Cyclization in Opioid Peptides

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798 Current Drug Targets, 2013, 14,798·816

Cyclization in Opioid Peptides

Justyna Piekielna, Renata Perlikowska, Katarzyna Gach and Anna Janecka'

Department oj Biomolecular Chemistry, Medical University of Lodz, Lodz, Poland

Abstraet: Endogenous opioid peptides have been studied extensively as potential therapeuties for the treatment ot"pain.The major problems of using natural opioid peptides as drug candidates are their poor receptor specificity, metabolic in-stability and inability to reaeh the brai n after systemie administration. A lot of synthetic efforts have been rnade to opioidanalogs with improved pharmacological properties. One important structural modification leading to sueh analogs is Cj-

elization ot"lincar sequences. Intramolecular cyclization has been shown to improve biological properties ot' various bioac-tive peptides, Cyclization reduces conformational freedom responsible tor the simultaneous activation of two or rnore re-ceptors, increases metabolic stability and lipophilicity which may result in a longer half-life and easier penetration acrossbiological membranes. This rev iew deals with various strategies that have been employed to synthesize cyclic analogs ofopioid peptides. Discussed are such bridging bonds as amide and amin e linkages, sulfur-containing bonds, includingmonosulfide, disulfide and dithioether bridges, bismethylene bonds, monosulfide bridges of lanthionine and, finally, car-bonyl and guanidine linkages. Opioid affinities and activities of cyclic analogs are given and compared with linear opioidpeptides. Analgesic activities ot' analogs evaluated in the in vivo pain tests are also discussed.

Keywords: Amide bonds, monosulfide bonds, disulfide bonds, carbonyl and guanidine bridges, opioid' receptor affiniry, anal-gesic activity.

1. INTRODUCTlON

Opium, the m ixture of about 50 alkaloids, was used forpain relief for thousands of years. The most abundant andpotent alkaloid of opium, morphine, is still in use in clinicsfor the treatment of severe postoperative ar cancer pain [I].However, serious side-effects which accornpany morphineadministration, such as constipation, respiratory depression,cardiovascular and gastrointestinal problem s, tolerance anddependence [2], are the driving force for seeking new, saferpain-ki lIers.

Morphine ac ts in the central nervous system (CNS)through the opioid receptors. Three major categories of socalled elassie opioid receptors have been identified (u, O, K

ar MOR, DOR, KOR, respectively), that differ both, in theirfunctions and binding characteristics [3-6]. Some years later,an additional opioid receptor like-I receptor (ORLI or NOR)has been discovered in brain sites that are involved in learn-ing and memory [7]. A II these receptors belong to the fam ilyot' G-protein-coupled receptors (GPCR) and share 'extensivestructural homologies between themselves and also withsomatostatin and ang iotens in receptors. The use of mo lecu larbiology methods allowed to assess the contribution of eachreceptor type in opioid induced analgesia [8-10].

Endogenous ligands of the olassie opioid receptors areopioid peptides, such as enkephalins, dynorphins,endomorphins which are smali molecules produced mostly inthe CNS but also in other glands throughout the body, that '

"Address correspondence to this author at the Department of BiornolecularChernistry, Medical University of Łodz, azowiecka 6/8,92-215 Lodz, Po-land; Tel: +4842 272 5706; Fax +4842 272 5694;Email: anna.janecka.EJumed.lodz.pl

1873-5592/13 $58.00+ .no

exert the same effects as morphine [II]. The accurnulateddata indicate that the most potent analgesic effects arernediated by MOR agonists. Unfortunately, rhey areaccompanied by the high level of abuse liability. DOR.agonists have reduced addictive potenrial but also lowerantinociceptive efficacy. KOR ligands have been shown [0

cause strong dysphoric effects and can be v iewed as potentialanalgesics only for peripheral use [12]. An endogenousligand ofNOR is nociceptin (NC), also called orphąnin (F'Q),which is an opioid-like neuropeptide of similar to dynorphinA structure. Nociceptin plays important roles in variousphysiological functions, most importantly in learn ing andmemory, locomotion, anxiety [13, 14]. The main srructuraldifference between nociceptin and other opioid peptides isthe replacement ofTyrl by Phel (Table I).

The role of opioid peptides as endogenous analgesicssuggests the ir potential use as new drugs for pain relief,devoid, at least in part, of undesired side-effects of opiatealkaloids [15, 16]. However, usefulness of opioids as drugcandidates is iim ited, as peptides in generał, by their pcormetabolic stability and low merubrane permeability [17, 18].To address these problems, a variety of chemical modifica-tions has been designed, to improve opioid peptide pharma-cokinetics.

One strategy for achieving analogs resistant to enzymariehydrolyses and with increased lipophilic character is cycliza-tion of linear sequences. In nature numerous exarnples ofcyclic peptides can be found and they often exhibit stabilityand potent biological activ ity [19-21 j. Cyclization is also animportant, well recognized metbod in peptide chernistry, lorgenerating analogs with improved bioactivities and bioavail-abilities [22]. In opioid peptidestudies cyclizanon of lincar

;Si 2013 Bentham Science Publishers

- id Peptides

T L Endogenous Opioid Peptides Discussed in this Review.

Current Drug Targets, 2013, VoL 14, No. 7 799

Endogeuous Peptide Amino Acid Sequence Affinity for Opioid Receptors

Mammalian

[Met]enkephalin Tyr-Gly-Gly-Phe-Met DOR,MOR[Leu]enkephalin Tyr-Gly-Gly-Phe-Leu (DOR»MOR)

dynorphin A (Dyn A) Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys- Trp-Asp-Asn-GlnKOR, MOR, DOR

(KOR»MOR and DOR)

endornorphin-I (EM-l) Tyr-Pro-Trp-Phe-NH, MORendomorphin-2 (EM-2) Tyr-Pro-Phe-Phe-NH, MOR

nociceptin/orphanin (NCIFQ) Phe-Gly-Gly-Phe- Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln NORlORL,

Amphibian

dermorphin Tyr- D-Ala- Phe-Gly -Tyr-Pro-Ser -NH, MORdeltorphin I Tyr-D-Ala-Phe-Asp- Val- Val-Gly-NH, DORdeltorphin II Tyr-D-Ala-Phe-Glu- Val- Val-Gly-NH, DOR

Milk-derived

bovine ~-casomorphin( 1-7) Tyr- Pro- Phe- Pro-Gly- Pro- Ile-OH MORmorphiceptin Tyr-Pro-Phe-Pro-NH, MOR

sequences has been used to reduce conformational flexibilityof molecules, preventing their adaptation to the differenttopographies of the various opioid receptor types. Cyclicpeptides, in comparison with their linear counterparts, haveoften been demonstrated to have greater potential as thera-peutic agents due to their increased receptor selectivity, en-zymatic and chemical stability, and improved pharmacody-namic properties [22].

Additionally, short cyclized peptides with restrictednumber of conformations are valuable tools for studyingreceptor-binding sites and spatial requirements for ligandbinding. Constrained peptides are also useful in identifyingpossible bioactive conformations and in developing pharma-cophoric madeis for receptor-ligand interactions. Further-mare, cyclization can be considered an important step for-ward in the design and synthesis of non-peptide mimeticswhich can be viewed as a new generation of peptide-baseddrugs.

[n this review, various strategies elaborated to obtain cy-clic analogs of opioid peptides are discussed. .

2. CYCLIZATION STRATEGIES

2.1. Amide Bond

2.1.1. Csterminus lo Side-Chain

The first cyclizations in opioid peptide field wereachieved by Schiller and co-workers via the C-terminus toside-chain amide bond linkage. Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu, l) analogs were synthesized through incorpo-ration of a D-a,co-diamino acid (e.g. A2pr, A2bu, Om, Lys)into position 2 of a peptide sequence and cyclization of theco-amino group with the C-terminal carboxyl group [23,24].The shortening or lengthening of the side-chain in position 2permitted subtle variations in conformational restriction.

Moderately DOR-selective Leu-enkephalin was thus eon-verted to MOR-selective analogs 7-10. In further studies thePhe residue in the forth position of analog 8 was rep lacedwith l-naphthylalanine (l-Nal), an amino acid containing amore extended aromatic ring system [25]. Resulting analog11 showed improved potency in DOR-representative mousevas deference (MVD) assay.

