Parallel Synthesis of Hexahydrodiimidazodiazepines Heterocyclic

Article pubs.acs.org/jmc

Parallel Synthesis of Hexahydrodiimidazodiazepines Heterocyclic Peptidomimetics and Their in Vitro and in Vivo Activities at μ (MOR), δ (DOR), and κ (KOR) Opioid Receptors Shainnel O. Eans, Michelle L. Ganno, Elisa Mizrachi, Richard A. Houghten, Colette T. Dooley, Jay P. McLaughlin, and Adel Nefzi* Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, Florida 34987, United States S Supporting Information *

ABSTRACT: In the development of analgesics with mixedopioid agonist activity, peripherally selective activity is expected to decrease side effects, minimizing respiratory depression and reinforcing properties generating significantly safer analgesic therapeutics. We synthesized diazaheterocyclics from reduced tripeptides. In vitro screening with radioligand competition binding assays demonstrated variable affinity for μ (MOR), δ (DOR), and κ (KOR) opioid receptors across the series, with the diimidazodiazepine 14 (2065-14) displaying good affinity for DOR and KOR. Central (icv), intraperitoneal (ip), or oral (po) administration of 14 produced dose-dependent, opioid-receptor mediated antinociception in the mouse, as determined from a 55 °C warm-water tail-withdrawal assay. Only trace amounts of compound 14 was found in brain up to 90 min later, suggesting poor BBB penetration and possible peripherally restricted activity. Central administration of 14 did not produce locomotor effects, acute antinociceptive tolerance, or conditioned-place preference or aversion. The data suggest these diazaheterocyclic mixed activity opioid receptor agonists may hold potential as new analgesics with fewer liabilities of use.

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INTRODUCTION The modification of peptides to peptidomimetics has included the manipulation of peptide side chains, amino acid extensions, deletions and substitutions, and most recently backbone modification.1−8 The side chain groups of amino acid residues in polypeptide hormones, neurotransmitters, growth factors, substrates, antigens, and other bioactive peptides have been demonstrated to be extremely important pharmacophores for receptor binding and for signal transduction.1−8 Continuing with our work on the generation of heterocyclic compounds from modified peptides,7,8 we presently combined solid-phase and solution-phase synthesis to synthesize fused diimidazodiazepines from reduced tripeptides and then characterized their opioid activity with in vitro and in vivo assays. Nitrogen heterocycles of different ring sizes with different substitution patterns and embedded in various molecular frameworks constitute important structure classes in the search for bioactivity. For example, the nitrogen-containing diazepine scaffold is an important pharmacophore that displays a wide range of biological activities9including inhibitors of TNF-α converting enzyme (TACE) and matrix metalloproteinase (MMP),10 antimicrobials,11 and inhibitors of human protein kinases and histamine H3 antagonists.12 Diazepam itself is used as an adjuvant analgesic to morphine for alleviating pain induced by the skeletal muscle spasms associated with painful vertebral metastases.13 To date, reported fused imidazodiazepines display a wide range of biological activities including antihepatitis C virus (HCV) activity, inhibitory activity against © 2015 American Chemical Society

the West Nile virus nucleoside triphosphatases (NTPase)/ helicase,14 inhibition of adenosine deaminase,15 inhibition of guanase16 and cannabinoid receptor modulators.17 Known opioid analgesics such as morphine remain the gold standard in efficaciously treating many types of pain, but their clinical use is limited by side effects mediated primarily through centrally located μ-opioid receptors (MOR),18 including respiratory depression, constipation, and addiction. From attempts to retain opioid analgesia while limiting side effects, a number of agonists developed have selectively targeted δ (DOR) and κ (KOR) opioid receptors. Unfortunately, this strategy is limited by inherent side effects mediated by DOR (notably seizures) and KOR (notably aversion and psychomimetic effects) located in the CNS.19 In contrast, the development of efficacious compounds possessing mixed opioid agonist activity at some or all three of the opioid receptors has resulted in clinically useful medications,20−25 suggesting that new, structurally diverse ligands with mixed opioid activity may prove to be effective new analgesics.18−25 Previous studies by our group have also shown the feasibility and utility of generating highly active compounds from peptidomimetics.7,26−30 A number of low molecular weight compounds derived from modified peptides have demonstrated good affinity for opioid receptors in radioligand competition binding assays.28−31 For instance, screening of permethylated Received: October 13, 2014 Published: May 21, 2015 4905

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Journal of Medicinal Chemistry Scheme 1. Parallel Combined Solid-Phase and Solution-Phase Synthesis of Diimidazodiazepine Compounds

with acetic acid and lyophilized to obtain solid white powder. The obtained crude tetraamine compounds were then separately treated with diisopropylethylamine (DIEA) and diethyl malonoimidate dihydrochloride in DMF at 85 °C for 48 h and then purified to generate the desired diimidazodiazepines in good yield and high purity. On the basis of previous studies from our laboratory and other groups,37−39 tyrosine and aromatic residues were universally found in the wide family of opioid peptide and peptidomimetic ligands. Thus, we selected Val and Phe for the first position of diversity (R1), Phe and Tyr for the second position of diversity (R2), Leu and Tyr for the third position of diversity (R3), and phenylacetic acid and H (no acylation) for the fourth and final position of diversity (R4). Using tea-bag technology,65 we performed the parallel synthesis of 16 diimidazodiazepines (Table 1; see also Figure 1). Compounds were purified by RP-HPLC, and purities of the tested compounds were determined to be at least 95% for each compound. Evaluation of Opioid Receptor Affinity and Selectivity by the Diimidazodiazepine Compounds. All synthesized compounds were tested in vitro with radioligand competition binding assays to measure their affinities for MOR, DOR, and KOR by determining the IC50 values for the inhibition of [3H]DAMGO, [3H]DPDPE, and [3H]U69,593, respectively, from their rat brain membrane binding sites. Calculated inhibitory constants (Ki) of the analogues are listed underneath their structures in Figure 1. A number of compounds displayed affinity for DOR, with compounds 5, 6, 21, and 22 demonstrating affinities of less than 70 nM for DOR (Figure 1). Of interest, incorporation of a phenylethyl group at the R4 position decreased DOR selectivity by increasing affinity for the KOR (Figure 1). Additionally compound 14 demonstrated equivalent affinity (>35 nM) for both κ and δ opioid receptors. Accordingly, for further functional characterization, we selected those analogs having an aliphatic amino acid (Val; compound 14) or aromatic amino acid (Phe; compound 6) side chain at R1, and the matching tripeptide analog lacking the phenylethyl at the R4 position (compound 5). Evaluation of Functional δ Opioid Receptor Activity for Diimidazodiazepine Compounds in Vitro. The functional activity of three diimidazodiazepine compounds found to have good (>60 nM) affinity for DOR was assessed in Chinese hamster ovary cells stably expressing the DOR (CHODOR) using a cAMP inhibition assay. The DOR agonist DPDPE was tested as a positive control. A dose−response curve for DOR-mediated activity was determined for each compound (Figure 2), with EC50 values (mean ± SEM) of 366 ± 170 nM, 1639 ± 530 nM, and 6365 ± 2580 nM for compounds 14, 6, and 5, respectively. Although the DOR

chiral tetramines derived from resin-bound tripeptides identified permethylated YYF, a high affinity (0.5 nM), selective MOR antagonist.32 Likewise, the screening of a series of bicyclic guanidines derived from reduced tripeptides led to the identification of a bicyclic guanidine with good affinity and selectivity for the KOR.26 Recently, we reported the identification of two novel, potent, low-liability antinociceptive compounds from the direct in vivo screening of a large mixturebased combinatorial library of pyrrolidine bis-cyclic guanidines derived from resin bound, proline containing acylated tetrapeptides,31 further suggesting the therapeutic potential of peptidomimetics. Finally, given that peptides generally cross the blood−brain barrier and penetrate the CNS poorly after peripheral administration, it was theorized that peripherally selective peptidomimetic opioid agonists would demonstrate analgesia without the liabilities associated with the activation of opioid receptors in the CNS.32 Continuing with our efforts toward the generation of heterocyclic peptidomimetics from modified resin bound peptides, we report the parallel synthesis of fused diimidazodiazepines, from reduced tripeptides. The diimidazodiazepine compounds were subjected to initial pharmacological screening in vitro for opioid receptor affinity with radioligand competition binding assays. The antinociceptive activity of a promising opioid ligand so identified, the diimidazodiazepine 14 (206514), was then characterized in vivo with the mouse 55 °C warm-water tail-withdrawal test after intracerebroventricular (icv), intraperitoneal (ip), or oral (po) administration, and opioid receptor agonist and antagonist profiles were assessed. Bioavailability of the diimidazodiazepine 14 in both blood and brain was evaluated up to 90 min after oral administration. Finally, the effect on acute antinociceptive tolerance, gastrointestinal transit, coordinated locomotor activity, and placeconditioning preference was investigated to evaluate potential liabilities of use.

