Synthesis and antinociceptive activity of orally active opioid peptides: improvement of oral bioavailability by esterification

To improve the oral bioavailability of a dermorphin tetrapeptide analog, N(alpha)-1-iminoethyl-Tyr-D-MetO-Phe-MebetaAla-OH (III), which has a potent analgesic activity after oral administration, various derivatives were synthesized to increase lipophilicity by esterification of the C-terminal carboxyl group and/or acylation of the phenolic hydroxyl group on Tyr1. Antinociceptive activity was evaluated after subcutaneous or oral administration using the mouse tail pressure test. As a result, increased antinociceptive activity after oral administration as well as an improved ED50(p.o.)/ED50(s.c.) ratio, which is an indicator of oral bioavailability, were found for some compounds. With regard to the improvement of bioavailability, derivatives with acylation of the phenolic hydroxyl group on Tyr1 showed better results than derivatives with esterification of the C-terminal carboxyl group. In particular, an ED50(p.o.)/ED50(s.c.) ratio equivalent to that of morphine was found for an acetylated derivative, N(alpha)-1-iminoethyl-Tyr(COMe)-D-MetO-Phe-MebetaAla-OH (7a), as well as for a methoxycarbonylated derivative, N(alpha)-1-iminoethyl-Tyr(CO2Me)-D-MetO-Phe-MebetaAla-OH (7l).     

Synthesis and structure-activity relationships of [MeTyr1, MeArg7]-dynorphin A(1-8)-OH analogues with substitution at position 8

A series of [MeTyr1, MeArg7]-Dynorphin A (Dyn)(1-8)-OH analogues, modified at position 8 with various amino acids, is described. Their biological activities were determined in the three bioassays [guinea pig ileum (GPI), mouse vas deferens (MVD), and rabbit vas deferens (RVD)] and in the mouse tail-pinch test after subcutaneous administration. None of the analogues tested displayed more potent kappa-opioid activity in the RVD than [MeTyr1, MeArg7, D-Leu8]-Dyn(1-8)-NHEt (1), which is a potent analgesic peptide with similar opioid receptor selectivity to that of Dyn. However, [MeTyr1, MeArg7, Melle8]-Dyn(1-8)-OH (11) showed about a twofold more potent analgesic effect than 1. Based on the obtained results it is conceivable that in the case of Dyn(1-8)-OH analogues both a lipophilic L-amino acid in position 8 and an unchanged 7-8 amide bond are essential to maintain potent kappa-opioid activity.     

Prodrugs of 9-benzyl-8-hydroxy-2-(2-hydroxyethylthio)adenine: potent interferon inducing agents in monkeys

In order to improve the oral bioavailability of 9-benzyl-8-hydroxy-2-(2-hydroxyethylthio)adenine (SM-295072), a potent interferon (IFN) inducing agent, we synthesized prodrugs of it by utilizing the hydroxy groups at the C(2)-side chain and/or the C(8)-position. The carbonate prodrug at the C(8)-position was more effective than that at the C(2)-side chain for oral absorption in rats. Among the compounds prepared, compound 6 demonstrated the most preferable prodrug properties, and the maximum plasma concentration of 6 was approximately 4-fold higher than that of SM-295072. Furthermore, compound 6 was dose-dependently absorbed in monkeys by oral administration, and exhibited a potent IFN-inducting activity that correlated well with its plasma drug concentration.     

Isomerization reaction of olefin using RuClH(CO)(PPh(3))(3)

When methyl 5-(tert-butyldiphenylsilyl)oxy-2-pentenoate was refluxed in toluene in the presence of RuClH(CO)(PPh(3))(3) (5 mol %), double-bond migration took place to afford methyl 5-(tert-butyldiphenylsilyl)oxy-4-pentenoate in high yield. This means that the double bond conjugated with the ester moiety migrates to a deconjugated position by a ruthenium catalyst. We planned to prepare an enol ether from alpha,beta-unsaturated compounds having an ether moiety in a tether using ruthenium-catalyzed isomerization of the double bond. As a result, silyl or benzyl enol ether was obtained from the alpha,beta-unsaturated ester having alcohol protected by the silyl or benzyl group in a tether in high yield. In this reaction, double bond migration of alpha,beta-unsaturated ketone and alpha,beta-unsaturated amide took place to produce deconjugated compounds. Moreover, the double bond of alpha, beta-unsaturated ester having a triple or double bond in a molecule migrated to produce conjugated enyne and diene. On the other hand, treatment of a bis-metalated compound having an alpha, beta-unsaturated ester moiety or the double bond in a tether with RuClH(CO)(PPh(3))(3) gave allyl bis-metalated compound in good yield. These compounds are useful units in synthetic organic chemistry.     

