Synthesis of aza and carbocyclic β-carbolines for the treatment of alcohol abuse . Regiospeci fi c solution to the problem of 3 , 6-disubstituted β-and aza-β-carboline speci fi city †

A novel two step protocol was developed to gain regiospecific access to 3-substituted βand aza-βcarbolines, 3-PBC (1), 3-ISOPBC (2), βCCt (3), 6-aza-3-PBC (4) and 6-aza-3-ISOPBC (5). These β-carbolines (1–3) are potential clinical agents to reduce alcohol self-administration, especially 3-ISOPBC·HCl (2·HCl) which appears to be a potent anti-alcohol agent active against binge drinking in a rat model of maternally deprived (MD) rats. The method consists of two consecutive palladium-catalyzed reactions: a Buchwald– Hartwig amination followed by an intramolecular Heck-type cyclization in high yield.

Introduction β-Carbolines, aza-β-carbolines and their derivatives are important targets in synthetic chemistry. 1 In addition, they are found in a large number of natural products, many of which demonstrate novel biological activity, especially in regard to the reduction of alcohol self-administration [binge drinking (BD)]. This is proposed to be due to the activity at the benzodiazepine site of the GABA A receptor. 2 Surprisingly, BD kills six people a day, most of which are men, and approximately 88 000 people die from alcohol related issues annually making it the third leading preventable cause of death in the United States. 3 In 2006, this alcohol misuse cost the US government approximately $223.5 billion dollars. 3 BD (Blood-alcohol level ≥0.08 g% in a 2 hour period) is one form of excessive drinking and because of it, alcohol addiction and dependence remain a significant public health concern. 4 Maternal separation and early life events can cause profound neurochemical and behavioral alterations in childhood that persist into adulthood, enhance the risk to develop alcohol use disorders and excessive drinking. [5][6][7] Consequently, the development of clinically safe and cost effective therapeutic agents to reduce alcohol addiction and dependence remain essential for the future treatment of alcoholism. 8,9 One influence on alcohol abuse is known to be mediated by GABA A receptors, the major inhibitory chloride ion gated channels with γ-aminobutyric acid (GABA) as the endogenous ligand in the central nervous system. It plays a vital role in several neuronal disorders including anxiety, epilepsy, insomnia, depression, bipolar disorder, schizophrenia, as well as mild cognitive impairments and Alzheimer's disease. [10][11][12][13][14][15] The pentameric structure of the GABA A receptor is made up of 2 α, 2 β and 1 γ subunits, with a higher distribution of the α1subunit in the mesolimbic system of the ventral pallidum (VP) possibly playing an important role in regulating alcohol abuse. [16][17][18][19][20] However, the precise neuromechanisms of regulating alcohol-seeking behavior remain unknown. In addition to the ventral pallidum, there is now compelling evidence that the GABA A receptors within the striatopallidal and extended amygdala system are involved in the 'acute' reinforcing actions of alcohol. [21][22][23] To evaluate the role of the α1 receptor in regulating alcohol reinforcement, the orally active β-carbolines 3-propoxy-β-carboline hydrochloride 1·HCl (3-PBC·HCl) and β-carboline-3-carboxylate-tert-butyl ester 3 (βCCt), the mixed benzodiazepine (BDZ) agonist-antagonists with binding selectivity at the α1 Bz/ GABA A receptor were developed (see Fig. 1). 18,24,25 Behavioral studies in several species (e.g., rats, mice, primates) show that these ligands were BDZ antagonists, at the α1 Bz/GABA A subtype exhibiting competitive binding-site interactions with BDZ agonists over a broad range of doses. 18,24,26 In studies which involved the α1 subtype, they were shown to selectively reduce alcohol-motivated behaviors and more importantly, 3-PBC·HCl significantly reduced alcohol self-administration and reduced craving in baboons. 26 β-Carbolines 1·HCl and 3 displayed mixed weak agonist-antagonist profiles in vivo in alcohol preferring (P) and high alcohol drinking (HAD) rats. 18,[26][27][28] Therefore, in addition to their use to study the molecular basis of alcohol reinforcement, α1 Bz β-carboline ligands which display mixed pharmacological antagonistagonist activity in alcohol P and HAD rats may be capable of reducing alcohol intake while eliminating or greatly reducing the anxiety associated with habitual alcohol, abstinence or detoxification. 18,[28][29][30] Consequently, these types of ligands may be ideal clinical agents for the treatment of alcohol dependent individuals.

