Asymmetric Synthesis of the Carbon-14-Labeled Selective Glucocorticoid Receptor Modulator using Cinchona Alkaloid Catalyzed Addition of 6-Bromoindole to Ethyl Trifluoropyruvate

We describe in this study the asymmetric synthesis of radioisotope (RI)-labeled selective glucocorticoid receptor modulator. This synthesis is based on optimization of the cinchona alkaloid catalyzed addition of 6-bromoindole to ethyl trifluoropyruvate and Negishi coupling of zinc cyanide to the 6-bromoindole moiety. [14C] Labeled (−)-{4-[(1-{2-[6-cyano-1-(cyclohexylmethyl)-1H-indol-3-yl]-3,3,3-trifluoro-2-hydroxypropyl}piperidin-4-yl)oxy]-3-methoxyphenyl}acetic acid (−)-1 was synthesized successfully with high enantioselectivity (>99% ee) and sufficient radiochemical purity.


Introduction
Catalytic asymmetric synthesis, which is one of the important methodologies in modern synthetic chemistry for the preparation of enantiomerically enriched compounds, requires the use of metal catalysts or organocatalysts [1]. Organocatalytic asymmetric reactions, which have recently been developed into a practical synthetic paradigm, have several significant advantages over metal-catalyzed reactions due to the following reasons: (1) minimal sensitivity to moisture and oxygen; (2) low cost and low toxicity; and (3) recyclable and reusable catalysts [2][3][4]. Since the first report of cinchona alkaloids-catalyzed asymmetric reactions by Bredig and Fiske [5], cinchona alkaloids and their OPEN ACCESS derivatives have been recognized as one of the most important organocatalysts in catalytic asymmetric reactions [6][7][8]. Recently, several applications of organocatalysts, including cinchona alkaloids, to Friedel-Crafts reaction have attracted chemists' attention as useful methods for carbon-carbon formation [9][10][11][12][13][14][15][16][17][18][19][20][21]. Encouraged by these findings, we have reported the use of cinchona alkaloids in the asymmetric addition of 6-cyanoindole to ethyl trifluoropyruvate and applied the reaction to the asymmetric synthesis of glucocorticoid receptor modulator (GRM) (−)-1 [22].
RI-labeled compounds are widely used in Drug Metabolism and Pharmacokinetics/Absorption, Distribution, Metabolism and Excretion (DMPK)/ADME studies. They help understand drug metabolism in different species, perform DMPK/ADME studies, such as Quantitative Whole Body Autoradiography (QWBA), and evaluate reactive metabolite formation by protein covalent binding tests [23]. Advances in radiochemistry with the expansion of the use of organocatalysts and understanding of the importance of DMPK/ADME studies have led us to synthesize RI-labeled drug candidates using asymmetric reactions with organocatalysts. In support of our drug discovery program for GRM, we required [ 14 C]-labeled (−)-1 with sufficient radiochemical purity. Here, we describe the successful asymmetric synthesis of [ 14 C]-labeled (−)-1.