Design,synthesis and insecticidal activities of novel 1-substituted-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide derivatives

A series of novel 5-(trifluoromethyl)-1H-pyrazole-4-carboxamide derivatives(6a–6n, 7a, 7b, and 8a-8f)were synthesised by placing the amide bond at the 4-position of the pyrazole ring. These derivatives differed from the structure of chlorantraniliprole analogues with the amide bond at the 5-position of the pyrazole ring. Preliminary bioassay results revealed that a few title compounds exhibited good insecticidal activities against lepidopteran pests, such as Plutella xylostella, Mythimna separate, Heliothis armigera, and Ostrinia nubilalis. Some title compounds also elicited broad-spectrum insecticidal activities against dipterous insects including Culex pipiens pallens after altering the amide position. Similar to pyrazole-5-carboxamide analogues, compounds 6b and 6e showed 100% insecticidal activity against P. xylostella, C. pipiens pallens, and M. separate at concentrations of 200, 2, and 200 mg/m L, respectively.This finding suggested that 5-(trifluoromethyl)-1H-pyrazole-4-carboxamide derivatives are potential alternative insecticides for management of agriculture pests.     

Design and synthesis of simple macrocycles active against vancomycin-resistant enterococci (VRE)

16-membered meta,para-cyclophanes mimicking the vancomycin binding pocket (D-O-E ring) were designed and synthesized. The structural key features of these biaryl ether containing macrocycles are (1) the presence of beta-amino-alpha-hydroxy acid or alpha,beta-diamino acid as the C-terminal component of the cyclopeptide and (2) the presence of a hydrophobic chain or lipidated aminoglucose at the appropriate position. Cycloetherification by an intramolecular nucleophilic aromatic substitution reaction (S(N)Ar) is used as the key step for the construction of the macrocycle. The atropselectivity of this ring-closure reaction is found to be sensitive to the peptide backbone and chemoselective cyclization (phenol versus primary amine) is achievable. Glycosylation of phenol was realized with freshly prepared 3,4,6-tri-O-acetyl-2-N-lauroyl-2-amino-2-deoxy-alpha-D-glucopyranosyl bromide under phase-transfer conditions. Minimum inhibitory concentrations for all of the derivatives are measured by using a standard microdilution assay, and potent bioactivities against both sensitive and resistant strains are found for some of these compounds (MIC (minimum inhibitory concentration) = 4 microg mL(-1) against VRE). From these preliminary SAR studies, it was anticipated that both the presence of a hydrophobic substituent and an appropriate structure of the macrocycle were required for this series of compounds to be active against VRE.     

Inhibitory effect of 2-(E-2-alkenoylamino)ethyl alkyl sulfides on gastric ulceration in rats. II. Structure and activity relationships of 2-(E-n or Z-n-Decenoylamino)ethyl alkyl sulfides

The analogues of 2-(E-n or Z-n-decenoylamino)ethyl carbamoylmethyl sulfide, including the modifications of sulfide portion, double bond in decenoyl chain and alkyl sulfide moiety, were synthesized and their inhibitory effects on stress-induced ulceration in rats were compared. Replacing the sulfura atom by methylene group or oxygen atom reduced the effect of potency. Saturation of the double bond in the decenoyl chain tended to reduce the anti-ulcerogenic activity in rats. There was no relationship between the position of double bond in decenoyl chain and the pharmacological activity. On the other hand, compounds with E-configuration showed stronger anti-ulcer activity than the corresponding Z-type of compounds. Among 9 kinds of S substituted alkyl groups for carbamoylmethyl, 2-(E-2-decenoylamino)ethyl 2-cyclohexylethyl sulfide showed the most potent anti-ulcerogenic activity in rats and also showed the lowest acute toxicity in mice.     