Results and discussion
Previously, the β-carbolines 1 and 3 have been synthesized from DL-tryptophan. The overall yield of 1 (via 6 steps) as reported previously was 8%, while the combined yield of 3 (5 steps) was 35%. A few key steps occurred in low yields which was something of which we sought to improve on 31-34 in a continued effort to find more potent subtype selective ligands for GABA A receptors. This interest resulted in a short and concise synthesis of 1 and 3. In 2011, a palladium catalyzed two-step protocol for the synthesis of 1, and 3 as well as analogs of 1 was reported. 35 In the search for a more potent subtype selec-tive ligand for the GABA A receptor, with the knowledge that many 3-substituted β-carbolines and more water soluble azaβ-carbolines might exhibit greater subtype selectivity at α1β 2/3 γ2 BZR/GABAergic receptors, [31][32][33][36][37][38] the ligands 3-ISOPBC (2), 6-aza-3-PBC (4), and 6-aza-3-ISOPBC (5) were designed (see Fig. 1) and synthesized using a two-step protocol (Scheme 1).
As shown in Scheme 1, bromopyridines 6a-c 39,40 were reacted with anilines 4a-b in toluene at 100-140°C in the presence of 5 mol% Pd(OAc) 2 and 7.5 mol% X-Phos to obtain the corresponding diarlyamines 7a-e in moderate to good yields. Unfortunately, the intramolecular Heck cyclization [Pd(OAc) 2 , (t-Bu) 3 ·HBF 4 , K 2 CO 3 , DMA, 120°C] of 7a-e afforded both the β-carbolines 1-5 (individually) and their regioisomeric δ-carbolines 9a-e, respectively. Carbolines 2, 3, 9a, and 9d were subjected to X-ray crystallographic analysis (see Fig. 2, Scheme 4, and the ESI †) to confirm the regiochemistry. Although this protocol permitted synthesis of β-carbolines on gram scale for in vivo studies, occasionally the first step in the Buckwald-Hartwig coupling failed to give complete conversion into the carboline. This complicated purification for the diarylamine was difficult to purify via column chromatography because the diarylamine and one of the starting anilines had almost identical R f values. Furthermore, in the case of the water soluble aza-β-carboline the yields (51%) were very poor and importantly, since the second step was not regiospecific, this required careful purification to remove the unwanted δ-carboline present in 30 to 62.5% yield (Scheme 1). Interest- ingly, the in vivo results (unpublished) for 3-isopropoxyβ-carboline hydrochloride 2·HCl (3-ISOPBC·HCl) carried out in maternally deprived rats for binge drinking decreased dramatically this self-administration compared to 1·HCl without affecting the overall activity of the rats (i.e. no sedation). This important finding led to the interest in a regiospecific synthesis of 3-ISOPBC (2) on large scale.
