant 23 behavioral obtundation resulting in catalepsy and frank respiratory depression at the highest doses (0.9 and 3.0 umoles/kg). The bradykinin antagonist CP-0127 (a potent BK2 selective antagonist--Fig. 3 A 5 and B) will reduce the time spent licking in both phases of the formalin test but at doses that are substantially greater than would be practical in a clinical setting. CP-0494 (Fig. 4 A and B) , however, not only blocks both phases of the pain 10 response, but does so at doses substantially lower (0.1 umples/kg) than for either oxymorphone (0.9 Umoles/kg) or CP-0127 (12.6 umoles/kg) alone and, of ecjual or greater importance, with no observable narcotic effects over several hours. These data 15 indicate that BKAn/mu-opioid receptor agonist heterodimers are pharmacologically qualitatively superior to either of the parent pharmacophores as would be expected from the theoretical considerations outlined above.

One skilled in the art will appreciate that the compounds described are representative of a wide variety of compounds in which each of the components of the heterodimer {BKAn, linker and/or mu-opioid receptor agonist) can be varied to produce the 25 optimal effect desired.

Example 5 (BKAn/NEI) A BKAn/NEI type of compound (CP-0502) of the structure shown in Synthetic Scheme 3, was synthesized to illustrate that this class of 30 compounds can be used as'a potent topical arid/or systemic anti-inflammatory agent. This compound is derived from CP-0126 and the prototype elastase inhibitor, CE-1218 (see Compound (6) , Synthetic Scheme 1 below).

PCT7US93/10222 CE-l2 J B The linking element used in this heterodimer was chosen so as to allow for unhindered'hydrolysis of the joining ester bond by serum esterases. Those skilled in the art will recognize that the linker 5 can be modified so as to provide different rates of hydrolysis varying from rapid to practically zero by altering the steric accessibility of the ester carbonyl carbon or by changing the chemistry to an amide linkage. Completely stable linker moieties 10 can also be used which are free from potential hyarolytic degradation.

Synthesis and analysis of BKAn/NEI heterodimer(s) The synthesis of these compounds is illustrated by reference to Synthetic Schemes 2 and 3 and the 15 following detailed synthesis description which includes the preparation of the elastase inhibitor CE-1218 according to Synthetic Scheme 1: SYNTHETIC SCHEME 1; Synthesis of 4 -tert-Butvlacetophenone (1) 2 0 To a dry 1-L flask was added CS2 (250 mL) and A1C13 (133.34 g, 0.56 .mol) with stirring. The suspension was cooled in an ice bath and a solution of tert-butylbenzene (50.00 g, 0.37 mol) and acetyl chloride (78.50 g, 0.41 mol) was added dropwise over 25 2 hr (not allowing the temperature to rise above 25°C) . The reaction was allowed to stir at room temperature overnight and then poured into a 2 L beaker filled with ice. After quenching with 200 mL of 6 N HCl the solution was saturated with NaCl and 30 separated.. The aqueous layer was washed with ether (2 x 100 mL) and combined with previous organics.

This new organic solution was washed with water (100 mL) , dried (MgS04) and evaporated to give an oil 26 which was distilled to give 52.1 g (79.3%) of 4-Cert-acetophenone as a clear colorless oil (bp0os mm 70-76°C) . :H NMR (CDC1,) 6 1.35 (s, 9 H) , 2.58 (s, 3 H) , 7.48 (d, J = 8.5 Hz, 2 H), 7.91 (d, J = 8.5 Hz, 5 2 H) . 13C NMR (CDCl3) <5 26.42,. 30.96, 34.95, 125.36, 128.16, 134.49, 156.64, 197.61.

