Nucleophilic Substitution on 2-Monosubstituted Quinoxalines Giving 2,3-Disubstituted Quinoxalines: Investigating the Effect of the 2-Substituent

An investigation on the effect of substituent at the 2-position of mono-substituted quinoxalines in the synthesis of di-substituted quinoxaline derivatives via nucleophilic substitution reactions, is reported. Di-substituted quinoxalines bearing aryl-alky, aryl-aryl, aryl-heteroaryl, aryl-alkynyl, and amino-alkyl substituents were prepared in moderate to good yields. 2-Monosubstituted quinoxalines bearing a phenyl and butyl substituent reacted readily with alkyl-, aryl-, heteroaryl- and alkynyl- nucluephiles, giving di-substituted quinoxalines. 2-Monosubstituted quinoxalines bearing an amine and alkynyl substituent only reacted with alkyl nucleophiles. Oxidative rearomatization to give 2,3-disubstituted quinoxaline products occurred in atmospheric O2.


Introduction
Quinoxaline derivatives possess extensive applications in medicinal chemistry, due to their broad spectrum of biological activity [1,2]. A large number of synthetic quinoxalines have been reported to exhibit anti-tubercular [3], anti-viral [4,5], anti-microbial [6,7], and neuroprotective [8,9] activity. Quinoxaline derivatives have been reported to be prepared, but not limited, by intramolecular cyclisation of N-substituted aromatic ortho-diamines [10], ring transformation of benzofurazans [11], and condensation of benzofuran-1-oxide to form quinoxaline-N-oxides [12]. The most common method for the preparation of quinoxaline derivatives relies on the condensation of an aryl 1,2-diamine with a 1,2-dicarbonyl compound, of which this type of reaction has limitations due to the use of pre-defined starting materials [13][14][15][16], which limit the number of substituents that can be added.
A more convenient approach for the synthesis of quinoxaline derivatives is the substitution of hydrogen at the 2-or 3-position of quinoxaline by C-nucleophiles. Substitution of hydrogen by C-nucleophiles on electron deficient arenes has been reported extensively [17][18][19]. The reaction of quinoxaline derivatives with aryl and alkyl nucleophiles has been investigated [20][21][22][23][24][25][26]. C-nucleophiles have been shown to add to 6-aminoquinoxaline [9,20] affording alkyl-and aryl-substituted quinoxalines in low to high yields. Although efficient, these reactions require the use of an oxidising agent to afford aromatic substitution products. Azev et al. [21], reported formation of resorcinol derivative, 2-(2,4-dihydroxyphenyl)quinoxaline after reacting quinoxaline and resorcinol in refluxing ethanol (EtOH) in the presence of an acid. The reaction of quinoxaline with this nucleophile continues to oxidative rearomatization of the compound without the need of having an oxidizing agent, but requires the use of concentrated HCl and high temperature. The use of nucleophilic substitution has been extended to other functional groups like aromatic amines and heteroaryls, but examples of quinoxaline derivatives containing these groups are limited. The reaction of 2-chloro-3-methylquinoxaline with

Results
Our investigation began with the preparation of 2-benzensulfonyloxyquinoxaline 1 which was prepared from our previously reported method [27]. 2-Phenylquinoxaline 2 was prepared via Negishi coupling of 1 and 1.14 M solution of phenyl-ZnCl, in 75% yield (Scheme 1). Treating 2 with 0.5 equivalents of n-BuLi gave 3-butyl-2-phenylquinoxaline 2a in 12% yield. The oxidative elimination of H2 occurs in atmospheric oxygen, to exclusively provide the aromatic product. Increasing the amount n-BuLi to 1.5 equivalents gave 2a in 66% yield, which is a significant improvement. This method was extended to other; aryl-, heteroaryl-, alkynyl-, and alkyl-nucleophiles on compound 2, of which the compounds 2a-2g were synthesised (Table 1).  