Synthesis and inhibitory effect of novel glycyrrhetinic acid derivatives on IL-1 beta-induced prostaglandin E(2) production in normal human dermal fibroblasts

Olean-11,13(18)-dien-3beta,30-diol dihemiphthalate (3), which was derived from glycyrrhetinic acid (GA), has been reported to produce a potent of anti-inflammatory effect in in vivo assays. Using 3 as a lead compound, we attempted to synthesize some modified compounds which varied in the following; i) the position of a carboxyl group in the phthalate moiety, ii) the number of carboxyls attached to the benzoyl group, iii) conversion of benzene ring to another ring system, iv) the linkage form between the benzene ring and oleanene skeleton at position 3 and/or 30. These were screened for their inhibitory activity against interleukin-1 beta (IL-1 beta)-induced prostaglandin E(2) (PGE(2)) production in normal human dermal fibroblasts (NHDF). Although conversion of the ortho-carboxyl group of 3 into the meta-position or the para-position led to an increase in inhibitory activity, the elimination or increase of the carboxyl group resulted in loss of the inhibitory activity. Conversion of the ester bond to the amide bond at position 3 and/or 30 of 3 did not contribute to a significant increase in inhibitory activity. On the other hand, among the derivatives possessing an anthranilic acid moiety at position 30 of 3beta-O-acetyl-olean-11,13(18)-dien-30-oic acid (20), 3beta-hydroxy-30-nor-olean-11,13(18)-dien-20 beta-[N-(2-carboxyphenyl)]carboxamide (30) showed the most potent inhibitory activity (IC(50) 1.0 microM) in this series.     

Bis-2-oxo amide triacylglycerol analogues: a novel class of potent human gastric lipase inhibitors.

A novel class of potent human gastric lipase inhibitors, bis-2-oxo amide triacylglycerol analogues, was developed. These analogues of the natural substrate of lipases were prepared starting from 1,3-diaminopropan-2-ol. They were designed to contain the 2-oxo amide functionality in place of the scissile ester bond at the sn-1 and sn-3 position, while the ester bond at the sn-2 position was either maintained or replaced by an ether bond. The derivatives synthesized were tested for their ability to form stable monomolecular films at the air/water interface by recording their surface pressure/molecular area compression isotherms. The inhibition of human pancreatic and gastric lipases by the bis-2-oxo amides was studied using the monolayer technique with mixed films of 1,2-dicaprin containing variable proportions of each inhibitor. The nature of the functional group (ester or ether), as well as the chain length, at the sn-2 position influenced the potency of the inhibition. Among the compounds tested, 2-[(2-oxohexadecanoyl)amino]-1-[[(2-oxohexadecanoyl)-amino]methyl]ethyl decanoate was the most potent inhibitor, causing a 50% decrease in HPL and HGL activities at 0.076 and 0.020 surface molar fractions, respectively.     

Studies on antihypertensive agents with antithrombotic activity. 2. Syntheses and pharmacological evaluation of pyrrolo[2,3-c]azepine derivatives

As an extension of our previous investigation, a series of 7-aminoalkylpyrrolo[2,3-c]azepine derivatives was synthesized and evaluated as alpha1-adrenergic- and serotonin 2 (5-HT2)-receptor antagonists, with the aim of finding a novel potent antihypertensive agent with both activities. Among the compounds obtained in this study, (E)-1-ethyl-7-[3-[4-(4-fluorophenyl)piperazin-1-yl]propyl]-4-hy droxyimino-1,4,5,6,7,8-hexahydropyrrolo[2,3-c]azepin-8-on e (16d) displayed potent alpha1-adrenoceptor blocking activity (pA2=7.83+/-0.20) and 5-HT2-receptor blocking activity (pA2=9.47+/-0.17) in isolated guinea pig arteries. At 3 mg/kg oral administration, compound 16d exhibited antihypertensive activity more potent than that of doxazosin in deoxycorticosterone acetate (DOCA)-salt hypertensive dogs. Furthermore, this compound reduced the rate of mouse acute pulmonary thromboembolytic death induced by collagen and serotonin at oral doses of 0.3 mg/kg or more, and its effect lasted for at least 6 h at 3 mg/kg.     

N[bond]C(alpha) bond dissociation energies and kinetics in amide and peptide radicals. Is the dissociation a non-ergodic process?