The revised synthetic strategy for the regiospecific synthesis of 2 began with the protection of the intermediate amine 7b (N a -H) with bulkier groups such as tert-butyloxycarbonyl (Boc) 10 or a fluorenylmethylenoxy group (Fmoc) 11, which might block the formation of the Pd II π-complex that is required to obtain the undesired regioisomeric δ-carboline. The Boc protected amine 10 was easily accessible by treating the amine 7b with di-tert-butyl dicarbonate (Boc) 2 O and 4-(dimethylamino)pyridine (DMAP) in good yield (85%). The Fmoc protected amine 11 was synthesized under solvent free conditions by reaction of the amine 7b and Fmoc-Cl by microwave irradiation at 80°C in moderate yield (65%, Scheme 2). 41 Once protected, diarylamines 10 and 11 were subjected to a palladium catalyzed Heck-type cyclization using similar conditions to those from above. Unfortunately, both reactions afforded the deprotected regioisomers 3-ISOPBC (2) and δ-isomer 9b in approximately the same 2 : 1 ratio, as compared to cyclization with the previously unprotected diarylamine 7b (see Scheme 1 above). It was felt that deprotection of the carbamate occurred once the indole ring had formed (Scheme 2) which provided the better indole leaving group. To test the thermal stability of the carbamate starting materials, diarylamines 10 and 11 were heated at 120°C in DMA; they were stable to these conditions. In addition, the cyclization with PdCl 2 (PPh 3 ) 2 as a palladium source was also attempted using standard Heck-type reaction conditions with a milder base (NaOAc), but this failed to give the cyclized product. We also explored the reaction by varying the water content using NaOAc·3H 2 O as a base; however, there was no cyclization (Scheme 2). The second approach rested on the important switch of the chlorine atom from the benzene ring to the pyridine ring in amine 7b. Retrosynthetically, it was envisioned that the core structure of 3,6-disubstituted β-carboline A could be obtained from diarylamine B via an intramolecular Heck cyclization and it was anticipated that diarylamine B could arise from a substituted aniline C and a substituted pyridine derivative D via a Buchwald-Hartwig amination (Scheme 3).
At this point it was decided to explore the regioselective palladium catalyzed Buchwald-Hartwig coupling between aniline and pyridine 14 42 for the synthesis of diarylamine 16 (Table 1). With the previous history in mind, 35 the initial attempt was made with 5 mol% Pd(OAc) 2 , 7.5 mol% X-Phos and Cs 2 CO 3 (1.5 equiv.) in toluene at 110°C which gave only 18% of the diarylamine 16 with a large excess of unreacted starting material even after heating for 24 hours ( Table 1, entry 1). However, the catalyst based on the combination of Pd 2 (dba) 3 , Xantphos and Pd(OAc) 2 , Xantphos with Cs 2 CO 3 in toluene and dioxane gave the desire product diarylamine 16 in up to 62% yield ( Table 1, entries 2-3). The ligand Xantphos has been shown to be efficient in cross coupling reactions of C-N bond formation because of a wider bite angle, 43 which facilitates the reductive elimination. In addition, the excess base may also play a role in the improvement of the yield. 43 In recent years rapid synthesis with microwave technology has attracted a considerable amount of attention for C-N bond formation. [44][45][46] All three previous cyclizations were attempted with microwave irradiation (for 1 hour) in order to decrease the duration of the reaction time, as well as increase the selectivity under similar reaction conditions. However, the results were the same except that in the Xantphos-based ligand systems the cyclizations were completed in 1 hour. During continuation of the study of this selective amination, recent reports from Buchwald and coworkers 47 demonstrated air-and moisture-stable palladacyclic precatalysts, when employed with aryl iodides and heteroaryliodides were attractive substrates in Pd-catalyzed C-N crosscoupling reactions. This process works by preventing formation of the stable bridging iodide dimers and also using a solvent system in which iodide salts were insoluble. These complexes easily undergo deprotonation and reductive elimination to generate LPd(0) along with relatively inert indoline (for generation of 1) or carbazole (for generation of 2 and 3). These conditions also permit the successful coupling of aryl iodides with amines at ambient temperature. [47][48][49][50] The first attempt in this modification was to use the Buchwald 3 rd generation palladacycle precatalyst (BrettPhos Pd G3) Scheme 3 Retrosynthetic analysis of 3,6-disubstituted β-carbolines. with the BrettPhos ligand in the presence of Cs 2 CO 3 or NaOt-Bu in toluene at room temperature. This failed to give the desired product and there was