Synthesis of Methvl 4-Cert-butvlphenvlacetate (2) A dry 1 L flask equipped with a mechanical stirrer containing Pb(OAc), (132.06 g, 0.298 mol) and 10 250 mL of benzene was purged with nitrogen and cooled in an ice bath. To this cooled slurry was added dropwise a solution of BF3-OEt2 (137.8 mL, 1.12 mol), 4-tert-butylacetophenone (50.00 g, 0.284 mol) in 70 mL of methanol over 1 hr. This mixture was 15 allowed to stir overnight, quench with water (500 mL), diluted with 250 mL ether and the layers separated. The organic layer was washed with water, diluted NaHC03 (carefully) and dried over MgSO,. The mixture was filtered, evaporated and distilled to 20 give 31.2 g (53.4%) of methyl 4-tert- butylphenylacetate as clear colorless oil (bp0 04 mm 75-80°C) . . 1H NMR (CDCl3) 1.32 (s, 9 H) , 3.62 (s, 2 H), 3.71 (s, 3 H), 7.23 (d, J = 8.4 Hz, 2 H), 7.37 (d, J = 8.4 Hz, 2 H) . 13C (CDC13) 31.33, 34.46, 25 40.67, 52.04, 125.53, 128.88, 130.91, 149.94, 172.26.

Synthesis of Methvl 4-tert-butviphenvlisobutvrate ill A solution of methyl 4-tert-butylphenylacetate 3 0 (30.00 g, 0.14 5 mol) and iodomethane (45.41 g, 0.320 mol) in 125 mL of dry JHF was added to a slurry of NaH (8.72 g, 0.363 mol) in 200 mL of THF dropwise over 30 minutes. After completion of the addition, per/ US93/10222 27 the reaction mixture was heated at reflux for 1.5 hr. The reaction was allowed to cool to room temperature, filtered through Celite and • concentrated. The residue was diluted in ether, 5 washed with H20, and dried over MgS04. Evaporation of the solvent afforded the desired product as an oil. A mixture of the crude methyl 4-tert-butylphenylisobutyrate and 4:1 EtOH/H2C> containing KOH (10.07 g, 0.179 mol) were heated to reflux for 4 10 hr. The EtOH was evaporated in vacuo, the residual solution was acidified to pH 2 with- 2 N HCl, and the precipitated solid filtered. The white solid was the dried (60°C, 1 mm Hg, 24 hr) to give the desired product (23.45 g, 73.2% from methyl 4-tert-15 butylphenylacetate).. *H NMR (CDC13) 1.34 (s, 9 H) , 1.62 (s, 6 H) , 7.37 (s, 4 H) , 11.4-l2.,4 (brs, 1 H) . *3C NMR (CDClj) 26.16, 31.30, 34.35^ 45/81, 125.31, 125.48, 140.64, 149.66, 183.57. , Synthesis of 4 -(3'-carbo-tert-butoxv-propvl 2 0 mercapto) phenyl 4 - trert-butvlphenvlisobutvrate (4): A mixture of 4 -tert-butylphenylisobutyric acid (2.00g, 0.0091mol) and. thionyl chloride (1.62 g, 0.0136 mol) in 16 ml of CH2C12 was allowed to stir overnight under Argon. The volatiles were removed 25 under vacuum and the resulting solid was dissolved into THF (15 mL).and a solution of tert-butyl-4-(4'-hydroxyphenyl)mercaptobutyrate (2.44 g, 0.0091 mol), TEA (2.5 mL) in THF (15 mL) was added dropwise over 10 min. The mixture was stirred for 3 days, diluted 3 0 with Et20 and extracted with 5% NaHC03. The organics were washed with H20, brine and dried (MgS04) . After evaporization the colored oil was separated (HPLC, silica gel 70:30 CH2Cl2/hexane to CH2Cl2 linear graauent) to give the desired product as an oil 28 (2.20 g, 51.5%). rH NMR <CDC13) 6 1.33 (s, 9 H) , 1.43 (s, 9 H) , 1.70 (s, 6 H) , 1.8 8 (tt, J = 7,2 Hz, 2 H) , 2.35 (t, J m 7.2 Hz, 2 H) , 2.90 (C, J = 7.2, 2 H) , 6.92 (d, J m 8.6 Hz, 2 H), 7.32 (d, J « 8.6 Hz, 2 5 H) , 7.34 - 7.4 0 (m, 4 H) . 13C NMR (CDC13) <5 24.50, 26.42, 28.08, 31.31, 33.70, 34.11, 34.39, 46.40, 80.40, 121.92, 125.23, 125.46, 13a.82, 133.03, 140.86, 149.58, 149.69, 172.21, 175.34.