~-Casomorphin(1-5) 2 analogs 12, 13, cyclized via a C-terminus to side-chain lactam bridge, in which the Pr02 resi-due was replaced by a dibasic amino acid, showed signifi-cantly improved binding affin ities compared to ~-casomorphin and strong antinociceptive activity in rats [26].Substitution of the Phe3 residue in these analogs by 2-Nalresulted in obtaining mixed MOR-agonistsfDOR-antagonists14, 15 [27]. A 15-membered ring of 14 was optimal for themanifestation of DOR antagonism. Cyclic analogs of mor-phiceptin 3 of a similar structure (16, 17) were found to bevery active in both, MOR-representative guinea pig ileum(GPI) and MVD assays [28].

Recently, the first C-terminus to side-chain cyclized EM-2 analogs were reported [29]. In the sequence of EM-2 (5)the presence of Pr02 residue is crucial for the pro per ligandconformation. Pro is a stereochemical spacer capable of in-ducing favorable spatial orientation of aromatic pharma-cophores. Hruby and co-workers [29] utilized cis-4-amino-Pro (cAmp) to replace the native Pro. The presence of cAmpdoes not significantly alter the peptide backbone but offersan additional amino group available for a cyclization reac-tion. In' the side-chain to tail ring closure, an am ide bondbetween the cAmp-yNH2 group and the Phe4 carboxyl wasformed. The new analog, Tyr-c[ c/vmp-Phe-Phe] 18, did notshow affinity for DOR and KOR and only very poor affinityfor MOR. Changing configuration of the Phe3 and Phe4 resi-dues present in the ring led to analogs 19 and 20, with in-creased, though still low affinities for MOR [30]. Highly

••••••" ••••• LJ'r~ Targeis. 2013, VoL 14,No. 7

alogs 18-20 are the onły known EM anałogs\JUl..GM..~=:;..:'" rirhout addition of an extra residue, in order to hełp

- n o these very short peptides.

" 1_ Side-Chain to Side-Chain

A ifferent type of enkephalin analogs cyclized throughformation of an amide bond between side-chain amino

d carboxyl groups of appropriate residues was describedb. Schiller et al. [31]. For example, analog 21 with D-Lys2and Glu), further modified by amidation of the C-terminus," as highly potent but non-selective, whereas transposition ofGlu and Lys residues resulted in analog 22, with moderatepreference for MOR [32].

Shorter, tetrapeptide cycl ic sequences (23-25) containinga Phe residue in the third position, as it is the case with mor-phiceptin, dermorphin and EM-2, were also synthesized [33].A cyclic analog, Tyr-cjfx-Orn-Phe-Asp'[NH, 23, whieh eon-tained a rigid 13-membered ring structure, showed moderateMOR affinity. Expansion of this 13- to 15-membered ringcontained in Tyr-cjn-Lys-Phe-Glujl-ll-l, 25 produced themore relaxed conformational eonstraints which resulted inbetter compatibility with DOR.

Cyclization of linear peptides via amide bridges usuallyresulted in obtaining cyelodimers as side-products [34, 35].The extend of eyelodimerization is most likely governed byconformational factors. Generally, with two residues in be-tween the residues engaged in the ring ciosure, eyeliemonomer. formation was favored over cyclodimerization[34]. In contrast to the high poteneies of the eyelie mono-mers, the activities ofthe eyclodimers were quite low [35].

Moleeular mechanies studies performed for some eyelieanalogs revealed that exoeyclic Tyrl residue, as well as thePhe3 side-ehain still show considerable eonformational free-dom [36, 37]. Since these moieties are crucial for the opioidactivity, Schiller et al [38] designed a series of cyclic ana-logs of Tyr-clu-Orn-Phe-Glujr-lH, 26 that contained variousconformationally restricted surrogates of Phe in position 3 ofthe peptide sequence. Comparison of the opioid receptoraffinity profile of analogs 27-30, containing Ca

_

methylphenylalanine (C"MePhe), ortho-methylphenylalanine(oMePhe), 2-aminoindan-2-carboxylic acid (Aic), and 2-aminotetralin-2-earboxylie aeid (Ate), respeetively, (Fig. 1)led to the demonstration that the side-chain conformationalrestriction may result in enhaneed receptor seleetivity of cy-clic opia id peptides.

~COOHV / 'NH2

COQH

C"- MePhe o-MePhe

COOH

CXJNH'AIC Atc

Fig. (1). The structure of confonnationally restricted amino acidsused for the synthesis ofanalogs 27-30.

Piekielna et al.

A cyclic enkephalin analog containmg a retro-inversopeptide bond between GIl and Phe4 was also synthesizedbut was compłeteły inaetive [39).

An interesting apgroach was used by Pil et al. [40] to ob-tain two new [D-Pro ]morphiceptin analogs. They introduceda 6-atom bridge between the C-terminal amide group andPr02 residue substituted in position 4 by an amine or hy-droxyl group. The resulting peptides, 31 and 32, eyelized viaan amide ar ester bridge, respeetively, are shown in Fig. 2.Their opioid receptor affinities were not tested but they werefound to be stable against dipeptidyl peptidase rv (DPPlV),the main enzyme responsible for morphiceptin cleavage inphysiological conditions.

o

H2N

~

HO#-

HN

31 X =NH(I ,

32X=O

Fig. (2). Structure of analogs 31 and 32, cyclized through amideand ester bridge, respectively.

In the search for strong antinociceptive agents, cyclicpentapeptide analogs (33-36) of a generał formula Tyr-cjXaa-Phe-Phe- Yaa]-NH2 ar Tyr-cj Xaa-Phe-D-Pro- Yaa]-NH2 (where X, Y = Lys ar Asp), based on EM-2 ar mor-phiceptin structure, respectively, were synthesized by Jan-eeka and eo-workers [41]. The strong analgesie effeet inmice after i.c.v. administration was observed only for ana-logs 34 and 36, with Asp in position 2. High analgesie activ-ity of these analogs was inconsistent with their poor MORbinding affinity. Surprisingly, antinociception produeed byAsp'' analogs was reversed not only by the concomitant ad-rninistration of the universal opioid antagonist, naloxone, butalso by the KOR antagonist, nor-binaltorphimine (nor-BNI).In the earlier studies Tseng [42] demonstrated that analgesiaindueed by EM-2 but not EM-I (4), was partially blocked bythe pretreatment with nor-BNl, indicating that EM-2 antino-eieeptive aetivity may be elieited, at least in part, by stimu la-tion of the KOR. Therefore, cyclic peptides 34 and 36 couldelicit their effects partially by MOR and partially through therelease of Dyn A which activated KOR. In the subsequentstudies, amino. acids of D-configuration (n-Lys or n-Asp)were introduced into position 2 of the eyelie analogs 33 and34. Thus obtained peptides 37 and 38, which showed a dras-tie increase in MOR affinity, were alsa resistant to enzymatiedegradation and produced remarkable antinoeieeptive activ-ity following intraeerebroventricular (i.c.v.) administration

.[43]. Antinociception elicited by the most patent cyclic ana-log, Tyr-cjp-Lys-Phe-Phe-Aspj Nl-l- 37 was much strongerand longer łasting than that of EM-2. This analog was found

gęsia also after systemie administration andabolished by the concomitant i.c.v. admini-

e _lOR antagonist, ~-funaltrexamine W-FNA),mat antinociception was mediated by the

_._~3. - ~e min. Central action of this cyclic analog indi---- -- -as able to cross the blood-brain barrier (BBB)

",,-,c::;-. __ evidence that cyclization might be a useful strategy= ec - cing bioavailability of opioid peptides. In order to-~~ r philicity, further modification of the cyclic ana-

entioned above was performed by substitution of Tyrl

_ Dm (Dmt = 2',6'-dimethyltyrosine). Obtained analogs~9-ndispla ed high MOR affinity and enzymatic stability

d ałso remarkable, long-Iasting analgesic activity fo llow-ing central but not peripheral administration [44]. It seemsthat lipophilicity may not be the only factor determiningbrain passagę ofthese analogs. .

A great num ber of cyclic Dyn A analogs obtained byside-chain to side-chain am ide bond formation was also re-ported but they were only moderately selective KOR-agonists [45-51].