Dissociations of aminoketyl radicals and cation radicals derived from beta-alanine N-methylamide, N-acetyl-1,2-diaminoethane, N(alpha)-acetyl lysine amide, and N(alpha)-glycyl glycine amide are investigated by combined density functional theory and M?ller-Plesset perturbational calculations with the goal of elucidating the mechanism of electron capture dissociation (ECD) of larger peptide and protein ions. The activation energies for dissociations of N[bond]C bonds in aminoketyl radicals decrease in the series N[bond]CH(3) > N-CH(2)CH(2)NH(2) > N[bond]CH(2)CONH(2) approximately N[bond]CH(CONH(2))(CH(2))(4)NH(2). Transition state theory rate constants for dissociations of N[bond]C(alpha) bonds in aminoketyl radicals and cation-radicals indicate an extremely facile reaction that occurs with unimolecular rate constants >10(5) s(-1) in species thermalized at 298 K in the gas phase. In neutral aminoketyl radicals the N[bond]C(alpha) bond cleavage results in fast dissociation. In contrast, N[bond]C(alpha) bond cleavage in aminoketyl cation-radicals results in isomerization to ion-molecule complexes that are held together by strong hydrogen bonds. The facile N[bond]C(alpha) bond dissociation in thermalized ions indicates that it is unnecessary to invoke the hypothesis of non-ergodic behavior for ECD intermediates.     

2-hydroxymethyl-4-[5-(4-methoxyphenyl)-3-trifluoromethyl-1H-1-pyrazolyl]-1-benzenesulfonamide (DRF-4367): an orally active COX-2 inhibitor identified through pharmacophoric modulation

Analogs of 1,5-diarylpyrazoles with a novel pharmacophore at N1 were designed, synthesized and evaluated for the in-vitro cyclooxygenase (COX-1/COX-2) inhibitory activity. The variations at/around position-4 of the C-5 phenyl ring in conjunction with a CF3 and CHF2 groups at C-3 exhibited a high degree of potency and selectivity index (SI) for COX-2 inhibition. The in-vivo evaluation of these potent compounds with a few earlier ones indicated the 4-OMe-phenyl analog and the 4-NHMe-phenyl analog with a CF3, and the 4-OEt-phenyl analog with a CHF2 group at C-3 to possess superior potency than celecoxib. In addition to its impressive anti-inflammatory, antipyretic, analgesic and anti-arthritic properties, compound (DRF-4367) was found to possess an excellent pharmacokinetic profile, gastrointestinal (GI) safety in the long-term arthritis study and COX-2 potency in human whole blood assay. Thus, compound was selected as an orally active anti-inflammatory candidate for pre-clinical evaluation.     

Conformational analysis of dehydrodidemnin B (aplidine) by NMR spectroscopy and molecular mechanics/dynamics calculations.

Dehydrodidemnin B (DDB or aplidine), a potent antitumoral natural product currently in phase II clinical trials, exists as an approximately 1:1 mixture of two slowly interconverting conformations. These are sufficiently long-lived so as to allow their resolution by HPLC. NMR spectroscopy shows that this phenomenon is a consequence of restricted rotation about the Pyr-Pro(8) terminal amide bond of the molecule's side chain. The same technique also indicates that the overall three-dimensional structures of both the cis and trans isomers of DDB are similar despite the conformational change. Molecular dynamics simulations with different implicit and explicit solvent models show that the ensembles of three-dimensional structures produced are indeed similar for both the cis and trans isomers. These studies also show that hydrogen bonding patterns in both isomers are alike and that each one is stabilized by a hydrogen bond between the pyruvyl unit at the terminus of the molecule's side chain and the Thr(6) residue situated at the junction betwen the macrocycle and the molecule's side chain. Nevertheless, each conformational isomer forms this hydrogen bond using a different pyruvyl carbonyl group: CO(2) in the case of the cis isomer and CO(1) in the case of the trans isomer.     

<Emphasis Type="Italic">N</Emphasis>-(4-(quinazolin-2-yl)phenyl)benzamide derivatives with potent anti-angiogenesis activities: synthesis and evaluation

Expanding our studies on the anti-angiogenesis activities of 2,4-disubstituted quinazoline derivatives [8], a series of novel N-(2-(quinazolin-2-yl)phenyl)benzamide (SZ) derivatives were designed and synthesized. Cytotoxicity assays indicated that most of these compounds displayed similar cytotoxicity against tumor cells in comparison with our previously reported, but showed a higher cytotoxicity against HUVECs. The SZ derivatives showed a remarkable inhibitive effect against the migration and adhesion of HUVECs, in addition to demonstrating significant in vivo anti-angiogenesis activities in the chick embryo chorioallantoic membrane (CAM) assay. The results proved that the introduction of an aryl group with a basic amide side chain on the 4′ position linked to the amide of the C-2 substituted quinazoline scaffold is an effective approach to improve the anti-angiogenic activity of quinazoline derivatives.     