Synthesis of 4-(3'-carboxv-propvlmercapto)phenyl 10 4 -tert-butvlohenvlisobutvrate (5) Trifluoroacetic acid (25 mL) was added to a stirred solution of 4-(3'-carbo-tert-butoxy-propylmercapto)phenyl 4- tert-butylphenylisobutrate (2.40 g, 0.00510 mol) in 20 mL of CH2C12 over 15 min. 15 After an additional 15 tnin the volatiles were removed and the oil crystallized (hexane) to give 1.94 g (91.8%) of desired product as a white solid, m.p. 86.0-87.0°C, lH.(CDCl3) 1.33 (s, 9 H) , 1.70 (s, 6 H) , 1.92 (tt, J= 7.0 Hz, 2 H), 2.50 (t, J = 7.0 Hz, 20 2 H), 2.93 (t, J = 7.0 Hz,-2 H), 6.93 (d, J = 8.7 Hz, 2 H) , 7.33 (d, J = 8.7 Hz, 2 H) 7.35-7.39 (m, 4 H) . 13C NMR (CDC13) 23.89, 26.45, 31.32, 32.34, 33.63, 34.42, 46.42, 122.03, 125.24, 125.48, 131.12, 132.63, 140.85, 149.74, 175.40, 178.65.

Synthesis of 4-(3'-carboxv-propvlsulfonvl)phenyl 4 -tert-butvlphenvlisobutvrate (6) To a 50 mL flask was added 4-(3'-carboxy-propylsulfonyl)phenyl 4 -tert-butylphenylisobutyrate (1.64 g, 0.00396 mol), HOAc (25 mL) and 15 mL of 30% 30 ,H202. The reaction was allowed to stir overnight, diluted with HjO (50 mL) and the resulting solid filtered. After drying (12 hr, 1 mmHg) the solid was recrystallized (CH2Cl2/hexane) to give 1.54 g 2 9 (37.1%) of the desired product as a white powder, mp 107-108.5°C. lH (CDClj) 1.33 (s, 9 H),,172. (s, 6 K), 2.02 (p, J= 7.0 Hz, 2 H), 2.52 (t, J = 7.0 Hz, 2 H), 3.17 (t, J = 7.0 Hz, 2 H), 7.2 0 (d, J = 8.7 Hz, 2 H), 7.36 (d, J =8.7 Hz, 2 H), 7.41 (d, J = 8.6 Hz, 2 H) , 7.90 (d, J = 8.6 Hz, 2 H) . 13C NMR (CDC13) 17.88, 26.29, 31.28, 31.79, 34.42, 46.53, 55.04, 122.56, 125.16, 125.61, 129.70, 135.81, 140.22, 150.02, 155.29, 174.73, 177.71.

Synthesis of 6-Maleimidohexanol (7): The synthesis of 6-maleimidohexanol to be used for linking is illustrated in Synthetic Scheme 2 below: SYNTHETIC SCHEME 2 NaHC03, aq MeO HO (7) To a 100 mL flask was added 6-aminohexanol 15 (0.76 g, 0.0064 mol) and 25 mL of saturated NaHC03. The homogenous was allowed to stir a RT and N-methoxycarbonylmaleimide (1.00 g, 0.0064 mol) was added as a solid. The mixture cleared shortly after the addition and was allowed to stir for 1 hr. The 20 mixture was extracted by EtOAc, dried (MgS04) and evaporated. The resulting mixture was separated on silica gel (CH2C1, to EtOAC) . To give the product as a white solid 0.32 g (25.2%), used without further purification. (CDC13) <5 1.25 - 1.45 (rn, 4 H) , 1.45 - 1.70 (m, 4 H) , 3.53 (t, J = 7.3 Hz,' 2 H) , 3.63 (t, J= 6.0 Hz), 6.71 (s, 2 H) .