2.13. Nsterminus to Side-Chain

The presence of a cationic amino group, a phenol moietyand an additional aromatic ring, all properly oriented inspace, are cons idered necessary for the man ifestation of bio-logical activity of opioid peptides through interactions withopioid receptors. The cyclic opioid analogs mentionedabove, obtained by the formation of lactam bridges betweenappropriate side-chain residues, were designed to preservethese crucial structural elements, most importantly the N-terminal protonable amine. Indeed, the free N-terminalamino group of opioid ligands is believed to play an essentialrole in signal transduction at both, MOR and DOR. The dele-tion of this positively charged amino group or its rep lace-ment is considered to be a generał structural modification toconvert opioid peptide agonists into antagonists [52]. Thereplacement of the N-terminal Tyr residue with 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methy 1-3-(2' ,6' -dimethyl-4' -hydroxypheny l)propionic acid(Mdp) (Fig. 3) converted selective peptide agonists intohighly selective antagonists [53-55].

COOH COOH

HO HO

Dhp Mdp

Fig. (3). Structure of des-amino acids used as Tyrl replacements.

In 2003, the first N-terminus to side-chain cyclized DynA analog, c[Tyr-Gly- Trp- Trp-Glu ]-Arg-Arg-Ile-Arg-Pro-Lys-NH2 42, lacking the basie N-terminus was reported [56].This analog showed KOR-affinity and selectivity similar tothat of the linear parent but an antagonist activity, eonfirm-ing crucial role ofthe amino group in opioid receptor activa-tion.

[n order to develop analogs of Dyn A that both, maintainthe basie character of the N-terminus and constrain the Tyrl

Current Drug Targets, 20J3, VoL /4, No. 7 801

residue, an acetyllinker between the N-terminal amine andthe N'-amine of Lys in position 3 or 5 was introduced (Fig.4) [57]. This strategy led to obtaining the desired head toside-chain cyclic peptides with a basie N-terminal amine.Cyclizations resulted in the 21-membered cycIO(N,5)[CO_CH2-Tyr1

, Lys[Dyn A(l-ll)NHz 43 or 21-membered cy-N" 1-clo "[CO-CHz-Tyr, Lysjl'iyn A(I-Il)NHz 44. However,

these peptides exhibited greatly decreased affin ity for opioidreceptors, suggesting unfavorable spatial orientation ofpharmacophoric groups.

2.1.4. N-to-C-Terminus

Another attempt to investigate receptor binding proper-ties of cyclic analogs lacking a cationizable N-terminalamino group was undertaken by Cardillo et al. [58], whoprepared peptides based on the sequence of EM-l, addition-alIy extended at the C-terminus by the Gly residue and cy-clized head to tai!. To determine the best spatial d ispositionof all aromatic side-chains for an optimal ligand-receptorinteraction, each residue was introduced in the D or L eon-figuration, generating a library of stereoisomeric, three-dimensionalIy distinct cyclopeptides of the general structurec[-LlD- Tyr-L/u-Pro-Lzo- Trp-LlD-Phe-G [y-l. AlI cyc lic ana-logs showed three rows of magnitude lower affinity totheMOR than EM-l with the exception of c[-Tyr-o-Pro-o- Trp-Phe-Gly-]45 which showed some weak affinity for the MOR(Kj=34 nM, versus 0.16 nM for EM-I), even though lackinga protonable nitrogen. What is even more interesting, th iscyclic analog displayed significant antinociceptive activity inthe mouse visceral pain model after peripheral administration[59]. The selective opioid antagonists confirmed the specific-ity of 45 to activate MOR in vivo. Increased lipophilic char-acter was most likely responsible for the good performanceofthis analog in terms of resistance to enzymatic degradationand permeation across biological barriers.

To provide insights into how analog 45 can interact withthe MOR,several new analogs 46-50 with various linkersbetween head and tail were synthesized [60J. The authorsspeculated that these cyclic compounds activated opioid re-ceptors via alternative interactions than typical opioids. Ac-cordingto the proposed model, the induced fit of ligand andreceptor can cause the transmission of a deformation fromthe bind ing site to the transmembrane domain and the stronginteraction between aromatic side-chain of the 0-Trp3 resi-due in the peptide with Trp293 of transmembrane loop TMVI ofthe opioid receptor.

Table 2 summarizes opioid receptor affinities of the cy-clic analogs with an amide bridge, ChemicaJ structures oftheselected analogs cyclized through an amide bond are shownin Fig. 5.

2_2_Amine Br~dge-

The use of a nitrogen heteroatom as a bridge in the de-sign of cyclic opioid peptides was reported by Goodman andco-workers [61]. The introduction of diamines aJlowed thesynthesis of the cycl ic enkephalin analogs, Tyr-c[(N~ -CH3)-

o-A2pr-Gly-Phe-NH-CHrCHr] 51 (MABE I), containing a.13-membered methylamine bridge and Tyr-c[(N~-CH3)-O-

Azpr-Gly-Phe-NH-CHz-CHz-] 52 (MABE Il) with a 14-membered bridge (Fig. 6) [62]. MABE I exhibited high

802 Current Drug Tan!DS, _ 13, YoL 14, No. 7 Piekielna et al.

Table 2. Opioid Receptor Affin ities and GPUMVD Inhibitory Potencies ofOpioid Peptide Analogs Cyclized Via an Amide Linkage.

Binding Affinity Potency

No. qne nce JC,o or K [nM] ECso [nM] Ref.

MOR DOR KOR GPI MVD

l Tyr-Gl) -Gły-Phe-Leu (Leu-enkephalin) 9.43 2.53 - u 246 11.4 [23]

2 Tyr-Pro-Phe-Pre-Gly (fkasomorphin l-S) 1160 6500 - 2260 11400 [24)

3 T_ -Pro-!'I;e-Pro-.'Hh (morphiceptin) 63 30000 - 318 4800 [25)

4 Tyr-Pro- Trp-Phe-l H, (EM-I) 0.16 - - - - [58)

5 I Tyr-Pro- Trp-Phe-. H, (EM-2) 0.79 >1000 >1000 - - [44)

6 I Dyn A(l-II)NH, 7.6 2.9 0.07 - - [61]

7 T, r-c[D-A,pr-Gly-Phe-Leu] 95.8 118 - 23.4 73.1 [23]

8 I Tyr-<:[D-A,bu-Gly-Phe-Leu) 24.9 256 - 14.1 81.4 [23)

9 I Tyr-c[D-Om-Gly-Phe-Leu) 56.6 221 - 48 475 [23)

10 I Tyr-c[D-Lys-Gly-Phe-Leu) 22.4 32.2 - 4.8 141 [23)

lJ Tyr-c[D-A,bu-Gly-l-Nal-Leu] - - - 156 6.2 [25]

12 Tyr-c[D-Om-Phe-Pro-Gly] 3.98 1280 - 13.4 63.9 [24]

13 Tyr-c[D-Orn-Phe-D-Pro-Gly] 0.88 13.2 - 2.14 4.89 [24)

14 Tyr-c[D-Orn-2-Nal-D-Pro-Gly] 5.89 17.2 - 3.84 antagonist [27]

15 Tyr-c[D-Lys-2-Nal-D-Pro-Gly] 17.1 62.6 - -- 609 antagonist [27]

16 Tyr-c[D-Lys-Phe-D-Pro ] - - - 5.41 60.6 [28)

17 Tyr-c[D-Orn-Phe-D-Pro] - - - 236 52.3 [28]

18 Tvr-cjcAmp-Phe-Phe] 660 - - 1.4%at1JlM - [29]

19 Tyr-c[cAmp-D-Phe-D-Phe] 63 1010 >10000 480 1400 [30]

20 Tyr-c[cAmp- D- Phe-Phe] 38 860 >10000 330 950 [30]

21 Tyr-c[D-Lys-Gly-Phe-Glu]NH, l.31 0.69 - 1.13 0.648 [32]

22 Tyr-c[D-Glu-Gly-Phe-Lys]NH, 2.42 1.26 - 2.51 1.34 [32]

23 Tyr-c[D-Orn-Phe-Asp]NH, 10.4 2220 - 36.2 3880 [33]

24 Tyr-c[D-Asp-Phe-Om]NH, 9.6 1320 - 522 8570 [33]

25 Tyr-c[D-Lys-Phe-Glu]NH, 1.43 4.36 - 2.93 5.21 [33]

26 Tyr-c[D-Orn-Phe-Glu)NH, 0.981 3.21 - 1.17 1.11 [33]

27 Tyr-c[D-Orn-C"MePhe-Glu)NH, 7.17 54.6 - 3.31 5.51 [38]

28 Tyr-e[D-Orn-oMePhe-Glu ]NH, 1.92 9.22 - 0.543 1.1 I [38]

29 Tyr-c[D-Orn-Aic-Glu]NH, 4.21 209 - 7.21 36.5 [38]

30 Tyr-e[D-Orn-Atc-Glu]NH, 8.26 1570 ~ 31.6 254 [38]-

33 Tyr-e[l ys-Phe-Phe-Asp ]NH, >1000 >1000 >1000 - - [41]

34 Tyr-c[Asp-Phe-Phe-Lys)NH, 289 >1000 >1000 - - [41]

35 Tyr-e[Lys-Phe-D-Pro-Asp]NH, >1000 >1000 >1000 - - [41]

36 Tyr-c[Asp-Phe-D-Pro-Lys]NH, 513 . >1000 >1000 - - [41]

37 Tyr-e[D-Lys-Phe-Phe-Asp]NH, 0.56 279 - - - [43]

Cyclization in Opioid Peptides Current Drug Targets, 2013, VoL /4, No. 7 803

(Ta ble 2) contd ....

