molecules Article
Ionic Liquid as an Efficient Medium for the Synthesis of Quinoline Derivatives via α-Chymotrypsin-Catalyzed Friedländer Condensation Zhang-Gao Le 1,2 , Meng Liang 2 , Zhong-Sheng Chen 2 , Sui-Hong Zhang 2 and Zong-Bo Xie 1,2, * 1 2
*
Jiangxi 2011 Joint Center for the Innovative Mass Spectrometry and Instrumentation, East China University of Technology, Nanchang 330013, China;
[email protected] School of Chemistry, Biology and Material Science, East China University of Technology, Nanchang 330013, China;
[email protected] (M.L.);
[email protected] (Z.-S.C.);
[email protected] (S.-H.Z.) Correspondence:
[email protected]; Tel.: +86-791-8389-6550
Academic Editor: Hua Zhao Received: 30 March 2017; Accepted: 4 May 2017; Published: 8 May 2017
Abstract: An efficient, convenient, and eco-friendly biocatalytic approach was developed for the synthesis of quinoline derivatives via the α-chymotrypsin-catalyzed Friedländer reaction. Interestingly, α-chymotrypsin exhibited higher catalytic activity in an ionic liquid (IL) aqueous solution as compared to that observed in our previous relevant study, which was conducted using an organic solvent, and a series of substrates gave similar excellent yields at lower reaction temperature and under reduced enzyme-loading conditions. Keywords: Friedländer reaction; quinolines; α-chymotrypsin; ionic liquid; biocatalysis; promiscuity
1. Introduction Quinoline derivatives are of great significance in medicinal chemistry [1] as they usually display a broad range of excellent pharmacological activities, including anticancer [2], antiviral [3], anti-microbial [4], antifungal [5], anti-inflammatory [6], and anti-platelet aggregation [7] activities, which render them important drug intermediates. In addition, quinoline derivatives are valuable synthons for the preparation of nano- and mesostructures with enhanced electronic and photonic properties [8,9]. Various methods were developed for the synthesis of this class of compounds, such as the Skraup synthesis [10], Doebner–Von Miller reaction [11], Combes synthesis [12], and the Friedländer method [13]. Among these, the Friedländer method, which involves a condensation reaction between a 2-aminoaryl ketone and a α-methylene ketone, is the most simple and straightforward. Generally, this process can be carried out in the presence of base or acid catalysts to yield quinoline derivatives. However, this method often suffers from poor selectivity, complicated procedures, or harsh conditions [14,15] and is not practically feasible. Over recent years, biocatalysis has attracted increasing interest in synthetic chemistry because it offers high efficiency and excellent selectivity and requires mild reaction conditions; thus, biocatalysis has been identified as an eco-friendly and sustainable alternative to traditional organic synthesis [16,17]. In particular, several valuable studies on enzymatic promiscuity have been reported; for instance, hydrolase catalyzes unconventional reactions such as the Aldol reaction [18,19], the Mannich reaction [20], and the Henry reaction [21]. In our previous work, we demonstrated the first α-chymotrypsin-catalyzed Friedländer condensation between a 2-aminoaryl ketone and an α-methylene ketone in an organic solvent [22], but the environmental problems related to the use of organic solvents are a matter of serious concern. Room-temperature ionic liquids (RTILs), superior alternatives to organic solvents, have many potential benefits for biochemical processes, especially where the reaction substrates and biocatalysts can be dissolved better, leading to
Molecules 2017, 22, 762; doi:10.3390/molecules22050762
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Molecules 2017, 22, 762
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environmental problems related to the use of organic solvents are a matter of serious concern. environmental problems related to the use of organic solvents are a matter of serious concern. Room-temperature Molecules 2017, 22, 762 ionic liquids (RTILs), superior alternatives to organic solvents, have many 2 of 8 Room-temperature ionic liquids processes, (RTILs), superior to organic solvents, many potential benefits for biochemical especiallyalternatives where the reaction substrates and have biocatalysts potential benefitsbetter, for biochemical especially where the reaction substrates and biocatalysts can be dissolved leading toprocesses, a more efficient reaction process. Recently, some hydrolases have a more efficienttoreaction process. some have been reported to catalyze organic can bereported dissolved better, leading toRecently, a more efficient reaction Recently, some hydrolases have been catalyze organic reactions for thehydrolases synthesisprocess. of peptides [23] and esters [24,25] in an reactions for the synthesis of peptides [23] and esters [24,25] in an ionic liquid (IL) aqueous solution. been reported to catalyze organic reactions for the synthesis of peptides [23] and esters [24,25] in ionic liquid (IL) aqueous solution. The enzyme activity in this medium was clearly enhanced an in The activity inin this medium was enhanced in comparison with thatclearly in organic solvents, ionicenzyme liquid with (IL) aqueous solution. The clearly enzyme activity inresults this medium was enhanced in comparison that organic solvents, and the excellent obtained suggested that ILs hold and the excellent results obtained suggested hold great potential as efficient reaction comparison with in organic solvents, andthat theILs excellent results obtained suggested that ILsmedia hold great potential as that efficient reaction media for biocatalysis. for biocatalysis. great potential efficient reaction media for biocatalysis. As part of as our continuing interest in enzymatic synthesis and green synthetic methodologies, we part ofand ourefficient continuing interestinin enzymatic synthesis and green synthetic methodologies, Asa part our continuing interest enzymatic and green synthetic we report mildof biocatalytic route for thesynthesis Friedländer reaction. Here, methodologies, α-chymotrypsin is we report a mild and efficient biocatalytic route for the Friedländer reaction. Here, α-chymotrypsin is report a mild and efficient biocatalytic route for the Friedländer reaction. Here, α-chymotrypsin reported for the first time to catalyze the condensation reaction between a 2-aminoaryl ketone and a reported for the firstfor time catalyze reactioninbetween ketone and a reported time to catalyze the the condensation between a 2-aminoaryl 2-aminoaryl ketone α-methylene ketone theto preparation ofcondensation quinoline derivatives an IL (1-ethyl-3-methylimidazolium of quinoline derivatives in 1). an α-methylene ketoneaqueous for the preparation quinoline derivatives an IL IL(1-ethyl-3-methylimidazolium (1-ethyl-3-methylimidazolium tetrafluoroborate) solution ([EMIM][BF 4]/H 2O) (Scheme ]/H22O) O)(Scheme (Scheme1). 1). tetrafluoroborate) aqueous solution solution ([EMIM][BF ([EMIM][BF44]/H