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RESULTS AND DISCUSSION Design Rationale and Chemistry. Our approach toward the parallel synthesis of fused diimidazodiazepines is outlined in Scheme 1. Starting from resin-bound acylated tripeptides, the amide bonds were exhaustively reduced in the presence of BH3−THF. Typical reaction conditions for the solid-phase reduction of polyamides consist of the treatment of resin-bound peptides with BH3−THF at 65 °C for 72 h.34,35 The generated resin-bound borane−amine complexes are then disproportionate following overnight treatment with neat piperidine at 65 °C. As reported earlier by our group and others, the reduction of polyamides with borane is free of racemization.34−36 The resulting resin bound tetraamines were cleaved from the solid support in the presence of anhydrous HF and then extracted 4906

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Journal of Medicinal Chemistry

To further determine the antinociceptive activity in an assay not based on centrally mediated reflexes, we characterized 14 in the acetic acid writhing assay after ip administration (Figure 3B). Morphine and U50,488 were again examined (ip) in comparison, producing equivalent antinociception (1.75 (0.31− 7.55) and 2.13 (0.04−49.8) mg/kg, ip, respectively). In contrast, 14 demonstrated an ED50 (and 95% confidence interval) value of 0.07 (0.03−0.18) mg/kg, ip, 25- and 30.4-fold more potent than morphine and U50,488, respectively. A ratio of antinociceptive potencies in the tail-withdrawal test versus the writhing test after peripheral administration has been utilized previously to generate an index for the identification of putative peripherally selective opioid agonists.33 Calculated from the current ED50 values, both morphine and U50,488 showed low potency ratios (of 2.23 and 3.76, respectively), consistent with opioids that readily cross the blood−brain barrier (BBB). In contrast, 14 displayed a potency ratio of 72.7, consistent with poor passage across the blood−brain barrier. The opioid receptor mediation and selectivity of 14 induced antinociception were determined following icv administration to opioid receptor knockout (MOR KO or KOR KO) mice or after pretreatment with either δ opioid receptor selective antagonist naltrindole or ICI 174,864 prior to testing in the 55 °C warm water tail-withdrawal assay (Figure 4A). The antinociceptive effect of 14 was significantly reduced, although not eliminated, in both MOR KO and KOR KO mice (F(5,74) = 174.6, P < 0.0001; one-way ANOVA and Tukey’s multiple comparisons post hoc test). Treatment of wild type mice with either DOR selective antagonist reduced 14-induced antinociception to levels not significantly different from baseline responses. Together, these results suggest that although compound 14 was DOR-preferring, it produced nonselective agonism at all three opioid receptors in vivo. The ability of 14 to cross the blood−brain barrier was further evaluated in the tail-withdrawal assay. Pretreatment with naloxone methiodide, the BBB-impermeable opioid antagonist, significantly reduced 14-induced antinociception only when administered on the same side of the blood−brain barrier as the diimidazodiazepine peptidomimetic ligand (F(6,89) = 129.8, P < 0.0001; one-way ANOVA and Tukey’s multiple comparisons post hoc test; Figure 4B). In contrast, naloxone methiodide pretreatment was without effect when the antagonist was administered on the other side of the BBB from 14. Potential opioid receptor-selective antagonist activity by 14 was evaluated after the dissipation of agonist activity. Intracerebroventricular pretreatment for 2.5 h with compound 14 (30 nmol) did not significantly block the antinociception induced by morphine (10 mg/kg, ip; 78.6 ± 9.15% vs 100 ± 0.0% after treatment), (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80) (100 nmol, icv; 73.3 ± 9.33% vs 89.0 ± 7.71% after treatment), or U50,488 (10 mg/kg, ip; 81.8 ± 8.80% vs 99.0 ± 0.98% after treatment). Oral Absorbance and CNS Penetration of Diimidazodiazepine 14. To further confirm that 14 was absorbed after oral administration, yet remained impermeable to the blood− brain barrier, blood and perfused brains were collected from mice 30, 60, and 90 min after po administration of the diimidazodiazepine peptidomimetic ligand (30 mg/kg, po). 14 was readily detected by LC−MS/MS analysis in serum samples within 30 min of administration (Figure 5), indicating oral absorption. In contrast, negligible levels of 14 were detected in perfused brain samples at any time point, further suggesting

Table 1. Analytical Data of Diimidazodiazepine Compounds compd

R1a

R2

a

R3a

b

R4

3 4

Phe Phe

Phe Phe

Tyr Tyr

5 6

Phe Phe

Tyr Tyr

Leu Leu

7 8

Phe Phe

Tyr Tyr

Tyr Tyr

9 10

Val Val

Phe Phe

Leu Leu

11 12

Val Val

Phe Phe

Tyr Tyr

13 14

Val Val

Tyr Tyr

Leu Leu

21 22

Tyr Tyr

Phe Phe

Tyr Tyr

23 24

Tyr Tyr

Tyr Tyr

Tyr Tyr

no acylation phenylacetic acid no acylation phenylacetic acid no acylation phenylacetic acid no acylation phenylacetic acid no acylation phenylacetic acid no acylation phenylacetic acid no acylation phenylacetic acid no acylation phenylacetic acid

mass calcd/found (MH+)

yieldc (%)

464.2/465.3 568.3/569.4

49 44

430.2/431.3 534.3/535.5

42 47

480.2/481.3 584.3/585.5

51 43

366.2/367.3 470.6/471.7

37 40

416.2/417.7 520.3/521.4

51 48

382.2/383.3 486.3/487.6

45 42

480.2/481.4 584.3/585.4

50 41

496.2/497.1 600.3/601.4

45 43

a

Corresponding amino acids. bCorresponding carboxylic acids. cThe yields are based on the weight of purified products and are relative to the initial loading of the resin. The purity of the purified compounds is higher than 95% for all the compounds.

agonist DPDPE proved more potent (EC50 value of 18 ± 10 nM), these data prove that each diimidazodiazepine analog tested produced full agonist efficacy. In Vivo Characterization of Diimidazodiazepine 14 in Mouse Assays. On the basis of the in vitro data, compound 14 was chosen for characterization in vivo. The antinociceptive potency of 14 was determined with the 55 °C warm-water tailwithdrawal assay. The opioid agonist morphine and KORselective agonist (±)-trans-3,4-dichloro-N-methyl-N-[2-(1pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488) were used as positive controls.40 Morphine, U50,488, and 14 each demonstrated antinociceptive activity after icv (Figure 3A, circles), ip (Figure 3A, triangles), or po administration (Figure 3A, squares). After icv administration, morphine and 14 exhibited equivalent dose-dependent antinociceptive potency, with ED50 (and 95% confidence interval) values of 2.35 (1.13− 5.03) nmol and 5.37 (3.84−8.00) nmol, respectively, whereas U50,488 proved modestly (but significantly) less potent (8.62 (5.74−11.9) nmol, (F(1,106) = 19.3, P < 0.0001 nonlinear regression analysis). The three compounds repeated this pattern of antinociceptive potency after ip administration (equivalent values of 3.91 (2.92−5.17) and 5.09 (3.79−7.55) mg/kg, ip for morphine and 14, respectively, whereas U50,488 (8.68 (6.36−11.0) mg/kg ip) was statistically less potent (F(1,98) = 5.44, P = 0.02 nonlinear regression analysis). In contrast, after oral administration, the ED50 (and 95% confidence interval) value for 14 was 23.2 (17.7−36.6), approximately 11.2-fold less potent than the ED50 value of morphine (2.07 (1.60−2.85) mg/kg, po). Antinociception produced by 14 lasted up to 80 min (after administration of 30 nmol, icv or 10 mg/kg, ip) or 110 min (after administration of 30 mg/kg, po) before returning to basal response levels. 4907

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Figure 1. Chemical structures of synthesized diimidazodiazepine compounds and their affinities for μ (MOR), δ (DOR), and κ (KOR) opioid receptors. Values represent the mean ± SD of two experiments with two replicates in each.

Assessment of Diimidazodiazepine 14 Effects on Gastrointestinal Transit. Mice were administered morphine, U50,488, or 14 (each at 10 mg/kg, ip) or saline (0.9%, ip), then fed a charcoal meal. After 3 h, morphine significantly reduced the distance traveled by the charcoal through the intestines, consistent with the action of a MOR agonist (13.0 ± 4.02 cm, compared to 26.4 ± 2.82 cm for saline-treated mice; F(3,27) = 16.8, P < 0.0001; one-way ANOVA with Tukey’s multiple comparisons post hoc test). In contrast, the KOR-selective agonist U50,488 and mixed-opioid-selective agonist compound 14 were without significant effect (32.5 ± 0.85 cm and 35.9 ± 0.72 cm, respectively; see also Supporting Information). Evaluation of Locomotor Effects of Diimidazodiazepine 14. The effect of 14 on locomotor activity was assessed for 60 min after administration of a maximally antinociceptive (30 nmol, icv) central dose in the rotorod assay (Figure 7). Additional mice were administered vehicle (50% DMSO, icv)

poor penetration across the blood−brain barrier consistent with peripherally restricted activity. Liability Assessment of Diimidazodiazepine 14: Evaluation of Antinociceptive Tolerance. As an initial evaluation of the ability of 14 to produce antinociceptive tolerance, an acute tolerance assay was performed with centrally administered (icv) 14. As reported previously,40 the ED50 value (and 95% CI) for a second administration of morphine (10− 100 nmol, icv) given 8 h after the first dose (3 nmol, icv) was shifted rightward 9.6-fold to 22.5 (8.48−61.9) nmol. In contrast, 14 did not demonstrate acute antinociceptive tolerance, as the antinociceptive ED50 value of a second administration of the peptidomimetic ligand given 8 h after a first dose (5 nmol, icv) was 2.32 (1.52−3.50) nmol, a modest ̈ mice to leftward shift as compared to the response of naive morphine or 14 (Figure 6, diamonds). 4908

