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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem. Author manuscript; available in PMC May 15, 2009.
Published in final edited form as:
PMCID: PMC2441872
NIHMSID: NIHMS53337
Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 6. Opioid receptor binding properties of cyclic variants of 8-carboxamidocyclazocine
Mark P. Wentland,*a Xufeng Sun,a Dana J. Cohen,b and Jean M. Bidlackb
aDepartment of Chemistry and Chemical Biology , Rensselaer Polytechnic Institute, Troy, NY 12180, USA
bDepartment of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA
*Corresponding author. Tel.: +1 518 276 2234; fax: +1 518 276 4887; e-mail: wentmp/at/rpi.edu
Abstract
A series of 7,8- and 8,9-fused pyrimidinone, aminopyridine and pyridone derivatives of 8-carboxamidocyclazocine (8-CAC) have been prepared and evaluated in opioid receptor binding assays. Targets were designed to corroborate a pharmacophore hypothesis regarding the bioactive conformation of the carboxamide of 8-CAC. In addition to the results from this study strongly supporting this pharmacophore hypothesis, a number of novel compounds with high affinity to opioid receptors have been identified.
We reported in 20011 our observation that the prototypic phenolic OH group of certain opioids can be replaced by a carboxamide group (CONH2) and retain high affinity binding to opioid receptors. For example, binding affinities for 8-carboxamidocyclazocine (8-CAC, 1) were Ki (nM) = 0.31, 5.2 and 0.06 for μ, δ and κ, respectively, while for cyclazocine (2),2 the Ki values were within 2-fold. For other 2,6-methano-3-benzazocines (a.k.a. benzomorphans)1 as well as quadracyclic morphinans [e.g., cyclorphan (3)],3 the ratio of binding affinities [Ki (CONH2)/Ki (OH)] for μ and κ receptors was also near unity in most cases. However, for pentacyclic 4,5α-epoxymorphinans [e.g., morphine (4) and naltrexone (5)], that ratio was much higher indicating the CONH2 derivative displayed much lower affinity than its corresponding phenolic OH counterpart. For example, the Ki (CONH2)/Ki (OH) ratio for μ was 35 and 7 for the morphine and naltrexone pairs, respectively.4 For the CONH2 partner 6 of naltrexone (5), we observed that the most stable conformation of the unbound ligand was that represented by 6 which is stabilized via intramolecuar H-bonding of the furan O to the carboxamido NH.5 Since this compound had much lower binding affinity than would be predicted from SAR studies, we reasoned that the putative bioactive conformation was 7 rather than 6 and that 6 must pay an energy penalty to adopt the putative bioactive conformation 7 resulting in lower affinity. For 2,6-methano-3-benzazocines and morphinans [e.g., 8-CAC (1)] the putative carboxamide bioactive conformation (as shown in 1) is among many a number of stable conformations and is one that can easily be attained since there is no barrier created by H-bonding to a neighboring ether bridge.
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To test this conformational hypothesis, we designed, prepared and evaluated the 4-hydroxy-3-caboxamido-naltrexone analogue 8 in which the newly introduced 4-OH was found to stabilize the carboxamide in the putative bioactive conformation shown in 8 and not the alternative conformation 9.5 Compound 8 displayed extraordinarily high affinity for μ receptors (Ki = 0.052 nM) and high affinity for δ and κ receptors. When compared to 6, compound 8 had binding affinities 14-, 212- and 50-fold higher against μ, δ and κ, respectively. We also showed that the benefit of the 4-OH was to stabilize the putative bioactive conformation and not through direct contact with the receptor.
We now report additional studies where the overall goal was to confirm or refute this conformational hypothesis. Objectives to meet this goal were a) the design and preparation of analogues where we constrained the carboxamido (or surrogate) group of 8-CAC in the putative bioactive conformation 1 through covalent bonds rather than through non-covalent H-bonds as discussed above and b) evaluation of new targets in opioid receptor binding assays. Previously reported carboxamide SAR studies revealed that the H-bond donating and accepting properties of the CONH2 group were important for recognition by opioid receptors and that highly basic groups at the 8-position were not tolerated.1,6 Keeping these SAR trends in mind, we designed the 8,9- and 7,8-fused 8-CAC derivatives 10 and 12, respectively, as mimics of the putative carboxamide bioactive conformation as shown is 1. We also made derivatives 11 and 13 as controls (i.e., forcing the carboxamide into a conformation believed not to be the bioactive one). Further validation of our pharmacophore hypothesis would be evident if 10 and 12 had higher affinity for opioid receptors than 11 and 13, respectively.
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2.1. Chemistry
The first step in the syntheses of racemic target compounds 10-13 involved the nitration of cyclazocine (2) under standard conditions to provide a mixture of nitro derivatives 18 and 19 easily separated using silica gel flash chromatography (Scheme 1). Compounds 18 and 19 were treated with PhN(SO2CF3)2 and triethylamine in CH2Cl2 to provide triflate esters 20 and 21, respectively, in high yields. Compounds 20 and 21 were then converted to nitriles 22 and 23, respectively, by the use of Zn(CN)2 and Pd(PPh3)4 in DMF under microwave radiation. Partial hydrolysis of nitriles 22 and 23, using KOH and t-BuOH gave carboxamides 24 and 25, respectively. Subsequent reduction of the nitro groups of 24 and 25 using standard conditions provided the corresponding amines 26 and 27 which upon treatment with formic acid under microwave radiation provided the target derivatives 10 and 11, respectively.
Scheme 1
Scheme 1
Reagents and conditions
Target compound 12 was prepared as shown in Scheme 2. Carboxamide derivative 26 (from Scheme 1) was dehydrated using POCl3 and pyridine under microwave radiation to provide nitrile intermediate 28 which was then treated with HC(OMe)3 to provide 29. Compound 29 was treated with ammonia to give the target pyrimidine derviative 12. To assess the effect of N-substitution of target 12, we made the (4′-phenyl)-phenethyl and benzyl derivatives 14 and 15 by treating intermediate 29 with (4′-phenyl)-phenethylamine and benzylamine, respectively.
Scheme 2
Scheme 2
Reagents and conditions
Target 13 having the same aminopyrimidine ring fusion as 12 but at the 8,9-positions was made using a slight modification of the methodology just described. As shown in Scheme 3 intermediate 23 (from Scheme 1) was reduced using hydrogen and 10% Pd/C in methanol to provide intermediate 30 which was then treated with trimethyl orthoformate to provide imidate 31. Exposure of 31 to ammonia and methanol under microwave radiation provided target 13.
Scheme 3
Scheme 3
Reagents and conditions
As shown in Scheme 4, we also made the (4′-phenyl)-phenethyl and benzyl derivatives 16 and 17, respectively, by treating intermediate 32 with (4′-phenyl)-phenethylamine and benzylamine, respectively. Using conditions similar to those previously reported,7 compound 32 was made by exposing 27 (from Scheme 1) to POCl3 and DMF under microwave radiation.
Scheme 4
Scheme 4
Reagents and conditions
Lastly, novel fused 8,9-pyridinone analogue 37 was prepared as shown in Scheme 5. Activated ester intermediate 338 was treated with methoxylamine hydrochloride in pyridine to afford N-methoxycarboxamide 34. Using a general method previously described for making pyridinones,9 intermediate 34 was lithiated at the sterically less encumbered 9-position and at nitrogen using sec-butyllithium in the presence of TMEDA at -20 °C. The resulting dianion was quenched with methyl iodide to provide the 9-methylated derivative 35. Lithiation of 35 with excess sec-butyllithium at -78 °C followed by a DMF quench gave the N-methoxypyridinone derivative 36. Titanium trichloride reduction of 36 provided target 37.