Scheme 1. Friedländer condensation reaction between 2-aminoaryl ketones and α-methylene ketone Scheme 1. Friedländer Friedländer condensation between 2-aminoaryl 2-aminoaryl ketones ketones and and α-methylene α-methylene ketone ketone in ionic liquid (IL) aqueous solution. reaction Scheme 1. condensation reaction between in ionic liquid liquid (IL) (IL) aqueous aqueous solution. solution. in ionic
2. Results and Discussion 2. Results and Discussion 2. Results and Discussion The reaction of 2-aminoacetophenone and ethyl acetoacetate was chosen as the model reaction The reactionthe of 2-aminoacetophenone and ethyl acetoacetate was chosen as the model reaction for determining conditions (Scheme 2). It acetoacetate is well known that enzyme activity is strongly The reaction of optimal 2-aminoacetophenone and ethyl was chosen as the model reaction for determining the optimal conditions (Scheme 2). It is well known that enzyme activity is strongly affected by the reaction medium. In order to select theis optimal reaction the initial for determining the optimal conditions (Scheme 2). It well known thatmedium, enzyme activity is affected by was the carried reactionoutmedium. In order toILs, select the optimal reaction medium, the initial experiment in six different dry including three tetrafluoroborate ILs (1-ethyl-3strongly affected by the reaction medium. In order to select the optimal reaction medium, the initial experiment was carried out in six different dry ILs, including three tetrafluoroborate (1-ethyl-3methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate,ILs and 1-hexylexperiment was carried out in six different dry ILs, including three tetrafluoroborate ILs (1-ethyl-3methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, and 1-hexyl3-methylimidazolium tetrafluoroborate) and three hexafluorophosphate ILsand(1-ethyl-3methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-hexyl-33-methylimidazolium tetrafluoroborate) and three hexafluorophosphate ILs (1-ethyl-3methylimidazolium hexafluorophosphate, and methylimidazolium hexafluorophosphate, tetrafluoroborate) 1-butyl-3-methylimidazolium and three hexafluorophosphate ILs (1-ethyl-3methylimidazolium hexafluorophosphate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-hexyl-3-methylimidazolium hexafluorophosphate). Unfortunately, α-chymotrypsin exhibited methylimidazolium hexafluorophosphate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-hexyl-3-methylimidazolium hexafluorophosphate). Unfortunately, α-chymotrypsin exhibited extremely poor catalytic activity, and the product could Unfortunately, not be detected α-chymotrypsin by TLC. This wasexhibited possibly 1-hexyl-3-methylimidazolium hexafluorophosphate). extremely poor viscosity catalytic of activity, and the productwhich couldlimited not be mass detected by TLC. Thisthe wassubstrates possibly due to the high the reaction medium, transfer between extremely poor catalytic activity, and the product could not be detected by TLC. This was possibly due due to the high viscosity of the reaction medium, which limited mass transfer between the substrates and active site ofofthe Then, 10% (H2O/(IL + Hmass 2O), v/v) water was added to the ILs to to thethe high viscosity theenzyme. reaction medium, which limited transfer between the substrates and and thetheir activeviscosity, site of the enzyme. Then,of10% (H2O/(IL + H2O), v/v) water wasenhanced. added to To the ILs to reduce and the activity α-chymotrypsin was found be the active site of the enzyme. Then, 10% (H2 O/(IL + H2 O), v/v) water wastoadded to the ILs to clearly reduce reduce theirbetween viscosity, and the activity of water α-chymotrypsin was found to be results enhanced. To clearly distinguish the various ILs, more was added to each IL; better were obtained their viscosity, and the activity of α-chymotrypsin was found to be enhanced. To clearly distinguish distinguish between various ILs,1),more water was added to each IL; At better werewe obtained when 50% water wasthe used (Figure andadded the best reached the results same time, found between the various ILs, more water was to yield each IL; better51%. results were obtained when 50% − whendifferent 50% water was used (Figureeffect 1), and best yield reached 51%. At the same time, wea cation found that ILs have a different on the enzyme activity, and the ILs with [BF 4] and water was used (Figure 1), and the best yield reached 51%. At the same time, we found that different that differentshort ILs have a different effect on theprovide enzyme activity, and the ILs with [BF 4]− and a cation containing could better results, 1-ethyl-3− and ILs have a different 1-alkyl effect ongroups the enzyme activity, and the ILs with [BF4 ]among a which cation containing containing short 1-alkyl groups could provide better results, among which 1-ethyl-3methylimidazolium to superior activity. The probable reason short 1-alkyl groupstetrafluoroborate could provide contributed better results, among enzyme which 1-ethyl-3-methylimidazolium methylimidazolium tetrafluoroborate contributed toa superior enzyme activity. The probable reason for this is that the IL with the cation containing shorter alkyl chain has higher thus tetrafluoroborate contributed to superior enzyme activity. The probable reason for thispolarity, is that the IL for this is that the IL with the cation containing a shorter alkyl chain has higher polarity, thus increasing the solubility of the substrates and enzyme, and eventually accelerating the reaction process. with the cation containing a shorter alkyl chain has higher polarity, thus increasing the solubility of the increasing the solubility ofchose the substrates and enzyme, and eventually accelerating [EMIM][BF the reaction]process. As indicated weand (1-ethyl-3-methylimidazolium aschose the substrates andabove, enzyme, eventually accelerating the reactiontetrafluoroborate) process. As indicated above, 4we As indicated above, we chose (1-ethyl-3-methylimidazolium tetrafluoroborate) [EMIM][BF4] as the optimal IL for further reactions. (1-ethyl-3-methylimidazolium tetrafluoroborate) [EMIM][BF4 ] as the optimal IL for further reactions. optimal IL for further reactions.
Scheme 2. Model reaction between 2-aminoacetophenone and ethyl acetoacetate. Scheme 2. Model reaction between 2-aminoacetophenone and ethyl acetoacetate.
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Yieldb (%)
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][B IM EM
]
F4
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]
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F6 F4 F4 F F [P [B [B [P 6 [P 6 M] M] M] M] M] MI MI MI MI MI [H [B [B [E [H
Figure 1. Reaction yields yields in in different differentIL ILaqueous aqueoussolutions solutionsa .a.aaReaction Reactionconditions: conditions: 2-aminoacetophenone 2-aminoacetophenone (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (10 mg) in 50% (H 2O/(IL++H H22O), O), v/v) v/v) mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (10 mg) in 50% (H 2 O/(IL ◦ b b IL aqueous solution at 60 °C C for 24 h. Isolated Isolated yield yield after after column column chromatography. chromatography.