Results and Discussion
Our approach to the radiosynthesis of (−)-1 is shown in Scheme 1. The cyano moiety of the indole structure was selected to introduce the most commonly used [ 14 C] as radioisotope, because of its chemical and metabolic stability and the availability of [ 14 C] cyanide. In addition, introduction of the [ 14 C] cyanide was optimized as a later step to avoid unnecessary waste of radioactive product. The coupling reaction of cyanide to haloindoles enables this synthetic hypothesis [24]. Based on the findings of our previous report, we used the cinchona alkaloid-catalyzed asymmetric addition of 6-bromoindole to ethyl trifluoropyruvate as a key step. In order to establish a feasible radiosynthetic route, we examined optimization of the cinchona alkaloid catalyzed addition and coupling reaction of cyanide to the 6-bromoindole moiety.
Based on these finding, we selected toluene as solvent, just as we did with 6-cyanoindole, and conducted further optimization of the reaction by examining the effect of temperature and the amount of catalyst (Table 2). Although the yield was maintained with increased reaction temperature, enantioselectivity decreased (entries 1-4). Unlike in the case of 6-cyanoindole, carrying out the reaction even at a very low temperature (−78 °C) afforded the product in high yield (89%), suggesting higher reactivity at the C(3)-position of the 6-bromoindole ring caused by a weaker electronwithdrawing effect of the bromo group than that of the cyano group. On the other hand, the use of 2.5 mol% or 15 mol% of catalyst gave comparable yields and enantioselectivity to the use of 7.5 mol% of catalyst (entries 7 and 8). When the amount of catalyst was reduced to 1 mol%, the yield was maintained, but the enantioselectivity decreased significantly to 71% (entry 6). Interestingly, the initial reaction rate in toluene without the catalyst was comparable to those with catalysts (entry 5) and seems even higher than that of the reaction catalyzed by 1 mol% CD. This result is different from that obtained by Török's group, in which it was indicated that application of CD as catalyst in Et 2 O significantly increases reaction rate by more than two orders of magnitude as compared to the use of no catalyst. This discrepancy may be caused by the effect of toluene, which remarkably increases the reaction rate compared to Et 2 O. From these observations, it seems that this asymmetric induction is not just a kinetic phenomenon in the reaction performed in toluene. Using quinine, a cinchona alkaloid, also provided the product (+)-4 with 83% ee in 90% yield (entry 9). Based on the findings above, we synthesized the [ 14 C]-labeled (−)-1. For a gram-scale synthesis of the enantiomerically pure 4, toluene as solvent, 2.5 mol% CD, and 0 °C were selected as reaction conditions (Scheme 2). Under these conditions, the product (+)-4 was obtained with 83% ee in 90% yield. Recrystallization of (+)-4 from diisopropyl ether-n-hexane gave the racemic crystalline 4, and the filtrate provided the enantiomerically pure (+)-4 (>99% ee). The following reaction was performed according to the procedure previously reported for the 6-cyanoindole derivative [22]. N-Boc protection of the nitrogen atom of (+)-4 with Boc 2 O gave (+)-5. Reduction of the ester (+)-5 with LiBH 4 gave the corresponding alcohol (+)-6. After that, compound (−)-7 was prepared by tosylation of (+)-6. Epoxidation of (−)-7 with aqueous 1 M NaOH in THF yielded (+)-8. Then, ring-opening reaction of 8 with the phenoxypiperidine 9 [22], followed by deprotection of the Boc group, provided (+)-10. Alkylation of (+)-10 with cyclohexylmethyl bromide gave (−)-11. Next, we considered establishing the cyano coupling with the 6-bromo group. For that, we selected CuCN and Zn(CN) 2 as coupling reagents, because the corresponding [ 14 C]-labeled reagents were available. As our attempt to perform the coupling reaction of CuCN to the 6-bromoindole moiety didn't work well, we tried Negishi coupling on the ester (−)-11 (Table 3). A simple attempt to introduce a cyano group by substitution reaction on the aryl bromide led only to decomposition of ester (−)-11 (entries 1 and 2), thus we tried Negishi coupling using Pd(t-Bu 3 P) 2 as catalyst (entries 3-5). Fortunately, running the reaction at 80 °C did not lead to the decomposition, but yielded 6-cyanoindole in 22% yield with recovery of (−)-11 in 75% yield (entry 3). This result indicated that decomposition of the starting materials did not occur, allowing us to examine an increase in reaction temperature. Even at the highest temperature (110 °C) Negishi coupling conditions did not lead to product decomposition, but instead gave the desired (−)-12 with recovery of (−)-11 (entry 4). Among the temperatures tested, performing Negishi coupling at 150 °C afforded the product in 83% yield (entry 5). Applying the less reactive catalyst Pd(PPh 3 ) 4 to the reaction resulted in a lower yield (entry 6). Optimization of the reaction was achieved using [ 14 C]-labeled Zn(CN) 2 and afforded the corresponding [ 14 C](−)-12 in 78% yield (entry 7). As expected, hydrolysis of the obtained compound (−)-12 resulted in the desirable enantiomer (−)-1 with >99% ee in an overall yield of 9.3%. These results indicate that the established synthetic route caused no racemization.

General
All reagents and solvents were used as obtained from commercial suppliers without further purification. Monitoring of reactions was carried out using Merck 60 F 254 silica gel, glass-supported TLC plates, and visualization with UV light (254 nm). NMR spectra were recorded on a JEOL JNM-AL400 spectrometer ( 1 H at 400 MHz and 13 C at 100 MHz) at room temperature. Chemical shifts were given in δ values (ppm), and following abbreviations were used: s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet and m = multiplet. IR spectra were recorded on a PerkinElmer Spectrum One using the attenuated total reflection (ATR) technology. Optical rotations were recorded on a JASCO Polarimeter P-1020. Melting points were recorded on a Yanako MP-J3 melting point apparatus without correction. Low-resolution mass spectra were recorded on a Shimadzu LCMS-2010EV instrument under electron spray ionization (ESI) conditions. Elemental analyses were obtained on a CE Instruments EA1110. [ 14 C] Zn(CN) 2 (5.0 Ci, 115 mCi/mmol) was purchased from American Radiolabeled Chemicals, Inc. Radio-TLC was scanned on a raytest Rita Star. Quantitation of radioactivity was recorded on an AB Sciex 4000QTrap MS instrument. The following abbreviations are used for reagents and solvents: TFA (trifluoroacetic acid), Boc 2 O (di-tert-butyl dicarbonate), DMF (N,N-dimethylformamide), EtOAc (ethyl acetate), THF (tetrahydrofuran), IPE (diisopropyl ether).
The mixture was extracted with EtOAc. The organic layer was washed with water and brine, dried over Na 2 SO 4 and concentrated to afford (+)-8 (4.51 g, 97%) as a white solid; Anal. Calcd for C 16

Conclusions
In this study we have optimized the asymmetric addition of 6-bromoindole (2) to ethyl trifluoropyruvate (3) and developed an efficient synthesis of enantiomerically pure key intermediate (+)-4 (>99% ee) by using easily available, recyclable, and inexpensive cinchona alkaloids as catalysts. In addition, we discovered that Negishi coupling of zinc cyanide to the 6-bromoindole moiety proceeds smoothly without decomposition. Based on these results, we successfully synthesized [ 14 C]-labeled GRM (−)-1. This study is the first example to apply organocatalyst-catalyzed reactions to asymmetric synthesis of an isotope labeled compound. This methodology is important in drug industry, because the numbers of enantiometically pure drug candidates is increasing and enantiomerically pure RI-labeled drug candidates are necessary for the drug development process. Our studies show that organocatalysts could be an important option for RI synthesis.