Scheme 5
Scheme 5
Reagents and conditions
2.2. Biology
Target compounds were evaluated for their affinity and selectivity for μ, δ and κ opioid receptors stably expressed in Chinese hamster ovary (CHO) cell membranes. The details of these assays are found in the experimental section and data are summarized in Table 1. Opioid binding affinity data for 8-CAC (1) and the two N-alkylated 8-CAC analogues 38 and 39 are also included. All compounds in Table 1 are racemic. Against the δ receptor, binding affinity for all new targets in Table 1 was low (Ki = 35 to >10,000 nM) relative to their affinities for μ and κ opioid receptors. Therefore, we focused our analysis of the data on the μ and κ receptors. For target compounds 10-13, binding affinities for the μ opioid receptor ranged from very high (e.g., Ki = 0.55 nM for 12) to very low (e.g., Ki = 890 nM for 13). Affinity for the κ receptor was good for targets 10 and 12 with Ki values of 12 nM and 1.0 nM, respectively, while for compounds 11 and 13, affinity was very low (Ki values of 160 nM and 560 nM, respectively). With the exception of target 14, binding affinities of the N-substituted aminopyrimidine analogues 14-17 were relatively weak for μ and κ receptors (Ki = 28-88 nM and 48-240 nM, respectively). For 14, however, affinities for μ and κ were good with Ki values of 6.9 nM and 8.6 nM, respectively. The pyridinone target 37 had high affinity μ and κ receptors (Ki values of 5.5 nM and 0.74 nM, respectively). Lastly, the binding affinities of a number of synthetic intermediates were assessed. For μ and κ receptors, 7-nitro-containing compounds 18 (Ki = 32 nM and 3.2 nM, respectively) and 24 (Ki = 20 nM and 34 nM, respectively) had reasonably good affinity, while the corresponding 9-nitro-containing compounds 19 and 25 had very poor affinities ((Ki values in the range of 110-3800 nM). The 7-amino variant 26 of 8-CAC had very high affinity for μ and κ with Ki = 0.55 nM and 0.70 nM, respectively, while affinities of its regioisomer 27 were much lower (Ki = 88 nM and 32 nM, respectively).
Table 1
Table 1
Opioid receptor binding data for 7,8- and 8,9-ring fused 2,6-methano-3-benzazocines and related compounds.
Three compounds, 12, 26 and 37, that displayed high affinity for μ and κ receptors were characterized in a [35S]GTPγS assay to assess functional activity. These data are summarized in Table 2. Due to the relatively poor binding affinity to δ receptors, these compounds were not evaluated for functional activity at δ. Target compound 12 was found to be an antagonist at both μ and κ receptors and 26 to be an antagonist at μ and moderate agonist at κ. Compound 37 displayed weak mixed agonist/antagonist properties at μ and for κ, it was an agonist. Functional activity for 38, another highly potent in the binding assays, has been previously reported.12,13 It is an antagonist at μ and an agonist at δ and κ receptors.
Table 2
Table 2
EC50 and Emax values for the stimulation of [35S]GTPγS binding and IC50 and Imax values for the inhibition of agonist-stimulated [35S]GTPγS binding to the human μ and κ opioid receptors.a
For the two 8,9- and 7,8-fused-pyrimindinone targets 10 and 11, respectively, our pharmacophore hypothesis predicts the former to have relatively high affinity for μ and κ receptors and the latter predicted to have low affinity. Against the κ receptor we do, in fact, observe a significant difference in the binding affinities of 10 and 11. As predicted, target 10 has reasonably high affinity (Ki = 12 nM) and compound 11 has low affinity (Ki = 160 nM). Against the μ receptor, however, we do not observe the same divergence. While target 11 did, as predicted, exhibit low affinity for μ (Ki = 270 nM) target 10 did so as well (Ki = 170 nM). It may well be that the carboxamide group embedded in 10 is in the proper bioactive conformation and its poor binding affinity is due to the substantial structural change at position-9 relative to 8-CAC (9-H). In other words, unlike the κ receptor, μ poorly accommodates substitution at position-9 of 2,6-methano-3-benzaocines. There are several data points in this study that support such an argument. As shown in Table 1, the 9-nitro variants 19 and 25 of cyclazocine and 8-CAC, respectively, have much lower affinity for μ and κ receptors than their 7-nitro counterparts 18 and 24. Also, the 9-amino variant 27 of 8-CAC displays considerable lower affinity for the receptors than its 7-amino counterpart 26. There are data, however, that contradict this argument. Opioids with a fused 8,9-fused aminothiazole ring (2,6-methano-3-benzaocine numbering) or aminooxazole ring are reported to have high affinity for μ receptors.10 In another study, cyclazocine derivatives with a 8,9-fused imidazole or triazole ring are also characterized by having high affinity for the μ receptor.11 Besides one based on an invalid pharmacophore hypothesis, the only other explanation that comes to mind regarding the poor activity of 10 at the μ receptor is that the electron withdrawing imine part of the pyrimidinone ring reduces the H-bond accepting ability of the carboxamide oxygen. To test this premise, we prepared the pyridinone derivative 37 where the imine N of 10 is replaced by a non-electron withdrawing CH. Physical data that support such a premise is seen in the downfield chemical shift [δ 8.05 (s, 1H)] of the CH on the pyrimidinone ring of 10 relative to that [δ 7.15 (d, 1H, J = 7.0 Hz)] of the corresponding CH of 37. Compound 37 has high affinity for the μ receptor (Ki = 5.5 nM) and is 31-fold more potent than the corresponding pyrimidinone 10. . Pyridinone 37 also has very high affinity for the κ receptor (Ki = 0.74 nM) and has 16-fold higher affinity than 10. These results fit nicely with the premise that an electron withdrawing group at position-9 is detrimental for binding. They also support of the overall hypothesis that the carboxamide structure embedded in 37 is in the bioactive conformation. We attempted to make the analogue of 37 having a 7,8-fusion, however, we were unsuccessful using a similar method to that used to make 37.
For the 7,8- and 8,9-fused-aminopyrimidine targets 12 and 13, respectively, our pharmacophore hypothesis predicts 12 to have relatively high affinity for μ and κ receptors and 13 predicted to have low affinity. This is precisely what we observe. Compound 12 has Ki values of 0.55 nM and 1.0 nM against μ and κ, respectively and 13 has Ki values of 890 nM and 560 nM against μ and κ, respectively. For an aminopyrimidine surrogate of a carboxamide, these data indicate our pharmacophore hypothesis is reinforced.