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DISCUSSION AND CONCLUSION

The current library of polyheterocyclic peptidomimetics contains great potential in the ongoing search for therapeutic compounds coupled with minimal side effects. The lead compound 14 produced opioid-receptor-mediated antinociception after both central (icv) and oral administration and displayed no antinociceptive tolerance, conditioned-place preference, or locomotor activity even after central administration. 14 demonstrated antinociception, slightly less potent than morphine but lasting longer, with activity for at least 120 min after po administration in the tail-withdrawal assay. When samples were analyzed by LC−MS/MS for 14, significant levels of these compounds were detected in blood, but not brain, samples analyzed up to 90 min after oral administration, strongly suggesting oral absorbance with negligible CNS penetration. Given that many of the liabilities of opioid analgesics stem from the centrally mediated effects, whereas analgesia is produced by interactions with both centrally and peripherally located receptors, there is renewed interest in developing peripherally restricted opioids; ligands that do not cross the BBB.41−43 Recent studies suggest that a large proportion (50− 100%) of the analgesic effects produced by systemic administration of conventional opioids can be attributed to activity at peripheral opioid receptors44−51 and that peripherally restricted opioids may be more advantageous than opioids working on CNS targets when treating orofacial muscular pain in rats.52 In the present study, local, peripheral activity of 14 was confirmed by multiple methods. An index of peripheral selectivity was calculated from the antinociceptive potency of 14 after ip testing in the tail-withdrawal assay (where antinociception is attributed to opioid activity at central spinal and supraspinal sites) and the acetic acid writhing assay (where antinociception is attributed to opioid activity at peripheral as well as central sites). Compounds with poor capability to cross the blood−brain barrier would be expected to possess high index values.33 In the current study, 14 demonstrated a high index value, 32.6-fold higher than the index value displayed by morphine, which is generally consistent with comparison values reported with H-D-Phe-D-Phe-D-Nle-D-Arg-NH-4-picolyl 25 (FE200665) and H-D-Phe-D-Phe-D-Leu-D-Orn-morpholine amide 26 (FE200666).33 Additionally, local activity of 14 was confirmed by administering a peripherally restricted opioid

Figure 2. Chinese hamster ovary cells stably expressing δ-opioid receptors (CHO-DOR) were plated at 100 000 cells/well and exposed to 1 μM forskolin (1× PBS and 1 mM IMBX) and increasing concentrations of peptides for 30 min at rt. The FRET reaction was analyzed using a Cisbio cAMP HiRange kit on a Tecan Safire2. Representative curves of mean ± SD of two replicates are shown. EC50 ± SEM values were determined from three experiments with two replicates each.

or morphine (30 nmol, icv) to serve as controls. Although all mice showed significant improvement in rotorod performance over time (F(2,187) = 16.9, P < 0.0001; two-way ANOVA and Tukey’s multiple comparisons post hoc test), 14-treated mice showed no significant deficit in coordinated locomotor activity across time tested (Figure 7). In contrast, morphine-treated mice showed significant impairment up to 40 min after central administration. Evaluation of Place-Conditioning Effects of Diimidazodiazepine 14. Mice were place-conditioned for 40 min each of 2 days with 50% DMSO, morphine, the KOR-selective agonist U50,488, or 14, using intracerebroventricular doses equivalent to or greater than their antinociceptive ED90 values. While morphine produced significant conditioned-place preference (CPP) and U50,488 produced conditioned-place aversion (CPA) (F(4,161) = 4.16; P = 0.003; two-way ANOVA), 14 produced place-conditioning responses similar to that of saline (Figure 8). These data suggest that compound 14 administered centrally at therapeutic doses was neither rewarding nor aversive. Notably, place conditioning of mice lacking the κ opioid receptor (KOR KO mice) with 14 resulted in a similar result, confirming the absence of aversive effects of 14 in the wild-type C57BL/6J mice.

Figure 3. Antinociceptive activity of morphine, U50,488, and diimidazodiazepine 14 was assessed in vivo following icv administration (circles), ip administration (triangles), or po administration (squares) in (A) the mouse 55 °C warm-water tail-withdrawal test or (B) the acetic acid writhing assay. All points represent antinociception at peak response, 30 min after administration in the tail-withdrawal test and 30−45 min after administration in the acetic acid writhing test. Each point represents the mean % antinociception ± SEM for eight mice. 4909

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Figure 4. In vivo opioid receptor selectivity (A) and blood−brain barrier impermeable activity (B) of diimidazodiazepine 14. (A) Opioid receptor selectivity of 14 (30 nmol, icv, −30 min) in MOR or KOR knockout mice or after pretreatment with the DOR selective antagonist naltrindole (0.5 mg/kg, sc, −20 min) or ICI174,864 (4 nmol, icv, −30 min). (B) Impermeability of 14 (left side, 30 nmol, icv; right side, 10 mg/kg, ip) across the blood−brain barrier demonstrated by pretreatment with naloxone methiodide (gray bar, 30 nmol, icv; yellow bar, 30 mg/kg, sc). All points represent antinociception 30 min after test compound administration. All data represent the mean % antinociception ± SEM from eight mice: ∗ = significantly different from baseline response; † = significantly different from response of 14 administered alone (P < 0.05); one-way ANOVA followed by Tukey’s post hoc test.

Figure 5. LC−MS/MS detection of diimidazodiazepine 14 in blood and perfused mouse brain after po administration. Samples were collected 30, 60, and 90 min after administration (30 mg/kg). Mean peak area ± SEM from two separate mice for each time point is presented.

Figure 7. Locomotor testing of 14 in the rotorod assay at therapeutic doses. Effects of vehicle (50% DMSO, icv; gray triangles), morphine (30 nmol, icv; circles), or diimidazodiazepine 14 (30 nmol, icv; squares) were assessed in the rotorod assay of evoked locomotor activity (indicated by latency to fall from a rotorod as the percent change from baseline performance/10 min) of mice (n = 8−16/ group): ∗ = significant difference from the vehicle treated control group (p < 0.05, two-way ANOVA with Tukey HSD post hoc test).

antagonist, naloxone methiodide, prior to injection of 14 and demonstrating an antagonism of antinociception only when the two drugs were administered on the same side of the blood− brain barrier. Both these methods and observed results were similar to the demonstration of peripherally restricted antinociceptive activity of the all D-amino acid tetrapeptides 25 and 26.33 Moreover, the current results further serve to validate the strategy of utilizing peptidomimetic compounds to convey peripherally restricted activity. As noted above, the antinociceptive action of opioids in the mouse-tail-withdrawal assay has been attributed to opioid ligands acting at the spinal or supraspinal levels but not in the periphery.33 This mechanism could suggest the present antinociception produced by 14 was due to CNS penetration by the compound. Some orally administered peptides such as the macrocyclic tetrapeptide [D-Trp]CJ-15,208,53 c[YpwFG] and related compounds, and acylated tripeptide Ac-wFG-NH2

Figure 6. Evaluation of acute antinociceptive tolerance capability of morphine and diimidazodiazepine 14 in the mouse 55 °C warm-water tail-withdrawal assay. Morphine (circles) or 14 (diamonds) was administered at time 0 (3 and 5 nmol, icv, respectively; open symbols) and again 8 h later (filled symbols). At 8 h, morphine demonstrated significant tolerance whereas 14 did not. Points represent n = 8 mice each.

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the promise of peripherally restricted opioids, further preclinical and clinical trials with novel peripherally restricted MOR, DOR, and KOR agonists will be needed in the future to evaluate their utility as analgesics in treating neuropathic pain, cancer pain, and painful inflammatory disorders. One of the major liability factors of the MOR-preferring agonist, morphine, is that it causes significant tolerance when administered repeatedly. To counter this limitation, it has been suggested that compounds possessing opioid agonist activity at multiple opioid receptors might produce less tolerance.20−25 Our lead compound 14 demonstrated mixed opioid receptor agonist activity, producing antinociception through the MOR, DOR, and KOR (though with a higher preference for DOR) in vivo. Because of this mixed opioid agonist activity, we theorized 14 might show limited tolerance when administered icv and tested in an acute tolerance paradigm.40 Although morphine demonstrated significant acute antinociceptive tolerance, the shift between the two dose−response curves was not statistically significant for 14, validating our theory. Moreover, upon central administration of doses producing maximal antinociception, 14 did not produce effects in placeconditioning or locomotor assays. Together, these results suggest possible advantages for mixed-activity opioid agonists in the face of clinical liabilities produced by morphine and other receptor-selective opioid agonists.

Figure 8. Diimidazodiazepine 14 demonstrates no conditioned place preference (CPP) or aversion (CPA). C57BL/6J mice were placeconditioned for 2 days with morphine (100 nmol/d, icv) displayed CPP (black striped bar), whereas place conditioning with U50,488 (100 nmol/d, icv) produced CPA (orange striped bar). Two days’ place conditioning with 50% DMSO (icv; white striped bar) or 14 (30 nmol, icv; blue striped bar) resulted in no significant place preference of any kind. Place conditioning of KOR KO mice with 14 (30 nmol, icv; purple striped bar) also resulted in no significant place preference or aversion. N = 11−24 mice/bar; ∗ = P < 0.05; significantly different from both initial preference and final response of saline-place conditioned mice; † = P < 0.05; significantly different from morphine-CPP response; two-way ANOVA with Tukey post hoc and Student’s paired t tests.