no consumption of starting material. Following this attempt, the temperature was raised to reflux, with the addition of 3 equivalents of Cs 2 CO 3 and the reaction went to completion within 5 hours. However, it only gave the desired amine 16 in 66% yield (Table 1, entry 5). When the same experiment was performed using only 1.5 equiv. of Cs 2 CO 3 the process took a longer time to go to completion with an isolated yield of 45% of the desired amine 16. This was accompanied by the diaminated product [6-isopropoxy-N 3 ,N 4 -diphenylpyridine-3,4-diamine] in ∼18% yield ( . It was found the Pd(OAc) 2 , rac-BINAP and K 2 CO 3 combination, unfortunately, did not lead to full conversion even after heating for 24 hours (Table 1, entry 10). Interestingly, the catalyst system Pd 2 (dba) 3 and Xantphos with a large excess of base [Cs 2 CO 3 (5 equiv.)] gave 74% yield of 16, whereas the catalyst system Pd(OAc) 2 , rac-BINAP under similar reaction conditions yielded 80% (Table 1, entry 8 and 9) of the desired amine 16. Remarkably, these data indicated a large excess of mild base was essential to obtain good yields, as well as selectivity. Furthermore, a rate-limiting interphase deprotonation of the Pd(II)-amine complex inter-mediate has occured in the catalytic cycle. [51][52][53] Encouraged by these promising results, efforts turned toward lowering the aniline loading from 1.2 equivalents to 1 equivalent for regioselectivity. In doing so we achieved selective amination of pyridine 14 with aniline. Interestingly, neither a 4-nor 4,5diaminated pyridine product was obtained. Using this catalystbase combination in refluxing toluene, the desired cross-coupling proceeded smoothly to provide the desired anilinopyridine 16 in excellent yield (92%, Table 1, entry 11). Interestingly, the same reaction conditions gave good yields in the case of the more polar starting 4-amino pyridine (Scheme 4); however, the temperature was necessarily increased to 140°C to increase the solubility of the starting material, 4-amino pyridine. In contrast, when a polar solvent such as DMA was employed, the result was either inferior yields and/or deiodination of pyridine 16, as mentioned above.
Once the diarylamines 15-18 were in hand in good to excellent yields, the previously applied Heck-type conditions [Pd(OAc) 2 , (t-Bu) 3 ·HBF 4 , K 2 CO 3 , DMA, 120°C] were employed for cyclization. Gratifyingly, this catalyst system gave excellent yields of 91-92% and 90-92% for β-carbolines 1-2 and azaβ-carbolines 4-5, respectively (Scheme 4). The switch of the chlorine position from the benzene ring to the pyridine ring worked regiospecifically and completely eliminated the corresponding unwanted δ regioisomer. This completely eliminated the difficult chromatography required to separate β and δ carbolines. The 3-ISOPBC 2 has now been prepared on 15-25 gram scale for studies in vivo (Scheme 5) and it is very easy to scale up to 50-100 gram level. Finally, the overall yield increased from 43% to 84% compared to the previous syntheses. 33,35 Conclusions In conclusion, a novel two-step regiospecific route to the four anti-alcohol agents of biological interest, 3-PBC (1), 3-ISOPBC (2), 6-aza-3-PBC (4) and 6-aza-3-ISOPBC (5), has been developed. The process provided improved yields when compared to the earlier reported syntheses. 33,35 This two-step protocol consists of the combination of a regioselective Buchwald-Hartwig amination and an intramolecular Heck-type cyclization. The first step, regioselective arylamination, was achieved by using a Pd-BINAP catalytic system in combination with a large excess of Cs 2 CO 3 , while the latter intramolecular Heck-type cyclization went smoothly with Pd(OAc) 2 in combination with the air-stable monodentate ligand (t-Bu) 3 ·HBF 4 and K 2 CO 3 . These conditions permit the presence of base sensitive functional groups in the substrates. Regiospecific synthesis of βand aza-β-carbolines was achieved by simply changing the chlorine position from the benzene ring to the pyridine derivatives. Importantly, these reactions are capable of scaleup to multigram quantities and were performed on 25 gram scale level for in vivo biology. We observed similar results except in the case of the Buchwald-Hartwig amination step, where it required an increase of the catalyst loading from 3 to 6 mol% whenever the starting material was not consumed. This new process reported here provides the material necessary to study alcohol self-administration and reduction thereof in MD rats and in primates. This regiospecific two-step synthetic protocol increased the overall yield from 43% to 84% in the case of β-carbolines 1-2 and from 16% to 66% for azaβ-carbolines 4-5 respectively, and negated the need for a difficult chromatographic step.