b Yield (%)
To explain explain the content was investigated in To the low low yield yield observed observedin insome somecases, cases,the theeffect effectofofwater water content was investigated further detail (Figure 2). The contentcontent was found have to a notable on the activity of in further detail (Figure 2). water The water was tofound have ainfluence notable influence on the α-chymotrypsin. An increase in the water content led to an enhancement in the synthetic activity of activity of α-chymotrypsin. An increase in the water content led to an enhancement in the synthetic α-chymotrypsin. When the water content reached 80% (H 2O/([BMIM][BF4] + H2O), v/v), α-chymotrypsin activity of α-chymotrypsin. When the water content reached 80% (H2 O/([BMIM][BF4 ] + H2 O), v/v), exhibited the bestexhibited catalytic the activity an excellent yield an of 87%; in other a 20% IL aqueous α-chymotrypsin best with catalytic activity with excellent yieldwords, of 87%; in other words, solution can give results that are superior to those observed previously when using an organic a 20% IL aqueous solution can give results that are superior to those observed previously when using solvent. From the discussion above, we can conclude that thethat IL the with waterwater content can an organic solvent. From the discussion above, we can conclude IL optimal with optimal content provide an appropriate environment for for thethe mass transfer ofofsubstrates the can provide an appropriate environment mass transfer substratestotothe theactive active site site of of the enzyme, in which the enzyme activity was improved to some degree, thus allowing for the efficient enzyme, in which the enzyme activity was improved to some degree, thus allowing for the efficient synthesis of of quinoline quinoline derivatives. derivatives. Thus, aqueous solution chosen as as the the optimum optimum synthesis Thus, the the 20% 20% IL IL aqueous solution was was chosen Molecules 2017, 22, 762 4 of 8 medium for the α-chymotrypsin-catalyzed Friedländer condensation. medium for the α-chymotrypsin-catalyzed Friedländer condensation. In our previous work, heating at 60 °C contributed to superior yields; hence, the above-mentioned reaction was performed at 60 °C. Since the reaction in the 20% IL aqueous solution presented a 90 superior trend in the yields, we decided to verify whether a lower temperature could afford 80 excellent results in IL aqueous 70 solution. Thus, we performed the model reaction at six different temperatures ranging from 40 to 60 70 °C for 24 h (Figure 3). It can be seen that the enzyme activity was lower at lower temperatures (4050°C and 45 °C). At 55 °C, the reaction gave superior results, with yields of up to 82% as compared40with our previous work, where the temperature was set at 60 °C. From this, we can see that the IL 30 aqueous solution had worked, and the result had reached our requirement. Although temperatures of 60 °C and 70 °C contributed to superior results, as a higher 20 temperature and IL [26] can also promote this reaction (in Figure 4, when the enzyme loading was 0 mg), 10 the key role of the enzyme as a catalyst in the60reaction was20not obvious in this case, and the enzyme 80 40 0 easily reduced its activity at higher temperature. On the other hand, the energy consumption, too, The concentration of IL aqueous solution (%) had to be considered. Thus, implementing the reaction at 55 °C was the best strategy in an IL a a Figure 2. aqueous solution. Figure 2. Reaction Reaction yield yield in inIL ILaqueous aqueoussolutions solutionswith withdifferent differentconcentrations concentrations a.. a Reaction Reaction conditions: conditions: 2-aminoacetophenone 2-aminoacetophenone (0.3 (0.3 mmol), mmol), ethyl ethyl acetoacetate acetoacetate (0.36 (0.36 mmol), mmol), and and α-chymotrypsin α-chymotrypsin (10 (10 mg) mg) in in IL IL aqueous solutions of different concentrations (80 to 0%, [EMIM][BF4 ]/(H2 O + [EMIM][BF4 ]), v/v) at aqueous solutions of different concentrations (80 to 0%, [EMIM][BF4]/(H2O + [EMIM][BF4]), v/v) at 60 °C 60 ◦ C for b24 h. b Isolated yield after column chromatography. for 24 h. Isolated yield after column chromatography.
Yieldb (%)
◦ In our previous work, heating 100 at 60 C contributed to superior yields; hence, the above-mentioned ◦ reaction was performed at 60 C. Since the reaction in the 20% IL aqueous solution presented a superior 90 trend in the yields, we decided to80verify whether a lower temperature could afford excellent results in IL aqueous solution. Thus, we performed the model reaction at six different temperatures ranging 70 from 40 to 70 ◦ C for 24 h (Figure 60 3). It can be seen that the enzyme activity was lower at lower 50 40 30 20 40
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◦ C). At 10
temperatures (40 and 45 55 ◦ C, the reaction gave superior results, with yields of up to 82% ◦ as compared with our previous work,80where60the temperature was 20 40 0 set at 60 C. From this, we can The concentration of IL result aqueous solution (%) see that the IL aqueous solution had worked, and the had reached our requirement. Although ◦ ◦ temperatures of 60 C and 70 C contributed to superior results, as a higher temperature and IL [26] Figure 2. Reaction yield in IL aqueous different loading concentrations . a Reaction conditions: can also promote this reaction (in Figuresolutions 4, when with the enzyme was 0 amg), the key role of the 2-aminoacetophenone (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (10 mg) in IL its enzyme as a catalyst in the reaction was not obvious in this case, and the enzyme easily reduced 4]/(H2O + [EMIM][BF 60 °C aqueous solutions of differentOn concentrations (80 tothe 0%,energy [EMIM][BF activity at higher temperature. the other hand, consumption, too, had4]),tov/v) be at considered. b Isolated yield after column◦chromatography. for 24 h. Thus, implementing the reaction at 55 C was the best strategy in an IL aqueous solution. 100 90 80
Yieldb (%)
70 60 50 40 30 20 40
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a Reaction Figure 3.3. Reaction yields conditions: 2-aminoacetophenone (0.3 Figure yields at at different differenttemperatures temperaturesa.a a. Reaction conditions: 2-aminoacetophenone mmol), ethyl acetoacetate (0.36 mmol), and 2O (0.3 mmol), ethyl acetoacetate (0.36 mmol), andα-chymotrypsin α-chymotrypsin(10 (10mg) mg)in in 20% 20% ([EMIM][BF44]/(H ]/(H 2 O+ 4]), h. bb Isolated yield yield after after +[EMIM][BF [EMIM][BF v/v)ILILaqueous aqueoussolution solutionatatdifferent different temperatures temperatures for for 24 h. 4 ]),v/v) column columnchromatography. chromatography.
Yieldb (%)
Encouragedby bythe the above results, further investigated the enzyme loading for enhanced Encouraged above results, we we further investigated the enzyme loading for enhanced results results (Figure 4). The yields were improved greatly by increasing the enzyme loading. The reaction (Figure 4). The yields were improved greatly by increasing the enzyme loading. The reaction in the in aqueous the IL aqueous any enzyme a low yield, awhereas yield of obtained 81% was IL solutionsolution withoutwithout any enzyme affordedafforded a low yield, whereas yield ofa81% was obtained when 10 mg of α-chymotrypsin loaded °C. When the was enzyme loading when 10 mg of α-chymotrypsin was loaded atwas 55 ◦ C. Whenatthe55enzyme loading increased fromwas 10 increased from 10 to 20 mg, only a small increase in the yield was observed. This trend may be due to to 20 mg, only a small increase in the yield was observed. This trend may be due to the fact that the the fact that the system with the IL hindered efficient dispersion of the enzyme and reduced the system with the IL hindered efficient dispersion of the enzyme and reduced the mass transfer rate of mass transfer[27]. rateIn of addition, the substrate [27]. In addition, considering problem of energy consumption, the substrate considering the problem of energythe consumption, 10 mg was chosen as Molecules 2017,chosen 22, 762 as the optimum catalyst dosage for the reaction. 5 of 8 10 mg was the optimum catalyst dosage for the reaction. Under the optimized reaction conditions, the generality and scope of the α-chymotrypsincatalyzed Friedländer condensation between 2-aminoaryl ketones and a number of α-methylene 90 ketones were examined; the results are summarized in Table 1. A wide range of substrates could 80 participate in the condensation reaction to give the corresponding products in moderate to excellent yields in the biphasic [BMIM][BF 4]/H2O medium. The corresponding quinoline derivatives were 70 synthesized in a superior way when using 2-aminobenzophenone (Table 1, Entries 8–14) compared 60 to those obtained with 2-aminoacetophenone (Table 1, Entries 1–7). In particular, 2-aminoaryl ketones and the α-methylene ketones with a reactive α-methylene group provided the desired 50 products in higher yields. However, in the case of the cyclic single ketone, which could not participate efficiently in the reaction, the yields were lower than 50% (Table 1, Entries 4–6, 11–12). 40 The low yields were probably due to the substantial steric hindrance, which retarded the reaction. 0
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Enzyme loading (mg)
Figure 4. Reaction yields at different enzyme loadings a . a Reaction conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings a. a Reaction conditions: 2-aminoacetophenone (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% ([EMIM][BF4 ]/(H2 O + [EMIM][BF4 ]), v/v) IL aqueous solution at 55 ◦ C for 24 h. bb Isolated yield after ([EMIM][BF4]/(H2O + [EMIM][BF4]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after column chromatography. column chromatography.
Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous solution a.
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bb(%) bbb(%) b(%) b(%) bb(%) bbb(%) bYield Yield Yield Yield Yield Yield Yield (%) Yield Yield Yield (%) Yield (%) Yield (%) (%) b(%) bYield bb(%) b(%) b(%) bYield b(%) Yield (%) Yield (%) Yield (%) Yield Yield (%) Yield Yield (%)
90 90 90 90 9090 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 Molecules 2017, 22, 762 5 of 8 80 70 70 70 70 7070 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 60 60 60 60 6060 60 60 60 60 60 60 Under the optimized reaction conditions, the generality and scope of the α-chymotrypsin-catalyzed 60 60 60 60 60 60 60 60 60 50 50 50 2-aminoaryl ketones and a number of α-methylene ketones were 50 50 Friedländer condensation between 50 5050 50 50 50 50 50 50 50 50 50 50 examined; the results are summarized in Table 1. A wide range of substrates could participate in the 50 50 50 40 40 40 40 4040 40 40 40 40 40 condensation reaction to give the corresponding products in moderate to excellent yields in the biphasic 40 40 40 40 40 40 40 00 0 555 5 10 15 20 10 15 20 40 10 15 20 40 40corresponding 0 5 10 15 20 0 1010 1515 2020 0 5 [BMIM][BF ]/H O medium. The quinoline derivatives were synthesized in a superior 0 5 10 15 20
4
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5 55 1010 1515 10 (mg) 15 10 15 10 15 55Enzyme 10 Enzyme loading Enzyme loading (mg) (mg) 5555Enzyme 10 15 10loading 1515 10 (mg) 15 loading Enzyme loading (mg)
2020 20 20 20 20 2020 20
Enzyme (mg) 0000 0 5555Enzyme 10 15 20 10loading 1515 8–14) 2020compared to those obtained with Enzyme loading (mg) Enzyme loading (mg) 10 Enzyme loading (mg) Enzyme loading (mg) 10 15 20 way when using 2-aminobenzophenone (Table 1, Entries 10 15 20 loading (mg) Enzyme loading (mg) 0 55Enzyme 10 15 20 loading (mg) Enzyme loading (mg) Enzyme loading (mg) Enzyme loading (mg) Enzyme loading (mg) Enzyme loading (mg) Enzyme loading (mg) Enzyme loading (mg) Enzyme loading (mg) Enzyme loading (mg)2-aminoaryl ketones and the α-methylene 2-aminoacetophenone (Table 1, Entries 1–7). In particular, a Reaction aaaa... aaaaa.Reaction Figure 4. Reaction yields at different enzyme loadings Reaction conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings Reaction conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings conditions: 2-aminoacetophenone a Reaction Figure 4. Reaction yields at different enzyme loadings conditions: 2-aminoacetophenone Figure 4.4. Reaction yields atat different enzyme loadings . Reaction Reaction conditions: 2-aminoacetophenone Figure Reaction yields different enzyme loadings conditions: 2-aminoacetophenone a Reaction Figure 4. Reaction yields at different enzyme loadings conditions: 2-aminoacetophenone aaa.. aaaaa..Reaction Figure 4. Reaction yields at different enzyme loadings Reaction conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings conditions: 2-aminoacetophenone aaaReaction ketones with a reactive α-methylene group provided desired products in higher yields. However, Figure 4. Reaction yields enzyme loadings . Reaction conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings Reaction conditions: 2-aminoacetophenone aaa...aathe a Figure 4. Reaction yields at different enzyme loadings . conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings Reaction conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings Reaction conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings Reaction conditions: 2-aminoacetophenone aa.. aa.Reaction (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% a Figure 4. Reaction yields at different enzyme loadings conditions: 2-aminoacetophenone (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% aa.. aaa.Reaction (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 2020 mg/mL) inin 20% Figure 4. Reaction yields at different enzyme loadings Reaction conditions: 2-aminoacetophenone Figure 4. Reaction yields at different enzyme loadings Reaction conditions: 2-aminoacetophenone (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, a Figure 4. Reaction yields at different enzyme loadings conditions: 2-aminoacetophenone (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% Figure 4. Reaction yields at different enzyme loadings . Reaction conditions: 2-aminoacetophenone (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% (0.3 mmol), ethyl acetoacetate mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% Figure 4. Reaction yields at different enzyme loadings . Reaction conditions: 2-aminoacetophenone (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20mg/mL) mg/mL) in20% 20%yields in the case of the cyclic single ketone, which could not participate efficiently in the reaction, the (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% b b b (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% b 4 ]/(H 2 O + [EMIM][BF 4 ]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF 4 ]/(H 2 O + [EMIM][BF 4 ]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF b 4 ]/(H 2 O + [EMIM][BF 4 ]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF b (0.3 mmol), ethyl (0.36 mmol), α-chymotrypsin 1, 2, 3, 5, 10, 15, mg/mL) in 20% 4ethyl 2acetoacetate [EMIM][BF 44]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF 4]/(H ]/(H 2O O22acetoacetate + [EMIM][BF [EMIM][BF ]), v/v) ILand aqueous solution at(0, 55 °C for 24 h. Isolated yield after ([EMIM][BF (0.3 mmol), acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3,for 5, 10, 15, 20 mg/mL) inafter 20% bb 15, (0.3 mmol), ethyl acetoacetate (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 20 mg/mL) in 20% 4]/(H O +[EMIM][BF [EMIM][BF 4mmol), ]), v/v) IL aqueous solution at 55 °C 24 h. yield after ([EMIM][BF b20 (0.3 mmol), ethyl (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% 4ethyl ]/(H 22acetoacetate O ++++(Table 44]), ]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield ([EMIM][BF b (0.3 mmol), acetoacetate (0.36 and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% 4]/(H ]/(H O ]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF bIsolated bh. 4]/(H ]/(H O 44]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF (0.3 mmol), ethyl (0.36 mmol), and α-chymotrypsin (0, 1, 2, 3, 5, 10, 15, 20 mg/mL) in 20% b 4 2 O + [EMIM][BF 4 v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF 4 2 O + [EMIM][BF IL aqueous solution at 55 °C for 24 Isolated yield after ([EMIM][BF 4 ]/(H 2 O [EMIM][BF 4 ]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF b were lower than 50% 1, Entries 4–6, 11–12). The low yields were probably due to the substantial b b Isolated 44]/(H 2O +[EMIM][BF [EMIM][BF 4]), v/v) IL aqueous solution at 55 °C for 24 h. yield after ([EMIM][BF 44]/(H 22O +O 44]), v/v) IL aqueous solution at 55 °C for 24 h. yield after ([EMIM][BF ]/(H O ]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF ]/(H +[EMIM][BF [EMIM][BF ]), v/v) IL aqueous solution at 55 °C for 24 h. yield after ([EMIM][BF bbIsolated column chromatography. column chromatography. column chromatography. b Isolated 44]/(H 22O +++++[EMIM][BF [EMIM][BF 44]), v/v) IL aqueous solution at 55 °C for 24 h. yield after ([EMIM][BF column chromatography. bb Isolated column chromatography. ]/(H O222O ]),444]), v/v) ILIL aqueous solution atat 5555 °C°C for 2424 h.h. Isolated yield after ([EMIM][BF 4]/(H O [EMIM][BF ]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF column chromatography. b Isolated 44]/(H 22O [EMIM][BF 44]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF column chromatography. ]/(H O ]), v/v) IL aqueous solution at 55 °C for 24 h. Isolated yield after ([EMIM][BF column chromatography. column chromatography. 4]/(H ++[EMIM][BF [EMIM][BF v/v) aqueous solution for yield after ([EMIM][BF column chromatography. column chromatography. column chromatography. steric hindrance, which retarded the reaction. column chromatography. column chromatography. column chromatography. column chromatography.