Synthesis of CP-0502: Compound (6) (CE-1218) was esterified with compound. (7) to form compound (8) which was then conjugated to CP-0126 to form the dimer CP-0502. These latter reactions are illustrated in Synthetic Scheme 3: iL. 31 SYNTHETIC SCHEIVIE 3 q \ -/ n (6) Bop-CI (7) TEA ' chici-i 0 \ / . N \ 1 O (8) CP-0126 DEA DMF H,N y= NH-TFA TFA 0 > ■\ II O NH, tfa-HN=^ , V , ^V^Vv-^Vv-^. ( " HN )*= NH-TFA h2n O CP-0502 h o 32 Referring more specifically to Synthetic Scheme 3, compound (6) (200 mg, 0.448 mmol), triethylamine (0.124 ml, 2 eqv), 6-maleimidohexanol (7) (97 mg. 1.1 eqv) were dissolved in 2 ml methylene chloride. 5 Bis(2-oxor3- oxazolidinyl)phosphinic chloride (122 mg, 1.0 eqv) was added to the stirred solution. The resulting suspension was stirred at room temperature for four hours. The reaction mixture was diluted with 25 ml methylene chloride and washed with 10 saturated NaHC03. The organic solution was dried over anhydrous MgS04 and the solvent was removed in vacuo. Silica gel chromatography (2X18 cm column); 3 5/65:acetone/hexane (Rf=0.4) as eluent provided the compound (8) as a colorless oil.

Compound (8) (50 mg, 0.08mmol) was dissolved in ml DMF containing 100 ul diisopropylethylamine. 100 mg (0.08mmols) of CP-0126 was added and reaction proceeded for 3 0 minutes, with occasional mixing. The reaction mixture was injected on a Vydac 1" C-18 2 0 reverse phase column, and eluted at lOml/min, %-90% acetonitrile in H20 over 35 minutes (Constant .1% TFA). The appropriate fractions were lyophilized to yield 52 mg (35%) -of a colorless white powder (CP-0502) Laser desorption mass 25 spectrometry M/Z =1890 (M+H), calculated 1890.

Automated amino acid sequence results confirmed the correct peptide sequence with no altered amino acids.

In vitro activity of BKAn/NEI heterodimer(s) 3 0 In vitro evaluation of BKAn and NEI activity of the following compounds was carried out according to standard protocols well known to those in the art. BKAn activity (pA2) was assessed using the rat uterus preparation and NEI (K^*.) activity was evaluated 33 using purified human neutrophil elastase (HNE) and a synthetic soluble chromogenic substrate, methoxysuccinyl-alanyl-alanyl-prolyl-valyl-paranitroaniline (MOS-AAPV-pNA) . The inhibitor was 5 mixed with MOS-AAPV-pNA (0.5 mM) in 0.05 M sodium phosphate, 0.1 M NaCl, 0.005% Triton X-.100, 5% DMSO, pH 7.5. HNE (10-20 nM) is then added. The . production of nitroaniline was monitored spectrophotometrically at a wavelength of 400-410 nm 10 at 25 C. An ENZFITTER program then automatically calculated standard enzyme kinetic parameters including K^8.

The following results were obtained: TABLE II Compound pA2--Rat Uterus Kx" (HNE) nM CP-0126 7.1 inactive CE-1218 inactive 10.5 CP-0487 8.4 inactive CP-0502 7.5 6.6 The data in Table II indicate that for NEI activity there is little difference between the intact heterodimer (CP-0502) and the free monomeric NEI moiety (CE-1218) as far as their respective Kj's 25 are concerned. This is not true for the activity of the BKAn portion of the intact heterodimer relative to its hydrolysis product, CP-0487 (the succinimidohexanol derivative of CP-0126) wherein the intact compound is almost a full log less potent 3 0 than the monomeric BKAn. Interestingly, the activity of the intact compound displayed a type of irreversible bradykinin antagonism and an apparently enhanced antagonist activity of bradykinin induced PCT/IJS93/10222 uterine contractions at longer incubation times. These types of receptor interactions are not well measured by standard pA2 analyses so the differences in activity observed between CP-0487 and CP-0502 5 with respect to BKAn activity may be more apparent than real. Regardless of the molecular pharmacologic mechanisms underlying these data it is clear that combined BKAn and NEI activity can be incorporated into a single molecule. 10 The above data suggest that allowing for in vivo hydrolysis of the intact compound may alter the behavior of the two moieties so as to enhance the overall in vivo activity of the primary compound. Unfortunately, there are no established animal 15 models that can be employed to assess combined BKAn and NEI activity in vivo. Therefore, in order to assess the potential for in vivo hydrolysis of the intact heterodimer, an in vitro "surrogate" system was employed wherein the parent heterodimer (CP-20 0502) was incubated with human plasma and the resulting metabolites analyzed by reverse phase HPLC.