General considerations
All reactions were carried out in oven-dried, round-bottom flasks or in resealable screw-cap test tubes or heavy-wall pressure vessels under an argon atmosphere. The solvents were anhydrous unless otherwise stated. Stainless steel syringes were used to transfer air-sensitive liquids. Organic solvents were purified when necessary by standard methods or purchased from commercial suppliers. Anhydrous solvents of toluene, dioxane and N,N-dimethylacetamide (DMA) were subjected to the freeze-thaw method to render them oxygen free to execute the Buckwald-Hartwig coupling and intramolecular Heck reactions. All chemicals purchased from commercial suppliers were employed as is, unless stated otherwise in regard to purification. Silica gel (230-400 mesh) for flash chromatography was utilized to purify the analogues. The 1 H and 13 C NMR data were obtained on an NMR spectrometer (300 MHz/ 500 MHz) instrument with chemical shifts in δ (ppm) reported relative to TMS. The HRMS were obtained on a LCMS-IT-TOF mass spectrometer by Dr Mark Wang.
General procedure for the Buchwald-Hartwig coupling reaction between substituted anilines and substituted pyridines: representative procedure for the synthesis of N-(2-chlorophenyl)-6-propoxypyridin-3-amine (7a) A heavy-wall pressure tube was equipped with a magnetic stir bar and fitted with a rubber septum. It was then charged with 5-bromo-2-propoxypyridine 6a (1.3 g, 6 mmol), Pd(OAc) 2 (67.4 mg, 0.3 mmol), X-Phos (214 mg, 0.45 mmol) and Cs 2 CO 3 (2.34 g, 7.2 mmol). The vessel was evacuated and backfilled with argon (this process was repeated a total of 3 times). The 2-chloroaniline 4a (0.8 g, 6.3 mmol) and freeze-thawed toluene (20 mL) was injected into the tube with a degassed syringe under a positive pressure of argon. The rubber septum was replaced with a screw-cap by quickly removing the rubber septum under the flow of argon and the sealed tube was introduced into a pre-heated oil bath at 110°C. The reaction mixture was maintained at this temperature for 15 h. At the end of this time period, the pressure tube was allowed to cool to rt. The reaction mixture was filtered through a short pad of celite, and the pad was washed with ethyl acetate (until no more product could be obtained; ≈100 mL; TLC, silica gel). The combined organic fractions were washed with water (100 mL), brine (100 mL), dried (Na 2 SO 4 ) and concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 20 : 1 hexanes/ethyl acetate) to afford 7a (0.64 g, 81%) as a pale yellow oil: 1

b]indole (3-PBC, 1) and 2-propoxy-5H-pyrido[3,2-b]indole (9a)
A heavy-wall pressure tube was equipped with a magnetic stir bar and fitted with a rubber septum and loaded with N-(2-chlorophenyl)-6-propoxypyridin-3-amine 7a (526 mg, 2.0 mmol), Pd(OAc) 2 (44.8 mg, 0.2 mmol), (t-Bu) 3 P·HBF 4 (116 mg, 0.4 mmol) and K 2 CO 3 (552 mg, 4.0 mmol). The vessel was evacuated and backfilled with argon (this process was repeated a total of 3 times) and degassed DMA (8 mL) was injected into the tube with a degassed syringe under a positive pressure of argon. The rubber septum was replaced with a screw-cap by quickly removing the rubber septum under the flow of argon and the sealed tube was introduced into a preheated oil bath at 120°C. The reaction mixture was maintained at this temperature for 16 h. At the end of this period, the reaction mixture was allowed to cool to rt. The dark brown mixture which resulted was then passed through a short pad of celite. The celite pad was further washed with ethyl acetate (150 mL) until no more product (TLC; silica gel) was detected in the eluent. The combined filtrate was washed with water (100 mL × 3), brine (100 mL), dried (Na 2 SO 4 ) and concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 5 : 1 hexanes/ethyl acetate) to afford 3-PBC (1) (235 mg, 52%) as an off white solid. mp 120.5-121.5°C (lit. 35