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MATERIALS AND METHODS

Chemistry: General. NMR spectra were recorded in DMSO-d6 with TMS as the internal reference solvent for 1H NMR (500 MHz) and 13C NMR (125 MHz). NMR chemical shifts are expressed in ppm relative to internal solvent peak, and coupling constants were calculated in hertz. The reported final purity of the compounds were verified by Shimatzu HPLC and mass spectra under the following conditions: column, Phenomenex Luna 150 mm × 21.20 mm, 5 μm, C18; mobile phase, (A) H2O (+0.1% formic acid)/(B) MeCN (+0.1% formic acid) and three gradient methods used based on compound hydrophobicity (2% B to 20% B, 11 min) (25% B to 45% B, 31 min) (45% B to 65% B, 21 min); flow rate, 12 mL/min; detection, UV 214 nm. The purity of all final compounds was >95%. All chirality were was generated from the corresponding amino acids. Under the reaction conditions described, no epimerization was observed. General Synthesis of the Diimidazodiazepine Compounds. All compounds were synthesized following the strategy outlined in Scheme 1. The solid phase synthesis was performed using the “tea bag” methodology.65 Parallel Synthesis of Resin Bound Tripeptides (1). Initially, 100 mg of p-methylbenzhydrylamine (pMBHA) resin per compound (1.1 mmol/g, 100−200 mesh) was sealed in a mesh “tea bag”, neutralized with 5% diisopropylethylamine (DIEA) in dichloromethane (DCM), and subsequently swelled with additional DCM washes. The first diversity position (R1) was introduced by the coupling of Boc amino acid (6 equiv) in dimethylformamide (0.1 M DMF) for 60 min in the presence of diisopropylcarbodiimide (DIC, 6 equiv) and 1hydroxybenzotriazole hydrate (HOBt, 6 equiv). The Boc protecting group was removed with 55% TFA/DCM for 30 min and subsequently neutralized with 5% DIEA/DCM (3×). The second and third diversity positions (R2 and R3) were introduced by the subsequent couplings of Boc-amino acids utilizing the same standard coupling procedures. The Boc protecting group was removed with 55% trifluoroacetic acid (TFA) in DCM for 30 min and subsequently neutralized with 5% DIEA/DCM (3×). Substitutions to the fourth diversity position (R4) were introduced following the coupling of phenylacetic acid (10 equiv for compounds 4, 6, 8, 10, 12, 14, 22, and 24) in the presence of DIC (10 equiv). All coupling reactions were monitored for completion by ninhydrin test. Synthesis of Chiral Tetraamines (3). The reduction was performed in a 1000 mL Wilmad LabGlass vessel under nitrogen in the presence

and related compounds54−56 readily cross the blood−brain barrier, so it is plausible that 14 entered the brain after oral administration to produce maximal antinociception in the tailwithdrawal assay. However, the present LC−MS/MS results confirmed minimal blood−brain barrier penetration by 14 after oral administration, more consistent with a peripherally restricted mechanism of activity. Moreover, the results of icv administration suggest that the levels of 14 detected in brain would be inadequate to produce full antinociception. Alternatively, it should be noted that the present data are consistent with the entry of potentially lipophilic 14 into the CNS and rapid efflux by P-glycoprotein (PGP), much as demonstrated by the “peripherally restricted” action of the lipophilic MOR agonist loperamide.57,58 It is also possible that the full pharmacological and behavioral effects reported here are mediated by a metabolite (or metabolites) of 14, presumably working through opioid receptors, as the effects were antagonized by opioid receptor antagonists. Although a full pharmacokinetic study of 14 was beyond the scope of this initial characterization, future studies determining the metabolites of 14 and including the evaluation of possible interactions with PGP are planned. Effective analgesia attributed to activation of peripheral opioid receptors has now been demonstrated in a variety of clinical settings. Human trials indicate that opioid agonists that do not readily cross the blood−brain barrier (BBB) can have the same analgesic efficacy as conventional opioids with significantly fewer CNS side effects.59−62 A peripherally selective KOR agonist has been reported to be effective in relieving the pain associated with chronic pancreatitis,63 and CR845 (an all D-amino acid tetrapeptide, H-D-Phe-D-Phe-DLeu-D-Lys-4-aminopiperidine-4-carboxylic acid, also known as CR665) has completed two phase II clinical trials for pain associated with hysterectomy.64 Although these results show 4911

DOI: 10.1021/jm501637c J. Med. Chem. 2015, 58, 4905−4917

Article

Journal of Medicinal Chemistry of 1.0 M borane−tetrahydrofuran (BH3−THF) complex solution. A 40-fold excess of BH3−THF was used for each amide bond. The vessel containing the 16 bags was heated to 65 °C, and the temperature was maintained for 72 h. The solution was then discarded, and the bags were washed with THF and methanol. Once completely dry, the bags were treated overnight with piperidine at 65 °C and washed several times with methanol, DMF, and DCM. Before proceeding, completion of the reduction was monitored by a control cleavage and analyzed by LCMS. The resulting tetraamines were cleaved from the solid support and then extracted with acetic acid and lyophilized to obtain solid white powder. Synthesis of the Desired Diimidazodiazepines. The crude tetraamines (0.01 mol) were then separately treated with DIEA (6 equiv) and diethyl malonoimidate dihydrochloride (1.1 equiv) in 2 mL of anhydrous DMF. The solutions were stirred in separate vials at 85 °C for 48 h to generate (following evaporation of DMF) oily solutions. The crude compounds were then dissolved in acetic acid and lyophilized. The obtained white powders were purified using preparative high performance liquid chromatography, and the desired diimidazodiazepines were obtained in good yield and high purity (Table 1). 4-(((2S,5S,8S)-5,8-Dibenzyl-2,3,5,6,8,9-hexahydro-1Hdiimidazo[1,2-d:2′,1′-g][1,4]diazepin-2-yl)methyl)phenol (3). 1 H NMR (500 MHz, DMSO-d6, 300 K) 8.50 (s, 1H), 8.08 (s, 1H), 7.98 (s, 1H), 7.41 (t, J = 7.1 Hz, 2H), 7.32 (m, 4H), 7.23 (m, 4H), 6.70 (d, J = 8.5 Hz, 2H), 6.62 (d. J = 8.4 Hz, 2H), 4.27 (s, 1H), 4.05(m, 2H), 3.84 (pent, J = 4.7 Hz), 3.77 (dd, JAB = 9.3 Hz, JBC = 4.7 Hz), 3.64(m, 1H), 3.43 (t, J = 9.2 Hz), 3.15 (dd, JAB = 11.2 Hz, JBC = 3.5 Hz, 1H), 2.80 (m, 3H), 2.61 (dd, JAB = 11.1 Hz, JBC = 2.0 Hz, 1H), 2.45 (dd, JAB = 6.5 Hz, JBC = 3.3 Hz, 1H), 1.89 (dd, JAB = 9.7 Hz, 3.4 Hz, 1H). 13C NMR (125 MHz, DMSO-d6, 300 K) 165.9, 162.4, 159.1, 156.2, 137.9, 136.8, 129.9, 129.4, 128.9, 128.7, 128.5, 126.7, 126.6, 126.4, 115.2, 79.1, 63.3, 57.5, 57.0, 56.1, 54.6, 47.2, 47.0, 37.9, 35.9. MS (ESI) m/z [M + H]+: 465.0. 4-(((2S,5S,8S)-5,8-Dibenzyl-1-phenethyl-2,3,5,6,8,9-hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepin-2-yl)methyl)phenol (4). 1H NMR (500 MHz, DMSO-d6, 300 K) 9.78 (sbroad, 1H), 8.50 (s, 1H), 8.07 (s, 1H), 7.42 (t, J = 7.1 Hz, 2H), 7.28 (m, 13H), 6.62−6.58 (m, 4H), 4.37 (s, 1H), 4.08 (m, 1H), 4.05 (pent, J = 4.7 Hz, 1H), 3.79 (dd, JAB = 9.3 Hz, JBC = 4.7 Hz), 3.65 (m, 1H), 3.43 (t, J = 9.8 Hz, 1H), 3.27 (t, J = 9.3 Hz, 1H), 3.15 (dd, JAB = 9.6 Hz, JBC = 3.5 Hz, 1H), 2.84 (m, 4H), 2.71 (dd, JAB = 9.6 Hz, JBC = 4.6 Hz, 1H), 2.61(dd, JAB = 11.0 Hz, JBC = 2.0 Hz, 1H), 2.47 (dd, JAB = 5.6 Hz, JBC = 7.9 Hz, 1H), 2.16 (dd, JAB =8.0 Hz, JBC = 5.4 Hz, 1H), 1.23 (s, 1H), 1.05 (t, J = 4.7 Hz, 1H). 13C NMR (125 MHz, DMSO-d6, 300 K) 165.8, 162.1, 157.5, 156.1, 138.4, 137.9, 136.8, 129.9, 129.4, 129.0, 128.9, 128.7, 128.5, 128.4, 126.8, 126.6, 126.4, 126.0, 115.1, 79.1, 63.3, 59.1, 57.2, 57.1, 54.0, 47.0, 46.9, 45.9, 37.6, 37.1, 35.1, 33.1. MS (ESI) m/z [M + H]+: 569.0. 4-(((2S,5S,8S)-8-Benzyl-2-isobutyl-2,3,5,6,8,9-hexahydro-1Hdiimidazo[1,2-d:2′,1′-g][1,4]diazepin-5-yl)methyl)phenol (5). 1 H NMR (500 MHz, DMSO-d6, 300 K) 8.50 (s, 1H), 7.94 (sbroad, 2H), 7.28 (m, 5H), 7.04 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 4.23 (s, 1H), 4.04 (m, 1H), 3.95 (pent, J = 4.8 Hz, 1H), 3.72 (dd, JAB = 4.7 Hz, JBC = 9.2 Hz, 1H), 3.68 (m, 1H), 3.60 (t, J = 9.7 Hz, 1H), 3.26 (t, J = 9.3 Hz, 1H), 3.18 (dd, JAB = 3.5 Hz, JBC = 9.6 Hz, 1H), 2.77 (m, 2H), 2.61 (t, J = 11.0 Hz, 1H), 2.46 (t, J = 6.3 Hz, 1H), 1.34 (pent, J = 6.8 Hz, 1H), 1.13 (m, 1H), 1.04 (t, J = 6.1 Hz, 1H), 0.94 (m, 1H), 0.79 (d, J = 6.6 Hz, 3H), 0.76 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO-d6, 300 K)165.6, 162.3, 159.0, 156.4, 136.9, 130.2, 129.0, 128.5, 127.4, 126.6, 115.3, 79.1, 67.3, 63.3, 57.4, 57.4, 57.1, 51.4, 47.2, 47.0, 43.9, 38.0, 35.2, 24.0, 22.8, 22.5, 22.0. MS (ESI) m/z [M + H]+: 431.0. 4-(((2S,5S,8S)-8-Benzyl-2-isobutyl-1-phenethyl-2,3,5,6,8,9hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepin-5-yl)methyl)phenol (6). 1H NMR (500 MHz, DMSO-d6, 300 K) 9.60 (sbroad, 1H), 8.50 (s, 1H), 7.98 (s, 1H), 7.28 (m, 10H), 7.03 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 8.5 Hz, 2H), 4.32 (s, 1H), 4.13 (m, 1H), 3.95 (pent, J = 4.8 Hz, 1H), 3.82 (dd, JAB = 4.7 Hz, JBC = 9.2 Hz, 1H), 3.49 (t, J = 9.7 Hz, 1H), 3.42 (t, J = 9.5 Hz, 1H), 3.21 (dd, JAB = 3.5 Hz, JBC

