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J. Med. Chem. 2005, 48, 3852-3857
Synthesis and Monoamine Transporter Binding Properties of 3r-(Substituted phenyl)nortropane-2β-carboxylic Acid Methyl Esters. Norepinephrine Transporter Selective Compounds F. Ivy Carroll,*,† Sameer Tyagi,† Bruce E. Blough,† Michael J. Kuhar,‡ and Hernn A. Navarro† Chemistry and Life Sciences, Research Triangle Institute, Research Triangle Park, North Carolina 27709, and Yerkes National Primate Center of Emory University, 954 Gatewood Road NE, Atlanta, Georgia 30329 Received January 10, 2005
3R-(Substituted phenyl)nortropane-2β-carboxylic acid methyl esters (8a-h) showed high affinity for the norepinephrine transporter (NET). The most potent and selective compound was 3R-(3-fluoro-4-methylphenyl)nortropane-2β-carboxylic acid methyl ester (8d), with a Ki of 0.43 nM at the NET and 21- and 55-fold selectivity relative to binding at the dopamine and serotonin transporters. The development of 8d makes available compounds selective for all three transporters from the same structural class. Monoamine neurotransmitter transporters are key targets for a variety of drugs. Compounds selective for the dopamine, serotonin, and norepinephrine transporters (DAT, 5-HTT, and NET, respectively) are all of interest. One of the earlier drugs developed was the NET selective antidepressant desipramine (1), a drug that has been used to treat attention deficit hyperactivity disorder (ADHD).1 However, side effect issues, including anticholinergic and antihistaminergic, impair the utility of this drug.1 Interest in NET selective drugs continues as evidenced by the development of atomoxetine (2), manifoxine (3), and reboxetine (4) as new NET selective compounds for treating ADHD and other CNS disorders such as depression.1,2 5 was also reported as an interesting new NET selective compound.3 The 3β-phenyltropane-2β-carboxylic acid methyl esters (6a-c) are interesting monoamine uptake inhibitors.4-7 We have reported that the 3R-(substitutedphenyl)tropane-2β-carboxylic acid methyl esters (7ac) possessed monoamine transporter binding and pharmacological properties similar to those of their corresponding 2β,3β-isomers.8,9 We previously reported that the 2β,3R-nortropane analogues 8a-c showed improved binding affinity at the NET relative to their corresponding 2β,3β-isomers 7a-c and that 8c showed higher affinity at the NET than at the DAT or 5-HTT.10 Here, we describe the synthesis and monoamine transporter binding properties of several 3R-(substituted phenyl)nortropane-2β-carboxylic acids (8a-h) and report that 3R-(3-fluoro-4-methylphenyl)nortropane-2β-carboxylic acid methyl ester (8d) possesses high potency and good selectivity for the NET relative to the 5-HTT and DAT. Chemistry Compounds 8a-h were synthesized by refluxing the appropriate 3R-(substituted phenyl)tropane-2β-carboxylic acid methyl ester (7a-h) with R-chloroethyl chloroformate (ACE chloride) in dichloroethane under * To whom correspondence should be addressed. Telephone: 919-541-6679. Fax: 919-541-8868. E-mail:
[email protected]. † Research Triangle Institute. ‡ Yerkes National Primate Center of Emory University.
a nitrogen atmosphere to give the intermediate (R-chloroethyl)urethane, which was not isolated but was converted directly to the nortropane analogues 8a-h by solvolysis with methanol (Scheme 1). The yield obtained and the analytical data are in Table 1. Compounds 7a-c were synthesized as previously reported.8 The synthesis of the 3R,2β-tropanes 7d-e is outlined in Scheme 2. Addition of a solution of (1R,5S)2-(3′-methyl-1′,2′,4′-oxadiazol-5′-yl)-8-methyl-8-azabicyclo[3.2.1]oct-2-ene (9)11 in anhydrous THF at -78 °C to a solution of the appropriate aryllithium (prepared from the appropriate aryl bromide and butyllithium) followed by quenching with 1 N hydrochloric acid at -78 °C formed the 3R-(substituted phenyl)tropane-2R-(3′-methyl-1′,2′,4′-oxadiazol-5′-yl)tropanes (10). In addition to the desired isomer, the 2R,3β-isomer was also formed, which was removed by flash chromatography or carried through
10.1021/jm058164j CCC: $30.25 © 2005 American Chemical Society Published on Web 04/30/2005
Synthesis and Monoamine Transporter Binding Properties
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3853
Scheme 1a
Scheme 3a
a Reagents: (a) ClCO CH(Cl)CH , (CH Cl) , heat; (b) CH OH, 2 3 2 2 3 heat.
Table 1. 3R-(Substituted phenyl)nortropane 2β-Carboxylic Acid Methyl Esters Yields and Analytical Dataa compd 8a 8b 8c 8d 8e 8f 8gd 8h
yield,b
[R]20D (concn in CH3OH, % g/100 mL)
76 65 31 66 25 74 33 22
-49.4 (0.41) -46.8 (0.47) -62.5 (0.49) -62.5 (12.5) -65.4 (2.2)a -63.3 (0.3) -49.4 (1.5) -45.1 (4.0)
saltc
molecular formula
analyses
HCl HCl HCl HCl HCl C7H7SO3H HCl free base
C16H22ClNO2 C15H19ClFNO2 C15H19Cl2NO2 C16H21ClFNO2 C16H21ClFNO2‚0.5H2O C22H26FNO5S C15H18ClF2NO2 C15H17F2NO2
C, H, N C, H, N C, H, N C, H, N C, H, N C, H, N C, H, N C, H, N
a A typical experimental is given in the Experimental Section. This is the overall yield of product. c Salts used to characterize the compound. d This compound was purified by conversion to the Boc-protected analogue, which was then purified and the Boc group removed by treatment with HCl/dioxane.