Synthesis and antibacterial evaluation of a new series of N-Alkyl-2-alkynyl/(E)-alkenyl-4-(1H)-quinolones

To gain further insight into the structural requirements of the aliphatic group at position 2 for their antimycobacterial activity, some N-alkyl-4-(1H)-quinolones bearing position 2 alkynyls with various chain length and triple bond positions were prepared and tested for in vitro antibacterial activity against rapidly-growing strains of mycobacteria, the vaccine strain Mycobacterium bovis BCG, and methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and -16. The compounds were also evaluated for inhibition of ATP-dependent MurE ligase of Mycobacterium tuberculosis. The lowest MIC value of 0.5 mg/L (1.2-1.5 μM) was found against M. fortuitum and M. smegmatis. These compounds displayed no or only weak toxicity to the human lung fibroblast cell line MRC-5 at 100 μM concentration. The quinolone derivatives exhibited pronounced activity against the epidemic MRSA strains (EMRSA-15 and -16) with MIC values of 2-128 mg/L (5.3-364.7 μM), and M. bovis BCG with an MIC value of 25 mg/L (66.0-77.4 μM). In addition, the compounds inhibited the MurE ligase of M. tuberculosis with moderate to weak activity showing IC50 values of 200-774 μM. The increased selectivity towards mycobacterial bacilli with reference to MRC-5 cells observed for 2-alkynyl quinolones compared to their corresponding 2-alkenyl analogues serves to highlight the mycobacterial specific effect of the triple bond. Exploration of a terminal bromine atom at the side chain of N-alkyl-2-(E)-alkenyl-4-(1H)-quinolones showed improved antimycobacterial activity whereas a cyclopropyl residue at N-1 was suggested to be detrimental to antibacterial activity.     

Synthesis and pharmacological evaluation of some 3-(4-methoxyphenyl)-2-substitutedamino-quinazolin-4(3H)-ones as analgesic and anti-inflammatory agents

A variety of novel 3-(4-methoxyphenyl)-2-substitutedamino-quinazolin-4(3H)-ones were synthesized by reacting the amino group of 2-hydrazino-3-(4-methoxyphenyl)-quinazolin-4(3H)-one with a variety of alkyl and aryl ketones. The starting material 2-hydrazino-3-(4-methoxyphenyl)-quinazolin-4(3H)-one was synthesized from 4-methoxyaniline. The title compounds were investigated for analgesic, anti-inflammatory and ulcerogenic index activities. While the test compounds exhibited significant activity, compounds 2-(1-methylpropylidene)-hydrazino-3-(4-methoxyphenyl)-quinazolin-4(3H)-one (A1), 2-(1-ethylpropylidene)-hydrazino-3-(4-methoxyphenyl)-quinazolin-4(3H)-one (A2) and 2-(1-methylbutylidene)-hydrazino-3-(4-methoxyphenyl)-quinazolin-4(3H)-one (A3) showed moderately more potent analgesic activity and the compound 2-(1-methylbutylidene)-hydrazino-3-(4-methoxyphenyl)-quinazolin-4(3H)-one (A3) showed moderately more potent anti-inflammatory activity when compared to the reference standard diclofenac sodium. Interestingly the test compounds showed only mild ulcerogenic potential when compared to aspirin.     

Synthesis and pharmacological evaluation of 2-(1-alkylpiperidin-4-yl)-n-[(1r)-1-(4-fluorophenyl)-2-methylpropyl]acetamide Derivatives as Novel Antihypertensive Agents

We synthesized and evaluated the inhibitory activity of a series of 2-(1-alkylpiperidin-4-yl)-N-[(1R)-1-(4-fluorophenyl)-2-methylpropyl]acetamide derivatives against T-type Ca(2+) channels. Structure-activity relationship studies revealed that the position of the amide structure was important for the potent inhibitory activity toward T-type Ca(2+) channels. In addition, the introduction of an appropriate substituent on the pendant benzene ring played a crucial role for the selectivity towards T-type Ca(2+) channels over L-type Ca(2+) channels and the potent bradycardic activity of these derivatives. Oral administration of N-[(1R)-1-(4-fluorophenyl)-2-methylpropyl]-2-(1-{2-[2-(2-methoxyethoxy)phenyl]ethyl}piperidin-4-yl)acetamide (4f), which had superior selectivity for T-type Ca(2+) channels over L-type Ca(2+) channels, lowered blood pressure in spontaneously hypertensive rats without inducing reflex tachycardia, which is often caused by traditional L-type Ca(2+) channel blockers.     