= 9.6 Hz, 1H), 2.87 (m, 1H), 2.76 (m, 2H), 2.63 (t, J = 11.0 Hz, 1 H), 2.36 (t, J = 6.3 Hz, 1H), 1.24 (m, 1H), 0.77 (m, 1H), 0.70 (d, J = 6.6 Hz, 3H), 0.67 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO-d6, 300 K)165.9, 162.0, 157.2, 156.4, 138.4, 136.8, 130.2, 129.0, 128.9, 128.6, 128.4, 127.4, 126.6, 126.4, 115.3, 79.1, 63.3, 57.6, 57.0, 56.3, 47.1, 45.7, 40.7, 37.8, 34.6, 33.1, 24.1, 23.1, 21.1. MS (ESI) m/z [M + H]+: 535.0. 4,4′-(((2S,5S,8S)-8-Benzyl-2,3,5,6,8,9-hexahydro-1Hd i i m i d a z o [ 1 , 2 - d : 2 ′ , 1′ - g ] [ 1 , 4 ]d i a z e p i n e - 2 ,5 - d i y l ) b i s (methylene))diphenol (7). 1H NMR (500 MHz, DMSO-d6, 300 K) 9.84 (sbroad, 2H), 8.52 (s, 1H), 8.03 (s, 1H), 7.91 (s, 1H), 7.33 (m, 5H), 7.08 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 4.24 (s, 1H), 4.03 (m, 1H), 3.95 (pent, J = 4.7 Hz, 1H), 3.78 (m, 1H), 3.75 (dd, JAB = 9.6 Hz, JBC = 4.3 Hz, 1H), 3.46 (t, J = 9.9 Hz, 1H), 3.26 (t, J = 9.2 Hz, 1H), 3.15 (dd, JAB = 4.0 Hz, JBC = 9.2 Hz, 1H), 2.72 (m, 2H), 2.61 (m, 2H), 2.17 (dd, JAB = 8.4 Hz, 5.0 Hz, 1H), 1.23 (s, 1H,). 13C NMR (125 MHz, DMSO-d6, 300 K)165.8, 162.3, 159.0, 156.5, 156.1, 136.9, 130.2, 130.0, 129.0, 128.5, 127.5, 126.6, 126.4, 115.5, 115.2, 79.1, 63.3, 57.4, 57.3, 56.1, 54.6, 47.1, 47.0, 38.0, 35.2. MS (ESI) m/z [M + H]+: 481.0. 4,4′-(((2S,5S,8S)-8-Benzyl-1-phenethyl-2,3,5,6,8,9-hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepine-2,5-diyl)bis(methylene))diphenol (8). 1H NMR (500 MHz, DMSO-d6, 300 K) 9.84 (sbroad, 2H), 8.51 (s, 1H), 8.02 (s, 1H), 7.31 (m, 8H), 7.24 (t, J = 7.0 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.66 (d, J = 8.5 Hz, 2H), 6.60 (d, J = 8.4 Hz, 2H), 4.34 (s, 1H), 4.10 (m, 1H), 3.93 (pent, J = 4.7 Hz, 1H), 3.79 (dd, JAB = 9.4 Hz, JBC = 4.3 Hz, 1H), 3.68−3.61 (m, 1H), 3.40 (t, J = 9.9 Hz, 1H), 3.25 (t, J = 9.2 Hz, 1H), 3.19 (dd, JAB = 4.0 Hz, JBC = 9.2 Hz, 1H), 3.17 (s, 1H), 2.92 (m, 1H), 2.77 (m, 4H), 2.61 (dd, JAB = 10.8 Hz, JBC = 2.0 Hz, 1H), 2.45 (dd, JAB = 9.6 Hz, JBC = 4.0 Hz, 1H, H6), 1.91 (dd, JAB = 10.0 Hz, JBC = 3.1 Hz, 1H). 13C NMR (125 MHz, DMSO-d6, 300 K)165.7, 162.0, 157.4, 156.6, 156.1, 138.4, 136.8, 130.2, 130.0, 129.0, 128.9, 128.5, 128.4, 127.5, 126.6, 126.4, 126.0, 115.5, 115.2, 63.3, 59.1, 57.5, 57.0, 54.1, 47.0, 46.9, 45.9, 37.7, 37.2, 34.4, 33.1. MS (ESI) m/z [M + H]+: 585.0. (2S,5S,8S)-5-Benzyl-2-isobutyl-8-isopropyl-2,3,5,6,8,9-hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepine (9). 1H NMR (500 MHz, DMSO-d6, 300 K) 8.47 (s, 1H), 7.93 (s, 1H), 7.91 (s, 1H), 7.34 (m, 2H), 7.27 (m, 3H), 4.21 (s, 1H), 4.01 (pent, J = 4.8 Hz, 1H), 3.81 (td, JAB = 2.4 Hz, JBC = 4.5 Hz, 1H), 3.65 (dd, JAB = 4.5 Hz, JBC = 9.2 Hz, 1H), 3.61 (d, J = 7.1 Hz, 1H), 3.53 (t, J = 9.5 Hz, 1H), 3.46 (t, J = 6.2 Hz, 1H), 3.44 (dd, JAB = 7.1 Hz, JBC = 7.0 Hz, 1H), 3.16 (s, 1H), 2.87 (m, 2H), 2.17 (dd, JAB = 4.5 Hz, JBC = 9.2 Hz, 1H), 2.11 (m, 1H), 1.05 (t, J = 9.7 Hz, 1H, H), 1.04 (m, 1H), 0.88 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 7.0 Hz, 3H), 0.78 (m, 1H), 0.75 (d, J = 6.7 Hz, 3H), 0.71 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO-d6, 300 K) 165.9, 162.7, 159.0, 138.1, 129.3, 128.6, 126.7, 79.1, 66.9, 57.5, 57.4, 57.1, 51.3, 47.7, 43.9, 41.4, 36.8, 26.6, 23.9, 22.4, 21.9, 18.7, 14.4. MS (ESI) m/z [M + H]+: 367.0. (2S,5S,8S)-5-Benzyl-2-isobutyl-8-isopropyl-1-phenethyl2,3,5,6,8,9-hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepine (10). 1H NMR (500 MHz, DMSO-d6, 300 K) 8.46 (s, 1H), 8.16 (s, 1H), 7.28 (m, 10H), 4.33 (s, 1H), 4.02 (pent, J = 4.8 Hz, 1H), 3.87 (tod, JAB = 2.4 Hz, JBC = 4.5 Hz, 1H), 3.67 (dd, JAB = 4.5 Hz, JBC = 9.2 Hz, 1H), 3.51 (t, J = 9.5 Hz, 1H), 3.44−3.26 (m/H2O signal, 6H) 2.88 (d, J = 7.8 Hz, 2H), 2.82 (m, 1H), 2.76 (m, 1H) 2.15 (m, 1H), 2.10 (dd, JAB = 6.2 Hz, JBC = 3.0 Hz, 1H), 1.18 (m, 1H), 0.88 (t, J = 6.7 Hz, 6H), 0.62 (t, J = 6.6 Hz, 6H). 13C NMR (125 MHz, DMSO-d6, 300 K)166.0, 162.5, 157.1, 138.4, 138.0, 129.3, 128.9, 128.6, 128.4, 126.7, 126.4, 66.9, 57.6, 57.2, 56.2, 54.6, 47.4, 45.7, 41.5, 40.6, 36.1, 33.1, 26.4, 23.9, 23.1, 21.0, 18.7, 14.4. MS (ESI) m/z [M + H]+: 471.0. 4-(((2S,5S,8S)-5-Benzyl-8-isopropyl-2,3,5,6,8,9-hexahydro1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepin-2-yl)methyl)phenol (11). 1H NMR (500 MHz, DMSO-d6, 300 K) 8.51 (s, 1H), 8.06 (s, 1H), 8.02 (s, 1H), 7.40 (t, J = 7.1 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 7.30 (d, J = 7.0 Hz, 2H), 6.62 (q, J = 8.6 Hz, 4H), 4.25 (s, 1H), 4.01 (q, J = 4.4 Hz, 1H), 3.80 (m, 2H), 3.62 (dd, JAB = 4.5 Hz, JBC = 9.2 Hz, 1H), 3.46 (t, J = 10.0 Hz, 1H), 3.35 (m/H2O peak, 3H), 2.85 (d, J = 7.4 Hz, 2H), 2.42 (m, 2H), 2.08 (m, 2H), 0.88 (d, J = 6.7 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO-d6, 300 K) 4912