b
Scheme 2a
a Reagents: (a) C H N(Tf) , NaN[Si(CH ) ] , THF, -78 °C, 6 5 2 3 3 2 12 h; (b) ArB(OH)2, Pd[P(C6H5)3]4, K2CO3, toluene 90 °C, 5 h; (c) Sml2, CH3OH, 40 °C, 6 h; (d) 10% HCl.
butyloxycarboxy (boc) protected amine, the desired 2β,3R-stereoisomer was separable from the 2β,3β-isomer by chromatography. Removal of the boc group was accomplished by treatment with hydrochloric acid in dioxane to yield the desired 2β,3R-7g. The structural and stereochemical assignments for 7a-h were made using the same methods as previously reported for other 2β,3R-tropanes.8,14 Biology
a Reagents: (a) C H Li, (C H ) O, -78 °C, 4 h; (b) 1 N HCl, 4 9 2 5 2 -78 °C; (c) Ni2B, CH3OH, HCl.
and removed at the next reaction. Transformation of the oxadiazoles 10 to the desired methyl esters 7d-e was accomplished by reduction with nickel boride (generated in situ by reaction of sodium borohydride and nickel tetraacetate) and hydrochloric acid in refluxing methanol to form 7d-e. Under such conditions, a complete epimerization of C-2 to form the 3R,2β-stereoisomer was observed. Attempts to prepare 2β,3R-tropanes 7f-h by the procedures used to synthesize 7d-e were unsuccessful. In these cases, product formation occurred in only ∼3% yield. The desired compounds 7f-h were prepared as shown in Scheme 3. Addition of N-phenyltrifluoromethanesulfonimide to a THF solution of (1R)-2-carbomethoxy-3-tropinone (11)12 containing sodium bis(trimethylsilyl)amide yielded triflate 12. Reaction of 12 with the appropriate phenylboronic acid using tetrakis(triphenylphosphine)palladium(0) and sodium or potassium carbonate followed by purification gave the corresponding coupled products 13a-c.13 Reduction of 13a-c using samarium(II) iodide at 40 °C and methanol as a proton source followed by quenching with hydrochloric acid gave a mixture of 3R-(substituted phenyl)2β-carboxylic acid methyl esters as the major products and the 2β,3β-isomer, which were separated by chromatography. In the case of 7g, the mixture was inseparable by chromatography. We found that when 8g was reacted with di-tert-butyl dicarbonate to form the tert-
The binding affinities of 7a-e and 8a-h along with cocaine at the DAT, 5-HTT, and NET were determined via competition binding assays using previously reported procedures.14 The results are listed in Table 2. Some of the data are from previous reports. The final concentration of radioligands in the assays was 0.5 nM [3H]WIN 35,428 for the DAT, 0.2 nM [3H]paroxetine for the 5-HTT, and 1.0 nM [3H]nisoxetine for the NET. For these assays, rat brain homogenate (added last) was incubated with 1 of 10 different concentrations of test compound. Prism (version 3.0, GraphPad Software, San Diego, CA) was used for nonlinear fitting of the binding data. The IC50 values from at least three independent experiments were used to calculate the Ki using the formula Ki ) IC50/(1 + [L]Kd),15 where [L] is the assay concentration of radioligand and Kd is the apparent dissociation constant for the radioligand determined under identical conditions as the competition binding assays. Results and Discussion In our first paper on 3β-(substituted phenyl)tropane2β-carboxylic acid methyl esters, we reported that the 4-methyl and 4-chloro analogues 6a and 6c, respectively, were 52 and 80 times more potent at the DAT than cocaine.16 Moreover, they were 9 and 14 times more potent at the DAT than the 4′-fluoro analogue 6b (WIN 35,428) originally reported by Clarke et al.6 Later studies showed that 6a-c were also considerably more potent at the 5-HTT and NET than cocaine.17 In another study, we reported that 7a-c showed decreased binding at all three transporters relative to the corresponding 2β,3β-isomer 6a-c; however, binding at the DAT and NET was affected less than the 5-HTT transporter.8 A comparison of the data for 6a-c to that of 7a-c showed that binding at the DAT and NET decreased by 0.13 to 6-fold and by 1.5 to 4.4-fold, whereas binding at the 5-HTT decreased by 7 to 22-fold. A study to determine the effect of replacing the N-methyl group of 6a-c with
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Table 2. Comparison of Transporter Binding Properties of 3-Phenyltropane and 3-Phenylnortropane 2β-Carboxylic Acid Methyl Esters Analogues
IC50, nM (Ki, nM)a
a
compd
isomer 3
R
cocaineb 6ab 6bb 6cb 7ac 7bc 7cc 7d 7e 8ad 8bd 8cd 8d 8e 8f 8g 8h 14ad 14bd 14cd
β β β R R R R R R R R R R R R R β β β
CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 H H H H H H H H H H H
X, Y, Z
NE [3H]nisoxetine
DA [3H]WIN 35,428
5-HT [3H]paroxetine