Alkyl nucleophiles afforded 2,3-disubstituted quinoxalines in higher yields compared to the aryl and alkynyl nucleophiles (entries 1, 2 vs. entries 3-7). The high electron density found on the aryl and alkynyl nucleophiles could be responsible for the low to moderate yields observed.  iodide, forming the corresponding 2-arylamino-3-methylquinoxalines, has been described by Badr et al. [22]. This method only describes 2-arylaminoquinoxaline derivatives containing a methyl-substituent at the 3-position. A more recent strategy by Zhou entails direct heteroarylation of N-heteroarenes by heteroaryl Grignard reagents. Although this method gives high yields and is efficient, it focuses solely on heterobiaryl preparation for double functionalization [23,25,26]. Phenylacetylide nucleophile was also reported to add to quinoxaline via nucleophilic substitution, affording mono-substituted and di-substituted quinoxaline [24]. The influence of substituents at the 2-position on nucleophilic substitution by C-nucleophiles has not been fully investigated. Herein, we wish to report nucleophilic substitution reactions on mono-substituted quinoxalines, bearing an aryl, alkyl, alkynyl, and amino substituents at the 2-position. By starting with quinoxaline bearing a benzenesulfonyloxy leaving group, which we previously reported to be an efficient leaving group in coupling of pteridines [27,28], we envisaged that we can prepare mono-substituted quinoxaline derivatives, employing the Negishi [29], Sonogashira [30], and Buchwald coupling conditions [31].
Alkyl nucleophiles afforded 2,3-disubstituted quinoxalines in higher yields compared to the aryl and alkynyl nucleophiles (entries 1, 2 vs. entries 3-7). The high electron density found on the aryl and alkynyl nucleophiles could be responsible for the low to moderate yields observed. Alkyl nucleophiles afforded 2,3-disubstituted quinoxalines in higher yields compared to the aryl and alkynyl nucleophiles (entries 1, 2 vs. entries 3-7). The high electron density found on the aryl and alkynyl nucleophiles could be responsible for the low to moderate yields observed.
We also envisaged that the phenyl group attached at the 2-position could be affecting the yields, presumably due to steric hindrance. We then synthesised 2-butylquinoxaline 3, which contain a less bulky substituent, by Negishi coupling of 1a and butyl-ZnCl. Reacting compound 3 with 1.5 eq of n-BuLi, gave 2,3-dibutylquinoxaline 3a in 76% yield. When the number of equivalents of n-BuLi was increased to 3.0 eq, the target compound 3a was isolated in 97% yield. We were also able to synthesize 2-butyl-3-(furan-2-yl)quinoxaline 3b in 91% yield, 3-butyl-2-phenylquinoxaline 2a in 95% yield and 3-butyl-2-(2-phenylethynyl)quinoxaline 3c in 65% yield. The use of 2-butylquinoxaline 3 for the synthesis of 3a-c and 2a (Table 2) further supports the assumption that the low yields are due to the phenyl-group. The use of a less bulky substituent, in this case, makes nucleophilic substitution occur readily, affording products in good yields. We also envisaged that the phenyl group attached at the 2-position could be affecting the yields, presumably due to steric hindrance. We then synthesised 2-butylquinoxaline 3, which contain a less bulky substituent, by Negishi coupling of 1a and butyl-ZnCl. Reacting compound 3 with 1.5 eq of n-BuLi, gave 2,3-dibutylquinoxaline 3a in 76% yield. When the number of equivalents of n-BuLi was increased to 3.0 eq, the target compound 3a was isolated in 97% yield. We were also able to synthesize 2-butyl-3-(furan-2-yl)quinoxaline 3b in 91% yield, 3-butyl-2-phenylquinoxaline 2a in 95% yield and 3-butyl-2-(2-phenylethynyl)quinoxaline 3c in 65% yield. The use of 2-butylquinoxaline 3 for the synthesis of 3a-c and 2a (Table 2) further supports the assumption that the low yields are due to the phenyl-group. The use of a less bulky substituent, in this case, makes nucleophilic substitution occur readily, affording products in good yields.