Binding Affinity Potency

No. Sequence rcso or K;[nM] ECso [nM] Ref.

MOR DOR KOR GPI MVD38 Tyr-c[D-Asp-Phe-Phe-Lys]NH, 1.14 112 - - - [43]

39 Dml-c[D-Lys-Phe-Phe-Asp]NH, 0.26 400 - - - [44]

40 Dml-c[D-Lys-Phe-D-Pro-Asp]NH, 5.6 132 - - - I [44],41 Dmt-c[D-Asp-Phe-D-Pro-Lys]NH, 4.4 58.3 - - - (44]

42 c[Tyr-Gly-Trp- Trp-Glu]-Arg-Arg-Ile-Arg-Pro-Lys-NH, 331 >8900 26.8KOR-antagonist in adenylyl cyclase

[56]assay

cyclo .s[CO-CH,-Tyr', Łys[Dyn A,

43 813 >7190 290 - - [57](I-II)NH,

44cycloN.3[CO-CH,- Tyr', Lys'[Dyn A

5690 >10000 106 [57]- -(l-II)NH,

45 e[-Tyr-D-Pro-D-Trp-Phe-Gly-] 34 - - MOR-agonist in forskolin-stimulated[58]

cAMP inhibition test

46 e[ -Tyr-D-Pro-D- Trp-Phe-BvAla-] 6100 - - - - [60]

47 e[ -Tyr- D- Pro-D- T rp- Phe-GABA-] 3200 - - - - [60],---

48 e[ -Tyr-D-Pro-D- Trp- Phe-Aib-] 2900 - - - I - ! [60]I

49 e[ -Tyr-D-Pro- D-Trp- Phe-D- Pro-] 32000 - - - - I [60]--

50 e[ -Tyr-D-Pro-D- Trp-Phe-D-Pro-] 720 - - - - ! [60]

a not deterrnine

NH-Tyr-Gly-Gly-Phe-Lys-Arg-Arg-I1e-Arg-Pro-Lys-NH2

~H2CONH I

iH-Tyr-G1Y-LYS-Phe-LeU-Arg-Arg-Ile-Arg-pro-LyS-NH2

CH2CONHJ

Fig. (4). Structure of cyclic N-terminal to side-chain Dyn. A analogs 43 and 44 maintaining a protonaole amine.

OH

7 (C-terminus to side-chain) 39 (side-chain to side-chain)

Fig. (5). Examples of analogs cyclized through an amide bond.

HO r9'

%1

O H NH

HNgN O "'"

~ j

45 (N- to C-terminus)

04 Current Drug Targets, 2013, VoL 14, No. 7

affinity for the MOR and DOR and a modest affinity for theKOR. Conformational analysis of MABE I indicated that itsmethylamine bridge was highly flexible which was probablythe reason of the poor selectivity of this compound. How-ever, in the in vivo thermal test MABE l, aft er intrathecal(i.t.) administration, produced stronger antinociception thanDPDPE and morphine.

The main advantage of using amines as a bridging motifis that they contain a site for protonation and a trivalentamine can be used as a "handle" for functionalization of thebridge without disturbing the pharmacophores. Such modifi-cations can modulate biological activity, as well as biodis-tribution of the targ et structures.

Opioid activities ofanalogs cyclized via an amine bridgeare shown in Table 3.

2.3. Sulfur-Containing Bridges

2.3.1. Disulfide Bridge

The first cyclic enkephalin analogs containing a disulfidebridge were obtained by either side-chain to the C-terminus[23, 24] or side-chain to side-chain linkage [63-65]. Intro-duction of Cys residues caused moderate MOR selectivity[63]. The major success in developing highly DOR-selectiveanalogs came with introduction of penicillamine (P,P-dimethylcysteine, Pen) (Fig. 7) [65-68]. D-Pen was intro-duced into enkephalin sequence in the place of GIl and D-Pen ar Pen in the place of Leu" residue, resulting in twoDOR-selective disulfide peptides: Tyr-cju-Pen-Gly-Phe-u-Pen], DPDPE 53 and Tyr-c[D-Pen-Gly-Phe-Pen], DPLPE 54[65, 66, 69]. These enkephaIin analogs are conformationallyrestricted not only via a disulfide linkage but also because ofthe effect of the gem-dimethyl groups of Pen [70]. Molecularmodel ing and x-ray diffraction studies of DPDPE indicatedthat the gem-dimethyl substitution in position 2, whichcauses more severe steric interference at the MOR than at theDOR, is responsible for the exceptional DOR-selectivity ofthis analog [71, 72]. DPDPE turned out to be a useful bio-logical tool to study the interactions between DOR ligandsand DOR. Its exceptionaJ stability against chemical and en-zymatic degradation is one of the important features [73, 74],also observed in natural peptides, such as crotamine [75].

Substitution of the Phe4 aromatic ring in DPDPE withhalogen s in the para position gave interesting analogs 55-57that showed even higher potency and selectivity thanDPDPE [76]. Further modification of DPDPE, with evengreater degree of structural rigidity was also designed. A

Piekielna et al.

disulfide tetrapeptide Tyr-cjn-Cys-Phe-o-Pen] (JOM-13) 58,which can be viewed as des-Gly' DPDPE, showed high af-finity for the DOR but decreased selectivity compared toDPDPE [77]. To assess the effect of altered size, lipophilic-ity and electronic character of the third residue in JOM-l3,analogs containing bigger ring systems 59-61 or more hy-drophilic character 62-64 were also synthesized but did notbring interesting compounds [78].

OH

~ O

~Vl~lr~O (~n O

/N~NH

O

51 MABE I, n=152 MABE II. n=2

Fig. (6). Structure of MABE [ and MABE II cyclized through themethylamine bridge.

O

HS~OH

l-penicillamine D-penicillamine

Fig. (7). Structure of L- and D-penicillamine.

An enkephalin cyclic analog Tyr-cju-Cys-Gly-Phe-u/t-Cys]NH2 was modified by replacing Tyr with Dmt and N-methy lation at the Phe4 and/ar Cys" residues [79]. As couldbe expected, analogs 65-72, obtained by introduction ofDmt! showed high binding affinities at all three opioid recep-tors and high agonist potencies in the functional GPIIMVDassays.

In the search for new analogs with improved pharrnacol-ogical properties, same chimeric peptides were also designed[80]. The C-terminal fragments of deJtorphins (-Val-Gly-NH2 or -Nle-Gly-NH2) were linked to the highly DOR-selective cyclic peptides DPDPE or DPLPE. These studiesdemonstrated that a major structural feature determin ing high

Table 3. Opioid Receptor Affinities and GPlIMVD Inhibitory Potencies of Opioid Peptide Analogs Cyclized Via an Am ine Link-age.

-Binding Affioity Potency

No. Sequence ICso or KiloM] EC5<llnM] Ref.