CH3, H, H F, H, H Cl, H, H CH3, H, H F, H, H Cl, H, H CH3, F, H F, CH3, H CH3, H, H F, H, H Cl, H, H CH3, F, H F, CH3, H H, H, F F, F, H H, F, F CH3, H, H F, H, H Cl, H, H
3300 ( 290 (1900 ( 170) 60.0 ( 0.50 (36 ( 0.30) 835 ( 45 (503 ( 27) 37 ( 2.1 (22 ( 1.3) 270 ( 24 (160 ( 14) 1200 ( 91 (741 ( 54.8) 60 ( 2.4 (36 ( 1.5) 44.7 ( 9.0 (22.4 ( 1.1) 148 ( 26 (74 ( 13) 9.0 ( 0.30 (5.20 ( 0.18) 9.8 ( 0.7 (5.9 ( 0.40) 5.41 ( 1.08 (3.1 ( 0.60) 0.86 ( 0.03 (0.43 ( 0.02) 4.23 ( 0.48 (2.12 ( 0.24) 9.7 ( 1.6 (4.0 ( 0.70) 6.3 ( 1.1 (2.6 ( 0.5) 4.10 ( 0.68 (2.05 ( 0.34) 7.20 ( 0.45 (4.40 ( 0.27) 18.8 ( 0.68 (11.3 ( 0.41) 5.45 ( 0.21 (3.28 ( 0.13)
89.1 ( 4.8 1.70 ( 0.30 15.7 ( 1.4 1.12 ( 0.10 10.2 ( 0.80 21 ( 0.50 2.4 ( 0.2 7.38 ( 1.7 13.7 ( 2.4 33.6 ( 4.1 32.6 ( 26 3.1 ( 1.0 9.0 ( 2.5 9.38 ( 2.0 2.36 ( 0.24 5.61 ( 0.41 2.35 ( 0.27 0.84 ( 0.09 4.4 ( 0.2 0.62 ( 0.09
1050 ( 89 (95 ( 8) 240 ( 27 22 ( 2.5 760 ( 47 (69 ( 4) 45 ( 1.3 (4.0 ( 0.12) 4250 ( 422 (390 ( 38) 5060 ( 485 (460 ( 44) 998 ( 120 (91 ( 11) 1150 ( 96 (282 ( 24) 1161 ( 76 (258 ( 11) 500 ( 30 (46 ( 3) 92.4 ( 7.7 (8.40 ( 0.70) 53.3 ( 3 (4.8 ( 0.26) 97.4 ( 18.1 (23.8 ( 4.4) 69.8 ( 28.5 (17.1 ( 7.0) 110 ( 12 (26.8 ( 2.9) 226 ( 18 (55.3 ( 4.5) 55.8 ( 5.5 (13.7 ( 1.3) 135 ( 28 (12 ( 3) 68.6 ( 2.0 (6.24 ( 0.18) 4.13 ( 0.62 (0.38 ( 0.06)
Numbers in parentheses are the Ki values. b IC50 values are from ref 7. c IC50 values are from ref 10.
d
IC50 and Ki values are from ref
18.
a hydrogen to give the corresponding 3β-(4-substituted phenyl)nortropane-2β-carboxylic acid methyl esters (14a-c) revealed that binding affinity at the NET and 5-HTT increased with little change in binding affinity at the DAT.18 This can be seen by
comparing the affinity of the 3-phenyltropane analogues 6a-c to the 3-phenylnortropane analogues 14a-c (Table 2). In all three cases, binding at the DAT remained relatively constant while binding affinity to the NET increased 8.3-, 44.4-, and 5.8-fold and binding affinity for the 5-HTT increased 2-, 11-, and 11-fold for the 4-methyl, 4-fluoro, and 4-chloro analogues, respectively. Taken together, the monoamine binding results from 7a-c and 14a-c suggested that 8a-h might have their greatest potency at the NET with selectivity relative to the DAT and 5-HTT. Indeed, examination of the binding data in Table 2 shows that 8a-h possessed Ki values at the NET of 0.43-5.2 nM with lower potency at the DAT and 5-HTT. The most potent and selective compound was the 3-fluoro-4-methyl analogue, 8d, which had a Ki of 0.43 nM at the NET and was 21- and 55-fold selective for the NET relative to the DAT and 5-HTT, respectively. The 4-methyl analogue 8a has a Ki of 5.2 nM for the NET and is 7- and 9-fold selective for the NET relative to the DAT and 5-HTT. Thus, the addition of the 3-fluoro to 8a to give the 3-fluoro-4methyl analogue 8d resulted in a 12-fold increase of affinity at the NET (0.43 compared to 5.2 nM) and increased selectivity relative to DAT and 5-HTT. Similar to the 2β,3β-analogues, removal of the N-methyl group
from 7a-e to give the nortropane analogues 8a-e resulted in increased affinity at NET and 5-HTT with a modest effect at the DAT. The NET (5-HTT) increases were 31(9)-, 126(54)-, 12(19)-, 52(12)-, and 35(15)-fold in going from 7a-e to 8a-e, respectively. In summary, we have compared the monoamine transporter binding properties of the 2β,3β- and 2β,3Risomers of 3-(4-substituted phenyl)tropane-2-carboxylic acid methyl esters to the corresponding 3-(4-substituted phenyl)nortropane-2-carboxylic acid methyl esters and have shown that the 2β,3R-nortropane analogues 8a-h possess high affinity for the NET. Compound 8d is a highly potent and selective compound at the NET relative to the DAT and 5-HTT. We have previously reported the development of 3β-(substituted phenyl)tropane and nortropane-2β-carboxylic acid methyl esters selective for the DAT relative to the NET and 5-HTT and analogues selective for the 5-HTT relative to the DAT and NET.4 The development of the NET selective 8d now makes available compounds selective for the NET, DAT, and 5-HTT from the same structural class. These compounds will be highly useful for characterizing the animal behavioral profile of monoamine transporter inhibitors. The high NET potency and selectivity of 8d suggests that this compound should be considered as a lead for the development of a new structural type of drug for treating ADHD and depression. In addition, since 8d possess a fluorine, the fluorine-18 analogue may prove useful as a positron emission tomography imaging agent. Experimental Section 1 H and 13C NMR spectra were recorded on a 300 MHz (Bruker AVANCE 300) spectrometer. Chemical shift data for the proton resonances were reported in parts per million (δ) relative to internal (CH3)4Si (δ 0.0). Melting points were determined on a Thomas-Hoover capillary tube apparatus. All