DOI: 10.1021/jm501637c J. Med. Chem. 2015, 58, 4905−4917

Article


4,4′,4″-(((2S,5S,8S)-2,3,5,6,8,9-Hexahydro-1H-diimidazo[1,2d:2′,1′-g][1,4]diazepine-2,5,8-triyl)tris(methylene))triphenol (23). 1H NMR (500 MHz, DMSO-d6, 300 K) 9.75 (sv.broad, 3H), 8.52 (s, 1H), 7.99 (s, 1H), 7.85 (s, 1H,), 7.07 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 8.5 Hz, 2H), 6.79 (d, J = 8.345 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 4.23 (s, 1H,), 3.93 (m, 2H,), 3.83 (pent, J = 4.0 Hz, 1H), 3.71 (dd, JAB = 4.6 Hz, JBc = 9.3 Hz, 1H), 3.36 (m/H2O signal, XH), 3.24 (t, J = 9.2 Hz, 1H), 3.03 (dd, JAB = 3.4 Hz, JBC = 9.8 Hz, 1H), 2.72 (m, 2H), 2.61 (dd, JAB = 6.2 Hz, JBC = 3.5 Hz, 1H), 2.18 (dd, JAB = 8.3 Hz, JBC = 5.0 Hz, 1H). 13C NMR (125 MHz, DMSO-d6, 300 K)165.7, 162.4, 159.0, 156.5, 156.2, 156.1, 130.2, 130.0, 129.8, 127.5, 126.6, 126.4, 115.5, 115.4, 115.2, 63.6, 57.4, 57.3, 56.1, 54.6, 47.1, 47.0, 37.2, 35.2. MS (ESI) m/z [M + H]+: 497.0. 4-(((2S,5S,8S)-2-Isobutyl-8-isopropyl-1-phenethyl2,3,5,6,8,9-hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepin-5-yl)methyl)phenol (24). 1H NMR (500 MHz, DMSOd6, 300 K) 9.75 (sv. broad, 2H), 8.49 (s, 1H), 7.93 (s, 1H), 7.31 (m, 4H), 7.24 (t, J = 6.9 Hz, 1H), 7.18 (sbroad, 1H), 7.06 (d, J = 3.2 Hz, 2H), 7.04 (d, J = 3.3 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.5 Hz, 2H), 6.66 (d, J = 8.5 Hz, 2H), 6.60 (d, J = 8.4 Hz, 2H), 4.32 (s, 1H), 4.07 (m, 1H,), 3.92 (pent, J = 4.8 Hz, 1H), 3.74 (dd, JAB = 4.1 Hz, JBc = 9.2 Hz, 1H), 3.36 (m, 3H), 3.05 (dd, JAB = 3.8 Hz, JBC = 9.7 Hz, 1H), 2.87 (m, 1H), 2.82−2.65 (m, 4H), 1.91 (dd, JAB = 2.9 Hz, JBC = 9.8 Hz, 1H), 1.24 (s, 1H). 13C NMR (125 MHz, DMSO-d6, 300 K)165.9, 162.0, 157.4, 156.5, 156.2, 156.1, 138.4, 130.2, 130.0, 129.9, 128.9, 128.4, 127.6, 126.5, 126.4, 126.1, 115.4, 115.4, 115.1, 63.6, 59.1, 57.5, 56.9, 54.1, 46.9, 45.9, 37.2, 36.9, 34.4, 33.1. MS (ESI) m/z [M + H]+: 487.0. In Vitro Assessment of Opioid Receptor Affinity, Selectivity and Function. Radioligand Competition Binding Assays. MOR Binding Assay. Rat cortices were homogenized using 50 mM Tris, pH 7.4, and centrifuged at 16 500 rpm for 10 min. The pellets were resuspended in fresh buffer and incubated at 37 °C for 30 min. Following incubation, the suspensions were centrifuged as before, the resulting pellets resuspended in 100 volumes of 50 mM Tris, pH 7.4, plus 2 mg/mL bovine serum, and the suspensions combined. Each assay tube contained 0.5 mL of membrane suspension, 2 nM [3H]DAMGO, and 0.02 mg/mL mixture in a total volume of 0.65 mL. KOR Binding Assay. Guinea pig cortices and cerebella were homogenized using 50 mM Tris, pH 7.4, 10 mM MgCl2·6H2O, 200 μM, and centrifuged and incubated as above. Each assay tube contained 2 nM [3H]U69,593. Assay tubes were incubated for 2 h at 25 °C. Unlabeled U50,488 was used as a competitor to generate a standard curve and determine nonspecific binding. DOR Binding Assay. Rat cortices were homogenized using 50 mM Tris, pH 7.4, 10 mM MgCl2·6H2O, 200 μM PMSF, centrifuged, and incubated as above. Each assay tube contained 2 nM [3H]DPDPE. Assay tubes were incubated for 2.5 h at 25 °C. Unlabeled DPDPE was used as a competitor to generate a standard curve and determine nonspecific binding. All three binding assays were terminated by filtration through GF/B filters, soaked in 5 mg/mL bovine serum albumin, 50 mM Tris, pH 7.4, on a Tomtec Mach II Harvester 96. The filters were subsequently washed with 6 mL of assay buffer. Bound radioactivity was counted on a Wallac Betaplate liquid scintillation counter. Experiments were conducted using two replicates and repeated twice. Values in Figure 1 represent the mean ± SD of two experiments with two replicates in each test. cAMP Inhibition Assay. CHO cells expressing δ opioid receptors were plated at 20 000 cells/well and exposed to vehicle with or without DPDPE (10 μM) and a DOR opioid standard, a diimidazodiazepine sample, and 4 μM forskolin in assay buffer (1× PBS and 1 mM IMBX) for 30 min at rt. The cells were incubated with europium cryptate and a fluorescence acceptor dye (d2), both diluted in a conjugate lysis buffer for 1 h (Cisbio cAMP HiRange kit, as specified by the manufacturer’s instructions). The FRET reaction was analyzed using a Tecan Safire2 as specified by the manufacturer’s instructions. Experiments were conducted using two replicates and repeated three times.

165.7, 162.7, 159.1, 156.2, 138.1, 129.8, 129.2, 128.7, 126.8, 126.4, 115.1, 66.9, 57.6, 57.3, 56.3, 54.6, 47.7, 41.4, 36.7, 26.6, 18.8, 14.4. MS (ESI) m/z [M + H]+: 418.0. 4-(((2S,5S,8S)-5-Benzyl-8-isopropyl-1-phenethyl-2,3,5,6,8,9hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepin-2-yl)methyl)phenol (12). 1H NMR (500 MHz, DMSO-d6, 300 K) 8.50 (s, 1H), 8.17 (s, 1H), 7.41 (t, J = 7.1 Hz, 2H), 7.35 (t, J = 7.3 Hz, 1H), 7.30 (d, J = 7.0 Hz, 2H), 6.62 (q, J = 8.6 Hz, 4H), 4.25 (s, 1H), 4.01 (q, J = 4.4 Hz, 1H), 3.79 (m, 2H), 3.62 (dd, JAB = 4.5 Hz, JBC = 9.2 Hz, 1H), 3.46 (t, J = 10.0 Hz, 1H), 3.35 (m/H2O peak, 3H), 2.85 (d, J = 7.4 Hz, 2H), 2.42 (m, 2H), 2.08 (m, 2H), 0.88 (d, J = 6.7 Hz, 3H), 0.85 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO-d6, 300 K)165.8, 162.5, 157.5, 156.2, 138.4, 138.1, 129.8, 129.3, 129.0, 128.7, 128.4, 126.8, 126.4, 126.0, 115.1, 79.1, 66.9, 59.1, 57.4, 57.2, 54.2, 47.3, 46.0, 41.5, 37.3, 35.9, 33.1, 26.4, 18.7, 14.4. MS (ESI) m/z [M + H]+: 521.0. 4-(((2S,5S,8S)-2-Isobutyl-8-isopropyl-2,3,5,6,8,9-hexahydro1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepin-5-yl)methyl)phenol (13). 1H NMR (500 MHz, DMSO-d6, 300 K) 8.49 (s, 1H), 7.91 (s, 1H), 7.90 (s, 1H), 7.01 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 8.4 Hz, 2H), 4.20 (s, 1H), 3.90 (pent, J = 4.8 Hz, 1H), 3.79 (tod, JAB = 2.4 Hz, JBC = 4.5 Hz, 1H), 3.62 (m, 2H), 3.52 (t, J = 9.5 Hz, 1H), 3.33 (m/H2O peak, 2H), 2.75 (d, J = 7.3 Hz, 2H), 2.29 (dd, JAB = 4.5 Hz, JBC = 9.2 Hz, 1H), 2.10 (m, 1H, H5), 1.31 (m, 1H), 1.07 (m, 1H), 0.88 (t, J = 7.0 Hz, 6H), 0.77 (d, J = 6.6 Hz, 3H), 0.74 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz, DMSO-d6, 300 K) 165.6, 162.6, 159.0, 156.4, 130.1, 127.7, 115.3, 79.1, 66.9, 57.6, 57.5, 57.2, 51.3, 47.6, 44.0, 41.3, 36.0, 26.6, 24.0, 22.5, 22.0, 18.7, 14.4. MS (ESI) m/z [M + H]+: 383.0. 4-(((2S,5S,8S)-2-Isobutyl-8-isopropyl-1-phenethyl2,3,5,6,8,9-hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepin-5-yl)methyl)phenol (14). 1H NMR (500 MHz, DMSOd6, 300 K) 8.48 (s, 1H), 8.08 (s, 1H), 7.28 (m, 5H), 7.01 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.5 Hz, 2H), 4.31 (s, 1H), 3.91 (pent, J = 4.8 Hz, 1H), 3.86 (td, JAB = 2.4 Hz, JBC = 4.5 Hz, 1H), 3.63 (dd, JAB = 4.5 Hz, JBC = 9.2 Hz, 1H), 3.52 (t, J = 9.5 Hz, 1H), 2.78 (m, 4H), 2.20 (dd, JAB = 6.1 Hz, JBC = 3.0 Hz, 1H) 2.15 (m, 1H), 1.20 (m, 1H), 0.95 (m, 1H), 0.89 (d, J = 2.9 Hz, 3H), 0.88 (d, J = 3.1 Hz, 3H), 0.70 (m, 1H), 0.67 (d, J = 6.6 Hz, 3H), 0.64 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO-d6, 300 K)165.9, 162.4, 157.1, 156.5, 138.4, 130.1, 128.9, 128.4, 127.5, 126.4, 115.3, 66.8, 57.8, 57.1, 56.3, 54.7, 47.2, 45.7, 41.5, 40.8, 35.3, 33.1, 26.4, 24.0, 23.1, 21.1, 18.7, 14.3. MS (ESI) m/z [M + H]+: 488.0. 4,4′-(((2S,5S,8S)-5-Benzyl-2,3,5,6,8,9-hexahydro-1Hd i i m i d a z o [ 1 , 2 - d : 2 ′ , 1 ′ - g ] [1 , 4 ] d i a z e p i ne - 2 , 8 - d i y l ) b i s (methylene))diphenol (21). 1H NMR (500 MHz, DMSO-d6, 300 K) 9.84 (sbroad, 2H), 8.52 (s, 1H), 7.89 (s, 1H), 7.84 (s, 1H), 7.04 (d, J = 8.2 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 4.21 (s, 1H), 3.96 (m, 2H), 3.74 (dd, JAB = 9.4 Hz, JBC = 4.3 Hz, 1H), 3.67 (m, 1H), 3.60 (t, J = 9.5 Hz, 1H), 3.37 (m/H2O peak, 5H), 3.24 (t, J = 9.3 Hz, 1H), 3.05 (dd, JAB = 4.5 Hz, JBC = 9.2 Hz, 1H), 2.77 (m, 2H), 2.47 (m, 2H), 1.35 (pent, J = 6.8 Hz, 1H), 1.09 (m, 1H), 0.93 (m, 1H), 0.79 (d, J = 6.6 Hz, 3H), 0.76 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz, DMSO-d6, 300 K)165.6, 162.3, 159.0, 156.3, 156.7, 130.2, 129.8, 127.5, 126.6, 115.3, 115.3, 63.6, 57.4, 57.3, 57.1, 51.4, 47.1, 47.0, 44.0, 37.2, 35.2, 24.0, 22.5, 22.0. MS (ESI) m/z [M + H]+: 481.0. 4,4′-(((2S,5S,8S)-5-Benzyl-1-phenethyl-2,3,5,6,8,9-hexahydro-1H-diimidazo[1,2-d:2′,1′-g][1,4]diazepine-2,8-diyl)bis(methylene))diphenol (22). 1H NMR (500 MHz, DMSO-d6, 300 K) 9.84 (sbroad, 2H), 8.50 (s, 1H), 7.94 (s, 1H), 7.28 (m, 5H), 7.06 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 8.3 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 4.31 (s, 1H), 4.05 (m, 1H), 3.95 (pent, J = 4.3 Hz, 1H), 3.78 (dd, JAB = 9.4 Hz, JBC = 4.3 Hz, 1H), 3.48 (t, J = 9.7 Hz, 1H), 3.34 (m, 4H), 3.08 (dd, JAB = 4.5 Hz, JBC = 9.2 Hz, 1H), 2.82 (m, 1H), 2.71 (m, 3H), 2.35 (dd, JAB = 6.2 Hz, JBC = 2.9 Hz, 1H), 1.23 (m, 2H), 1.06 (t, J = 7.0 Hz, 1H), 0.80−0.74 (m, 1H), 0.70 (d, J = 6.6 Hz, 3H, H), 0.67 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO-d6, 300 K) 165.9, 162.0, 157.2, 156.4, 156.2, 138.4, 130.2, 129.9, 128.9, 128.4, 127.4, 126.5, 126.4, 115.6, 115.4, 115.3, 79.1, 63.6, 57.7, 57.0, 56.3, 54.6, 47.1, 46.9, 45.7, 40.7, 37.0, 34.6, 33.1, 24.1, 23.2, 21.1. MS (ESI) m/z [M + H]+: 585.0. 4913

