Synthesis of New Functionalized Indoles Based on Ethyl Indol-2-carboxylate

Successful alkylations of the nitrogen of ethyl indol-2-carboxylate were carried out using aq. KOH in acetone. The respective N-alkylated acids could be obtained without separating the N-alkylated esters by increasing the amount of KOH and water. The use of NaOMe in methanol led to transesterification instead of the alkylation, while the use of NaOEt led to low yields of the N-alkylated acids. Hydrazinolysis of the ester gave indol-2-carbohydrazide which then was allowed to react with different aromatic aldehydes and ketones in ethanol catalyzed by acetic acid. Indol-2-thiosemicarbazide was used in a heterocyclization reaction to form thiazoles. The new structures were confirmed using NMR, mass spectrometry and X-ray single crystal analysis.


Introduction
Indole derivatives have been a topic of substantial research interest and continue to be one of the most active areas of heterocyclic chemistry, particularly due to their natural occurrence and pharmacological activities [1]. Indole derivatives also occur widely in many natural products such as those obtained from plants [2], fungi [3], and marine organisms [4]. The isolation, biological evaluation, and chemical properties of natural products have attracted the attention of organic chemists, medicinal chemists, biologists and pharmacists as well as led to optimization of highly efficient and economical synthetic routes.
The indazoline A is an indole derivative inhibitor of acetylcholinesterase used to treat Alzheimer's disease [16]. The indole derivative eletriptan (B) is an anti-migraine drug. A process route for the synthesis of eletriptan published by Pfizer starts from a preformed bromoindole [17]. Fluvastatin (C) is a member of the statin drug class, used to treat hypercholesterolemia and to prevent cardiovascular diseases. It has also been shown to exhibit antiviral activity against hepatitis C [18]. Ondansetron Bis-indole alkaloids are an important structural class due to their high degree of biological activity. For example, the nortopsentins Ei-iii exhibit in vitro cytotoxicity against P388 cells with IC50 (inhibitory concentration) values of 7.6, 7.8, and 1.7 µg/mL, respectively [21][22][23].
Given the significant pharmacological activities associated with these heterocycles, and in order to contribute to the development of the chemistry of indole [24][25][26][27][28][29][30][31][32], we were interested in the synthesis of new heterocyclic polyfunctional indole derivative systems using alkylation reactions.

Results
Alkylation of the nitrogen of the indole ring in indole-containing compounds requires strong bases to generate the indole anion [33]. Protecting the nitrogen of the indole ring in ethyl 1H-indol-2-carboxylate requires special care to avoid the ester hydrolysis before the alkylation. KOH in anhydrous DMSO was used for the alkylation of nitrogen of the indole esters [34]. Herein, we describe the alkylation of the indole nitrogen using aq. KOH in acetone. In this method we can control the reaction to give the alkylated esters or the alkylated acids in the same reaction process, with the additional benefit of the ease of solvent removal after reaction completion.
Reaction of ethyl indol-2-carboxylate (1) with allyl bromide and benzyl bromide in the presence of aq. KOH (3.0 mmol/0.1 mL H2O/10 mL acetone) and stirring for two hours at 20 °C afforded ethyl 1-allyl-1H-indole-2-carboxylate (2) and ethyl 1-benzyl-1H-indole-2-carboxylate (3) in excellent yields. The corresponding alkylated carboxylic acids 1-allyl-1H-indol-2-carboxylic acid (5) and 1-benzyl-1Hindol-2-carboxylic acid (6) were obtained in high yields directly without separating the alkylated esters by increasing the amount of aq. KOH and refluxing for one hour. Alkylation with amyl bromide seems to be slow, since it took about eight hours to give ethyl 1-pentyl-1H-indole-2-carboxylate (4) under the same conditions. Moreover, a considerable amount of 1H-indol-2-carboxylic acid (9) was detected. The alkylated acids 5-7 were also obtained in excellent yields from the hydrolysis of the respective esters 2-4 using aqueous KOH in acetone. The use of NaOMe in methanol led to transesterification to afford methyl indol-2-carboxylate (8) instead of NH alkylation, whereas using NaOEt in ethanol gave the acids 5 and 6 in low to moderate yields in the case of the of 1 with allyl and benzyl bromides whereas, in case of amyl bromide a high yield of 1H-indol-carboxylic acid 9 was obtained (Scheme 1, Table 1).
Moreover, hydrazide 10 was reacted with terephthalaldehyde under the same conditions to give the bis-indolyl product 15. A thiosemicarbazide 16 was obtained from the hydrazide 10 and served as adduct for cyclization with two phenacyl bromides to afford indolylcarbonylhydrazino-thiazoles 17 and 18 (Scheme 3). Bis-indole alkaloids are an important structural class due to their high degree of biological activity. For example, the nortopsentins Ei-iii exhibit in vitro cytotoxicity against P388 cells with IC 50 (inhibitory concentration) values of 7.6, 7.8, and 1.7 µg/mL, respectively [21][22][23].
Given the significant pharmacological activities associated with these heterocycles, and in order to contribute to the development of the chemistry of indole [24][25][26][27][28][29][30][31][32], we were interested in the synthesis of new heterocyclic polyfunctional indole derivative systems using alkylation reactions.