MOR DOR KOR GPI MVD

51 Tyr-c((N~-CH1)-O-A,pr-Gly-Phe-NHCH,-CH,- J (MABE I) 1.6 2.1 340 - a - [61)

52 Tyr-c[(NII-CH,)-O-A,bu-Gly-Phe-NHCH,-CH,-) (MABE II) - - - 1.39 I [62)

'I not determ ined

C/tI.::!::::'lIDm.3! Opioid Peptides

_ O~ such hybrid analogs is the chirality of the aminosidue in position 5. Chimeric peptides of DPLPE, but

- _: »:-DPDPE, retained high DOR-selectivity and affinity.

omatostatin, a 14 amino acid long peptide hormone eon-ining a disulfide bond, was shown to bind weakly to the

CNS opioid receptors [81]. This finding led Hruby and co-workers to examine the possibility of developing soma-tostatin-like analogs with opioid activity [82]. Utilizing vari-ous structural considerations and conformational constraintsthey modified a somatostatin analog, oetreotide, o-Phe-c[Cys-Phe-o- Trp-Lys- Thr-Cysj'I'hr-Nl-I-, by substitutingPhe3 by Tyr and Cys? by Pen and amidating the C-terminus.The obtained octapeptide amide, o-Phe-cj Cys-Tyr-n-Trp-Lys-Thr-Penj'Thr-Nl-I, (CTP) 73 containing a disulfidebridge [82], and two other similar analogs, o-Phe-c[Cys-Tyr-D-Trp-Arg-Thr-Pen]Thr-NH2, (CTAP) 74 and o-Phe-c[Cys-Tyr-D-Trp-Orn-Thr-Pen]Thr-NHz, (CTOP) 75 [83, 84],turned out to be extremely selective and potent MOR an-tagonists in a variety of assays. These peptides are stableagainst enzymatie degradation and have been extensivelyused to study the physiological and pharmacological proper-ties ofthe MOR [85].

2.3.2. Dithioether Linkage

As a further modification of a disulfide bond, ethylenedithioether linkage was introdueed by Mosberg and co-workers [77, 86, 87]. A tetrapeptide amide, Tyr-e(S-CHz-CH2-S)[o-Cys"Phe-D-Pen]NHz (JOM-6) 76, was MOR-selective [88]. Analogs with increased selectivity for KOR,such as Tyr-e(S-CHz-S)[o-Cys-Phe-Phe-Cys]NHz were ob-tained by introduction of a methylene dithioether linkage[89). Analog 69 displayed fuli agonism at MOR but onlypartial agonism at DOR. The growing body of evidence im-plicates a role of DOR in modulating or even preventingMOR-induced tolerance [90-92]. Opioid ligands with similaraffinities at MOR and DOR but displaying agonism at MORand antagonism at DOR might be of great potential as a newclass of analgesics. To increase efficacy at the DOR whileincreasing affinity for this receptor Purington et al. [93], onthe bases ofmolecular modeling studies [94,95], designed aseries of analogs with bulkier side-chains in positions 3 or 4.A cyclic analog Tyr-c(S-CHz-S)[D-Cys-Phe-2-Nal-Cys]NH278 displayed fuli agonism at the MOR (99% eompared withthe ful1 MOR agonist, DAMGO), while acting as an antago-nist at the DOR in the [35S]GTPyS assay and partial agonistin the adenylyl cyclase inhibition assay.

2.3.3. Bismethylene Bridge as a Disulfide Bond Modifica-tion

Replacement of a disulfide bridge in the cysteine-containing peptides for olefinie (-CH=CH-) or bismethylene(-CHrCHr) moieties represents yet another eyclizationstrategy. Resulting analogs showed altered conformationalproperties and therefore altered biological activity profiles,In the past, this structural modification was synthetieallydemanding and seldom used [96]. The use of ring-elosingmetathesis pawed the way to a relatively straightforwardproeedure for the preparation of such diearba analogs ofopioid peptides [97]. The dermorphin-derived eyclictetrapeptide analogs Tyr-cjb-Cys-Phe-Cys ]NH2 79 and Tyr-C[D-Cys-Phe-O-Cys]NH2 80 with agonist aetivity at both,

Current Drug Targets, 2013, Vol. 14, No. 7 805

MOR and DOR, were used as parent eompounds for thepreparation of dicarba anaIogs 81-85, in which both Cysresidues were replaced by allylGly residues, foliowed byring-cios ing metathesis between the side-chains of allylGlyresidues [98]. In the ease of a peptide with Cys" (79) the re-suiting olefinie peptide was formed as a mixture of cis (81)and trans (82) isomers, whereas the cis isomer (83) was theonly produet when D-allylGly was present in position 4.Catalytie hydrogenation yielded saturated -CHz-CHz-bridged peptides 84, 85 (Fig. 8). Continuing the studies ondiearba compounds, the same gro up used Cys-containingeyclic peptides, Tyr-e[o-Cys-Gly-Phe-Llo-Cys]-NH2 86 and87 with larger ring systems, as parent eompounds for thesynthesis of analogs 88-96 whieh showed retained highMOR and DOR affinities and potencies [99].

Tyr-D-Gly-Phe-D-Gly-NH2I ICH2-X-X-CH2

-x-x-

79 -S-S- 80

81 -CH=CH- (cis) 83

82 -CH=CH- (trans) -

84 85

Fig. (8). Structure of analogs eyelized through olefinie (-CH=CH-)and bismethylene (-CH2-CH2-) bridges.

Dicarba analogs of the cyclic peptide Tyr-c[o-Cys-Gly-Phe-D-Cys]OH, with the free C-terminal carboxylie group,were also reported and showed high MOR and DOR agonistactivities [100].

Interesring diearba analogs were obtained when the MORagonist peptide Tyr-c(S-CH2-S)[ D-Cys-Phe-Phe-Cys]N H269 [88] was used as a parent compound and modified at theN-terminus by replacing Tyr either by Dmt ar by Dhp orMdp [101]. The substitution ofDmt for Tyrl in opioid pep-tides is a well reeognized strategy for achieving inereasedbioactivities [54]. Indeed, replacement of Tyrl by Dmt indicarba analogs 97-99 resulted in the very potent but less

. Iselective analogs. On the other hand, replacement of Tyr byDhp and Mdp has been shown to eonvert opioid agonists intoantagonists. As expected, dicarba analogs 100-105, laekingan amino group at the N-terminus were all antagonists at theMOR.

2.3.4. Monosulfide Bridge oj Lanthionine

Lanthionine (Fig. 9) is an unnatural amino acideomposed of two alan ine residues that are eross-linked ontheir ~-earbon atoms by a thioether linkage. The monosulfidebridge of lanthionine is a thioether side-chain to side-chainbond which provides more constrained peptide structureswith greater stability towards enzymatic degradation com-pared to the disulfide bridge of cystein e [102].

g Targets, 2013, VoL 14, No. 7

HO~S~OH

NH2 NH2

Fig. (9). Structure of lanthionine.

Rew et al. [103] synthesized a series of monosulfide-bridged analogs of DPDPE (106-109) of the following struc-ture: Tyr-c[D-AAL-Gly-Phe-D-(or L-)AAL], , where AALdesignates the lanthionine amino acid ends linked by athioether linkage to form the lanthionine structure. Theseanalogs contained either D-AlaL or D-Val., residue in position2 and D-AlaL or Ala-, in position 5. The analog containing D-Val., residue is constrained by the presence of gem-dimethylgroups at the ~-carbon atom (similarly to Pen). Binding stud-ies and in vitro functional tests revealed that the dimethyl-lanthionine analogs (107 and 109) showed substantial selec-tivity towards DOR, while unsubstituted analogs (106 and108) were quite non-selective. The in vivo therrnal escapeassay was perfonned following i.t. administration of pep-tides. This assay measures the magnitude of the spinał anti-nociceptive effect. The presence of MOR and DOR in thespinal dorsal horn was demonstrated previously [104] andthe activation of each resulted in antinociception [105]. Alifour lanthionine analogs produced a dose-dependent increasein the thermal escape latency and had much lower EDso val-ues (dosage necessary to produce a halfmaximai effect) thanDPDPE and morphine. Dimethylated analogs 107 and 109with high affinities at the DOR are among the most potentligands producing antinociception through this receptor.

The structures of elassie opioid analogs containing sulfuratom/atoms in a bridging unit are shown in Fig. 10 and

53 DPDPE

o

NH HN y'H NH-NH2 <.. 'o Os, HN

s~ O-rNH2O

HO

69

Fig. (10). Examples of analogs with sulfur-contain ing bridges.

Piekielna et al.

opioid activities of such analogs are summarized in Table 4.

2.3.5. Disulfide Bridge in Nociceptin Analogs

To investigate structural and conformational requirements ofnociceptin interactions with its receptor, a series of NC(J-13)-NH2 110 or NC(I-14)-NH2 111 analogs 112-117 cy-clized via a disulfide bridges either at the N-terminal, C-'terminal parts of the peptide or in the middle, were obtained[106]. The best analog of the series, C[CyslO, CysI4]NC(1-l4)-NH2 117 sustained high affinity at the NOR and goodpotency in the eSS]GTPyS functional assay, similar to that ofthe parent peptide, NC. The analogs cyclized in the middlepart (114, 115) were slightly less potent, while introductionof the conformational constraint at the N-term inus was det-rimental for activity (112, 113). Further modification of 117,performed by substitution ofPhe1 by Nphe ' (were Nphe = N-benzylglycine) produced an antagonist (118) in the[3sS]GTPyS functional assay [107].