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optical rotations were determined at the sodium D line using a Rudolph Research Autopol III polarimeter (1 dm cell). Thinlayer chromatography was carried out on Whatman silica gel 60 TLC plates. Visualization was accomplished under UV or in an iodine chamber. Microanalyses were carried out by Atlantic Microlab Inc. The compounds were purified by flash chromatography on silica gel 60 (230-400 mesh) or preparative-layer chromatography (radial PLC) on a chromatotron (Harrison Association, Palo Alto, CA) using glass plates coated with 1-, 2-, or 4 mm thickness of Kissel gel 60 pF254 containing gypsum. Glassware was oven- or flame-dried and cooled under nitrogen. All reactions were performed under nitrogen or argon. Samarium iodide was purchased from Fluka Chemical Corp. All other chemicals were purchased from Aldrich Chemical Company, Inc. THF and ether were freshly distilled from sodium benzophenone. All other reagents were used without further purification. CMA is a mixture of 95% chloroform, 4% methanol, and 1% concentrated ammonium hydroxide. General Procedure for the Synthesis of 3r-(Substituted phenyl)nortropane-2β-carboxylic Acid Methyl Esters (8a-h). A representative experimental procedure is given below. The percent yields, salts prepared, and analytical data for each compound are in Table 1. 3r-(3-Fluoro-4-methylphenyl)nortropane-2β-carboxylic Acid Methyl Ester (8d) Hydrochloride. To a solution of tropane 7d (0.820 g, 2.817 mmol) in 1,2-dichloroethane (20 mL) was added 1-chloroethyl chloroformate (2.01 g, 14.1 mmol). The mixture was refluxed for 12 h under N2. Cooling to room temperature was followed by concentration of the solvent in vacuo to afford a light-yellow oil. The oil was dissolved in anhydrous CH3OH (15 mL), and the mixture was refluxed for 4 h. The mixture was concentrated in vacuo, and the residue was dissolved in CH2Cl2 (25 mL). The solution was washed with NH4OH (5%, 10 mL). The aqueous layer was separated and extracted with CH2Cl2 (2 × 15 mL). The combined organic layer was dried (Na2SO4). Concentration in vacuo gave a light-yellow oil. Purification of the oil via radial PLC (silica, 10:1 Et2O/Et3N) gave the product (0.515 g, 66%) as a light-yellow oil. The free base was converted to the hydrochloride salt: mp 204-205 °C; [R]20D -62.50° (c 12.5 CH3OH); 1H NMR (CDCl3) δ 7.02-7.16 [m, 2 H], 6.84-6.98 [m, 2 H], 3.59-3.65 [m, 4 H], 3.10 [q, 1 H], 2.45 [d, 1 H, J ) 9.0 Hz], 2.21-2.28 [m, 4 H], 1.91-2.00 [m, 1 H], 1.85-1.90 [m, 2 H], 1.57-1.68 [m, 3 H], 1.32 [t, 1 H, J ) 12.0 Hz]; 13C NMR ((CDCl3) δ 175.45, 162.82, 159.58, 143.47, 131.28, 123.05, 114.27, 114.20, 113.97, 113.91, 56.20, 55.57, 51.74, 51.55, 36.13, 36.11, 32.83, 32.38, 14.15. Anal. C16H21ClFNO2‚0.5H2O) C, H, N. 3r-(3-Fluoro-4-methylphenyl)-2β-(3′-methyl-1′,2′,4′-oxadiazol-5′-yl)tropane (10a). A flame-dried 100 mL roundbottomed flask equipped with a magnetic stirrer was charged with a solution of 4-bromo-2-fluorotoluene (0.92 g, 4.9 mmol) in 20 mL of anhydrous ether, and the solution was cooled to -78 °C via a dry ice/acetone bath. To this was added slowly a solution of n-BuLi (2.5 M in hexane, 1.95 mL, 4.9 mmol) over 15 min, maintaining the temperature below -70 °C. The solution was stirred at -78 °C for 30 min, and then a solution of anhydroecgonine oxadiazole (9, 0.500 g, 2.43 mmol) in anhydrous ether (10 mL) was added slowly over 10 min. The resulting solution was stirred at -78 °C for 4 h and quenched with 1 N HCl at the same temperature. The mixture was allowed to warm to room temperature, then washed with ether. The ether layer was discarded, and the aqueous layers were basified to pH 11 with concentrated NH4OH at 0 °C (via an ice bath) and extracted with chloroform (3 × 15 mL). The combined organic layer was dried (Na2SO4), filtered, and concentrated under reduced pressure to afford the product as a light-yellow oil. Purification by flash column chromatography [silica, 1:1 CMA/(1:1 hexane/ethyl acetate)] gave the pure product (0.278 g, 36%) as a light-yellow oil: 1H NMR (CDCl3) δ 7.02 [m, 1 H], 6.81 [m, 2 H], 3.43 [m, 1 H], 3.34 [m, 2 H], 3.05 [d, 1 H, J ) 9 Hz], 2.49 [m, 1 H], 2.72 [s, 3 H], 2.27 [s, 3 H], 2.18 [s, 3 H], 2.14 [m, 2 H], 1.68 [m, 1 H], 1.55 [m, 1 H], 1.41 [m, 1 H]; 13C NMR 167.34, 163.17, 159.93, 143.31, 143.22,