Results
Alkylation of the nitrogen of the indole ring in indole-containing compounds requires strong bases to generate the indole anion [33]. Protecting the nitrogen of the indole ring in ethyl 1H-indol-2-carboxylate requires special care to avoid the ester hydrolysis before the alkylation. KOH in anhydrous DMSO was used for the alkylation of nitrogen of the indole esters [34]. Herein, we describe the alkylation of the indole nitrogen using aq. KOH in acetone. In this method we can control the reaction to give the alkylated esters or the alkylated acids in the same reaction process, with the additional benefit of the ease of solvent removal after reaction completion.
Reaction of ethyl indol-2-carboxylate (1) with allyl bromide and benzyl bromide in the presence of aq. KOH (3.0 mmol/0.1 mL H 2 O/10 mL acetone) and stirring for two hours at 20˝C afforded ethyl 1-allyl-1H-indole-2-carboxylate (2) and ethyl 1-benzyl-1H-indole-2-carboxylate (3) in excellent yields. The corresponding alkylated carboxylic acids 1-allyl-1H-indol-2-carboxylic acid (5) and 1-benzyl-1H-indol-2-carboxylic acid (6) were obtained in high yields directly without separating the alkylated esters by increasing the amount of aq. KOH and refluxing for one hour. Alkylation with amyl bromide seems to be slow, since it took about eight hours to give ethyl 1-pentyl-1H-indole-2-carboxylate (4) under the same conditions. Moreover, a considerable amount of 1H-indol-2-carboxylic acid (9) was detected. The alkylated acids 5-7 were also obtained in excellent yields from the hydrolysis of the respective esters 2-4 using aqueous KOH in acetone. The use of NaOMe in methanol led to transesterification to afford methyl indol-2-carboxylate (8) instead of NH alkylation, whereas using NaOEt in ethanol gave the acids 5 and 6 in low to moderate yields in the case of the of 1 with allyl and benzyl bromides whereas, in case of amyl bromide a high yield of 1H-indol-carboxylic acid 9 was obtained (Scheme 1, Table 1).

Structural Analysis
All NMR spectra showed the indole CH protons between δ 7.00 and 7.70 ppm and all indole carbons from δ 103.0 to 138.0 ppm.