4-Chloro-6-isopropoxy-N-phenylpyridin-3-amine (16)
A heavy-wall pressure tube was equipped with a magnetic stir bar and fitted with a rubber septum that had been charged with 4-chloro-5-iodo-2-isopropoxypyridine 14 (75 mg, 0.252 mmol), aniline (27.6 µL, 0.256 mmol) and Cs 2 CO 3 (410 mg, 1.26 mmol). The vessel was evacuated and backfilled with argon (this process was repeated a total of 3 times) and degassed toluene (1 mL) was injected into the tube with a degassed syringe under a positive pressure of argon. In another round bottom flask fitted with a rubber septum, Pd(OAc) 2 (1.7 mg, 0.0076 mmol) and rac-BINAP (4.7 mg, 0.0076 mmol) was charged. This flask was evacuated and backfilled with argon (this process was repeated a total of 3 times) and then degassed toluene (0.5 mL) was added under a positive pressure of argon. This mixture was stirred for 10 min and then the mixture which resulted was added to the above pressure tube. The rubber septum was replaced with a screwcap by quickly removing the rubber septum under the flow of argon and the sealed tube was introduced into a pre-heated oil bath at 110°C. The reaction mixture was maintained at this temperature for 5 h. At the end of this time period the pressure tube was allowed to cool to rt. The reaction mixture was filtered through a short pad of celite, and the pad was washed with ethyl acetate (until no more product could be obtained; ≈50 mL). The combined organic eluents were washed with water (50 mL), brine (50 mL), dried (Na 2 SO 4 ) and concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel 4.0 mmol) were heated to give a solid which was purified by a wash column (silica gel, 5 : 1 hexanes/ethyl acetate) to yield 3-ISOPBC 2 (412.30 mg, 91%).
Step 2: Synthesis of 3-isopropoxy-9H-pyrido[3,4-b]indole (2). A heavy-wall pressure tube was equipped with a magnetic stir bar and fitted with a rubber septum loaded with 4-chloro-6-isopropoxy-N-phenylpyridin-3-amine 16 (19.86 g, 75.58 mmol), Pd(OAc) 2 (1.70 g, 7.558 mmol), (t-Bu) 3 P·HBF 4 (4.39 g, 15.12 mmol) and K 2 CO 3 (20.89 g, 151.16 mmol). The vessel was evacuated and backfilled with argon (this process was repeated a total of 3 times) and degassed DMA (200 mL) was added to this vial via a cannula. The rubber septum was replaced with a screw-cap by quickly removing the rubber septum under the flow of argon and the sealed tube was introduced into a pre-heated oil bath at 120°C. The reaction mixture was maintained at this temperature for 16 h. At the end of this period, the reaction mixture was allowed to cool to rt. The dark brown mixture which resulted was then passed through a short pad of celite. The celite pad was further washed with ethyl acetate until no product (TLC; silica gel) was detected in the eluent. The combined filtrate was washed with water, brine, dried (Na 2 SO 4 ) and concentrated under reduced pressure. The solid product was purified by a wash column (silica gel, 5 : 1 hexanes/ethyl acetate) to afford 3-ISOPBC (2) (15.74 g, 92%) as an off white solid.