In earlier SAR studies, we reported that N-substitution of the carboxamide of 8-CAC (1), with groups such as methyl, OH, NH2 or phenyl greatly reduced binding affinity, however, when the substituent was a (4′-phenyl)-phenethyl group (38) binding affinity for μ and κ receptors was very high (Ki values of 0.30 nM and 1.8 nM, respectively).1,12,13 We hypothesized that the N-(4′-phenyl)-phenethyl appendage occupies a previously unexplored hydrophobic pocket in opioid receptors.12,13 We also made the corresponding N-benzyl 8-CAC analogue 39 which had much lower affinity for μ and κ receptors (Ki values of 27 nM and 36 nM, respectively).12 To study the additivity of SAR between the 8-CAC and 7,8-fused-aminopyrimidine platforms, we prepared and evaluated analogues 14 and 15 of aminopyrimidine target 12 by appending (4′-phenyl)-phenethyl and benzyl groups to the exocyclic nitrogens which correspond to the carboxamide N of 8-CAC. Data from Table 1 reveal that introduction of a (4′-phenyl)-phenethyl group in the 7,8-fused aminopyrimidine core (compare 12 and 14) results in a 13- and 48-fold decrease in binding affinity against μ and κ, respectively. These data contrast the 8-CAC core μ results (compare 1 and 38) where N-substitution with a (4′-phenyl)-phenethyl group results in comparable binding affinity; against κ there is a similar decrease (30-fold) in binding affinity. With a N-benzyl substituent (compare 12 and 15), there is a 80- and 240-fold decrease in binding affinity against μ and κ, respectively. This decrease in binding affinity parallels the decrease observed upon introduction of an N-benzyl group into 8-CAC (compare 1 and 39) where an 87- and 600-fold decrease in binding affinity against μ and κ, respectively, is observed. We also made and tested the N-(4′-phenyl)-phenethyl and N-benzyl analogues in the much less active 8,9-fused-aminopyrimidine platform 13. In contrast to the 7,8-fused system 12, the addition of the hydrophobic appendages to 13 enhances binding affinity for μ and κ 32- and 4-fold, respectively, for the (4′-phenyl)-phenethyl derivative 16 and 10- and 12-fold, respectively, for the benzyl analogue 17. Absolute binding affinities, however, are relatively weak.
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Examination of binding data for synthetic intermediates 18, 19 and 24-27, revealed that nitro substitution on the 7- or 9-positions of the aromatic ring was detrimental to binding for both cyclazocine (compare 2 to 18 and 19) and 8-CAC (compare 1 to 24 and 25). It is noteworthy that when nitro is at position-7 in cyclazocine and 8-CAC (i.e., 18 and 24, respectively), binding affinities are much higher than the corresponding 9-nitro derivatives 19 and 25. The observation that the 7-amino variant 26 of 8-CAC had very high affinity for μ and κ (Ki = 0.55 nM and 0.70 nM, respectively suggests the amino group stabilizes the carboxamide in the putative bioactive conformation as depicted in 26a. While the proton NMR of 26 in CDCl3 suggests the presence of an intramolecular H-bond between the CONH2 and adjacent NH2, we can not tell whether the most stable form is 26a or 26b. Abraham aromatic H-bond structural constants for ArCONH2 are 0.49 (H-bond acidity) and 0.53 (H-bond basicity) and for ArNH2, they are 0.26 (H-bond acidity) and 0.27 (H-bond basicity).14 These data would lead one to conclude that 26a and 26b would have similar stabilities, however, the ArCONH2 and ArNH2 groups are considered in isolation in this analysis. In 26, the groups are, of course, conjugated which may well tip the scale on favor of 26a; this would be highly consistent with our view on stabilization of the carboxamide group in the bioactive conformation by H-bonding with an adjacent OH group (e.g., 8). The relatively poor activity of the 9-amino regioisomer 27 (Ki = 88 nM and 32 nM, respectively) may be due to a) poor tolerance of the receptor to 9-substitution and/or b) the 9-amino group stabilizing the carboxamide in a conformation other than the bioactive one.
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All three compounds, 12, 26 and 37, that that were characterized in the [35S]GTPγS assay, were found to be antagonists at the μ receptor, although 37 was a mixed agonist/antagonist. For the three compounds at the κ receptor, however, a divergence in functional activity was observed. Whereas 26 and 37 were agonists at κ, compound 12 was an antagonist.
Opioid receptor binding affinity data for novel target compounds 10-14 and 37 were used in this SAR study to substantiate and strengthen our pharmacophore hypothesis that the carboxamide bioactive conformation of 8-CAC and related opioids is that depicted by 1 versus rotomers where the carboxamide is rotated about the C-C bond to the aryl ring. This conclusion is based on the observed (and predicted) high affinity binding of a) compound 12 for μ and κ relative to 13 and b) compound 10 for κ relative to 11. The poor affinity of 10 for μ was not as predicted and seemingly contradicts our underlying hypothesis. However, by the design and evaluation of 37, a highly active close analogue of 10, we now believe the poor μ affinity of 10 is due to a weakening of the H-bond accepting ability of its carboxamide due to presence of the electron withdrawing imine moiety embedded in the heterocyclic ring. Not only does the observed high μ affinity of 37 help explain the poor activity of 10, it also strengthens our underlying pharmacophore hypothesis since this compound rigidifies the carboxamide group in the putative bioactive conformation.
From our data, it is apparent that target 12 and 8-CAC (1) share a common pharmacophore. Therefore, we expected that the effect of substituting the carboxamide N of 8-CAC with, for example, a (4′-phenyl)-phenethyl group (i.e., 38) would be very similar to that same substitution on the exocyclic N of 12 (e.g., 14). While the impact on κ affinity is similar across both platforms (i.e., 30-fold decrease for the 138 conversion and 9-fold decrease for 1214), there is a significant divergence on μ affinity (i.e., 1 and 38 have same affinity and a 13-fold decrease is seen for the 1214 conversion). This divergence may be a consequence of a conformational change of the carboxamide group of 38 (relative to 8-CAC) to facilitate interaction of the N-(4′-phenyl)-phenethyl group with its putative complimentary hydrophobic binding site. For 14, the aminopyrimidine surrogate of the carboxamide can’t undergo conformational change due to its rigidified nature. This may weaken the stability of the putative hydrophobic interaction between the (4′-phenyl)-phenethyl group and receptor. In summary, there appears to be no benefit in binding affinity when a (4′-phenyl)-phenethyl or benzyl group is appended to the exocyclic nitrogen of 12. While binding affinity is increased when the groups are attached to 14, absolute potency is relatively weak.
Assuming the intramolecular H-bond between the neighboring amino and carboxamide groups of 26 and 27 is due to H-bond donation by the amine and accepting by the carboxamide oxygen, the observation that 26 has much higher affinity for μ and κ receptors than 27 adds further credence to our pharmacophore hypothesis.
In summary, the value of the SAR data generated in this study is not only the strengthening of our underlying pharmacophore hypothesis, but also in the identification of a number of novel opioids having high affinity to μ and κ receptors. These novel compounds (e.g., 12 and 26) have drug-like structures suitable for additional studies to aid in the selection of clinical candidates. Additional SAR studies in this area are ongoing in our laboratories and will be the subject of future communications.
5.1. Chemistry
Proton NMR spectra and in certain cases 13C NMR were obtained on a Varian Unity-300 or 500 NMR spectrometer with tetramethylsilane as an internal reference for samples dissolved in CDCl3. Samples dissolved in CD3OD and DMSO-d6 were referenced to the solvent. Proton NMR multiplicity data are denoted by s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets), and br (broad). Coupling constants are in hertz. Direct insertion probe chemical ionization mass spectral data were obtained on a Shimadzu GC-17A GC-MS mass spectrometer. Direct infusion electrospray ionization (in positively charged ion mode) mass spectral data were obtained on an Agilent 1100 series LC/MSD system (Germany). Melting points were determined on a Meltemp capillary melting point apparatus and were uncorrected. Infrared spectral data were obtained on a Perkin-Elmer Paragon 1000 FT-IR spectrophotometer. Reactions performed under microwave radiation were done on a Personal Chemistry Creator system (20W). The assigned structure of all test compounds and intermediates were consistent with the spectral data. Carbon, hydrogen, and nitrogen elemental analyses for all novel targets were performed by Quantitative Technologies Inc., Whitehouse, NJ, and were within ± 0.4% of theoretical values except as noted; the presence of water or other solvents was confirmed by proton NMR. Reactions were generally performed in an argon or nitrogen atmosphere. Commercially purchased chemicals were used without purification unless otherwise noted. Silica gel (Bodman Industries, ICN SiliTech 2-63 D 60A, 230-400 Mesh) was used for all flash chromatography. Where indicated, an Isco CombiFlash Companion was used for purification of reaction products. Toluene and Et2O were distilled from sodium metal. THF was distilled from sodium/benzophenone ketyl. Pyridine was distilled from KOH. Methylene chloride was distilled from CaH2. DMF and DMSO were distilled from CaH2 under reduced pressure. Methanol was dried over 3Å molecular sieves prior to use.
Cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-7-nitro-2,6-methano-3-benzazocine-8-ol (18) and cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-9-nitro-2,6-methano-3-benzazocin-8-ol (19)
A solution of 69% nitric acid (0.20 g) in 2.0 mL glacial acetic acid was added to a solution of cyclazocine2 (1; 0.542 g, 2.0 mmol) in 3.0 mL glacial acetic acid at 25 °C. After stirring at 25 °C for 3 h, TLC indicated the presence of starting material and an additional 0.10 gm of 69% nitric acid was added. After stirring 2 h at 25 °C, TLC indicated all starting material was consumed and the reaction mixture was poured into a mixture of ice and excess concentrated ammonium hydroxide. The mixture was treated with ethyl acetate and the organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated to give a crude solid product which was purified by gradient silica gel flash chromatography (CH2Cl2:CH3OH; 20:1 → 10:1) to give 18 (0.26 g, 40%) as a brownish solid and 19 (0.35 g, 54%) as a brownish foam: Recrystallization from MeOH/CH2Cl2 gave yellow crystals having mp 145 °C and mp 175 °C, respectively.
For 18: 1H NMR (500 MHz, CDCl3) δ 6.98 (d, 1H, J = 8.3 Hz), 6.83 (d, 1H, J = 8.5 Hz), 3.10 (m, 1H), 2.84 (d, 1H, J = 18.8 Hz), 2.81-2.57 (m, 2H), 2.46 (m, 1H), 2.32 (m, 1H), 2.03 (m, 3H), 1.86-1.66 (m, 1H), 1.31 (s, 3H), 1.25 (m, 1H), 0.87 (m, 4H), 0.51 (m, 2H), 0.11 (m, 2H); MS (ESI) m/z 317 (M+H)+; Anal. Calcd. for C18H24N2O3·0.75 H2O: C 65.53, H 7.79, N 8.49. Found: C 65.27, H 7.41, N 8.23.
For 19: 1H NMR (500 MHz, CDCl3) δ 10.36 (s, 1H), 7.80 (s, 1H), 7.03 (s, 1H), 3.16 (m, 1H), 2.95 (d, 1H, J = 18.8 Hz), 2.79-2.56 (m, 2H), 2.48 (m, 1H), 2.32 (m, 1H), 1.96 (m, 3H), 1.39 (s, 3H), 1.36 (m, 1H), 0.85 (m, 4H), 0.52 (m, 2H), 0.11 (m, 2H); MS (ESI) m/z 317 (M+H)+; Anal. Calcd. for C18H24N2O3·0.5 H2O: C 66.44, H 7.74, N 8.61. Found: C 66.03, H 7.33, N 8.48.
Trifluoromethanesulfonic acid, cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-7-nitro-2,6-methano-3-benzazocine-8-yl ester (20)
Triethylamine (0.22 g, 2.22 mmol) was added to a solution of 18 (0.47 g, 1.48 mmol) dissolved in 20 mL of CH2Cl2. PhN(SO2CF3)2 (0.58 g, 1.63 mmol) was then added and the resulting mixture stirred at 25 °C for 4 h. The solvent was removed on a rotary evaporator and the resulting mixture was purified by gradient silica gel flash chromatography (CH2Cl2:CH3OH; 80:1 → 40:1) to give 20 (0.59 g, 88%) as a yellow foam. 1H NMR (500 MHz, CDCl3) δ 7.30 (d, 1H, J = 8.5 Hz), 7.24 (d, 1H, J = 8.6 Hz), 3.56 (m, 1H), 3.17 (m, 1H), 3.05 (m, 2H), 2.81 (m, 1H), 2.66 (m, 1H), 2.29-2.04 (m, 2H), 1.90 (m, 1H), 1.34 (m, 4H), 0.87 (m, 4H), 0.69 (m, 2H), 0.28 (m, 2H).
Trifluoromethanesulfonic acid, cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-9-nitro-2,6-methano-3-benzazocine-8-yl ester (21)
Using a procedure similar to that used to prepare 20, compound 19 was converted to 21 (93%) as a yellow foam. 1H NMR (500 MHz, CDCl3) δ 7.94 (s, 1H), 7.27 (s, 1H), 3.60 (m, 1H), 3.22-2.94 (m, 3H), 2.84 (m, 1H), 2.68 (m, 1H), 2.30 (m, 1H), 2.11 (m, 2H), 1.41 (s, 3H), 1.38 (m, 1H), 0.84 (m, 4H), 0.69 (m, 2H), 0.29 (m, 2H).
Cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-7-nitro-2,6-methano-3-benzazocine-8-carbonitrile (22)
To a tube containing 20 (0.27 g, .061 mmol) was added under a N2 blanket, Zn(CN)2 (0.14 g, 1.22 mmol) and Pd(PPh3)4 (0.07 g, 0.061 mmol). DMF (degassed with N2, 3.0 mL) was then added via a cannula under N2. The resulting mixture was irradiated with microwaves at 150 °C for 15 min. The resulting mixture was partitioned between water and EtOAc. The organic phase was washed with water (X2) and brine, and then dried over Na2SO4, filtered, and concentrated to give a crude product which was purified by silica gel flash chromatography (CH2Cl2:CH3OH:NH4OH;80:1:0.1) to give 22 (0.14 g, 70%) as an off-white foam. 1H NMR (500 MHz, CDCl3) δ 7.52 (d, 1H, J = 8.1 Hz), 7.35 (d, 1H, J = 8.1 Hz), 3.20 (m, 1H), 3.04 (d, 1H, J = 19.1 Hz), 2.86 (m, 1H), 2.68 (m, 2H), 2.47 (m, 1H), 2.34 (m, 1H), 2.10-1.74 (m, 3H), 1.34 (m, 4H), 0.89 (m, 1H), 0.84 (d, 3H, J = 7.1 Hz), 0.54 (m, 2H), 0.12 (m, 2H). MS (ESI) m/z 326 (M+H)+.
Cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-9-nitro-2,6-methano-3-benzazocine-8-carbonitrile (23)
Using a procedure similar to that used to prepare 22, compound 21 was converted to 23 (quantitative yield) as an off-white foam. 1H NMR (500 MHz, CDCl3) δ 8.05 (s, 1H), 7.75 (s, 1H), 3.22 (m, 1H), 3.08 (d, 1H, J = 19.8 Hz), 2.79 (m, 2H), 2.47 (m, 1H), 2.32 (m, 1H), 2.10-1.78 (m, 3H), 1.46 (s, 3H), 1.33 (m, 1H), 0.87 (m, 1H), 0.83 (d, 3H, J = 7.1 Hz), 0.54 (m, 2H), 0.12 (m, 2H).
Cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-7-nitro-2,6-methano-3-benzazocine-8-carboxamide (24)
A solution of 22 (0.11 g, 0.33 mmol) dissolved in t-BuOH (2.0 mL) was heated at 58 °C and KOH (0.056 g, 1.0 mmol) was added. After stirring at 58 °C for 1 h, brine and EtOAc were added. The organic phase was dried over Na2SO4, filtered, and concentrated to give a crude product which was purified by silica gel flash chromatography (CH2Cl2:CH3OH:NH4OH; 20:1:0.1) to give 24 as an off-white solid (0.098 g, 85%). Crystallization of this solid from acetone followed by a recrystallization from i-PrOH/t-BuOH gave crystals having mp 190 °C. 1H NMR (500 MHz, CDCl3) δ 8.03 (s, 1H), 7.54 (s, 1H), 7.38 (s, 2H), 3.04 (m, 1H), 2.96 (d, 1H, J = 19.5 Hz), 2.76 (m, 2H), 2.38 (m, 1H), 2.24 (m, 1H), 2.06-1.56 (m, 3H), 1.19 (m, 4H), 0.79 (m, 1H), 0.73 (d, 3H, J = 6.8 Hz), 0.44 (m, 2H), 0.07 (m, 2H); MS (ESI) m/z 344 (M+H)+; Anal. Calcd. for C19H25N3O3·0.5 H2O: C 64.75, H 7.44, N 11.92. Found: C 64.47, H 7.21, N 11.56.
Cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-9-nitro-2,6-methano-3-benzazocine-8-carboxamide (25)
Using a procedure similar to that used to prepare 24, compound 23 was converted to 25 (45%) as an off-white foam. 1H NMR (500 MHz, CDCl3) δ 7.80 (s, 1H), 7.42 (s, 1H), 5.89 (m, 2H), 3.20 (m, 1H), 3.03 (d, 1H, J = 19.0 Hz), 2.75 (m, 2H), 2.47 (m, 1H), 2.33 (m, 1H), 2.06-1.82 (m, 3H), 1.43 (s, 3H), 1.34 (m, 1H), 0.87 (m, 1H), 0.83 (d, 3H, J = 7.1 Hz), 0.53 (m, 2H), 0.12 (m, 2H); MS (ESI) m/z 344 (M+H)+; Anal. Calcd. for C19H25N3O3·0.25 H2O: C 65.59, H 7.39, N 12.08. Found: C 65.39, H 7.38, N 11.93.
Cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-7-amino-2,6-methano-3-benzazocine-8-carboxamide (26)
To a solution of 24 (0.15 g, 0.44 mmol) dissolved in MeOH (20 mL) was added 10% Pd/C (0.093 g). The resulting mixture was subjected to 55 psi H2 in a Parr shaker for 3d at 25 °C. The mixture was filtered and concentrated to give a crude product that was purified by silica gel flash chromatography (CH2Cl2:CH3OH:NH4OH; 30:1:0.1) giving 26 (0.060 g, 44%) as a white foam. 1H NMR (500 MHz, CDCl3) δ 7.14 (d, 1H, J = 8.1 Hz), 6.42 (d, 1H, J = 8.1 Hz), 6.12 (s, 1H), 5.58 (br s, 2H), 3.09 (m, 1H), 2.76 (m, 3H), 2.24 (m, 1H), 2.28 (m, 1H), 2.06-1.70 (m, 3H), 1.59 (s, 3H), 1.58 (m, 1H), 0.91 (d, 3H, J = 7.1 Hz), 0.86 (m, 1H), 0.51 (m, 2H), 0.10 (m, 2H); MS (ESI) m/z 314 (M+H)+; Anal. Calcd. for C19H27N3O·0.25 H2O: C 71.78, H 8.72, N 13.22. Found: C 72.00, H 8.84, N 12.98.
Cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-9-amino-2,6-methano-3-benzazocine-8-carboxamide (27)
Using a procedure similar to that used to prepare 26, compound 25 was converted to 27 (63%) as an off-white foam. 1H NMR (500 MHz, CDCl3) δ 7.20 (s, 1H), 6.42 (s, 1H), 5.61 (br s, 2H), 5.42 (s, 2H), 3.10 (m, 1H), 2.84 (d, 1H, J = 18.8 Hz), 2.75-2.53 (m, 2H), 2.46 (m, 1H), 2.30 (m, 1H), 2.06-1.80 (m, 3H), 1.35 (s, 3H), 1.27 (m, 1H), 0.87 (m, 1H), 0.84 (d, 3H, J = 7.1 Hz), 0.51 (m, 2H), 0.10 (m, 2H); MS (ESI) m/z 314 (M+H)+; Anal. Calcd. for C19H27N3O·0.25 H2O: C 71.78, H 8.72, N 13.22. Found: C 72.00, H 8.73, N 13.27.
7,8-Fused pyrimidinone derivative 11
A mixture of 26 (0.035 g, 0.11 mmol) and 2.0 mL of 88% formic acid was heated at 120 °C under microwave radiation for 30 min. The reaction mixture was basified using excess NH4OH and the organic material was extracted into ethyl acetate. The organic phase was washed with brine, dried over Na2SO4 and concentrated giving a crude product that was purified by silica gel chromatography (Combiflash - CH2Cl2:CH3OH:NH4OH; 20:1:0.1) giving 11 (0.020 gm, 54%) as an off-white foam. Further crystallization from acetone gave white crystals (mp 220 °C): 1H NMR (500 MHz, CDCl3) δ 10.90 (br s, 1H), 8.08 (d, 1H, J = 8.1 Hz), 7.99 (s, 1H), 7.25 (d, 1H, J = 8.1 Hz), 3.19 (m, 1H), 2.93 (m, 2H), 2.77 (m, 1H), 2.48 (m, 1H), 2.29 (m, 1H), 2.06 (m, 1H), 1.90 (m, 2H), 1.81 (s, 3H), 1.64 (m, 1H), 0.90 (d, 3H, J = 7.1 Hz), 0.88 (m, 1H), 0.51 (m, 2H), 0.10 (m, 2H); MS (ESI) m/z 324 (M+H)+; Anal. Calcd. for C20H25N3O: C 74.27, H 7.79, N 12.99. Found: C 73.95, H 7.86, N 12.78.
8,9-Fused pyrimidinone derivative 10
Using a procedure similar to that used to prepare 11, compound 27 (0.021 g, 0.067 mmol) was converted to 10 (0.010 gm, 46%) as an off-white foam: 1H NMR (500 MHz, CDCl3) δ 11.10 (br s, 1H), 8.19 (s, 1H), 8.05 (s, 1H), 7.48 (s, 1H), 3.23 (m, 1H), 3.14 (d, 1H, J = 19.3 Hz), 2.91-2.72 (m, 2H), 2.51 (m, 1H), 2.35 (m, 1H), 2.08-1.86 (m, 3H), 1.52 (s, 3H), 1.38 (m, 1H), 0.90 (m, 1H), 0.87 (d, 3H, J = 7.1 Hz), 0.88 (m, 1H), 0.53 (m, 2H), 0.13 (m, 2H); MS (ESI) m/z 324 (M+H)+; Anal. Calcd. for C20H25N3O·0.25 H2O: C 73.25, H 7.84, N 12.81. Found: C 73.14, H 7.90, N 12.38.
Cis-(±)-7-Amino-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-2,6-methano-3-benzazocine-8-carbonitrile (28)
A mixture of 26 (0.22 g, 0.70 mmol), POCl3 (0.11 gm, 0.70 mmol), and pyridine (2.0 mL) was heated at 100 °C for 20 min under microwave radiation and concentrated. The residue was dissolved in 1.0 N HCl and stirred for 1 h at 25 °C. The reaction mixture was made basic with saturated NaHCO3/crushed ice and the organic material was extracted into CH2Cl2. The organic layer were washed with brine, dried over Na2SO4, filtered and concentrated to give a crude product that was purified by silica gel chromatography (Combiflash - CH2Cl2:CH3OH:NH4OH) to give 28 as a brownish oil (0.11 g) in 54% yield: 1H NMR (500 MHz, CDCl3) δ 7.07 (d, 1H, J = 8.5 Hz), 6.41 (d, 1H, J = 8.8 Hz), 3.16 (m, 1H), 2.70 (m, 4H), 2.46 (m, 1H), 2.30 (m, 1H), 2.07-1.70 (m, 3H), 1.64 (m, 4H), 0.94 (d, 3H, J = 6.8 Hz), 0.88 (m, 1H), 0.53 (m, 2H), 0.12 (m, 2H).