CP-0502-was added to- freshly obtained normal human plasma and allowed to incubate at 37°C for 25 varying amounts of time. At the designated time the samples were treated with acidified (0.1 N HCl) acetonitrile in order to precipitate the plasma proteins. Aliquots (75 ul) of the supernatents were then analyzed on a Vydac C-18 reverse phase HPLC 30 column using 24% to 80% acetonitrile gradient in 0.1% TFA. The eluent was monitored at 214 nm.

Figures 5 a and b are representative reverse phase HPLC chromatograms illustrative of this type of analysis. As can be seen from these 3 5 chromatograms, the parent compound appears to be 94/11021 readily hydrolyzed to the succinimidohexanol modified monomer, CP-0487 and its des-Args derivative (Plasma carboxypeptidase will cleave the terminal arginine residue from both the intact heterodimer, CP-0502, as well as CP-0487.) The apparent T1/2 of this hydrolysis reaction is approximately 113 minutes. The NEI component of the heterodimer is an active ester and undergoes hydrolysis as well. However, the intact NEI monomer as well as its hydrolysis products are obscured by the plasma derived peaks seen in the middle of this tracing and cannot be visualized using this system. Since the NEI is equally active as a component of the heterodimer as it is as a monomer, the dissociation of the heterodimer into its two component parts will have less of an effect on its activity than that for the BKAn component.

Those skilled in the art will appreciate that the hydrolysis rate of the heterodimer can be influenced by the steric and electronic environment of the "linking" ester moiety and that the type of chemistry used is only a single example of the types of chemistry, that can be .employed to adjust the rate of dissociation (or lack thereof) of the two components of the heterodimer.

Example 6 (BKAn/COI) Synthesis and Analysis of BKAn/COI Heterodimers A representative BKAn/COI heterodimer (CP-0460) was synthesized according to Synthetic Schemes 4 and 5 below: wo 94/11021 94/11021 37 Synthesis of 6-Maleimidchsxanvl 1-(4-chlorobenzovl) - 5-methoxv-2-methvl-3-inaonvlacetate (9) To a 100 mL flask was added indomethacin (1.90 g, 0.00532 mol), 25 mL of CH2C12 and DCC (0.55 g, 0.002S6 mol). After 2 hr, the mixture filtered, the DCU washed with 15 mL of CH2C12 and to this new solution was added 6-maleimidohexanol (0.50 g, 0.00253 mol) as a solid followed by anhydrous Na2C03 (0.32 g, 0.00304 mol). After 4 days the mixture was filtered diluted with Et20 and washed with 5% NaHC03, H,0 and dried (MgSO«) . The resulting yellow oil was purified in a HPLC (silica gel; CH2C12 to 80:20 CH2Cl2/EtOAc, linear gradient 60 min.) to give 0.79 g \58.0%) of the desired product as a yellow oil. *H (CDC13) 1.20 - 1.35 (m, 4 H), 2.38 (s, 3 H), 3.47 (t, J = 7.3 Hz, 2 H), 3.66 (s, 2 H), 3.83 (s, 3 H) , 4.08 (t, J m 6.6 Hz, 2 H), 6.66 .(J - 9.0 Hz, J" = 2.5 Hz, 1 H), 6.68 (s, 1 H), 6.87 (d, J = 9:0 Hz, 1 H), 6.96 (d, J = 2.5 Hz, 1 H), 7.47 (d, J = 8.5 Hz, 2 H), 7.66 (d, J = 8.5 Hz, 2 H).

Conjugation of compound (9) with CP-0126 to form CP-0460 is illustrated in Synthetic Scheme 5 and described thereafter: wo 94/11021 PCX/US93/10222 38 synthetic scheme 5 J NH-TFA H2N nh, tfa \ ' tfa-hn nh ri «h H« o H 0 H O »-V ■ hn nh-tfa OH h o ^ h o vh o sh CP-0126 h,n ch30 dmf diea NH4HCO3 h,n y= NH-TFA nh, TFA h h o f h o f h o TFA-HN^ nh •HjN N ^ n-Sr ^ ^ n-Sr ^ n I Avn ' u . • hn )c=z nh-tfa h2n ch3o OH CP-0460 39 CP-0126 (100 mg, 0.08 mmol) was reacted with compound (9) (0.12 mmol, 1.5 eqv) in 2 mL 95% DMF/5% 0.1M ammonium bicarbonate containing 50 ul diisopropylethylamine, for 30 minutes, with 5 occasional mixing. The reaction mixture was purified in 1 injection on a Vydac 1" C-18 reverse phase column at lOml/min, using a gradient running from 15% acetonitrile/0.1% TFA to 40% acetonitrile/0.1% TFA in 2 0 minutes. Appropriate fractions were 10 iyophilized to yield 64 mg (45%) of a colorless powder (CP-0460). Laser desorption mass spectrometry: M/Z= 1802 (M+H), calculated 1802.