Entry
Product The successful synthesis of compounds 2a-2g, and 3a-c, encouraged us to further investigate the possibilities of nucleophilic addition on compounds with a different functional group on the 2-position of the quinoxaline moiety. We wanted to investigate the possibilities for nucleophilic substitution if the quinoxaline moiety at the 2-position had a Csp 2 -Csp bond. We thus employed Sonogashira crosscoupling conditions to synthesise 2-(2-phenylethynyl)quinoxaline 4. Treating 4 with 3.0 equivalents of n-BuLi gave 3-butyl-2-(2-phenylethynyl)quinoxaline 3c in 20% yield. Increasing the reaction time from 18 h to 36 h improved the percentage yield to 56% (Scheme 2). Attempts to extend the scope to other C-nucleophiles employed on quinoxaline 2, were unsuccessful as only the starting material was recovered. We postulate that nucleophilic substitution on 2-(2phenylethynyl)quinoxaline 4 was difficult due to the electron donating nature of the phenylalkynyl attached at the 2-position of the quinoxaline moiety. This is possibly due to the high electron density caused by highly delocalized pi-electrons from the phenyl ring and the acetynyl moiety, which could make the -N=C-H site less electrophilic. This suggests that only very strong nucleophiles, such as n-BuLi, can work efficiently. We then proceeded to investigate the possibility of nucleophilic attack if the bonding atom on the 2-position of the quinoxaline was C-N. We used Buchwald-Hartwig cross-coupling reaction to synthesize quinoxaline derivatives containing an arylamine.
The coupling reaction between 2-benzenesulfonyloxyquinoxaline 1 and aniline, under Buchwald-Hartwig conditions, formed N-phenylquinoxalin-2-amine 5. When we extended nucleophilic substitution on the compounds synthesized by Buchwald-Hartwig coupling, we found that the results were similar to those on the Sonogashira coupled product 4. When N-phenylquinoxalin-2-amine 5 was reacted with n-BuLi (3.0 eq) and left to stir for 18 h, the target compound 3-butyl-N-phenylquinoxalin-2-amine 5a was isolated in 15% yield and there was also recovery of the starting material 5. When the reaction time was increased to 36 h, compound 5a was isolated in 32% yield (Table 3), and there was still recovery of the starting material. Reacting 5 with a less bulky nucleophile, Me-MgCl, gave 3-methyl- The successful synthesis of compounds 2a-2g, and 3a-c, encouraged us to further investigate the possibilities of nucleophilic addition on compounds with a different functional group on the 2-position of the quinoxaline moiety. We wanted to investigate the possibilities for nucleophilic substitution if the quinoxaline moiety at the 2-position had a Csp 2 -Csp bond. We thus employed Sonogashira cross-coupling conditions to synthesise 2-(2-phenylethynyl)quinoxaline 4. Treating 4 with 3.0 equivalents of n-BuLi gave 3-butyl-2-(2-phenylethynyl)quinoxaline 3c in 20% yield. Increasing the reaction time from 18 h to 36 h improved the percentage yield to 56% (Scheme 2). We also envisaged that the phenyl group attached at the 2-position could be affecting the yields, presumably due to steric hindrance. We then synthesised 2-butylquinoxaline 3, which contain a less bulky substituent, by Negishi coupling of 1a and butyl-ZnCl. Reacting compound 3 with 1.5 eq of n-BuLi, gave 2,3-dibutylquinoxaline 3a in 76% yield. When the number of equivalents of n-BuLi was increased to 3.0 eq, the target compound 3a was isolated in 97% yield. We were also able to synthesize 2-butyl-3-(furan-2-yl)quinoxaline 3b in 91% yield, 3-butyl-2-phenylquinoxaline 2a in 95% yield and 3-butyl-2-(2-phenylethynyl)quinoxaline 3c in 65% yield. The use of 2-butylquinoxaline 3 for the synthesis of 3a-c and 2a (Table 2) further supports the assumption that the low yields are due to the phenyl-group. The use of a less bulky substituent, in this case, makes nucleophilic substitution occur readily, affording products in good yields.