Cis-(±)-9-Amino-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-6,11-dimethyl-2,6-methano-3-benzazocine-8-carbonitrile (30)
A mixture of 23 (0.180 g, 0.55 mmol), 10% Pd/C and CH3OH (20 mL) was subjected to 40 psi H2 in a Parr shaker at 25 °C for 15 h. The mixture was filtered and concentrated to give 30 as a crude product that was purified by silica gel chromatography (Combiflash - CH2Cl2:CH3OH:NH4OH) to give a brownish solid (0.070 g, 47%): 1H NMR (500 MHz, CDCl3) δ 7.25 (s, 1H), 6.47 (s, 1H), 4.18 (s, 2H), 3.15 (m, 1H), 2.86 (d, 1H, J = 19.0 Hz), 2.80-2.58 (m, 2H), 2.48 (m, 1H), 2.33 (m, 1H), 1.94 (m, 3H), 1.32 (s, 3H), 1.25 (m, 1H), 0.90 (m, 1H), 0.81 (d, 3H, J = 7.1 Hz), 0.53 (m, 2H), 0.12 (m, 2H); MS (ESI) m/z 296 (M+H)+.
7,8-Fused aminopyrimidine derivative 12
A mixture of 28 (0.11 g, 0.38 mmol), CH(OCH3)3 (2 mL) and 4Å molecular sieves was heated at 140 °C for 48 h. The reaction mixture was filtered and concentrated to give imidate intermediate 29 (0.120 g) which, without further purification, was combined with methanol saturated with ammonia gas. The resulting mixture was heated for 1 h at 100 °C under microwave radiation and then made basic with concentrated ammonia. After dilution with H2O, the organic material was extracted into CH2Cl2 and the organic layer was washed with brine, dried over Na2SO4 and concentrated to give mixture that was purified by silica gel chromatography (Combiflash - CH2Cl2:CH3OH:NH4OH) and crystallization. The desired product 12 (0.074 gm) was obtained in 56% yield (2 steps) as an off-white solid: mp 190 °C: NMR (500 MHz, CDCl3) δ 8.56 (s, 1H), 7.49 (d, 1H, J = 8.3 Hz), 7.20 (d, 1H, J = 8.3 Hz), 5.54 (s, 2H), 3.20 (m, 1H), 2.92 (m, 2H), 2.76 (m, 1H), 2.48 (m, 1H), 2.29 (m, 1H), 2.19 (m, 1H), 1.94 (m, 4H), 1.89 (s, 3H), 0.91 (d, 3H, J = 7.1 Hz), 0.89 (m, 1H), 0.51 (m, 2H), 0.10 (m, 2H); MS (ESI) m/z 323 (M+H)+; Anal. Calcd. for C20H26N4·0.25 H2O: C 73.47, H 8.17, N 17.14. Found: C 73.59, H 8.04, N 16.92.
8,9-Fused aminopyrimidine derivative 13
Using a procedure similar to that used to prepare 12, compound 30 was converted to imidate interemdiate 31 which was then converted to 13 (86%) as an off-white foam: 1H NMR (500 MHz, CDCl3) δ 8.56 (s, 1H), 7.62 (s, 1H), 7.58 (s, 1H), 6.00 (s, 2H), 3.23 (m, 1H), 3.18 (d, 1H, J = 19.0 Hz), 2.89 (m, 1H), 2.73 (m, 1H), 2.51 (m, 1H), 2.35 (m, 1H), 2.01 (m, 3H), 1.48 (s, 3H), 1.35 (m, 1H), 0.89 (m, 1H), 0.87 (d, 3H, J = 7.3 Hz), 0.53 (m, 2H), 0.13 (m, 2H); MS (ESI) m/z 323 (M+H)+; C20H26N4·0.25 H2O: C 73.47, H 8.17, N 17.14. Found: C 73.33, H 8.03, N 16.85.
7,8-Fused biphenylethylaminopyrimidine derivative 14
Using a procedure similar to that used to prepare 12 (except acetic acid was added), compound 29 was treated with 4-biphenylethylamine to provide to 14 (71%) as an off-white foam: NMR (500 MHz, CDCl3) δ 8.64 (s, 1H), 7.58 (m, 4H), 7.45 (m, 2H), 7.34 (m, 3H), 7.29 (d, 1H, J = 8.5 Hz), 7.13 (d, 1H, J = 8.5 Hz), 5.56 (m, 1H), 3.93 (m, 2H), 3.19 (m, 1H), 3.06 (t, 2H, J = 6.6 Hz), 2.89 (m, 2H), 2.77 (m, 1H), 2.47 (m, 1H), 2.28 (m, 1H), 2.21 (m, 1H), 1.90 (s, 3H), 1.87 (m, 1H); 1.63 (m, 2H), 0.90 (d, 3H, J = 7.1 Hz), 0.88 (m, 1H), 0.51 (m, 2H), 0.10 (m, 2H); MS (ESI) m/z 503 (M+H)+; Anal. Calcd. for C34H38N4·0.5 H2O: C 79.81, H 7.68, N 10.95. Found: C 79.88, H 7.66, N 10.83.
7,8-Fused benzylaminopyrimidine derivative 15
Using a procedure similar to that used to prepare 12, compound 29 was treated with benzylamine to provide 15 (69%) as an off-white foam: NMR (500 MHz, CDCl3) δ 8.64 (s, 1H), 7.44 (d, 1H, J = 8.5 Hz), 7.40-7.30 (m, 5H); 7.15 (d, 1H, J = 8.3 Hz), 5.81 (m, 1H), 4.83 (d, 2H, J = 5.4 Hz), 3.20 (m, 1H), 2.91 (m, 2H), 2.77 (m, 1H), 2.48 (m, 1H), 2.29 (m, 1H), 2.22 (m, 1H), 1.93 (m, 2H), 1.90 (s, 3H), 1.88 (m, 1H); 0.90 (d, 3H, J = 7.1 Hz), 0.88 (m, 1H), 0.51 (m, 2H), 0.10 (m, 2H); MS (ESI) m/z 413 (M+H)+; Anal. Calcd. for C27H32N4·0.5 H2O: C 76.92, H 7.89, N 13.29. Found: C 76.77, H 7.99, N 12.90.