As mentioned previously, for the COI to work it may need to be dissociated from the BKAn so as to 15 allow for its intracellular penetration.

Therefore, in order to evaluate the functional activity of a BKAn/COI heterodimer, CP-0460 was exposed to rat lung parenchymal strips which were then challenged with arachidonic acid. This tissue 20 is known to contain both non-specific esterase activity as well as to convert arachidonic acid to thromboxane (via a cyclooxygenase dependent pathway) which is then ultimately responsible for the smooth muscle contraction observed in this assay. 25 Using this system, the log dose ratio shifts for indomethacin and CP-0460 were found to be 0.998 +/- 0.425 and 1.029 +/- 0.042 respectively indicating that both indomethacin alone and CP-0460 will prevent the contraction, produced in response to 30 exogenously applied arachidonic acid with .qual potency. BKAn's have no effect on this system in and of themselves. These data indicate that the COI component of BKAn/COI heterodimer is functionally active in a tissue containing both esterolytic and 35 cyclooxygenase activities. 40 Intact CP-0460 was also tested for BKAn activity using the standard rat uterus assay .and the pA2 of the CP-0460 was found to be approximately 7.8. Again, CP-0460 (similarly to CP-0502) did not behave 3 as a classical competitive antagonist of bradykinin induced uterine contraction but rather as a type of "pseudo-non'-competitive" antagonist, particularly at higher concentrations. This atypical behavior cannot be attributed to COI activity per se as free 10 indomethacin has no effect on this assay at any concentration.

Regardless of the explanation for the observed data, one skilled in the art will appreciate that, as in the other two classes of compounds illustrated 15 herein, pharmacologically important BKAn/COI heterodimers can be made using a variety of appropriate linking moieties to provide a free hydroxyl and the carboxyl group (a common feature of many COIs) of the COI monomer to form a hydroiyzable 20 ester based heterodimer. Compounds such,as these may be used in the treatment of a variety of inflammatory or painful conditions as well as in the treatment of dysfunctional uterine smooth muscle activity.

While the invention has been exemplified above by the use of Y components which are non-peptides, this component may equally comprise in whole or part a peptide as exemplified in the afore-mentioned WO 92/17201, including the heterodimers therein 3 0 described.

The dimers of the invention may be used in the form of conventional pharmaceutical compositions comprising the active component and a pharmaceutically acceptable carrier. Such 25 compositions may be adapted fcr topical, oral, 41 aerosolized, intramuscular, subcutaneous or intravenous administration. The amount of active component present in such compositions will range from, for example, about 0.0 01 to 90.0% by weight 5 depending on the application and mode of administration although more or less of the active component may be used. Conventional dosages will vary considerably on the basis of the intended application and mode of administration. Usually, 10 however, an effective dose is in the order of 0.1 to 1000 micrograms per kg body weight.

The scope of the invention is defined in the-following claims wherein: ( > What is claimed is: ' 1. A heterodimer of the formula: (BKAn)(X)(Y) where BKAn is a bradykinin antagonist peptide; Y is a mu-opioid receptor agonist, neutrophil elastase inhibitor, cyclooxygenase inhibitor or a neurokinin receptor antagonist; and X is a linking moiety chemically joining the BKAn and Y components.