Entry
Product The successful synthesis of compounds 2a-2g, and 3a-c, encouraged us to further investigate the possibilities of nucleophilic addition on compounds with a different functional group on the 2-position of the quinoxaline moiety. We wanted to investigate the possibilities for nucleophilic substitution if the quinoxaline moiety at the 2-position had a Csp 2 -Csp bond. We thus employed Sonogashira crosscoupling conditions to synthesise 2- Attempts to extend the scope to other C-nucleophiles employed on quinoxaline 2, were unsuccessful as only the starting material was recovered. We postulate that nucleophilic substitution on 2-(2phenylethynyl)quinoxaline 4 was difficult due to the electron donating nature of the phenylalkynyl attached at the 2-position of the quinoxaline moiety. This is possibly due to the high electron density caused by highly delocalized pi-electrons from the phenyl ring and the acetynyl moiety, which could make the -N=C-H site less electrophilic. This suggests that only very strong nucleophiles, such as n-BuLi, can work efficiently. We then proceeded to investigate the possibility of nucleophilic attack if the bonding atom on the 2-position of the quinoxaline was C-N. We used Buchwald-Hartwig cross-coupling reaction to synthesize quinoxaline derivatives containing an arylamine.
The coupling reaction between 2-benzenesulfonyloxyquinoxaline 1 and aniline, under Buchwald-Hartwig conditions, formed N-phenylquinoxalin-2-amine 5. When we extended nucleophilic substitution on the compounds synthesized by Buchwald-Hartwig coupling, we found that the results were similar to those on the Sonogashira coupled product 4. When N-phenylquinoxalin-2-amine 5 was reacted with n-BuLi (3.0 eq) and left to stir for 18 h, the target compound 3-butyl-N-phenylquinoxalin-2-amine 5a was isolated in 15% yield and there was also recovery of the starting material 5. When the reaction time was increased to 36 h, compound 5a was isolated in 32% yield (Table 3), and there was still recovery of the starting material. Reacting 5 with a less bulky nucleophile, Me-MgCl, gave 3-methyl- Attempts to extend the scope to other C-nucleophiles employed on quinoxaline 2, were unsuccessful as only the starting material was recovered. We postulate that nucleophilic substitution on 2-(2-phenylethynyl)quinoxaline 4 was difficult due to the electron donating nature of the phenylalkynyl attached at the 2-position of the quinoxaline moiety. This is possibly due to the high electron density caused by highly delocalized pi-electrons from the phenyl ring and the acetynyl moiety, which could make the -N=C-H site less electrophilic. This suggests that only very strong nucleophiles, such as n-BuLi, can work efficiently. We then proceeded to investigate the possibility of nucleophilic attack if the bonding atom on the 2-position of the quinoxaline was C-N. We used Buchwald-Hartwig cross-coupling reaction to synthesize quinoxaline derivatives containing an arylamine.
The coupling reaction between 2-benzenesulfonyloxyquinoxaline 1 and aniline, under Buchwald-Hartwig conditions, formed N-phenylquinoxalin-2-amine 5. When we extended nucleophilic substitution on the compounds synthesized by Buchwald-Hartwig coupling, we found that the results were similar to those on the Sonogashira coupled product 4. When N-phenylquinoxalin-2-amine 5 was reacted with n-BuLi (3.0 eq) and left to stir for 18 h, the target compound 3-butyl-N-phenylquinoxalin-2-amine 5a was isolated in 15% yield and there was also recovery of the starting material 5. When the reaction time was increased to 36 h, compound 5a was isolated in 32% yield (Table 3), and there was still recovery of the starting material. Reacting 5 with a less bulky nucleophile, Me-MgCl, gave 3-methyl-N-phenylquinoxalin-2-amine 5b in 42% yield. The low yields could be due to the competition between deprotonation of the free N-H and nucleophilic substitution at the -N=C-H site. The N-H group on compound 5, was methylated to give N-methyl-N-phenylquinoxalin-2-amine 6. N-phenylquinoxalin-2-amine 5b in 42% yield. The low yields could be due to the competition between deprotonation of the free N-H and nucleophilic substitution at the -N=C-H site. The N-H group on compound 5, was methylated to give N-methyl-N-phenylquinoxalin-2-amine 6. Reacting quinoxaline 6 with n-BuLi gave 3-butyl-N-methyl-N-phenylquinoxalin-2-amine 6a in 92% yield and no starting material was recovered. With the protecting group (methyl-) on the nitrogen, the n-BuLi was able to fully react with the starting material 6 with no other competing reactions (Table 3). Attempts to use other nucleophiles on the mono-substituted Buchwald-Hartwig coupled products were not successful. Similarly to the Sonogashira coupled product 4, only strong alkyl nucleophiles can readily react at the 3-position of the quinoxaline moiety. This could be due to steric hindrance accompanied by electron density on nitrogen atom, making the -N=C-H site less reactive towards nucleophiles.