8,9-Fused biphenylethylaminopyrimidine derivative 16
A mixture of 27 (0.084 g, 0.27 mmol), POCl3 (0.41 g, 2.7 mmol), and DMF (3.0 mL) was heated at 100 °C under microwave radiation for 10 min and concentrated. The resulting dark oil was dissolved in H2O, made basic with Na2CO3 and extracted (X3) with CH2Cl2. The combined organic extracts were dried over Na2SO4 and concentrated to give mixture that was purified by silica gel chromatography (CH2Cl2:CH3OH:NH4OH) giving the desired amidine intermediate 32 in 89% yield. Compound 32 (0.15 g, 0.43 mmol) was treated with 4-biphenylethylamine (0.10 g, 0.51 mmol) and 30% HOAc in CH3CN (3.0 mL) at 160 °C under microwave radiation for 5 min. The reaction mixture was cooled down and partitioned between saturated Na2CO3 and CH2Cl2. The organic phase was washed with brine, dried over Na2SO4 and concentrated to give a crude product that was purified by silica gel chromatography (Combiflash – Hexane:EtOAc:Et3N 80:20:0.5 to 50:50:0.5) to provide to 16 (0.18 g, 86%) as an off-white foam: NMR (500 MHz, CDCl3) δ 8.63 (s, 1H), 7.58 (m, 4H), 7.54 (s, 1H), 7.45 (m, 2H), 7.36 (m, 4H), 5.72 (m, 1H), 3.96 (m, 2H), 3.20 (m, 1H), 3.16 (d, 1H, J = 19.1 Hz), 3.09 (t, 2H, J = 7.1 Hz), 2.80 (m, 1H), 2.71 (m, 1H), 2.50 (m, 1H), 2.34 (m, 1H), 2.04-1.94 (m, 3H), 1.43 (s, 3H), 1.30 (m, 1H), 0.88 (m, 1H), 0.85 (d, 3H, J = 7.1 Hz), 0.52 (m, 2H), 0.12 (m, 2H); MS (ESI) m/z 503 (M+H)+; Anal. Calcd. for C34H38N4·0.5 H2O: C 79.81, H 7.68, N 10.95. Found: C 79.52, H 7.64, N 10.83.
8,9-Fused benzylaminopyrimidine derivative 17
Using a procedure similar to that used to prepare 16, compound 32 was treated with benzylamine to provide to 17 (92 %) as an off-white solid: NMR (500 MHz, CDCl3) δ 8.64 (s, 1H), 7.56 (m, 1H), 7.50 (s, 1H), 7.44 (d, 2H, J = 7.3 Hz), 7.39 (t, 2H, J = 7.3 Hz), 7.34 (d, 1H, J = 7.3 Hz), 5.94 (br s, 1H), 4.90 (m, 2H), 3.22 (m, 1H), 3.17 (d, 1H, J = 19.0 Hz), 2.88 (m, 1H), 2.72 (m, 1H), 2.51 (m, 1H), 2.34 (m, 1H), 1.99 (m, 3H), 1.47 (s, 3H), 1.33 (m, 1H), 0.88 (m, 1H), 0.86 (d, 3H, J = 7.1 Hz), 0.52 (m, 2H), 0.12 (m, 2H); MS (ESI) m/z 413 (M+H)+; C27H32N4·H2O: C 75.31, H 7.96, N 13.01. Found: C 75.64, H 7.73, N 13.02.
Cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-N-methoxy-6,11-dimethyl-2,6-methano-3-benzazocine-8-carboxamide (34)
A solution of 338 (240 mg, 0.606 mmol) and methoxylamine hydrochloride (61 mg, 0.727 mmol) in 3 mL of dry pyridine was stirred at room temperature for 12 h. The solvent was removed in vacuo and the residue was taken up in methylene chloride (40 mL), and washed with saturated sodium bicarbonate solution, water, and brine. The organic phase was dried over sodium sulfate, filtered, and concentrated to give a brown residue, which was purified by flash chromatography (CH2Cl2:CH3OH:NH4OH 20:1:0.1) to give 34 as an off-white foam (159 mg, 0.485 mmol, 80%): 1H NMR (500 MHz, CDCl3) δ 8.64 (bs, 1H), 7.64 (d, 1H, J = 1.5 Hz), 7.42 (dd, 1H, 7.8, 2.5 Hz), 7.12 (d, 1H, J = 7.8 Hz), 3.89 (s, 3H), 3.15 (m, 1H), 2.96 (d, 1H, J = 18.6 Hz), 2.70 (m, 2H), 2.46 (m, 1H), 2.32 (m, 1H), 1.91 (m, 3H), 1.42 (s, 3H), 1.33 (m, 1H), 0.86 (m, 1H), 0.82 (d, 3H, J = 7.2 Hz), 0.51 (m, 2H), 0.11 (m, 2H); MS (ESI) m/z 329 (M+H)+; IR (CH2Cl2) νmax 3464, 3196, 1643 cm-1; Anal. Calcd. for C20H28N2O2·0.25H2O: C 72.15, H 8.63, N 8.41. Found: C 72.15, H 8.67, N 8.13.
Cis-(±)-3-(cyclopropylmethyl)-1,2,3,4,5,6-hexahydro-N-methoxy-6,9,11-trimethyl-2,6-methano-3-benzazocine-8-carboxamide (35)
Conditions of Fisher and coworkers were used.9 sec-Butyllithium (1.4 M in cyclohexane, 4.6 mL, 4.57 mmol) was added to a mixture of 34 (100 mg, 0.305 mmol) and TMEDA (530 mg, 4.57 mmol) in dry THF at −78 °C under nitrogen atmosphere. The resulting mixture was warmed to −20 °C, stirred for 10 min and cooled to −78 °C again. Iodomethane (649 mg, 4.57 mmol) was added dropwise and the resulting mixture was stirred for 5 min. The reaction was quenched with 20 mL of saturated ammonia chloride and extracted with EtOAc (3×20 mL). The combined organic phases were washed twice with water and once with brine, dried over sodium sulfate, filtered, and concentrated to give 35 as a brown oil (146 mg, estimated yield 74%). There was small amount of starting material in this oil and it is difficult to separate starting material from product using flash chromatography because these two compounds have the same Rf value on silica gel TLC plate. Therefore, 35 was used in next step without further purification. 1H NMR (500 MHz, CDCl3) δ 7.19 (s, 1H), 6.90 (s, 1H), 3.86 (s, 3H), 3.12 (m, 1H), 2.87 (d, 1H, J = 19 Hz), 2.64 (m, 2H), 2.42 (m, 1H), 2.36 (s, 3H), 2.28 (m, 1H), 1.86 (m, 3H), 1.34 (s, 3H), 1.26 (m, 1H), 0.85 (m, 1H), 0.85 (d, 3H, J = 6.8 Hz), 0.50 (m, 2H), 0.10 (m, 2H); MS (ESI) m/z 343 (M+H)+.
8,9-Fused N-methoxypyridinone derivative 36
Conditions of Fisher and coworkers were used.9 sec-Butyllithium (1.4 M in cyclohexane, 2.3 mL, 2.99 mmol) was added dropwise to a solution of 35 (146 mg, 0.427 mmol) in 2 mL of dry THF under argon at −78 °C. The resulting mixture was stirred for 5 min. DMF (0.23 mL) was added and the reaction mixture was stirred for additional 5 min. Then the reaction was quenched with 20 mL of saturated ammonia chloride and extracted with EtOAc (3×20 mL). The combined organic phases were washed twice with water and once with brine, dried over sodium sulfate, filtered, and concentrated to give a brown oil, which was mixed with 1 mL of concentrated HCl and stirred for 1 h. The reaction mixture was then made basic using 5 N NaOH solution and extracted with EtOAc (3×20 mL). The combined organic phases were washed twice with water and once with brine, dried over sodium sulfate, filtered, and concentrated to give a brown oil, which was purified by flash chromatography (CH2Cl2:CH3OH:NH4OH 25:1:0.1) to give 36 as a white foam (0.054 g, 0.153 mmol) in 50% overall from 34: 1H NMR (500 MHz, CDCl3) δ 8.34 (s, 1H), 7.25 (d, 1H, J = 8.0 Hz), 7.24 (s, 1H), 6.37 (d, 1H, J = 8.0 Hz), 4.08 (s, 3H), 3.19 (m, 1H), 3.08 (d, 1H, J = 19 Hz), 2.81 (m, 1H), 2.71 (m, 1H), 2.49 (m, 1H), 2.32 (m, 1H), 1.97 (m, 3H), 1.52 (s, 3H), 1.39 (m, 1H), 0.88 (m, 1H), 0.84 (d, 3H, J = 7.0 Hz), 0.52 (m, 2H), 0.11 (m, 2H); MS (ESI) m/z 353 (M+H)+.