Claims (36)

1. 42 CLAIMS 25 7 4
2. 2. A heterodimer according to claim l wherein Y is a mu-opioid receptor agonist. (
3. 3. A heterodimer according to claim 1 wherein Y is a neutrophil elaptase inhibitor.
4. 4. A heterodimer according to claim 1 wherein Y is a cyclooxygenase inhibitor.
5. 5. A heterodimer according to claim l wherein Y is a NK; receptor antagonist or NK, receptor antagonist.
6. WO 94/11021 PCT/US93/10222 25 7 4 43
7. 7. A heterodimer according to claim 1 wherein X is non-hydroiyzable.
8. 8. A heterodimer according to claim 1 wherein X comprises an amino acid or an amino acid analog 5 incorporated into the BKAn.
9. 9. A heterodimer according to claim 1 wherein X comprises a maleimide/succinimide-based linkage.
10. 10. A heterodimer according to claim l wherein X comprises a bissuccinimidoalkane. 10 n.
11. A heterodimer according to claim 1 wherein X comprises the S atom of a BKAn peptide sulfhydryl group.
12. 12. A heterodimer according to claim 1 wherein Y is selected from the group consisting of oxycodone 15 or oxymorphone.
13. 13. A heterodimer according to claim 1 wherein Y is indomethacin
14. 14. A heterodimer according to claim 1 wherein Y is a neutrophil elastase inhibitor and X includes 20 a succinimide group attached to BKAn through the sulfur atom of a sulphydryl group of the BKAn peptide chain.
15. 15. A heterodimer according to claim 14 . wherein the neutrophil elastase inhibitor is 4 -(3'-25 carboxy-propylsulfonyl)phenyl-4 -tert-butylphenyl isobutyrate. «fc .. 25 7 4
16. 16. A compound comprising a pharmacophore selected from a mu-opioid receptor agonist, neutrophil elastase inhibitor, cyclooxygenase inhibitor or a neurokinin receptor antagonist and a linker group for linkage to the BKAn peptide. 5
17. 17. A compound according to claim 16 wherein the linker group' comprises an amine, a carboxylic acid, a hydroxyl, a sulfhydryl or a maleimide group or an amino acid capable of incorporation into a peptide. 10
18. 18. A compound comprising a BKAn peptide and a linker group.
19. 19. A compound according to claim 18 wherein the linker group comprises a sulfhydryl group, a maleimide group or an alkane chain terminating in a hydroxyl, amine or carboxylic acid group. 15
20. 20. A compound according to claim 16 wherein the pharmacophore is a mu-opioid receptor agonist.
21. 21. A compound according to claim 16 wherein the pharmacophore is 2 0 a neutrophil elastase inhibitor.
22. 22. A compound according to claim 16 wherein the pharmacophore is a cyclooxygenase inhibitor. 25
23. 23. A compound according to claim 16 wherein the pharmacophore is a NK, receptor antagonist or NK2 receptor antagonist.
24. 24. A heterodimer according to claim 1 wherein BKAn is DR-R-P-J-G-F-C-DF-L-R or DR-R-P-J-G-Thi-G-D lic-Oic-R. WO 94/11021 PCT/US93/10222 25 7 4 77 45
25. 25. A heterodimer according to claim 24 wherein the pharmacophore is a mu-opioid receptor. agonist.
26. 26. A heterodimer according to claim 24 5 wherein the pharmacophore is a neutrophil elastase inhibitor.
27. 27. A heterodimer according to claim 24 wherein the pharmacophore is a cyclooxygenase inhibitor. 10
28. 28. A heterodimer according to claim 24 wherein the pharmacophore is NKi antagonist or NK2 antagonist.
29. 29. A heterodimer according to claim 26 wherein the pharmacophore is 4-(3'-carboxy- 15 propylsulfonyl)phenyl-4-tert-butylphenyl isobutyrate
30. 30. A heterodimer according to claim 29 wherein X comprises a bissuccinimidoalkane group joined by -S- to the BKAn'component.
31. 31. The compound 4-(3'-carboxy- 2 0 propylsulphonyl)-phenyl-4-tert-butylphenyl isobutyrate.
32. 32. A pharmaceutical composition comprising a heterodimer according to claim l and a pnarmaceutically acceptable carrier. \oB6 WO 94/11021 PCT/US93/10222 25 74 77
33. 33. a heterodimer of the formula as defined in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.
34. 34 • A pharmacophore as defined in claim 16 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.
35. 35. A BKAn peptide as defined in claim is substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.
36. 36. A pharmaceutical composition as claimed in claim 32 substantially as herein described with reference to any example thereof and with or without reference to tiie accompanying drawings. (OftTFf-14 |r,r> By the auinorioto etyuntb A. J Park & Son Par