Alkylated Ester Analysis
The formation of ethyl N-alkylated indol-2-carboxylates 2-4 was confirmed by the disappearance of the indole NH proton signal from the 1 H-NMR of these compounds, the presence of ethoxy group signals (-OCH 2 CH 3 ) at δ 1.30 and 4.30 ppm and the respective carbons in 13 C-NMR at δ 15.0 and 46.7 ppm in addition to the ester carbonyl group around δ 162.0 ppm. Moreover, new signals appeared in the NMR spectra which are characteristic for the new groups and can be summarized as follows: in compound 2 the signals of the allyl group appeared as two doublets at δ 4.81 and 5.06 ppm for the olefinic CH 2 with coupling constants δ 16.8 and 10.2 Hz, respectively, the NCH 2 appeared at 5.23 ppm and the corresponding carbon (NCH 2 ) appeared at δ 46.7 ppm, whereas, the remaining olefinic CH appeared as multiplet at δ 5.97-6.07 ppm. The benzylated ester 3 showed the NCH 2 protons as a singlet at δ 5.87 ppm and the respective carbon at δ 47. 6

Hydrolyzed Ester Data Analysis
Hydrolysis of the esters with time was deduced from the disappearance of the signals of the ethoxy group in the NMR of 5-7 and instead a new broad signal appeared around δ 12.90 ppm for COOH, in addition to characteristic allyl, benzyl and pentyl signals.
Transesterification and formation of the methyl ester 8 was confirmed by 1 H-NMR by observing a singlet signal at δ 3.88 ppm for the ester methyl group and the indole NH at δ 11.91 ppm. The respective methyl carbon appeared in 13 C-NMR at δ 52.2 ppm and the ester carbonyl appeared at δ 162.3 ppm. The acid 9 lacked any alkyl signals and showed the C=O at δ 163.0 ppm, in addition to the remaining expected protons and carbons.

Hydrazinolysis of the Esters and the Related Products Analysis
Hydrazinolysis of either indol-2-carboxylate 1 or 2 led to the formation of indol-2-carboxylic acid hydrazide (10). The structure of 10 was confirmed by NMR which showed only two signals at δ 4.52 and 9.78 ppm for the -NH-NH 2 group, whereas the carbonyl group appeared at δ 161.7 ppm. The 1 H-NMR (DMSO-d 6 + D 2 O) of 11 showed the anomeric proton as a doublet at 3.87 ppm with a coupling constant value of 8.7 Hz which confirms the β-configuration. The corresponding anomeric carbon appeared in 13 C-NMR at 90.9 ppm and the carbonyl carbon signal appeared at 161.3 ppm. The NMR spectra of 12, 13 showed the -CH=N-proton around δ 8.60 ppm whereas, the NMR of 14 showed the methyl protons at 2.43 ppm and the respective methyl carbon at 15.5 ppm in addition to the remaining aromatic signals. The NMR spectra of 15 showed a singlet signal at δ 8.51 ppm for the two -CH=N-protons and two signals at δ 11.86 and 12.02 ppm for indole and hydrazide NHs, besides all aromatic protons and carbons signals.

Thiazole Structural Analysis
The 1 H-NMR spectra of thiazoles 17 and 18 showed all aromatic CH protons of thiazole, indole and phenyl in the range of δ 7.07-7.83 ppm, while the three NH protons appeared as broad signals at δ 9.69, 10.87 and 11.76 ppm. In addition, the 13 C-NMR of 17 and 18 showed the carbonyl carbons at δ 161.42 and 161.35 ppm, respectively.

X-ray Diffraction Analysis
The structure of 13 was confirmed by X-ray crystal structural analysis. The crystallographic data, conditions retained for the intensity data collection and some features of the structure refinements are listed in Table 2. Selected interatomic distances and bond angles are given in Table 3. The unit cell of the titled compound contains one molecule. All of the bond lengths and bond angles in the phenyl rings are in the normal range. The indole ring (C1-C8/N1) forms a dihedral angle of 28.05˝with the pyridine-3-yl ring (C11-C14/N4/C15). The title compound exists in trans configuration with respect to the C10=N3 bond [1.2805 (15) Å] as shown in Figure 2. In the crystal structure (Figure 3), molecules are linked via three intermolecular N1-H1N1¨¨¨N4, N2-H1N2¨¨¨O1 and C10-H10A¨¨¨O1 hydrogen bonds in b axis (Table 4).    (14) 144.00 Symmetry codes: (i) x − 1/2, −y + 3/2, −z + 1; (ii) -x + 3/2, y + 1/2, z.