131.72, 131.65, 123.34, 123.30, 123.05, 114.61, 114.31, 64.66, 59.79, 49.91, 41.42, 39.49, 37.88, 37.87, 29.31, 29.24, 14.51, 14.4611.96; [R]20D -68.29° (c 22.7 CH3OH). This material was used without further purification to prepare 7d. 3r-(4-Fluoro-3-methylphenyl)-2β-(3′-methyl-1′,2′,4′-oxadiazol-5′-yl)tropane (10b). A flame-dried 100 mL roundbottomed flask equipped with a magnetic stirrer was charged with a solution of 4-bromo-2-fluorotoluene (0.922 g, 4.87 mmol) in 20 mL of anhydrous ether, and the solution was cooled to -78 °C via a dry ice/acetone bath. To this was added slowly a solution of n-BuLi (2.5 M in hexane, 1.95 mL, 4.87 mmol) over 15 min, maintaining the temperature below -70 °C. The solution was stirred at -78 °C for 30 min, and then a solution of anhydroecgonine oxadiazole (9, 0.500 g, 2.43 mmol) in anhydrous ether (10 mL) was added slowly over 10 min. The resulting solution was stirred at -78 °C for 4 h and quenched with 1 N HCl at the same temperature. The mixture was allowed to warm to room temperature, then washed with ether. The ether layer was discarded, and the aqueous layer was basified to pH 11 with concentrated NH4OH at 0 °C (via an ice bath) and extracted with chloroform (3 × 15 mL). The combined organic layer was dried (Na2SO4), filtered, and concentrated under reduced pressure to afford the crude product as a light-yellow oil. Purification by flash column chromatography [silica, 1:1 CMA/(1:1 hexane/ethyl acetate)] gave the pure product (0.120 g, 16%) as a light-yellow oil: [R]20D -58.4° (c 12, CHCl3); 1H NMR (CDCl3) δ 6.91 [m, 3 H], 3.34 [m, 3 H], 3.03 [d, 1 H, J ) 9.0 Hz], 2.39 [m, 1 H], 2.29 [s, 3 H], 2.20 [s, 3 H], 2.16 [m, 4 H], 1.70 [m, 1 H], 1.55 [m, 1 H], 1.33 [m, 1 H]. This material was used without further purification to prepare 7e. 3r-(3-Fluoro-4-methylphenyl)tropane-2β-carboxylic Acid Methyl Ester (7d) Hydrochloride. A 100 mL roundbottomed flask equipped with a magnetic stirrer was charged with a solution of nickel acetate tetrahydrate (0.853 g, 3.42 mmol) in 15 mL of methanol. To this was added solid NaBH4 (0.129 g, 3.42 mmol) cautiously because the reaction is exothermic. A black colloidal suspension formed immediately. A solution of 10a (0.22 g, 0.68 mmol) in 5 mL of methanol was added, and the reaction mixture was refluxed for 4 h. The mixture was allowed to cool to room temperature, filtered through Celite, and washed with methanol. The filtrate was concentrated under reduced pressure to give a green solid, which was dissolved in 15 mL of H2O. The aqueous layer was basified at 0-5 °C to pH 11 with concentrated NH4OH and extracted with ether (3 × 10 mL). The organic layer was dried (Na2SO4), filtered, and concentrated under reduced pressure to give the product as a colorless oil: 0.170 g (85%); 1H NMR (CDCl3) δ 7.13 [m, 1 H], 6.84 [m, 2 H], 3.60 [s, 3 H], 3.29 [m, 3 H], 2.46 [m, 2 H], 2.24 [s, 3 H], 2.21 [s, 3 H], 2.10 [m, 1 H], 1.53 [m, 4 H]; 13C NMR 175.34, 163.16, 159.92, 144.47, 144.37, 131.54, 131.46, 123.34, 123.30, 114.54, 114.25, 63.49, 59.86, 56.75, 56.53, 52.11, 41.36, 39.42, 39.19, 35.96, 35.94, 35.70, 29.12, 29.04, 14.49, 14.45. The hydrochloride salt had [R]20D -18.6° (c 2.5, CHCl3). Anal. (C17H23ClFNO2) C, H, N. 3r-(4-Fluoro-3-methylphenyl)tropane-2β-carboxylic Acid Methyl Ester (7e) Hydrochloride. A 100 mL roundbottomed flask equipped with a magnetic stirrer was charged with a solution of nickel acetate tetrahydrate (1.97 g, 7.93 mmol) in 20 mL of methanol. To this was added solid NaBH4 (0.300 g, 7.9 mmol) cautiously because the reaction is exothermic. A black colloidal suspension formed immediately. A solution of 10b (0.500 g, 1.6 mmol) in 5 mL of methanol was added, and the reaction mixture was refluxed for 4 h. The mixture was allowed to cool to room temperature, filtered through Celite, and washed with methanol. The filtrate was concentrated under reduced pressure to give a green solid, which was dissolved in 15 mL of H2O. The aqueous layer was basified at 0-5 °C to pH 11 with concentrated NH4OH and extracted with ether (3 × 10 mL). The organic layer was dried (Na2SO4), filtered, and concentrated under reduced pressure to give the crude product as a yellow oil: 0.370 g (80%); 1 H NMR (CDCl3) δ 7.04 [m, 2 H], 6.87 [m, 1 H], 3.58 [s, 3 H], 3.29 [m, 3 H], 2.42 [m, 2 H], 2.23 [m, 7 H], 1.52 [m, 3 H], 1.31
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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Carroll et al.