The opio id activities of NC analogs cyclized via the di-sulfide bridge are summarized in Table 5.

2.4. Carbonyl (Ureido) Bridge

Yet another type of ring formation, elaborated by lzdeb-ski and co-workers, is based on the reaction of bis( 4-nitrophenyl)carbonate with side-chain amino groups of twodiamino acid residues, permitting preparation of a cyclicpeptide with a carbonyl brid?e (Fig. 11) [108]. Analog 119,containing D-Lys2 and Lys as the di-basic amino acids,showed a week preference for MOR over DOR. The samemethod was further used to synthesize cyclic enkephalinamides 120-128, containing various diamino acids in posi-tions 2 and 5 [109-11l]. Such analogs showed very high

68 JOM-6

86

Current Drug Targets, 2013, VoL 14, No. 7 807

Opioid Receptor Affinities and GPIIMVD Inhibitory Potencies of Analogs of Classic Opioid Peptides Containing Sulfur ina Bridging Unit.

II IBinding Affinity Potency

. '0'1 Sequence JC50 or K; [nM] ECso or K, [nM) Ref.

MOR DOR KOR GPI MVO

53 Tyr-c[D-Pen-Gly-Phe-D-Pen] (DPDPE) 720 1.2 - a 11300 3.9 [69]

54 Tyr-c[D-Pen-Gly-Phe-Pen] (DPLPE) 11400 4 - 5800 1.89 (69)

55 Tyr-c[D-Pen-Gly-Phe(pF)-D-Pen] 620 2.5 - 5000 0.84 [76]

56 Tyr-c[D- Pen-Gly- Phe(pCI)- D- Pen] 980 1.6 - 4800 0.89 [76]

57 Tyr -c[D- Pen-Gly- Phe(pBr)- D- Pen] 418 1.73 - 13400 1.5 [76]

58 Tyr-c[D-Cys-Phe-D-Pen] (lOM-13) 107 1.79 - - - [77]

59 Tyr-c[D-Cys-I-Nal-D-Pen] 91.5 2.85 - - - [78]

60 Tyr-c[D-Cys- 2- Nal- D- Pen] 590 2.23 - - - [78]

61 Tyr-c[D-Cys-Trp-D-Pen] 183 2.49 - - - [78]

62 Tyr-e[D-Cys-His-D-Pen] 10700 56.3 - - - [78]

63 Tyr-e[D-Cys- Pal-O-Pen] 1610 132 - - - [78]

64 Tyr-c[D-Cys-Cha- D- Pen) 20.2 4.89 - - - [78]

65 Dmt-c[D-Cys-Gly- Phe(NMe)- D-Cys ]NH, 0.496 2.29 0.447 1.07 1.11 [79]

66 Dmt-c [D-Cys-Gly- Phe(NMe )-Cys ]NH, 0.354 2.36 0.855 0.457 0.884 [79]

67 Dmt-c[D-Cys-Gly-Phe-D-Cys(NMe)]NH, 0.504 0.525 LOI 1.81 0.352 [79]

68 Dmt-c[D-Cys-Gly-Phe-Cys(NMe»)NH, 0.586 0.776 0.894 1.36 0.122 [79]

69Dmt-c[D-Cys-Gly-Phe(NMe)-D-

0.876 6.07 1.42 1.34 3.72 [79]Cys(NMe»)NH,

70Dmt-c[D-Cys-Gly-Phe(NMe)-

0.641 1.79 0.875 0.394 1.96 [79]Cys(NMe)]NH,

I Dmt-e[D-Cys-Gly-Phe-D-Cys]NH, 0.412 0.202 0.602 0.586 0.053 [79]

2 Dmr-e[D-Cys-Gly-Phe-Cys]NH, 0.282 0.306 0.677 0.812 0.115 [79]

3D-Phe e(Cys-Tyr-D-Trp-Lys- Thr-Pen)-Thr-

3.7 84000MOR-selective

[82]-H, (CTP) Antagonist

-4 ID-Phe e(Cys-Tyr-D- Trp-Arg-Thr-Penj-Thr-

3.5 4500MOR-selective

[83]-. H,(CTAP) Antagonist

- D-Phe e(Cys- Tyr-D-Trp-Om- Thr-Pen)- Thr-2.8 13500

MOR-selective[83]-

NH, (CTOP) Antagonist

6Tyr-c(S- Er-S)[D-Cys- Phe- D- Pen ]NH,

0.17 12 2650 - - (88](JOM-6)

77 Tyr -c(S-Me-S)[D-Cys- Phe- Phe-Cys ]NH, 0.016 1.8 2.5 - - [89]

99% agonist 'in (J5S)G,}:PySantagonist in [l5S] GTPyS

78 Tyr-c(S-Me-S)(D-Cys-Phe-2-Nal-Cys]NH, 0.47 0.48 13 assay compared with (93]

OAMGOassay

79 Tyr-c[D-Cys-Phe-Cys]NH, 11.0 373 - 64.7 740 [98J

80 Tyr-e[D-Cys-Phe-D-Cys]NH, - - - 20.0 28.8 [98]

81Tyr-e( -HC=CH- )[O-allyIG Iy-Phe- - - - 436 460 (98]

allyIGly]NH, (cis)

Targets, 2013, Vol. 14, No. 7 Piekielna et al.

~...

Binding Affinity PotencyIC,. ar K [nM] EC,o ar K. [nMI Ref_Sequence

MOR DOR KOR GPI MVD

Tyr-e(-HC=CH- )[D-allyIGly-Phe- - - - 162 444 [98]- allylGly]NH, (trans)

3Tyr-e( -HC=CH- )[D-aIlyIG Iy-Phe-D-

- - - 176 246 [98]aIlylGly)NH, (cis)

84Tyr-e( -CH,-CH2- )[D-allyIGly-Phe- - - - 76.5 655 [98]

aIlylGly]NH,

85 Tyr-e( -CH2-CHr )[D-aIlyIGly-Phe-D- - - - 208 786 [98]aIlylGly]NH,

86 Tyr-c[D-Cys-Gly-Phe-Cys]NH, 1.74 1.61 40.1 1.165 0.603 [99]

87 Tyr-c[D-Cys-Gly-Phe-D-Cys]NH2 0.550 0.822 44.9 1.30 0.562 [99]

88 Tyr-e( -HC=CH- )[D-aIlyIG Iy-Gly-Phe- 2.40 6.55 200 1.81 0.496 (99)aIlylGly]NH, (cis) I

89Tyr-e( -HC=CH- )[D-al lyIGly-Gly-Phe- 1.17 3.34 71.5 I 1.2 3.64 [99]

allylGly]NH2 (trans) I

90Tyr-e( -HC=CH- )[D-aJlyIGly-Gly-Phe-D- 2.27 7.04 498 I 1.9 9.91 (99)

allylGly)NH, (cis)

Tyr-e( -HC=CH- )[D-allyIGly-Gly-Phe-D- 1.05 1.92 30.4 5.63 4.38 [99]91allylGly]NH, (trans)

92Tyr-e( -CHl-CHr )[D-allyIGly-Gly-Phe-

2.34 5.87 309 1.02 3.J9 [99JallylGly]NH2

93Tyr-e(-CH,-CHl- )[D-al lyIGly-Gly-Phe-D- 1.17 3.34 71.5 n.z 3.64 (99)

allylGlyJNH,

94Tyr-c( -HC=CH- )[D-aIlyIGly-Phe-Phe- 0.281 0.910 9.36 0.401 0.851 (99)

allylGly)NH, (cis)-

9STyr-e( -HC=CH- )[D-allyIGly-Phe-Phe- 0.332 1.53 15.2 1.34 4.51 I (99)

allyIGly]NH, (trans)

96Tyr- e(-CHrCH,- )[D-allyIGly-Phe-Phe-

104 2.24 11.3 1.05 0.740 [99]allylGly]NH2

97Drut-et -HC=CH- )[D-al IyIGly-Phe-Phe-

0.528 0.491 12.8 0.161 0.191 [101]allylGly]NH, (cis)

98 Drnt-c(-HC=CH-)[D-allyIGly-Phe-Phe- 0.438 0.286 4.97 0.407 0.044 I [JOl]allylGly]NH, (trans) l

99 DMT -c (-CH,-CH,- )[D-a I1yIGly- Phe- Phe- 1.54 1.20 15.8 0.509 0.655 [101]allylGly]NHl

100Dhp-c(-HC=CH-)[D-allyIGly-Phe-Phe-

23.5 86.3 266 K,= 32.2 PAb [101]allylGly] H, (cis)

I Dhp-c( -HC=CH- )[D-allyIGly-Phe-Phe- -.238 [101]16.7 270 500 K,=44.3 -lal

aIlylGly)NH, (trans)

102Dhp-e e(-CH,-CH,- )[D-allyIGly-Phe-Phe-

8.95 39.2 500 K,=4 I.7 7500 [101]allylGly]NH,

--(2S)Mdp-c( -HC=CH- )[D-allyIGly-Phe-Phe-

744 7.67 185 K,=32.4 PA [101]103allylGly]NHl (cis) .