DOI: 10.1021/jm501637c J. Med. Chem. 2015, 58, 4905−4917

Article

Journal of Medicinal Chemistry Animals. Adult male C57BL/6J mice (N = 315), weighing 20−25 g obtained from the Jackson Laboratory (Bar Harbor, ME, USA), were selected for this study because of their established responses to opioid analgesics and place conditioning.40,53 μ-Opioid receptor genedisrupted (MOR KO) and κ-opioid receptor gene-disrupted (KOR KO) mice (N of 8 and 20, respectively) on a C57BL/6J genetic background were obtained from colonies established at Torrey Pines Institute for Molecular Studies from homozygous breeding pairs of mice obtained from the Jackson Laboratory. All mice were housed four to a cage and kept on a 12 h light−dark cycle (lights off at 19:00 h) in a temperature- and humidity-controlled room at the Torrey Pines Institute for Molecular Studies (Port Saint Lucie, FL, USA) vivarium with free access to food and water except during experimental sessions, in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals. All procedures involving animal testing were preapproved and carried out in accordance with the Institutional Animal Care and Use Committees at the Torrey Pines Institute for Molecular Studies as specified by the 2008 National Institutes of Health Guide for the Care and Use of Laboratory Animals. Consistent with these guidelines, ongoing statistical testing of data collected was used to minimize the number of animals used, within the constraints of necessary statistical power. Administration Techniques. Oral (po) injections were made as described previously, in a volume of 0.25 mL/25 g body weight.53 All intracerebroventricular (icv) injections were made directly into the lateral ventricle according to the method of Haley and McCormick.66 Compounds were delivered via the icv route to initially measure the inherent pharmacological activity in vivo with minimal material. The mice were lightly anesthetized with isoflurane, an incision made in the scalp, and the injection made 2 mm lateral and 2 mm caudal to bregma at a depth of 3 mm. A volume of 5 μL was injected, using a 10 μL Hamilton microliter syringe. For administration through the icv route, diimidazodiazepine 14 was solubilized in 50% DMSO/50% sterile saline (0.9%) as the vehicle. For administration through the ip or po route, 14 was solubilized in “1:1:8” vehicle, which itself is composed of 1 part ethanol, 1 part Tween-80, and 8 parts sterile saline (0.9%). This vehicle has been used previously to solubilize hydrophobic opiates for in vivo studies.67,68 In Vivo Antinociceptive Testing. 55 °C Warm-Water TailWithdrawal Assay. The 55 °C warm-water tail-withdrawal assay was performed on 176 mice as previously described,39 with the latency of tail withdrawal from the water taken as the end point. After determination of baseline tail-withdrawal latencies, mice (n = 8/ dose) were administered 14 icv in the 50% DMSO vehicle or po in the 1:1:8 vehicle and tested every 10 min thereafter for up to 120 min to determine direct antinociceptive effects. Opioid receptor-selective agonist activity of 14 was evaluated in vivo by testing the antinociceptive effect of the diimidazodiazepine in MOR KO or KOR KO mice or in wild-type C57BL/6J mice pretreated 20 min with the DOR-selective antagonists naltrindole (0.5 mg/kg, sc) or ICI174,864 (4 nmol, icv) prior to administration of 14. The blood−brain barrier permeability of 14 was evaluated by pretreating mice 30 min with naloxone methiodide (30 nmol, icv, or 30 mg/kg, sc) prior to 14 administration. The subsequent antinociceptive activity of 14 was then measured 30 min after injection of the diimidazodiazepine. Antagonist effects of 14 were determined by pretreatment with compound 14 for 2 h to preclude confounding effects of agonism produced by the fused diimidazodiazepine. To determine antagonist activity, mice were pretreated with 14 2 h before administration of the MOR-preferring agonist morphine (10 mg/kg, ip), KOR-selective agonist U50,488 (10 mg/kg, ip) or DOR-selective agonist SNC-80 (100 nmol, icv). Antinociception produced by these established agonists was then measured 30 min after their administration. Reference agonists and antagonists were administered using sterile saline (0.9%) as the vehicle except for SNC-80, which was dissolved in 35% DMSO in sterile saline (0.9%). A cutoff time of 15 s was used in this study. If the mouse failed to display a tail-withdrawal response during that time, the tail was removed from the water and the animal was assigned a maximal antinociceptive score of 100%. At each time point, antinociception was calculated according to the formula

% antinociception =

(test latency) − (control latency) × 100 15 − (control latency)