In conclusion, nucleophilic substitution on 2-phenylquinoxaline 2 and 2-butylquinoxaline 3, proceeded smoothly to give 2,3-disubstituted quinoxalines. Di-substituted quinoxalines bearing aryl-alky, aryl-aryl, aryl-heteroaryl, and aryl-alkynyl substituents were prepared in moderate to good yields. Nucleophilic substitutions on 2-acetylene and 2-amine quinoxaline, were only possible with strong alkyl nucleophiles (n-BuLi and Me-MgCl). This could be due to steric and electronic properties associated with these substituents. Molecular modelling studies are being conducted to understand the influence of the substituent attached at the 2-position, and the nature of the nucleophile.

General Information
Reagents were purchased from Sigma-Aldrich (Johannesburg, South Africa) and were used without further purification. Melting points were obtained using Lasec/SA-melting point apparatus from Lasec Company, SA (Johannesburg, South Africa). 1 H-and 13 C-NMR spectra were recorded at 400 MHz and 100 MHz, respectively, on Bruker Avance-400 spectrometer (Johannesburg, South Africa). Spectra were recorded in deuterated chloroform (CDCl3) unless otherwise specified. Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane as an internal standard at zero ppm. The 1 H-NMR chemical shifts are reported: value (number of hydrogens, description of signal, assignment) and the 13 C-NMR chemical shifts are reported: value (assignment). Abbreviations used: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, and m = multiplet. (1) In a round bottom flask, quinoxalinone (5 g, 34 mmol), DMAP (0.416 g, 3.4 mmol) and benzenesulfonyl chloride (8.72 mL, 68 mmol) were dissolved in DCM (100 mL), cooled to 0 °C and stirred for 5 min . Et3N (12 mL, 88 mmol) was added drop-wise over 5 min, the solution allowed to stir at room temperature for 1 h, and the reaction quenched with aqueous NaHCO3 (80 mL). The two layers Reacting quinoxaline 6 with n-BuLi gave 3-butyl-N-methyl-N-phenylquinoxalin-2-amine 6a in 92% yield and no starting material was recovered. With the protecting group (methyl-) on the nitrogen, the n-BuLi was able to fully react with the starting material 6 with no other competing reactions (Table 3). Attempts to use other nucleophiles on the mono-substituted Buchwald-Hartwig coupled products were not successful. Similarly to the Sonogashira coupled product 4, only strong alkyl nucleophiles can readily react at the 3-position of the quinoxaline moiety. This could be due to steric hindrance accompanied by electron density on nitrogen atom, making the -N=C-H site less reactive towards nucleophiles.

2-Benzensulfonyloxyquinoxaline
In conclusion, nucleophilic substitution on 2-phenylquinoxaline 2 and 2-butylquinoxaline 3, proceeded smoothly to give 2,3-disubstituted quinoxalines. Di-substituted quinoxalines bearing aryl-alky, aryl-aryl, aryl-heteroaryl, and aryl-alkynyl substituents were prepared in moderate to good yields. Nucleophilic substitutions on 2-acetylene and 2-amine quinoxaline, were only possible with strong alkyl nucleophiles (n-BuLi and Me-MgCl). This could be due to steric and electronic properties associated with these substituents. Molecular modelling studies are being conducted to understand the influence of the substituent attached at the 2-position, and the nature of the nucleophile.