Current Drug Targets, 2013, VoL 14, No. 7 809

(Table 4) contd ....

Binding Afłinity Potency

ICs• or KI [nMl ECs• or K., [nMl Ref.

MOR DOR KOR GPI MVD

- --_-- ""-~.t- [D-all IGly-Phe-Phe-323 35.3 635 K,= 157 [10 I]

!:Głyr- ::H~(uans)PA

- zz-c ~c-CHr )[D-allyIGly-Phe-- - 183 18.7 302 1(.=25.7 K.= 147 [101]:1 e IyIGlyJNH,

-. --cfD- ..>Ja,..-Gly-Phe-D-Alac] 2 2 1600 0.56 1.58 (103]

-_ -c[D- ak·Gly-Phe-D-AJaL] 630 0.93 >10000 730 2.33 (103]

~ Tyr-c[D-AIa..-Gly-Phe-Alad 2.3 0.63 6100 106 0.35 (103J

I II T. r-c[D-VaIL-Gly-Phe-Ala..] 130 0.79 >1000 82 0.26 (103]

Table 5. The Opioid Activities of NC Analogs Cyclized via the Disulfide Bridge.

:"0. SequenceNOR Binding Affinity [

J5SlGTPyS assay cAMP inhibition Ref.rcs• [oM] ECs• ruMI pEC"

llO 0.353 U2 (106]NC(I-13)-NH, 8.57

1016(107]

III NC( 1-14 l-NH, 0.520 1.97 - (106]

112 c[Cys·,Cys'] NC(I-13)-NH, 874 >10 000 - (106]

113 c(Cys·, Cys"] NC(I-13)-NH, 387 439 - (106]

114 C(Cys6, Cysl.] NC(I-13l-NH, 0.833 31.3 - (106]

115 c(Cys', Cysl'] NC(I-13)-NH, 3.04 156 - [106)

116 C[Cys7, Cysl4] NC(I-14)-NH, 4.97 25 - [106]

117 C[CyslO,Cysl') NC( 1-14)-NH, 0.1224.37

9.29(106]

8.29 (107)

118 c(Nphel,CysI0.14] NC(I-14)-NH, -c inactive 5.97 (107]

3 not determined

MOR activity which may be ascribed, at least in part, to theamidated C-terminus, characteristic for the MOR-selectiveopioids. Analog 124 with D-Lys2 and Dap ' is one ofthe mostpatent MOR-agonists among the enkephalin-derived ana-logs. In the in vivo studies this analog showed strong antino-ciception in the hot-plate and tail-flick tests after i.c.v. ad-ministration which was reversed not only by a MOR but pre-dominantly by DOR antagonist [112].

Cyclization via a carbonyl bridge was also used tó obtainanalogs of dermorphin(I-7) [113], dermorphin(1-4) [IIO,111] and deltorphins [114, 115]. Several of these com-pounds, between others c(N°, N°-carbonyl-D-Orn2

, D-Om''jdeltorphiru l-Z) (DEL-6) 129 showed high DOR po-tency and selectivity [115].

HNI(NHO

98 R=H112 R = NH-CHrCHrNH-CO-NH2.

Fig. (11). Structure of analogs cyclized through a urea bridge.

: 13, Vol. 14, No. 7 Piekielna et al.

ification of cyclic enkephalin analogs_ ;1 bridge, new analogs 130-133, contain-

- __;o-_.-"--'<:ll'VlamidatedC-terminus were synthesized.. z, Diverse opioid activities were observed, de-

~e size of the ring. However, the activities ofN-__::; ""--._ ides were usual!y lower than these of the cor-~::::""J_';""-~--,5amides. In comparison with [Leu '[enkephalin, al!

_ .. .. \'ere more active in the GPl assay, showing in-preference for MOR. Recently, anovel series of cy-

retrapeptide N-ureidoethy lamides based on deltorphin( 1-""'"structure was reported by Bańkowski et al. [117]. The best

alog, Tyr-elu- Lys- Phe-Dab ]CHz-CHz- NH -CO- NHz, eon-

taining a carbonyl bridge between side chains of o-Lys andDab, showed significant, stronger than morphine, antinoci-ceptive effect after systemie applieation and fuli stabilityagainst proteolytic enzymes.

A series of hybrid peptides containing an N-terminal se-quence of enkephalin and a C-terminal sequence of deltor-phin, restrieted via a urea bridge were recently obtained[118]. However, no special improvement in opioid activitywas observed. Opioid activities of analogs cyclized via acarbonyl bridge are shown in Table 6.

Table 6. GPIIMVD Inhibitory Potencies of Analogs Cyclized Via a Carbonyl Bridge.

Potency

No. Sequence EC" [oMI Ref.

GPI MVD

Tyr-D-Lys-G ly-Phe-Lys-Nl+,119 L-Co---.l 20.5 73.5 [108]

Tyr-n-Orn-G ly-Phe-Lys-Nl+,120 ~CO.....c..J 10.6 41.3 (109]

Tyr-D-Orn-Gly-Phe-Orn-NH2[109)121 ~CO.....c..J 4~68 20.2

Tyr-D-Orn-Gly-Phe-Dab-NHz122 ~O.....c..J 2.05 8.7 (109]

Tyr-D-Orn-Gly-Phe-Dap-NHz123 ~CO~ 1.64 12.5 [109]

Tyr-D-Lys-Gly-Phe-Dap-NH2124 L-co~ 0.212 0.651 [109]

Tyr-n-Lys-Gly-Phe-Orn-Nl-l ,[110]124 L-CO.....c..J 2.26 3.52

Tyr-o-Lys-Gly-Phe- Dab-NH2126 L-co~ 2.32 2.68 [110]

Tyr-D-Dab-Gly-Phe-Om-NH2127 ~co---.l 1.61 6~26 [110]

Tyr-D-Dap-Gly-Phe-Om-NH2128 ~co---.l 12~I 46.7 (110]

Tyr-p-Orn-Phe-Orn- Val- Val-Gly-NH2159 0.814 [114]129 L-CO....J

I Tyr-D-Lys-Gly-Phe-Dap-NH-CH2-CH)-NH-CO-NH?130 ~CO---.J - - 1.97 1.81 [116]

.~Tyr-D-Lys-Gly-Phe-Dab-NH-CH)-CH)-NH-CO-NH) -

131 L-ćO---.J - - - 5.55 6 [116]

Tyr-D-Lys-Gly-Phe-Orn-NH-CH2-CH2-NH-CO-NH2(116]132 ~CO~ 18.1 22.7

Tyr-D- Lys-Gly-Phe-Lys-NH -C HrC Hi- NH -CO- NH2133 ~CO-.J 20.2 26.6 [116]

- n in Opioid Peptides Curreni Drag Ti at." 13, VoL 14, _ 0.7 811

fil Vivo Bioactivities of Cyclic Opioid Analogs.

ED5łl ISeque nce Ref.Hot-plate Test Tail-flick Test \ rithi ag Test

II

i.t. (rat)Morphine 15 nM - a - [103,

2.4 ug 61)

i.t. (rat)

- Tyr-c[D-Pen-Gly-Phe-D-Pen] (DPDPE) 130 nM - - [103,54 ug 61]

rli.p. (mouse)

- c[-Tyr-D-Pro-D-Trp-Phe-Gly-) -1.24 mg/kg

[59)~ -s.c, (mouse)

II 2.13mg/kg

- Tyr -e [(W -CH,)- D- Aspr -Gly- Phe-NHCH,-CH,-) i.t. (rat)- [6 l)- -

(MABE I) 0.027 ug

Tyr-c[D-Alal.-G Iy-Phe-D-Aladi.t. (rat)

[103)0.0015 nM

- -

Tyr-e[D- Vall.-Gly-Phe-D-Aladi.t.(ra!)