[t, 1 H, J ) 6.0 Hz]. The hydrochloride salt had [R]20D -18.5° (c 4.0, CHCl3). Anal. (C17H23ClFNO2‚1.5H2O) C, H, N. (1R)-2-(Carboxymethyl)-3-[[trifluoromethylsulfonyl]oxy]trop-2-ene (12). (1R)-2-Carbomethoxytropinone (11, 3.21 g, 16.29 mmol) was dissolved in anhydrous THF (65 mL) under N2. After the mixture was cooled to -78 °C, sodium bis(trimethyl)silylamide (1.0 M solution in THF, 20.4 mL, 20.4 mmol) was added dropwise via an addition funnel. The mixture was stirred at -78 °C for approximately 45 min. N-Phenyltrifluoromethane sulfonimide (6.98 g, 19.6 mmol) was dissolved in anhydrous THF (40 mL) and added dropwise over a period of 15 min. The mixture was stirred at -78 °C for 30 min, warmed to 0 °C, and stirred for 2 h. The mixture was further stirred at room temperature overnight and quenched with water (35 mL) and extracted with CH2Cl2 (3 × 50 mL). The organic layer was washed with saturated NaCl solution, dried (Na2SO4), and concentrated in vacuo to give the crude product as a brown oil. Purification of the oil was performed by flash column chromatography (silica, 3:2 hexane/ethyl acetate) to give the product (4.31 g, 80%) as a light-goldenreddish oil. 1H NMR (CDCl3) δ 3.94 [d, 1 H, J ) 6.0 Hz], 3.82 [s, 3 H], 3.44 [t, 1 H, J ) 9.0 Hz], 2.83 [dd, 1 H, J1 ) 3.0 Hz, J2 ) 6.0 Hz], 2.41 [s, 3 H], 2.18 [m, 2 H], 1.96-2.02 [m, 2 H], 1.63-1.26 [m, 1 H]. (1R)-3-(3-Fluorophenyl)-2-(carboxymethyl)trop-2ene (13a). Nitrogen gas was bubbled through a suspension of the triflate 12 (1.18 g, 3.57 mmol), 3-fluorophenylboronic acid (1.0 g, 7.2 mmol), anhydrous powered potassium carbonate (0.74 g, 5.3 mmol), and toluene (12 mL) for 30 min. Tetrakis(triphenylphosphine)palladium(0) was added, and the mixture was heated at 90 °C for 3 h (via an oil bath). The mixture was cooled to room temperature and diluted with ethyl acetate (10 mL) followed by H2O (10 mL). The mixture was basified by addition of NH4OH (pH 9), and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (10 mL), and the organic layers were combined, washed with saturated NaCl solution, dried (Na2SO4), and concentrated in vacuo to give the product as a light-brown oil. Purification of the product was achieved by flash column chromatography (silica, 9:1:10 Et2O/Et3N/hexane) to give the product (0.70 g, 71%) as a light-yellow oil: 1H NMR (CDCl3) δ 7.72-7.24 [m, 1 H], 6.97-6.83 [m, 3 H], 3.85 [d, 1 H, J ) 6.0 Hz], 3.50 [s, 3 H], 3.35 [t, 1 H, J ) 12.0 Hz], 2.73 [dd, 1 H], 2.45 [s, 3 H], 2.20 [m, 2 H], 2.03-1.95 [m, 2 H], 1.63 [m, 1 H]. This material was used without further purification to prepare 7f. (1R)-3r-(3-Fluorophenyl)tropane-2β-carboxylic Acid Methyl Ester (7f). Tropene 13a (0.650 g, 2.36 mmol) was dissolved in anhydrous CH3OH (3 mL) under Ar. After the solution was heated to 40 °C, the SmI2 solution (0.1 M in THF, 97 mL, 9.7 mmol) was added dropwise via a syringe. The mixture was stirred at 40 °C for 2 h. After 2 h, the reaction was quenched with HCl solution (10%). Water and Et2O were added, the mixture was basified to pH 11 with NH4OH and filtered through Celite. Et2O and saturated Na2S2O3 were added, and the layers were separated. The aqueous layer was extracted with CHCl3. The organic layers were combined and dried over Na2SO4. The solvent was removed in vacuo to afford a light-yellow oil. Purification of the oil was accomplished by radial PLC (silica, 5:1:30 CH2Cl2/Et3N/hexane) to give the product (0.26 g, 40%) as a light-yellow oil. 1H NMR (CDCl3) δ 7.24-7.02 [m, 1 H], 7.01-6.82 [m, 3 H], 3.58 [s, 3 H], 3.34 [m, 2 H], 2.92 [m, 1 H], 2.50 [m, 2 H], 2.20 [m, 4 H], 1.57 [m, 4 H]. This material was used without further purification to prepare 8f. 3,4-Difluorophenylboronic Acid. 3,4-Difluorophenylmagnesium bromide (0.5 M in THF, 10.00 mmol) was added to anhydrous THF (20 mL) and cooled to -40 °C under N2. Triisopropyl borate (3.76 g, 20 mmol) was added via a syringe gradually, maintaining the temperature at -40 °C. The mixture was allowed to warm to 0 °C and stirred for 1 h. The reaction was quenched with NH4Cl and H2O, followed by extraction with Et2O. The organic layers were combined, washed with HCl (10%), and dried (Na2SO4). Concentration of the solvent under reduced pressure gave a tan solid.