General Details
Melting points were measured with a Stuart melting-point apparatus (SMP10, Bibby Scientific Ltd., Staffordshire, UK) in open capillaries and are uncorrected. Flash chromatography was done on silica gel 60 (230-400 mesh ASTM). TLC was performed on silica gel 60 F254 (Merck Millipore, Darmstadt, Germany) and spots were detected by absorption of UV light. 1 H-NMR spectra were recorded on Advanced NMR spectrometers (Bruker Biospin, Fallanden, Switzerland) at 300-600 MHz whereas 13 C-NMR spectra were recorded on the same instruments at 75-150 MHz, with TMS as internal standard. Mass spectra were obtained using MAT312 (ThermoFinnigan GmbH, Tokyo, Japan) and a JMS.600H (Jeol, Tokyo, Japan) instruments for EIMS; HRMS spectra were recorded on a Thermo Finnigan MAT 95XP and Jeol JMS HX110 and ESI on an Ion Trap 6320 mass detector (Agilent Technologies, Wilmington, DE, USA). IR spectra were recorded using KBr discs on a Bruker FT-IR IFS 48 spectrophotometer (Bruker Optics, Ettlingen, Germany).    on silica gel 60 (230-400 mesh ASTM). TLC was performed on silica gel 60 F 254 (Merck Millipore, Darmstadt, Germany) and spots were detected by absorption of UV light. 1 H-NMR spectra were recorded on Advanced NMR spectrometers (Bruker Biospin, Fallanden, Switzerland) at 300-600 MHz whereas 13 C-NMR spectra were recorded on the same instruments at 75-150 MHz, with TMS as internal standard. Mass spectra were obtained using MAT312 (ThermoFinnigan GmbH, Tokyo, Japan) and a JMS.600H (Jeol, Tokyo, Japan) instruments for EIMS; HRMS spectra were recorded on a Thermo Finnigan MAT 95XP and Jeol JMS HX110 and ESI on an Ion Trap 6320 mass detector (Agilent Technologies, Wilmington, DE, USA). IR spectra were recorded using KBr discs on a Bruker FT-IR IFS 48 spectrophotometer (Bruker Optics, Ettlingen, Germany).

General Procedure for the Alkylation of Ethyl Indol-2-carboxylate (1)
A solution of ethyl indol-2-carboxylate (1, 1.0 mmol) and aq. KOH (3.0 mmol) in acetone (10 mL) was stirred at 20˝C for half hour, then the appropriate alkylating agent (1.1 mmol) was added and stirring was continued for 2 h to give 2 and 3 and for eight hours to give 4. The solvent was removed, water was added and organic layer was extracted using ethyl acetate. The products were purified using column chromatography (ethyl acetate/hexane 1:9).

Hydolysis of the Ester and Formation of Acids 5-7
Method a: the above procedure was followed until the alkylation was complete, then KOH (6.0 mmol in 1.0 mL¨H 2 O) was added and the reaction mixture refluxed one hour, the solvent removed, cold water added, and acidified. The ppt was collected and purified by crystallization from ethanol in the case of 5 and 6 and from hexane in the case of 7.
Method b: a solution of the appropriate ester 2-4 and KOH (6.0 mmol in 1.0 mL¨H 2 O) and acetone (10 mL) was refluxed for one hour, then the above purification process was followed.
Method c: A mixture of ethyl indol-2-carboxylate (1, 1.0 mmol) and NaOEt (3.0 mmol) in ethanol (10 mL) was stirred for half an hour then, alkylating agent was added and the mixture heated under reflux for two hours and the above purification process followed.