column chromatography. column chromatography. column chromatography. column chromatography. column chromatography. column chromatography. Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate scope ofof α-chymotrypsin-catalyzed Friedländer condensation inin anan ILIL aqueous Table 1. Substrate scope α-chymotrypsin-catalyzed Friedländer condensation aqueous Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1.1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous a a a Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous solution .. aa.Substrate solution a..Substrate . solution Table 1. scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous solution solution Table 1.aaa1. scope ofof α-chymotrypsin-catalyzed Friedländer condensation inin anan ILIL aqueous aSubstrate Table 1. scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous solution Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous . solution Table 1. Substrate scope of α-chymotrypsin-catalyzed Friedländer condensation in an IL aqueous . solution a a . solution a Table Substrate scope α-chymotrypsin-catalyzed Friedländer condensation aqueous a solution solution aa... a... solution solution solution solution solution aa.. a. solution aa.. a . solution solution solution . . solution solution
Entry 2-Aminoaryl Ketone (1) α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl Ketone (1) α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl 2-Aminoaryl Ketone (1) α-Methylene α-Methylene Ketone (2) Product Product (3) Entry Ketone (1) Ketone (2) (3) Entry 2-Aminoaryl Ketone (1)(1) α-Methylene Ketone (2)(2) Product (3)(3) Entry 2-Aminoaryl Ketone α-Methylene Ketone Product Entry 2-Aminoaryl Ketone (1) α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl Ketone (1) α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl 2-Aminoaryl Ketone (1) α-Methylene α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl Ketone (1) α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl Ketone (1) α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl Ketone (1) α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl α-Methylene Ketone (2) Product (3) Entry Ketone (1) Ketone (2) Product (3) Entry 2-Aminoaryl Ketone (1) α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl Ketone (1) α-Methylene Ketone (2) Product (3) Entry Ketone (1) Ketone (2) (3) Entry 2-Aminoaryl Ketone (1)(1) α-Methylene Ketone (2)(2) Product (3)(3) Entry 2-Aminoaryl 2-Aminoaryl Ketone (1) α-Methylene α-Methylene Ketone (2) Product Product (3) Entry 2-Aminoaryl Ketone (1) α-Methylene Entry α-Methylene Ketone (2) Product (3) Entry 2-Aminoaryl Ketone (1) Ketone (2) Product (3) Entry 2-Aminoaryl Ketone α-Methylene Ketone Product
bb//% b % b Yield Yield % b% Yield b//% Yield Yield % bb///% Yield b/% Yield b Yield /% % Yield b Yield Yield / % bb b//% b//b Yield % Yield /% Yield Yield / % Yield bb/% b % Yield bb//% Yield % Yield %b /% b//% Yield Yield Yield /% Yield
11 1 1111 1 11111 1111 1111 11
81 81 81 81 8181 81 81 81 81 81 81 81 81 81 81 81 8181 81 81 81 81
22 2 2222 2 22222 2222 2222 22
83 83 83 83 8383 83 83 83 83 83 83 83 83 83 83 8383 838383 83 83
3333 3 3333 3333 3333 333 333
91 91 91 91 9191 91 91 91 91 91 91 91 91 91 91 9191 919191 91 91
44444 44 44444 44 44444 44 44
45 45 45 45 4545 45 45 45 45 45 45 45 45 45 45 45 4545 4545 45 45
55 5 5555555 555 555 5 555 555
40 40 40 40 4040 40 40 40 40 40 40 40 40 40 40 40 4040 4040 40 40
6 66666 66 6 66666 666 666 666
49 49 49 49 49 4949 49 49 49 49 49 49 49 49 49 49 4949 4949 49 49
7 762 7777722, Molecules 2017, Molecules 2017, 762 Molecules 2017, 762 7 22, 7777 722, 777 77 777 777
74 74 74 74 74 7474 74 74 74 74 74 74 74 74 74 74 7474 7474 74 74
888 8
90 9090 90
99 9
90 9090
10 1010
94 9494
66of of 88 6 8of
Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, 22, 762 Molecules 2017, Molecules 2017, 22, 22, 762 762
88 8 8888 88 888888 88 8 Entry
66 of 88 8 of 6 of of of 6of 66666of 88888 88 of 66 of of 6 of 88 of 6666of 6 8of of of 888 88 of 6 of 6 of 8
90 90 90 90 90 90 9090 90 90 90 90 90 90 90 90 9090
Table 1. Cont. 2-Aminoaryl Ketone (1)
α-Methylene Ketone (2)
Product (3)
Yield b /%
99 9 999999 99 9 9 9999 99
90 90 90 90 90 90 9090 90 90 90 90 90 90 90 90 90 9090
10 10 10 10 10 10 1010 10 10 10 10 10 10 10 10 10 1010
94 94 94 94 94 94 9494 94 94 94 94 94 94 94 94 94 9494
11 11 11 11 11 11 1111 11 11 11 11 11 11 11 11 11 1111
48 48 48 48 48 48 4848 48 48 48 48 48 48 48 48 48 4848
12 12 12 12 12 12 1212 12 12 12 12 12 12 12 12 12 1212
42 42 42 42 42 42 4242 42 42 42 42 42 42 42 42 42 4242
13 13 13 13 13 13 13 1313 13 13 13 13 13 13 13 13 1313
94 94 94 94 94 94 94 9494 94 94 94 94 94 94 94 94 9494
14 14 14 14 14 14 14 1414 14 14 14 14 14 14 14 14 1414
80 80 80 80 80 80 80 8080 80 80 80 80 80 80 80 80 8080
a Reactionconditions: Reaction 2-aminoaryl ketones α-methylene ketone (0.36 mmol), Reaction conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and conditions: 2-aminoaryl ketones (0.3 (0.3 mmol), α-methylene ketone (0.36 mmol), and α-chymotrypsin Reactionconditions: conditions: 2-aminoaryl ketones (0.3mmol), mmol), α-methylene ketone (0.36 mmol),and and a Reaction Reaction 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and aReaction Reaction conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone mmol), and Reaction conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and Reaction conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and aReaction ◦ C(0.36 b Isolated Reaction conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and a Reaction conditions: 2-aminoaryl ketones mmol), α-methylene ketone (0.36 mmol), and (10 mg) inconditions: 20% ([EMIM][BF ]/(H + ketones [EMIM][BF v/v) IL aqueous solution at 55 for 24 h. yield Reaction 2-aminoaryl (0.3 α-methylene ketone (0.36 mmol), and 420% 2O 4(0.3 444]/(H 2]), + [EMIM][BF 444]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF Reaction conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and ]/(H 2O O + [EMIM][BF ]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in ([EMIM][BF Reaction conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and 4(0.3 ]/(H 2mmol), O + [EMIM][BF 4]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF a ]/(H 2 O + [EMIM][BF ]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF Reaction conditions: 2-aminoaryl ketones mmol), α-methylene ketone (0.36 mmol), and Reaction conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and 4]/(H ++[EMIM][BF [EMIM][BF 4]), v/v) aqueous solution °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF ]/(H O22O +[EMIM][BF [EMIM][BF ]), v/v) ILIL aqueous solution atat 5555 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF Reaction conditions: 2-aminoaryl ketones (0.3 mmol), α-methylene ketone (0.36 mmol), and 444]/(H 222O ++++ 444]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF ]/(H O ]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF 4]/(H O [EMIM][BF 4]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF after column chromatography. 