8,9-Fused pyridinone derivative 37
Conditions of Fisher and coworkers were used.9 A solution of titanium(III) chloride (0.197 g, 1.28 mmol) in 2.5 mL of 6 N HCl was added to a solution of 36 (45 mg, 0.128 mmol) in 1 mL of EtOH. The resulting mixture was irradiated with microwaves at 100 °C for 30 min. The cooled reaction mixture was poured onto a mixture of ice and water and basified with 5 N NaOH to approximately pH 13. Air was bubbled through the solution until the blue color disappeared. White precipitants were observed in solution. The mixture was extracted with EtOAc (3×20 mL). The combined organic phases were washed twice with water and once with brine, dried over sodium sulfate, filtered, and concentrated to give a brown residue, which was purified by flash chromatography (CH2Cl2:CH3OH:NH4OH 25:1:0.1) to give 37 as a white foam (0.026 g, 0.0819 mmol, 64%): 1H NMR (500 MHz, CDCl3) δ 11.89 (bs, 1H), 8.33 (s, 1H), 7.29 (s, 1H), 7.15 (d, 1H, J = 7.0 Hz), 6.49 (d, 1H, J = 7.0 Hz), 3.22 (m, 1H), 3.11 (d, 1H, J = 18.5 Hz), 2.84 (m, 1H), 2.74 (m, 1H), 2.51 (m, 1H), 2.35 (m, 1H), 2.00 (m, 3H), 1.54 (s, 3H), 1.41 (m, 1H), 0.89 (m, 1H), 0.87 (d, 3H, J = 7.0 Hz), 0.52 (m, 2H), 0.11 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 164.81, 143.04, 142.86, 135.93, 127.41, 124.74, 124.48, 124.22, 106.29, 60.11, 57.05, 45.91, 42.72, 41.86, 37.12, 26.01, 24.43, 14.44, 9.58, 4.22, 3.82; MS (ESI) m/z 323 (M+H)+; Anal. Calcd. for C21H26N2O·0.5H2O: C 76.10, H 8.21, N 8.45. Found: C 76.20, H 8.36, N 8.01.
5.2. Opioid Receptor Binding Assays
Binding assays used to screen compounds are similar to those previously reported.15 Membrane protein from CHO cells that stably expressed one type of the human opioid receptor were incubated with 12 different concentrations of the compound in the presence of either 1 nM [3H]U69,593 (μ), 0.25 nM [3H]DAMGO (δ) or 0.2 nM [3H]naltrindole (κ) in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25°C. Incubation times of 60 min were used for [3H]U69,593 and [3H]DAMGO. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 μM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. For [3H]naltrindole and [3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for at least 60 min before use. IC50 values will be calculated by least squares fit to a logarithm-probit analysis. Ki values of unlabeled compounds were calculated from the equation Ki = (IC50)/1+S where S = (concentration of radioligand)/(Kd of radioligand)16 The Kd values for [3H]DAMGO, [3H]U69,593, and [3H]naltrindole were 0.56 nM, 0.34 nM, and 0.10 nM, respectively. Data are the mean ± SEM from at least three experiments performed in triplicate.
5.3. [35S]GTPγS Binding Assays
Procedure similar to those previously reported was used.12 In a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 15 μg (κ) or 7.5 μg (μ) of CHO cell membranes that stably expressed either the human κ, δ or μ opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 μM GDP, and 100 mM NaCl. The final concentration of [35S]GTPγS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 μM GTPγS. Binding was initiated by the addition of the membranes. After an incubation of 60 min at 30°C, the samples were filtered through Schleicher & Schuell No. 32 glass fiber filters. The filters were washed three times with cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL of Ecoscint scintillation fluid. Data are the mean Emax and EC50 values ± S.E.M. from at least three separate experiments, performed in triplicate. For calculation of the Emax values, the basal [35S]GTPγS binding was set at 0%. To determine antagonist activity of a compound at the μ opioid receptors, CHO membranes expressing the μ opioid receptor, were incubated with 12 different concentrations of the compound in the presence of 200 nM of the μ agonist DAMGO. To determine antagonist activity of a compound at the κ opioid receptors, CHO membranes expressing the κ opioid receptor, were incubated with the compound in the presence of 100 nM of the κ agonist U50,488.
Acknowledgments
We gratefully acknowledge the contributions of Rensselaer’s mass spectroscopist Dr. Dmitri Zagorevski and the technical assistance provided by Brian I. Knapp of the University of Rochester. Funding of this research was from NIDA (DA12180 and KO5-DA00360) and the NSF (Agilent 1100 series LC/MSD system).
Footnotes
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1. Wentland MP, Lou R, Ye Y, Cohen DJ, Richardson GP, Bidlack JM. Bioorg Med Chem Lett. 2001;11:623. [PubMed]
2. Archer S, Glick SD, Bidlack JM. Neurochem Res. 1996;21:1369–1373. [PubMed]
3. Zhang A, Xiong W, Bidlack JM, Hilbert JE, Knapp BI, Wentland MP, Neumeyer JL. J Med Chem. 2004;47:165. [PubMed]
4. Wentland MP, Lou R, Dehnhardt CM, Duan W, Cohen DJ, Bidlack JM. Bioorg Med Chem Lett. 2001;11:1717. [PubMed]
5. Wentland MP, Lu Q, Lou R, Knapp BI, Bidlack JM. Bioorg Med Chem Lett. 2005;15:2107. [PubMed]
6. Wentland MP, Sun X, Bu Y, Lou R, Cohen DJ, Bidlack JM. Bioorg Med Chem Lett. 2005;15:2547. [PubMed]
7. (a) Bachmann WE, Brockway CE. J Org Chem. 1948;13:384. (b) Zhang Q, Chen Y, Zheng Y, Xia P, Xia Y, Yang Z, Bastow KF, Morris-Natschke SL, Lee K-H. Bioorg Med Chem. 2003;11:1031. [PubMed]
8. Lou R, VanAlstine M, Sun X, Wentland MP. Tet Lett. 2003;44:2477.
9. Fisher LE, Caroon JM, Jahangir S, Stabler R, Lundberg S, Muchowski JM. J Org Chem. 1993;58:3643.
10. Peng X, Knapp BI, Bidlack JM, Neumeyer JL. Bioorg Med Chem. 2007;15:4106. [PMC free article] [PubMed]
11. Ganorkar RR, Lu Q, Wentland MP, Bidlack JM. Abstracts of Papers, 234th ACS National Meeting; Boston, MA, United States. August 19-23, 2007; MEDI-140.
12. Wentland MP, VanAlstine M, Kucejko R, Lou R, Cohen DJ, Parkhill AL, Bidlack JM. J Med Chem. 2006;49:5635. [PubMed]
13. VanAlstine MA, Wentland MP, Cohen DJ, Bidlack JM. Bioorg Med Chem Lett. 2007;17:6516. [PMC free article] [PubMed]
14. Abraham MH, Platts JA. J Org Chem. 2001;66:3484. [PubMed]
15. Neumeyer JL, Zhang A, Xiong W, Gu X, Hilbert JE, Knapp BI, Negus SS, Mello NK, Bidlack JM. J Med Chem. 2003;46:5162. [PubMed]
16. Cheng YC, Prusoff WH. Biochem Pharmacol. 1973;22:3099. [PubMed]