Recrystallization from hexanes gave the product as a tan solid (0.64 g, 41%). The product was used without further purification for the synthesis of 13b. (1R)-3-(3,4-Difluorophenyl)-2-(carboxymethyl)trop-2ene (13b). Nitrogen was bubbled through a suspension of the triflate 12 (0.66 g, 2.0 mmol), 3,4-difluorophenylboronic acid (0.64 g, 4.1 mmol), anhydrous powered potassium carbonate (0.42 g, 3.0 mmol), and toluene (8 mL) for 30 min. Tetrakis(triphenylphosphine)palladium(0) (0.16 g) was added, and the mixture was heated at 90 °C for 5 h (via oil bath). The mixture was cooled to room temperature and diluted with ethyl acetate (8 mL) followed by H2O (10 mL). The mixture was basified by addition of NH4OH (pH 9), and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (2 × 10 mL), and the organic layers were combined, washed with saturated NaCl solution, dried (Na2SO4), and concentrated in vacuo to give the product as a light-brown oil. Purification of the product was achieved by flash column chromatography (silica, 9:1:20 Et2O/Et3N/hexane) to give the product (0.43 g, 73%) as a light-yellow oil: 1H NMR (CDCl3) δ 7.11-6.85 [m, 3 H], 3.85 [d, 1 H, J ) 6.0 Hz], 3.52 [s, 3 H], 3.35 [t, 1 H, J ) 12.0 Hz], 2.74 [dd, 1 H, J1 ) 12.0 Hz, J2 ) 12.0 Hz], 2.44 [s, 3 H], 2.20 [m, 2 H], 1.98 [m, 2 H], 1.63 [m, 1 H]. This material was used to prepare 7g without further purification. (1R)-3r-(3,4-Difluorophenyl)tropane-2β-carboxylic Acid Methyl Ester (7g). Tropene 13b (0.512 g, 1.74 mmol) was dissolved in anhydrous CH3OH (4 mL) under Ar. After the solution was heated to 40 °C, the SmI2 solution (0.1 M in THF, 72 mL, 7.2 mmol) was added dropwise via a syringe. The mixture was stirred at 40 °C for 6 h. After 6 h, the reaction was quenched with HCl solution (10%). Water and Et2O were added, and the mixture was basified to pH 11 with NH4OH and filtered through Celite. Et2O and saturated Na2S2O3 were added, and the layers were separated. The aqueous layer was extracted with CHCl3. The organic layers were combined and dried over Na2SO4. Solvent was removed in vacuo to afford a light-yellow oil. Purification of the oil was accomplished by radial PLC (silica, 10:1 Et2O/Et3N) to give the desired 3R,2β-isomer and also the 2β,3β-isomer (0.15 g, 30%) as a lightyellow oil. All attempts to separate the isomers on silica gel and alumina were unsuccessful. Hence, this isomeric mixture was carried onto the next step: 1H NMR (CDCl3) δ 7.07-6.92 [m, 3 H], 3.52 [s, 3 H], 3.35-3.28 [m, 2 H], 2.87 [m, 1 H], 2.472.38 [m, 2 H], 2.24-2.16 [m, 4 H], 1.67-1.43 [m, 3 H], 1.25 [m, 1 H]. (1R)-3-(3,5-Difluorophenyl)-2-(carboxymethyl)trop-2ene (13c). A stirred suspension of triflate 12 (1.0 g, 3.0 mmol), 3,5-difluorobenzene boronic acid (1.43 g, 4.55 mmol), LiCl (0.25 g, 6.1 mmol), Na2CO3 (2.0 M, 6.1 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.125 g) in DME (6 mL) was heated at 80 °C for 12 h (via an oil bath). The mixture was cooled to room temperature and filtered through a short Celite pad, eluting with CHCl3. The filtrate was washed with 1:1 solution of NH4OH/H2O (100 mL). The aqueous phase was reextracted with CHCl3. The combined organic extracts was washed with H2O and dried (Na2SO4). The solution was filtered and concentrated in vacuo to give a brown oil. Purification of the product was achieved by flash column chromatography (silica, 9:1:20 Et2O/Et3N/hexane) to give the product (0.37 g, 42%) as a colorless oil: 1H NMR (CDCl3) δ 6.80-6.58 [m, 3 H], 3.85 [d, 1 H, J ) 6.0 Hz], 3.53 [s, 3 H], 3.36 [t, 1 H, J ) 9.0 Hz], 2.78 [dd, 1 H, J1 ) 3.0 Hz, J2 ) 6.0 Hz], 2.44 [s, 3 H], 2.20 [m, 2 H], 2.00 [m, 2 H], 1.65 [m, 1 H]. This material was used to synthesize 7h without further purification. (1R)-3r-(3,5-Difluorophenyl)tropane-2β-carboxylic Acid Methyl Ester (7h). Tropene 13c (1.35 g, 4.60 mmol) was dissolved in anhydrous CH3OH (6 mL) under Ar. After the solution was heated to 40 °C, the SmI2 solution (0.1 M in THF, 188.90 mL, 18.890 mmol) was added dropwise via a syringe. The mixture was stirred at 40 °C for 6 h. After 6 h, the reaction was quenched with HCl solution (10%). Water and Et2O were added, and the mixture was basified to pH 11 with NH4OH and filtered through Celite. Et2O and saturated Na2S2O3 were
Synthesis and Monoamine Transporter Binding Properties added, and the layers were separated. The aqueous layer was extracted with CHCl3. The organic layers were combined and dried over Na2SO4. The solvent was removed in vacuo to afford a light-yellow oil. Purification of the oil was accomplished by radial PLC (silica, 5:1:30 CH2Cl2/Et3N/hexane) to give the product (0.22 g, 16%) as a light-yellow oil: 1H NMR (CDCl3) δ 6.78-6.72 [m, 2 H], 6.61-6.58 [m, 1 H], 3.53 [s, 3 H], 3.36 [m, 2 H], 2.92 [d, 2 H, J ) 3.0 Hz], 2.44 [q, 2 H, J ) 12.0 Hz], 2.23-2.15 [m, 4 H], 1.71-1.33 [m, 3 H]. This material was used without further purification to prepare 8h. Ligand Binding. Brains from male Sprague-Dawley rats weighing 200-250 g (Harlan Labs, Indianapolis, IN) were removed, dissected into striata (DAT) and cerebral cortex (5-HTT and NET), and rapidly frozen. Ligand binding experiments for the dopamine transporter are conducted in assay tubes containing 0.5 mL of buffer (10 mM sodium phosphate containing 0.32 M sucrose, pH 7.40) on ice for 120 min. Each assay tube contained 0.5 nM [3H]WIN 35,428 and 0.1 mg of striatal tissue (original wet weight). The nonspecific binding of [3H]WIN 35,428 was defined using 30 µM (-)-cocaine. Ligand binding experiments for the serotonin transporter are conducted in assay tubes containing 4 mL of buffer (50 mM Tris, 120 mM NaC1, 5 mM KC1, pH 7.4 at 25 °C) for 90 min at room temperature. Each assay tube contained 0.2 nM [3H]paroxetine and 1.5 mg of midbrain tissue (original wet weight). Nonspecific binding of [3H]paroxetine was defined by 1 µM citalopram. Ligand binding experiments for the norepinephrine transporter were conducted in Tris buffer (50 mM Tris, 120 mM NaC1, 5 mM KC1, pH 7.4 at 4 °C) at a total volume of 0.5 mL. Each assay tube contained 0.5 nM [3H]nisoxetine and 8 mg of rat cerebral cortex. The nonspecific binding of [3H]nisoxetine was defined using 1 µM desipramine. Incubations were terminated by filtration with three 5 mL washes of ice-cold buffer through GF/B filters that were previously soaked in 0.05% polyethylenimine. The results were analyzed using Prism (version 3.0, GraphPad software, San Diego, CA).