44]/(H 22O [EMIM][BF 44]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF ]/(H O [EMIM][BF ]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF b 4 ]/(H 2 O + [EMIM][BF 4 ]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF b b 44]/(H 22O [EMIM][BF 44]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF bbIsolated yield after column chromatography. for 24 h. 4]/(H 2+ O + [EMIM][BF 4]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF Isolated yield after column chromatography. for 24 h. 4 ]/(H 2 O + [EMIM][BF 4 ]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF Isolated yield after column chromatography. for 24 h. b Isolated yield after column chromatography. for 24 h. ]/(H O + [EMIM][BF ]), v/v) IL aqueous solution at 55 °C α-chymotrypsin (10 mg) in 20% ([EMIM][BF 4]/(H 2O + [EMIM][BF 4]), v/v) ILIL aqueous solution atat 5555 °C°C α-chymotrypsin (10 mg) inafter 20% ([EMIM][BF bbIsolated yield column chromatography. for Isolated yield after column chromatography. for 2424 h.h. 4]/(H 2O + [EMIM][BF 4]), v/v) aqueous solution α-chymotrypsin (10 mg) in 20% ([EMIM][BF b Isolated yield after column chromatography. for 24 h. Isolated yield after column chromatography. for 24 h. b Isolated yield after column chromatography. for 24 h. bb Isolated yield after column chromatography. for 24 h. bbb Isolated yield after column chromatography. for 24 h. Isolated yield after column chromatography. for 24 h. bIsolated yield after column chromatography. for 24 h. Isolated yield after column chromatography. for 24 h. Isolated yield after column chromatography. for 24 h. b b yield after column chromatography. for 24 h. Isolated yield after column chromatography. for 2424 h.h.Isolated Isolated yield after column chromatography. for aaa aa a a aaa aa
3. Experimental 3. Experimental 3. Experimental 3. Experimental 3. Experimental 3. Experimental 3. Experimental 3. Experimental 3. Experimental 3. Experimental 3. Experimental 3. Experimental Experimental 3. Experimental 3. Experimental 3.3. Experimental 3. Experimental 3. Experimental 3. Experimental 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information 3.1. General Information All chemicals were purchased from commercial suppliers and used without further purification. 3.1. General Information 3.1. General Information 3.1. General Information All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. All chemicals were purchased from commercial suppliers and used without further purification. α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from All chemicals were purchased from commercial suppliers and used without further purification. α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from All chemicals were purchased from commercial suppliers and used without further purification. α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from All chemicals were purchased from commercial suppliers and used without further purification. α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). α-Chymotrypsin was obtained from Sigma-Aldrich (Shanghai, China). All ILs were purchased from Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). 13 1 H-NMR Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). 1 13 1 13 1H-NMR 13 Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). 1 13 Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). H-NMR and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, H-NMR and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, 1 13 1 13 Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). H-NMR and spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). 11H-NMR 13 and C-NMRspectra spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, Shanghai ChengJie Chemical Co. Ltd. (Shanghai, China). H-NMR and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, 13C-NMR 11H-NMR 13 and were recorded on Bruker AVANCE III HD 500 (Fällanden, H-NMR and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, 13 1H-NMR 13C-NMR and spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, 13 and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, Switzerland) Bruker AV400 spectrometer, respectively, using CDCl asas solvent. Chemical shifts 111H-NMR 13 H-NMR and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, 1H-NMR 13C-NMR H-NMR and C-NMR spectra were recorded Bruker AVANCE III HD 500 (Fällanden, 13 and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, 13 H-NMR and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, H-NMR and C-NMR spectra were recorded onon Bruker AVANCE III HD 500 (Fällanden, Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 3333as solvent. Chemical shifts 11H-NMR 13 Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl as solvent. Chemical shifts Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 3 as solvent. Chemical shifts(δ) 1H-NMR 13C-NMR and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl as solvent. Chemical shifts H-NMR and C-NMR spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, Switzerland) Bruker AV400 spectrometer, respectively, using CDCl 3 solvent. Chemical shifts and spectra were recorded on Bruker AVANCE III HD 500 (Fällanden, Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 3 as solvent. Chemical shifts Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 33as solvent. Chemical shifts Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl as solvent. Chemical shifts Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 3 as solvent. Chemical shifts Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 33as as solvent. Chemical shifts Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl as solvent. Chemical shifts were expressed in ppm with TMS as the internal standard, and coupling (J) were(J) reported Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 33constants as solvent. Chemical shifts Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 3coupling solvent. Chemical shifts Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl as solvent. Chemical shifts Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl as solvent. Chemical shifts (δ) expressed in with TMS as internal standard, and constants were (δ) were expressed in ppm with TMS as the internal standard, and constants (J) were (δ)were were expressed inppm ppm with TMS asthe the internal standard, andcoupling coupling constants (J) werein Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 333 coupling as solvent. Chemical shifts (δ) were expressed in ppm with TMS as the internal standard, and coupling constants (J) were Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl as solvent. Chemical shifts (δ) were expressed ppm with TMS the internal standard, and constants were Switzerland) and Bruker AV400 spectrometer, respectively, using CDCl 3 as solvent. Chemical shifts (δ) were expressed inin ppm with TMS asas the internal standard, and coupling constants (J)(J) were (δ) were expressed in ppm with TMS as the internal standard, and coupling constants (J) were (δ) were expressed in ppm with TMS as the internal standard, and coupling constants (J) were (δ) were expressed in ppm with TMS as the internal standard, and coupling constants (J) were (δ) were expressed in ppm with TMS as the internal standard, and coupling constants (J) were (δ) were expressed in ppm with TMS as the internal standard, and coupling constants (J) were Hz. Melting points were measured using a WRS-1B Digital Melting Point Apparatus (Shanghai, China). (δ) were expressed in ppm with TMS as the internal standard, and coupling constants (J) were (δ) were expressed in ppm with TMS as the internal and coupling constants (J) were (δ) were expressed in ppm with TMS as the internal standard, and coupling constants (J) were (δ) were expressed inin ppm with TMS asas the internal standard, and coupling constants (J)(J) were reported in Hz. Melting points were measured using aastandard, WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using aWRS-1B WRS-1B Digital Melting Point Apparatus (δ) were expressed in ppm with TMS as the internal and coupling constants (J) were reported in Hz. Melting points were measured using Digital Melting Point Apparatus (δ) were expressed in ppm with TMS as the internal standard, and coupling constants (J) were reported Hz. Melting points were measured using aaWRS-1B WRS-1B Digital Melting Point Apparatus (δ) were expressed ppm with TMS the internal standard, and coupling constants were reported in Hz. Melting points were measured using WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using aaaaa Digital Melting Point Apparatus reported inin Hz. Melting points were measured using astandard, WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using a WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using a WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using a WRS-1B Digital Melting Point Apparatus reported in Hz. Melting points were measured using a WRS-1B Digital Melting Point Apparatus (Shanghai, China). (Shanghai, China). (Shanghai, China). reported in Hz. points were using (Shanghai, China). reported inChina). Hz. Melting were measured using WRS-1B Digital Melting Point Apparatus (Shanghai, China). reported in Hz.Melting Melting points weremeasured usingaa aWRS-1B WRS-1BDigital DigitalMelting MeltingPoint PointApparatus Apparatus (Shanghai, China). (Shanghai, (Shanghai, China). 3.2. General Procedure forpoints the Synthesis ofmeasured Bis(Indolyl)Methane (Shanghai, China). (Shanghai, China). (Shanghai, China). (Shanghai, China). (Shanghai, China). (Shanghai, China). (Shanghai, China). (Shanghai, China). (Shanghai, China). (Shanghai, China). 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane A mixture of 2-aminoaryl ketone (0.3 mmol, 1 equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis ofof Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane and α-chymotrypsin (10 mg) in 1 mL of 20% IL aqueous solution (20%, [EMIM][BF 3.2. General Procedure for the Synthesis of Bis(Indolyl)Methane 3.2.A General Procedure for the Synthesis of Bis(Indolyl)Methane 4 ]/([EMIM][BF mixture of 2-aminoaryl ketone (0.3 mmol, 1111 equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), A mixture of 2-aminoaryl ketone (0.3 mmol, α-methylene ketone (0.36 mmol, 1.2 equiv), A mixture of 2-aminoaryl ketone (0.3 mmol, equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), 4 ] A mixture of 2-aminoaryl ketone (0.3 mmol, equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), A mixture of 2-aminoaryl ketone (0.3 mmol, 111equiv), equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), A mixture of 2-aminoaryl ketone (0.3 mmol, equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), ◦ A mixture of 2-aminoaryl ketone (0.3 mmol, 1 equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), A mixture of 2-aminoaryl ketone (0.3 mmol, 1 equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), A mixture of 2-aminoaryl ketone (0.3 mmol, equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), A mixture of 2-aminoaryl ketone (0.3 mmol, equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), +and Hα-chymotrypsin O), v/v) of was incubated in a constant temperature shaker (55 C, 260 (0.36 r/min, 24 h). The process A mixture of 2-aminoaryl ketone (0.3 mmol, equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), A mixture 2-aminoaryl (0.3 mmol, 1equiv), equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), 2α-chymotrypsin A mixture 2-aminoaryl ketone (0.3 mmol, 11111 equiv), α-methylene ketone 1.2 equiv), A mixture of 2-aminoaryl (0.3 mmol, equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), A mixture ofof 2-aminoaryl ketone (0.3 mmol, 1aqueous equiv), α-methylene ketone (0.36 mmol, 1.2 equiv), and (10 mg) in 11ketone mL of 20% IL aqueous solution (20%, [EMIM][BF 44mmol, ]/([EMIM][BF 444] and α-chymotrypsin (10 mg) in of 20% IL aqueous solution (20%, [EMIM][BF 4mmol, ]/([EMIM][BF (10 mg) in mL of 20% IL aqueous solution (20%, [EMIM][BF ]/([EMIM][BF ]+ A mixture of 2-aminoaryl ketone (0.3 mmol, α-methylene ketone (0.36 1.2 equiv), and α-chymotrypsin (10 mg) in mL of 20% IL aqueous solution (20%, [EMIM][BF ]/([EMIM][BF ]]]4++4++]+ A mixture of 2-aminoaryl ketone (0.3 mmol, α-methylene ketone (0.36 mmol, 1.2 equiv), and α-chymotrypsin (10 mg) in 111mL mL 20% solution (20%, [EMIM][BF 44mmol, ]/([EMIM][BF A mixture of 2-aminoaryl ketone (0.3 mmol, 11equiv), equiv), α-methylene ketone (0.36 1.2 equiv), and α-chymotrypsin (10 mg) in 11ketone mL of 20% IL aqueous solution (20%, [EMIM][BF ]/([EMIM][BF and α-chymotrypsin (10 mg) in 111chromatography mL of 20% IL aqueous solution (20%, [EMIM][BF 444]/([EMIM][BF 444]] and α-chymotrypsin (10 mg) in mL ofof 20% ILIL aqueous solution (20%, [EMIM][BF ]/([EMIM][BF +]] ++ and α-chymotrypsin (10 mg) in mL of 20% IL aqueous solution (20%, [EMIM][BF 44]/([EMIM][BF 44++ and α-chymotrypsin (10 mg) in mL of 20% IL aqueous solution (20%, [EMIM][BF 4 ]/([EMIM][BF was monitored by thin layer (TLC). After completion of the reaction, the product and α-chymotrypsin (10 mg) in 1 mL of 20% IL aqueous solution (20%, [EMIM][BF 4 ]/([EMIM][BF 4]]+ and α-chymotrypsin (10 mg) in 1 mL of 20% IL aqueous solution (20%, [EMIM][BF ]/([EMIM][BF and α-chymotrypsin (10 mg) in 1 mL of 20% IL aqueous solution (20%, [EMIM][BF 44]/([EMIM][BF 444] α-chymotrypsin (10 mg) in 1 mL of 20% IL aqueous solution (20%, [EMIM][BF 4]/([EMIM][BF 4+ ] ++ and α-chymotrypsin (10 mg) in 1 mL of 20% IL aqueous solution (20%, [EMIM][BF 4]/([EMIM][BF ]/([EMIM][BF 4]] ] + H 22and v/v) was incubated in a constant temperature shaker (55 °C, 260 r/min, 24 h). The process was H 2O), O), v/v) was incubated in a constant temperature shaker (55 °C, 260 r/min, 24 h). The process was H 2 O), v/v) was incubated in a constant temperature shaker (55 °C, 260 r/min, 24 h). The process was and α-chymotrypsin (10 mg) in 1 mL of 20% IL aqueous solution (20%, [EMIM][BF H O), v/v) was incubated in a constant temperature shaker (55 °C, 260 r/min, 24 h). The process was and α-chymotrypsin (10 mg) in 1 mL of 20% IL aqueous solution (20%, [EMIM][BF 4 ]/([EMIM][BF 4 + 2O), v/v) was incubated in aaconstant constant temperature shaker (55 °C, 260 r/min, 24 h). The process was and α-chymotrypsin (10in mg) in 1 mL temperature of 20% IL aqueous solution (20%, [EMIM][BF 4]/([EMIM][BF 4] + H 22O), O), v/v) was incubated in aaconstant temperature shaker (55 °C, 260 r/min, 24 h). The process was H 2H v/v) was incubated aaa× shaker (55 °C, 260 r/min, 24 h). The process was Hwas O), v/v) was incubated in constant temperature shaker (55 °C, 260 r/min, 24 h). The process was H 22O), v/v) was incubated in constant temperature shaker (55 °C, 260 r/min, 