General Information
Reagents were purchased from Sigma-Aldrich (Johannesburg, South Africa) and were used without further purification. Melting points were obtained using Lasec/SA-melting point apparatus from Lasec Company, SA (Johannesburg, South Africa). 1 H-and 13 C-NMR spectra were recorded at 400 MHz and 100 MHz, respectively, on Bruker Avance-400 spectrometer (Johannesburg, South Africa). Spectra were recorded in deuterated chloroform (CDCl 3 ) unless otherwise specified. Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane as an internal standard at zero ppm. The 1 H-NMR chemical shifts are reported: value (number of hydrogens, description of signal, assignment) and the 13 C-NMR chemical shifts are reported: value (assignment). Abbreviations used: s = singlet, d = doublet, dd = doublet of doublets, t = triplet, and m = multiplet.

2-Benzensulfonyloxyquinoxaline (1)
In a round bottom flask, quinoxalinone (5 g, 34 mmol), DMAP (0.416 g, 3.4 mmol) and benzenesulfonyl chloride (8.72 mL, 68 mmol) were dissolved in DCM (100 mL), cooled to 0 • C and stirred for 5 min. Et 3 N (12 mL, 88 mmol) was added drop-wise over 5 min, the solution allowed to stir at room temperature for 1 h, and the reaction quenched with aqueous NaHCO 3 (80 mL). The two layers were separated and the aqueous layer washed twice with DCM (2 × 60 mL). The combined organic layers were dried over MgSO 4 , filtered, concentrated, and dissolved in EtOAc (15 mL). The filtrate was concentrated to give 2-benzenesulfonyloxyquinoxaline as a brown solid (8.  (2) ZnCl 2 (0.95 g, 6.99 mmol, 2.0 eq, taken directly from the oven) was added to an oven dried 2-neck flask with a stirrer bar, which was further dried under vacuum for 15 min. Dry THF (10 mL) was added to the flask under dry nitrogen. The solution was left to stir until all dried ZnCl 2 was dissolved. Phenyl-MgBr (6.3 mL, 6.29 mmol, 1 M) was added to the solution and left to stir for 1 h. To a solution of Phenyl-ZnCl (6.29 mmol), 2-benzenesulfonyloxyquinoxaline 1 (1 g, 3.49 mmol) and PdCl 2 (PPh 3 ) 2 (122.6 mg, 0.18 mmol, 5 mol %) were added and set to reflux while stirring under nitrogen for 20 h. The solution was allowed to cool to room temperature, diluted with EtOAc (25 mL), quenched with sat. NaHCO 3 (15 mL). The two layers were separated and the aqueous layer diluted with EtOAc (15 mL). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated, and purified on silica gel eluting with 20% EtOAc/Hexane to give 2-phenylquinoxaline as a yellow solid (0.537 g, 75%); m.p. General Methods for Nucleophilic Substitution on 2-Phenylquinoxaline Method 1: A solution of n-BuLi or Mg-aryl/alkyl was treated with 2-phenylquinoxaline 2 (100 mg, 0.485 mmol), and the solution allowed to stir at room temperature for 18 h. The final solution was diluted with EtOAc (10 mL), quenched with sat. NaHCO 3 (10 mL). The organic layers were combined and dried over Na 2 SO 4 , filtered, concentrated, and purified by flash column chromatography on silica gel.

Method 2:
A solution of either an alkyne or hetero-aromatic substrate (0.97 mmol) in THF (5 mL) was lithiated with n-BuLi (0.4 mL, 0.97 mmol, 2 eq, 2.5 M) and left to stir for 15 min under a nitrogen atmosphere. 2-Phenylquinoxaline 2 (100 mg, 0.485 mmol) was added to the flask, and the solution allowed to stir at room temperature for 18 h. The final solution was diluted with EtOAc (10 mL), quenched with sat. NaHCO 3 (10 mL). The organic layers were combined and dried over Na 2 SO 4 , filtered, concentrated, and purified by flash column chromatography on silica gel.