Acknowledgment. This research was supported by the National Institute on Drug Abuse, Grant DA05477. Supporting Information Available: Results from elemental analysis. This material is available free of charge via the Internet at http://pubs.acs.org.
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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3857 (4) Carroll, F. I. Medicinal Chemistry Division Award address: Monoamine transporters and opioid receptors. Targets for addiction therapy. J. Med. Chem. 2003, 46, 1775-1794. (5) Clarke, R. L. The Tropane Alkaloids. In The Alkaloids; Academic Press: New York, 1977. (6) Clarke, R. L.; Daum, S. J.; Gambino, A. J.; Aceto, M. D.; Pearl, J.; Levitt, M.; Cumiskey, W. R.; Bogado, E. F. Compounds affecting the central nervous system. 3β-Phenyltropane-2-carboxylic esters and analogs. J. Med. Chem. 1973, 16, 1260-1267. (7) Carroll, F. I.; Kotian, P.; Dehghani, A.; Gray, J. L.; Kuzemko, M. A.; Parham, K. A.; Abraham, P.; Lewin, A. H.; Boja, J. W.; Kuhar, M. J. Cocaine and 3β-(4′-substituted phenyl)tropane-2βcarboxylic acid ester and amide analogues. New high-affinity and selective compounds for the dopamine transporter. J. Med. Chem. 1995, 38, 379-388. (8) Holmquist, C. R.; Keverline-Frantz, K. I.; Abraham, P.; Boja, J. W.; Kuhar, M. J. K.; Carroll, F. I. 3R-(4′-Substituted phenyl)tropane-2β-carboxylic acid methyl esters: Novel ligands with high affinity and selectivity at the dopamine transporter. J. Med. Chem. 1996, 39, 4139-4141. (9) Carroll, F. I.; Runyon, S. P.; Abraham, P.; Navarro, H.; Kuhar, M. J.; Pollard, G. T.; Howard, J. L. Monoamine transporter binding, locomotor activity, and drug discrimination properties of 3-(4-substituted-phenyl)tropane-2-carboxylic acid methyl ester isomers. J. Med. Chem. 2004, 47, 6401-6409. (10) Blough, B. E.; Holmquist, C. R.; Abraham, P.; Kuhar, M. J.; Carroll, F. I. 3R-(4-Substituted phenyl)nortropane-2β-carboxylic acids methyl esters show selective binding at the norepinephrine transporter. Bioorg. Med. Chem. Lett. 2000, 10, 2445-2447. (11) Triggle, D. J.; Kwon, Y. W.; Abraham, P.; Rahman, M. A.; Carroll, F. I. Synthesis of 2-(3-substituted-1,2,4-oxadiazol-5-yl)8-methyl-8-azabicyclo[3.2.1]octanes and 2R-(3-substituted-1,2, 4-oxadiazol-5-yl)-8-methyl-8-azabicyclo[3.2.1]oct-2-enes as potential muscarinic agonist. Pharm. Res. 1992, 9, 1474-1479. (12) Lewin, A. H.; Naseree, T.; Carroll, F. I. A practical synthesis of (+)-cocaine. J. Heterocycl. Chem. 1987, 24, 19-21. (13) Keverline, K. I.; Abraham, P.; Lewin, A. H.; Carroll, F. I. Synthesis of the 2β,3R- and 2β,3β-isomers of 3-(p-substituted phenyl)tropane-2-carboxylic acid methyl esters. Tetrahedron Lett. 1995, 36, 3099-3102. (14) Carroll, F. I.; Gray, J. L.; Abraham, P.; Kuzemko, M. A.; Lewin, A. H.; Boja, J. W.; Kuhar, M. J. 3-Aryl-2-(3′-substituted-1′,2′, 4′-oxadiazol-5′-yl)tropane analogues of cocaine: Affinities at the cocaine binding site at the dopamine, serotonin, and norepinephrine transporters. J. Med. Chem. 1993, 36, 2886-2890. (15) Cheng, Y.-C.; Prusoff, W. H. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which cause 50% inhibition (I50) of an enzyme reaction. Biochem. Pharmacol. 1973, 22, 3099-3108. (16) Carroll, F. I.; Gao, Y.; Rahman, M. A.; Abraham, P.; Lewin, A. H.; Boja, J. W.; Kuhar, M. J. Synthesis, ligand binding, QSAR, and CoMFA study of 3β-(p-substituted phenyl)tropane-2β-carboxylic acid methyl esters. J. Med. Chem. 1991, 34, 2719-2927. (17) Kotian, P.; Abraham, P.; Lewin, A. H.; Mascarella, S. W.; Boja, J. W.; Kuhar, M. J.; Carroll, F. I. Synthesis and ligand binding study of 3β-(4′-substituted phenyl)-2β-(heterocyclic)tropanes. J. Med. Chem. 1995, 38, 3451-3453. (18) Boja, J. W.; Kuhar, M. J.; Kopajtic, T.; Yang, E.; Abraham, P.; Lewin, A. H.; Carroll, F. I. Secondary amine analogues of 3β-(4′-substituted phenyl)tropane-2β-carboxylic acid esters and N-norcocaine exhibit enhanced affinity for serotonin and norepinephrine transporters. J. Med. Chem. 1994, 37, 1220-1223.
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