Investigating the effect of hydrogen bonding environments in amide cleavage reactions at zinc(II) complexes with intramolecular amide oxygen co-ordination

Amide oxygen co-ordination to a zinc(II) ion around a hydrogen bonding microenvironment is a common structural/functional feature of metalloproteases. We report two strategies to position hydrogen bonding groups in the proximity of a zinc(II)-bound amide oxygen, and we investigate their effect on the stability of the amide group. Polydentate tripodal ligands (6-R1-2-pyridylmethyl)-R2 (R1= NHCOtBu, R2= N(CH2-py-6-X)2 X = H L1, X = NH2, H L2, X = NH2 L3) form [(L)Zn]2+ cations (L =L1, 1; L2, 2; L3, 3) with intramolecular amide oxygen co-ordination (1-3), and intramolecular N-H...O=C(amide) hydrogen bonding (2, 3) rigidly fixed by the ligand framework. 1-3 undergo cleavage of the tert-butyl amide upon addition of Me4NOH.5H2O (1 equiv.) in methanol at 50(1) degrees C. Under these conditions the half-life, t(1/2), of the amide bond is 0.4 h for 1, 9 h for 2 and 320 h for 3. Mononuclear zinc(II) complexes of (6-NHCOtBu-2-pyridylmethyl)-R2(R2= N(CH2CH2)2S) L4 and chelating N2 ligands without hydrogen bonding groups (1,10-phenanthroline L5, 2-(aminomethyl)pyridine L6) as control compounds, and with an amino hydrogen bonding group (6-amino-2-(aminomethyl)pyridine L7) have been synthesised. Amide cleavage is in this case faster at the zinc(II) complex with the amino hydrogen bonding group. Thus, hydrogen bonding environments can both accelerate and slow down amide bond cleavage reactions at zinc(II) sites. Importantly, the magnitude of the effect exerted by the hydrogen bonding environments was found to be significant; 800-fold rate difference. This result highlights the importance of hydrogen bonding environments around metal centres in amide cleavage reactions, which may be relevant to the chemistry of natural metalloproteases and applicable to the design of more efficient artificial protein cleaving agents.     

Synthesis and anti-inflammatory-analgesic activity of 2'',4''-difluoro-3-(carbamoyl)biphenyl-4-yl benzoates

Eighteen 2',4'-difluoro-3-(carbamoyl)biphenyl-4-yl benzoates were synthesized from diflunisal in three steps with total yields from 72% to 86%. All compounds were identified by IR, 1H NMR, MS and elemental analysis. The anti-inflammatory activity and analgesic activity for 18 compounds were evaluated. The preliminary assay results showed that compounds 4a and 4p exhibited potent anti-inflammatory-analgesic activity.     

Intestinal permeability of cyclic peptides: common key backbone motifs identified

Insufficient oral bioavailability is considered as a key limitation for the widespread development of peptides as therapeutics. While the oral bioavailability of small organic compounds is often estimated from simple rules, similar rules do not apply to peptides, and even the high oral bioavailability that is described for a small number of peptides is not well understood. Here we present two highly Caco-2 permeable template structures based on a library of 54 cyclo(-D-Ala-Ala(5)-) peptides with different N-methylation patterns. The first (all-trans) template structure possesses two β-turns of type II along Ala(6)-D-Ala(1) and Ala(3)-Ala(4) and is only found for one peptide with two N-methyl groups at D-Ala(1) and Ala(6) [(NMe(1,6)]. The second (single-cis) template possesses a characteristic cis peptide bond preceding Ala(5), which results in type VI β-turn geometry along Ala(4)-Ala(5). Although the second template structure is found in seven peptides carrying N-methyl groups on Ala(5), high Caco-2 permeability is only found for a subgroup of two of them [NMe(1,5) and NMe(1,2,4,5)], suggesting that N-methylation of D-Ala(1) is a prerequisite for high permeability of the second template structure. The structural similarity of the second template structure with the orally bioavailable somatostatin analog cyclo(-Pro-Phe-NMe-D-Trp-NMe-Lys-Thr-NMe-Phe-), and the striking resemblance with both β-turns of the orally bioavailable peptide cyclosporine A, suggests that the introduction of bioactive sequences on the highly Caco-2 permeable templates may result in potent orally bioavailable drug candidates.