Acetic Acid Writhing Assay. Because antinociception induced by opioid agonists acting at peripheral, spinal, and supraspinal sites can be detected in the acetic acid induced writhing assay,69 we also investigated the action of morphine, U50,488, and 14 in the mouse acetic acid writhing test. Twenty-five minutes after receiving a single, graded ip dose of opioid agonist, an ip injection of 0.9% acetic acid (0.25 mL/25 g body wt) was administered to each of eight mice. Five minutes after administration, the number of writhing signs displayed by each mouse was counted for an additional 15 min. Antinociception for each tested mouse was calculated by comparing the test group to a control group in which mice were treated with ip the 1:1:8 vehicle, using the formula % antinociception = {[(average writhes in the vehicle group) − (number of writhes in each test mouse)] /(average writhes in vehicle group)} × 100 Acute Opioid Antinociceptive Tolerance. For quantitative measurements of acute opioid tolerance, a standardized state of tolerance was induced by administration of the opioid agonist at the approximate ED50 dose in the tail-withdrawal assay, as shown to be effective with morphine.40 To begin this protocol (time = 0 h), the first dose of morphine (3 nmol, icv) or compound 14 (5 nmol, icv) was administered 8 h before administration of a second dose of morphine or compound 14 (of varying dose but between 0.3 and 100 nmol, icv) to 8 mice/dose (for a total of 32 mice). Opioid-induced antinociception was assessed in the tail-withdrawal assay as described above, 30 min after the second injection of opioid. Previous reports have indicated that this dosing schedule induced relatively rapid, reliable, acute morphine antinociceptive tolerance.40 Assessment of Gastrointestinal Transit. C57BL/6J mice (8 per drug treatment) were administered morphine, U50,488, or 14 (each at 10 mg/kg, ip) or saline (0.9%, ip; 7 mice) 20 min prior to oral gavage with 0.3 mL of a 5% aqueous solution of charcoal meal. After 3 h, mice were euthanized and the intestines removed. The progression of charcoal through the intestines was measured as distance traveled from the jejunum to the cecum as utilized elsewhere.70 Rotorod Assay To Determine Locomotor Activity. Possible sedative or hyperlocomotor effects of vehicle, morphine, or compound 14 were assessed by rotorod performance, as modified from previous protocols.71 Following seven habituation trials (the last utilized as a baseline measure of rotorod performance), mice were centrally (icv) administered vehicle (50% DMSO/50% saline; 16 mice), morphine (30 nmol, 8 mice) or 14 (30 nmol, 8 mice) and assessed after 10 min in accelerated speed trials (180 s max latency at 0−20 rpm) over a 60 min period. Decreased latencies to fall in the rotorod test indicate impaired motor performance. Data are expressed as the percent change from baseline performance. Sample Preparation for LC−MS/MS Analysis. Mice (N = 6) were orally administered the diimidazodiazepine 14 (30 mg/kg, po in 1:1:8 vehicle) and euthanized at 30, 60, or 90 min after administration. Serum was obtained from blood samples (200−250 μL), and the proteins were precipitated by adding 4 volumes of ice-cold acetonitrile (MeCN) followed by centrifugation at 10 000 rpm for 5 min. The supernatants were collected, dried under vacuum, and reconstituted (20% MeCN, 70 μL) for analysis. After blood collection, brains were perfused with cold Dulbecco’s PBS (DPBS, 40 mL) to remove any residual blood, isolated, and frozen. The brains were weighed, washed with ice-cold DPBS (4 × 1 mL), and homogenized in ice-cold DPBS (500 μL), followed by addition of ice-cold MeCN/0.1% formic acid (1 mL) and homogenized again. The homogenates were centrifuged, the supernatants collected, dried under vacuum, and reconstituted (20% MeCN, 70 μL) for LC−MS/MS analysis as described below. Instrumentation and Analytical Conditions. The LC−MS/MS system consisted of a 3200 Q TRAP triple-quadrupole linear ion trap 4914

DOI: 10.1021/jm501637c J. Med. Chem. 2015, 58, 4905−4917

Article

Journal of Medicinal Chemistry mass spectrometer fitted with a TurboIonSpray interface (Applied Biosystems/MDS Sciex, Darmstadt, Germany) and a Shimadzu Prominence HPLC system. Separation was carried out on a C-18 reversed phase column (Luna 5 μm, 100 Å, 50 mm × 4.6 mm) with a C-18 reversed phase guard cartridge (Phenomenex, 4 mm × 3.0 mm), and the peptide eluted [retention time (tR) = 6.4 min] using a gradient of solvents A (10 mM ammonium formate) and B (0.1% formic acid in MeCN) at 0.5 mL·min−1 flow rate (0−3 min, 50% B; 3−6 min, 50− 95% B; 6−8 min, 95% B; 8−9 min, 95−50% B; 9−14 min, 50% B). MS instrument parameters were spray voltage 5.5 kV, curtain gas 25 psi, source temperature 700 °C, ion source gas 170 psi, and gas 260 psi. The ion transitions monitored were 578.2/70.2, 578.2/217.2, 600.2/572.2, and 600.2/425.3 with 150 ms dwell time and 5 ms pause time between the transitions; the counts for the ion transitions were summed to give the peak area for 14. Blank solvent injections were run between each sample to minimize analyte carryover from one LC− MS/MS run to the next. Conditioned Place Preference (CPP) Testing. Mice were conditioned with a counterbalanced CPP paradigm using similar timing as detailed previously.53 Prior to place conditioning, mice were allowed free access to all three chambers of the apparatus for 30 min to determine initial outer chamber preference. Time spent in each chamber was recorded using a computerized system (San Diego Instruments, San Diego, CA). Prior to place conditioning, the animals did not demonstrate significant differences in their time spent exploring the left (554.6 ± 17.4 s) versus right (543.1 ± 12.7 s) compartments (P = 0.65; Student’s t-test). Each day for the next 2 days, mice were administered blank vehicle (0.9% saline, sc) and consistently confined in a randomly assigned outer compartment (i.e., half of each group in the right chamber, half in the left chamber). Six hours later, C57BL/6J mice were administered compound vehicle (50% DMSO/50% sterile saline (icv, 24 mice), morphine (100 nmol, icv, 16 mice), U50,488 (100 nmol, icv, 24 mice), or diimidazodiazepine 14 (30 nmol, icv, 11 mice) and confined to the opposite compartment for 40 min. Note that an additional set of mice lacking the κ opioid receptor (KOR KO mice) were also place-conditioned with diimidazodiazepine 14 (30 nmol, icv, 12 mice). All place conditioning was repeated for a second day, and final place preference was determined 24 h later. Results were compared to the preconditioning responses and postconditioning responses between sets. Statistical Analysis. All data are presented as the mean ± SEM with significance set at p < 0.05. All tail-withdrawal latency data are reported as percent antinociception to control for each animal’s baseline latency response. Percent antinociception is calculated by the following equation:

% antinociception =

calculated from the shift in ED50 value from the singly treated to repeatedly treated condition.71 Data for intestinal transit was analyzed via one-way ANOVA with the Tukey post hoc test. Rotorod data were analyzed via repeated measures ANOVA, with drug treatment condition as a between-groups factor. For all repeated measures ANOVA, simple main effects and simple main effect contrasts are presented following significant interactions. Data for conditioned place preference experiments were analyzed with two-way ANOVA, with analyses examining the main effect of conditioned place preference phase (e.g., pre- or postconditioning) and the interaction of drug pretreatment (morphine, vehicle, or compound 14). Significant effects were further analyzed using Tukey’s HSD or Student’s t test post hoc testing as appropriate. CPP data are presented as the difference in time spent in drug- and vehicle-associated chambers.

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ASSOCIATED CONTENT

S Supporting Information *

LCMS, 1H, and 13 C NMR spectra of all compounds and an additional graph detailing the gastrointestinal effects of the lead compound. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ jm501637c.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 772-345-4739. Fax: 772-345-3649. E-mail: adeln@ tpims.org. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded in part through the Florida Drug Discovery Acceleration Program by the State of Florida, Department of Health and by the National Institute on Drug Abuse (Grant R01-DA31370 to R.A.H.).

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ABBREVIATIONS USED BBB, blood−brain barrier; cAMP, cyclic adenosine monophosphate; CHO, Chinese hamster ovary; CI, confidence interval; CPP, conditioned-place preference; DAMGO, H-TyrD-Ala-Gly-NMe-Phe-Gly-ol; DMSO, dimethyl sulfoxide; Dmt, 2′,6′-dimethyltyrosine; DOR, δ opioid receptor; DPDPE, c[DPen2,D-Pen5]encephalin; EC50, concentration for 50% of maximal effect; ED50, dose for 50% of maximal effect; ESIMS, electron spray ionization mass spectrometry; HBSS, Hanks’ balanced salt solution; HCV, hepatitis C virus; 1H NMR, H nuclear magnetic resonance; icv, intracerebroventricular; ip, intraperitoneal; KOR, κ opioid receptor; KOR KO, κ opioid receptor gene-disrupted mice; LC−MS, liquid chromatography coupled to mass spectrometry; MMP, matrix metalloproteinase; MOR, mu opioid receptor; MOR KO, mu opioid receptor gene-disrupted mice; MRM, multiple reaction monitoring; MS, mass spectrometry; nor-BNI, nor-binaltorphimine; NTPase, Nile virus nucleoside triphosphatase; PBS, phosphate buffered saline; PMSF, phenylmethanesulfonyl fluoride; RP-HPLC, reverse phase high-performance liquid chromatography; sc, subcutaneous; TACE, tumor necrosis factor α converting enzyme; TFA, trifluoroacetic acid; UV, ultraviolet

(test latency) − (baseline latency) × 100 15 − (baseline latency)

All acetic acid writhing data are reported as percent antinociception, calculated by the following equation: % antinociception = {[(average writhes in the vehicle group) − (number of writhes in each test mouse)] /(average writhes in vehicle group)} × 100 All dose−response lines were analyzed by regression, and D50 (dose producing 50% antinociception) values and 95% confidence limits were determined using each individual data point with Prism 5.0 software (GraphPad software, La Jolla, CA) and compared via linear or nonlinear regression modeling as appropriate with Prism 5.0 software. Latency to withdraw tail, rather than percent antinociception, was used to determine within group effects and to allow comparison to baseline latency in tail-withdrawal experiments. Responses were analyzed with a one-way ANOVA followed by Dunnett’s or Tukey’s post hoc test as appropriate for comparisons of multiple groups with one saline control group, and Student’s t tests for comparing baseline and post-treatment tail-withdrawal latencies for all tail-withdrawal data. The degree of tolerance in experiments of acute antinociceptive tolerance was

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ADDITIONAL NOTE Note that amino acids are the L-isomer unless otherwise specified. Abbreviations for amino acids follow IUPAC-IUB 4915

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Joint Commission of Biochemical Nomenclature. The BIND Profiler is a proper name for the equipment utilized.

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Parallel Synthesis of Hexahydrodiimidazodiazepines Heterocyclic

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