24 h). The process was H 2 O), v/v) was incubated in constant temperature shaker (55 °C, 260 r/min, 24 h). The process was H 2 O), v/v) was incubated in constant temperature shaker (55 °C, 260 r/min, 24 h). The process was extracted with 5 mL 3 EtOAc. Then, the combined organic layer was concentrated under H O), v/v) was incubated in a constant temperature shaker (55 °C, 260 r/min, 24 h). The process was H 2H v/v) was incubated in aaaaconstant temperature shaker (55 °C, 260 r/min, 24 h). The process was 2O), v/v) was incubated in constant temperature shaker (55 °C, 260 r/min, 24 h). The process H 2O), O), v/v) was incubated in constant temperature shaker (55 °C, 260 r/min, 2424 h). The process monitored by thin layer chromatography (TLC). After completion of the reaction, the product was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was H 22O), v/v) was incubated in constant temperature shaker (55 °C, 260 r/min, 24 h). The process monitored by thin layer chromatography (TLC). After completion of the reaction, the product was H O), v/v) was incubated in temperature shaker (55 °C, 260 r/min, 24 h). The process was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was H 2O), v/v) was incubated in aaconstant constant temperature shaker (55 °C, 260 r/min, h). The process monitored by thin layer chromatography (TLC). After completion of the reaction, the product monitored by thin layer chromatography (TLC). After completion of the reaction, the product was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was reduced pressure to afford the crude product, which was purified by silica gel column chromatography monitored by thin layer chromatography (TLC). After completion of the reaction, the product was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was monitored by thin layer chromatography (TLC). After completion of the reaction, the product was extracted with 5 mL × 3 EtOAc. Then, the combined organic layer was concentrated under reduced extracted with 5 mL ×× 3××33 EtOAc. Then, the combined organic layer was concentrated under reduced extracted with mL EtOAc. Then, the combined organic layer was concentrated under reduced monitored by layer chromatography (TLC). After completion of reaction, product was extracted with Then, the combined organic layer was concentrated under reduced monitored by thin layer chromatography (TLC). After completion of the reaction, the product was extracted with 555mL mL 333EtOAc. EtOAc. Then, the combined organic layer was concentrated under reduced monitored bythin chromatography (TLC). After completion ofthe the reaction,the the product was extracted with mL EtOAc. Then, the combined organic layer was concentrated under reduced extracted with 555555thin mL ×××layer Then, the combined organic layer was concentrated under reduced extracted with mL ×33to 3EtOAc. EtOAc. Then, the combined organic layer was concentrated under reduced extracted with mL × EtOAc. Then, the combined organic layer was concentrated under reduced extracted with mL EtOAc. Then, the combined organic layer was concentrated under reduced extracted with mL × 3 EtOAc. Then, the combined organic layer was concentrated under reduced extracted with 5 mL × 3 EtOAc. Then, the combined organic layer was concentrated under reduced (PE/EtOAc = 10:1) yield the pure product. All products were known compounds that were extracted with 5555mL ××××the 33××33crude EtOAc. Then, the combined organic layer was concentrated under reduced extracted with mL EtOAc. Then, the combined organic layer was concentrated under reduced extracted with mL EtOAc. Then, the combined organic layer was concentrated under reduced pressure to afford the product, which was by silica gel column pressure to afford the crude product, which was purified by silica gel column chromatography pressure to afford the crude product, which waspurified purified by silica gel columnchromatography chromatography extracted with mL EtOAc. Then, the combined organic layer was concentrated under reduced pressure to afford the crude product, which was purified by silica gel column chromatography extracted with Then, the combined organic layer was concentrated under reduced pressure to afford crude product, which was purified by silica gel column chromatography extracted with 55mL mL 33EtOAc. EtOAc. Then, the combined organic layer was concentrated under reduced pressure to afford the crude product, which was purified by silica gel column chromatography pressure to afford crude product, which was purified by silica gel column chromatography pressure to afford the crude product, which was purified by silica gel column chromatography 1the pressure to afford the crude product, which was purified by silica gel column chromatography pressure to afford the crude product, which was purified by silica gel column chromatography pressure to afford the crude product, which was purified by silica gel column chromatography pressure to afford the crude product, which was purified by silica gel column chromatography characterized by H-NMR. pressure to afford the crude product, which was purified by silica gel column chromatography pressure to afford the crude product, which was purified by silica gel column chromatography pressure toto afford the crude product, which was purified byby silica gel column chromatography pressure to afford the crude product, which was purified by silica gel column chromatography pressure to afford the crude product, which was purified by silica gel column chromatography pressure afford the crude product, which was purified silica gel column chromatography
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4. Conclusions In conclusion, we had successfully demonstrated an efficient approach for the synthesis of quinoline derivatives via Friedländer condensation using α-chymotrypsin as an efficient, environmentally friendly catalyst in an IL with moderate water content. The methodology developed required a low reaction temperature and reduced enzyme loading, and afforded excellent yields. A series of substrates was investigated, and the results were found to be better than those obtained with organic solvents. Importantly, the proposed methodology avoids the use of hazardous acids or bases and harsh reaction conditions, thereby stimulating the development of a green synthetic methodology. This method not only extends the application of proteases as non-specific biocatalysts, but also confirms the potential use of ILs as better alternatives to organic solvents for the Friedländer condensation between a 2-aminoaryl ketone and an α-methylene ketone. Acknowledgments: We gratefully acknowledge the financial support of the National Natural Science Foundation of China (Nos. 21462001, 21465002 and 51362002), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13054), and the Natural Science Foundation of Jiangxi Province (No. 20142BAB203008). Author Contributions: Zhang-Gao Le and Zong-Bo Xie conceived and designed the experiments; Meng Liang, Zhong-Sheng Chen, Sui-Hong Zhang performed the experiments. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all the compounds are available from the authors. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