- - [103)0.26 nM

Tyr-c[D-AlaL-Gly-Phe-AlaL]i.t. (rat)

[103)0.0018 nM

- -

Tyr-e[D- VaIL-G ly-Phe-Ala.]i.t. (rat)

- - [103)0.12 nM--. Tyr-c[Asp-Phe-Phe-Lys]NH,

i.c.v. (mouse) - - (41)0.45 J,lg

- l Tyr-c[Asp-Phe-D-Pro-Lys)NH,i.c.v. (mouse)

- - [41)0.22 J.lg,

i.c.v. (mouse)

~ Tyr-c[D-Lys-Phe-Phe-Asp )NH,<0.001 J,lg

- - [43]i.v. (mouse)

I I 1000 ug

Tyr-c[D-Asp-Phe-Phe-Lys]NH,i.c.v. (mouse) - - [43]

<0.01 J.lg

oJ Dmt-c[D-Lys-Phe-Phe-Asp]NH,i.c.v. (mouse) - - [44)

0.02 ug

Dmt-c[D-Lys-Phe-D-Pro-Asp]NH,i.c.v. (mouse)

- [44)<0.01 r'g -

ł Dmt-c[D-Asp-Phe-D-Pro-Lys)NH,i.c.v. (mouse)

- - [44)<Ofl l ug

Tyr-D-Lys-G ly-Phe- Dap-NH 2 i.c.v. (rat) i.c.v. (rat)1J... l...:....-c O ---.J - [112]0.0792 nM 03526 nM

Tyr-D-Orn-Phe-Orn-Val- Val-G Iy-NH 2-

1.9 i.c.v. (rat)

l[115]l__-CO....J - - -

20 nmol

" ~ derermined

_. S, Guanidine Bridge

A new type of cyclization was recently reported, incorpo-rating a guan idine bridge formed between the side-chains ofrwo diaminoacyl residues [119]. Two nitrogen atoms of the

guanidine group belong to the cyclic backbone, while thethird one is extra-cyclic and can be nono, mono or disubsti-tu ed (Fig. 12).

-.::= - assumed to be able to adopt four~S:::== ~=ri:"::lS - - e the peptide cycle. NMR and

== r: g-Gl -Asp-related cyclic peptides-= -=.:: O! o guanidine substitution (R , R2=H,

o;...--::=.;~::::: --e ridgeorientation and, therefore, the~~-=::z:a::r.::;r li 9].

O O

./-0pePtide/~0Y

(~m . Y)nHNyN/N,

R1 R2

R1,R2-Horalkyl

Fig. (12). Structure of analogs cyc1ized through a guanidine bridge,

s. LY VlVO ACTIVITlES OF THE CYCLIC OPIOmPEPTIDES

ming the difficulty of delivering therapeuticross the BBB to specific regions of the brain

. resen - a major ehaJ lenge. Specific properties of the BBBgeneralł- pre ent the entry of peptides into the brain. Cycli-zarion '- belie ed to improve permeation of opici id peptideanalos through biological barriers [120]. However, out ofthe numerous c elic opioid analogs synthesized so far onlyfew were tested in vivo, Table 7 summarizes bioactivities ofthe cyclic analogs which were tested for their analgesic ac-tivity in in vivo pain tests, such as hot-plate test, tail-flick testor writh ing test. Some analogs showed greatly increasedanalgesic activity, as compared to morphine, after direct ad-ministration to the brain (i.c.v.) or to spinal cord (i.t.) buteither they were not tested after peripheral administration orcould not cross the BBB. Analog 45, lacking a protonableamine, at 10 mglkg (i.p.) was shown to produce antinocicep-tion on acetic acid-induced writhes through peripheral opioidreceptors. A t high doses (20 mg/kg, i.p.) this analog pro-duced antinociception through both, peripheral and centralopioid receptors, indicating that it was able to reach the brainafter systemie administration.

lnteresting resuJts were obtained for analog Tyr-cjn-Lys-Phe-Phe-Asp'[Nl-l- 37. Analgesic effect of this analog in-jected i.c.v. was much stronger and longer lasting than thatof EM-2. What is more important, this analog elicited anal-gesia also after peripheral administration and this effect wasreversed by concomitant i.c.v. injection of the selectiveMOR antagonist, ~-FNA which indicated that antinocicep-tion was mediated by the MOR in the brain. Central action ofthis cyclic analog gave evidence that it was able to cross theBBB, most likely due to the increased lipophilicity and en-zymatie stability. These resuJts demonstrate that eyclizationmight be a promising strategy in the development of new,opioid-based pain-killers.

Piekielna et al.

4. SYNTHESIS OF CYCLIC PEPTIDES

As was diseussed above, cyclization of peptides can beperformed through various bridging bonds and in differentpositions (peptide ends and/or side ehains). Classically, cy-elie peptides were prepared in solution or through on-resinassembly of the linear sequences, folIowed by cyclization insolution. However, solution-phase cyclization can be af-fected by the formation of cyclo-dirners or oligomers that areformed even in high dilutions. Solution-phase cyclization isstilł the only option when head to tailor side-chain to tailbridges are designed [58,28, 29].

The development of a variety of protecting groups andnew solid-phase peptide synthesis protoeols facilitatedcyclization of peptides when they are still attached to theresin and provided new tools for the more effieient synthesisof bridged peptides. Side-chain to side-ehain or head to side-chain cyclization through an amide bridge ean be achievedentirely on the solid support. In such cases attachment of theC-terminal amino aeid residue to the resin and assembly ofthe linear peptide is folIowed by selective deprotection of theside-chain groups and on-resin cyelization [43, 57]. A vari-ety of orthogonaJly protected amino acids is eommerciallyavailable and cyelization strategies for both, Boc and Fmoeehemistry have been elaborated [121-127].

Disulfide bonds can be formed also on the resin [128] arin solution, following solid-phase synthesis and c1eavage ofthe crude peptides from the resin [95]. Similarly, earbonylbridges used to be obtained in solution after the solid-phaseassembly of the linear sequenees [III]. More recently, cycli-zation via a carbonyl bridge when the peptides are still at-tached to the resin was also described [115].

CONCLUSIONS

A lot of synthetie effort has gone into elaboration ofvarious cyclization procedures and obtaining cyclic analogsof opioid peptides. However, not many of these new analogshas been tested in vivo, leaving a field for eollaboration withbiologists and pharmacologists who should demonstrate howsuccessful the introduced modifieations are in the develop-ment of novel analgesics with increased therapeutie poten-tial.

CONFLICT OF INTEREST

The authors eonfirm that this article eontent has no eon-flicts of interest.

ACKNOWLEDGEMENTS

Declared none.

ABBREVIA TI,ONS

Aic 2-aminoindan-2-earboxylie acid

2-aminotetralin-2-earboxylie acid

a,y-diaminobutyrie aeid

Dap-u.ji-diaminopropionic acid

Blood-brain barrier

Ate

Cyclization in Opioid Peptides

C"MePhe C"-methy lphenylalan ine

CNŚ Central nervous system [9)

Dhp 3-(2,6-dimethy 1-4-hydroxypheny l)propanoicacid

[10)Dmt 2' ,6' -dimethyltyrosine

DOR Delta opioid receptor [II)

DPPIV Dipeptidyl peptidase IV [(2)

Dyn A Dynorphin A

EM-I Endomorphin-l [13)

EM-2 Endomorphin-2[14]

~-FNA ~-funaltrexamine

FQ Orphanin [15)

GPCR G-protein-coupled receptor[16J

GP] Guinea pig illeum

i.c.v. lntracerebroventricular [17]

i.t, lntrathecal

KOR Kappa oioid receptor [18J

Mdp C_S)-2-methyl-3-(2' ,6' -dimethyl-4'- [l9]hydroxyphenyl)propionic acid

o ePhe ortho-methylphenylaJanine [20]

OR Mu opioid receptor

MVD Mouse vas deferens [21]

l-l al l-naphthylalanine[22)

2-Nal 2-naphthylalanine[23J

NC Nociceptin[24J

NOR ORLI-opioid receptor like-l

nor-BNI Nor-binaltorphimine

Nphe N-benzylglycine[25)

Pen r= ~,~-dimethy lcysteine [26)

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Received: March 19,2013 Accepted: April22, 2013Revised: April 21,2013

PMID: 23621510