Metal-free 1,3-dipolar cycloaddition approach towards the regioselective synthesis of β-carboline and isoxazole based molecular hybrids

Abstract

Nature has nourished β-carboline and isoxazole derivatives as privileged scaffolds and consequently they are ubiquitously found in alkaloids isolated from various sources. Moreover, several drug molecules based on them have been released in the market. Considering their immense impact, novel β-carboline–isoxazole-based molecular hybrids have been designed and a 1,3-dipolar cycloaddition strategy was devised to prepare the desired prototypes. A library of compounds with a wide range of diversity have been developed by employing β-carboline-containing dipolarophiles as well as dipoles. The current method represents a simple, efficient and easy-to-execute protocol towards the regioselective synthesis of β-carboline–isoxazole conjugates.

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Introduction

The literature shows that the β-carboline nucleus is one of the most prominent heterocyclic frameworks in chemistry and constitutes more than one-third of the total alkaloids isolated from various natural sources, such as from terrestrial and aquatic plants, marine species, insects and mammals, including human beings (Fig. 1).¹ β-Carboline-containing alkaloids exhibit a broad spectrum of pharmacological properties, such as sedative, anticonvulsant, anxiolytic, anti-tumour, anti-malarial, anti-leishmanial, anti-microbial and anti-HIV.² Interestingly, the majority of these alkaloids display potent cytotoxic properties by multiple action mechanisms, such as intercalation into DNA strands, inhibiting cyclin-dependent kinases and topoisomerases and interacting with MK-2, kinesin-like protein Eg5 and PLK1, etc.³ More importantly, the β-carboline framework is also represented in commercial drugs, such as Tadalafil and Abecarnil.⁴

Fig. 1 Selected examples of β-carboline- and isoxazole-containing bioactive natural products and drug molecules.

The isoxazole nucleus is represented by a large number of commercial drugs and a few natural products, such as AMPA, ibotenic acid.⁵ Interestingly, isoxazole derivatives also demonstrate a wide range of biological properties, such as being inhibitors of heat shock protein 90 activity, a broad spectrum of antibiotics, neurone generative agents, antiviral agents, NSAIDs, antiproliferative and immunosuppressive agents (Fig. 1).⁶ In addition to this, isoxazole derivatives also serve as a latent precursor for the synthesis of enaminones and α-cyanoaldehydes, which in turn act as templates for natural products and other heterocycles, such as quinolines, oxazoles and indoles.⁷

The excellent activity profile of these pharmacophores motivated us to develop a new molecular hybrid of these two frameworks for our anticancer project. Interestingly, an exponential growth towards the construction of molecular hybrids across the globe has been observed owing to their high affinity and selectivity, better efficacy and a different or dual mode of action.⁸

The growing resistance against the existing chemotherapeutic agents is another driving force for this paradigm shift towards the development of new molecular hybrids.

With the objective to synthesize the desired prototype, a retrosynthetic analysis revealed that there may be two possible pathways for the construction of our designed molecular hybrid, as depicted in Fig. 2. The first route involves the conventional approach, where the Pictet–Spengler condensation of tryptamine/L-tryptophan ester (I) with isoxazole-5-carbaldehyde (II), followed by oxidation of the resulting product may furnish the desired prototype.⁹ However, the synthesis of isoxazole-5-carbaldehyde (II) with substitution at the C-3 and C-4 positions is deemed difficult and there are only limited reports of it available in the literature.¹⁰ Furthermore, it is anticipated that steric hindrance offered by the C-3 and C-4 substituents (bulky nature) may hamper the Pictet–Spengler condensation. Alternatively, a more facile and straightforward approach may be 1,3-dipolar cycloaddition between β-carboline-containing dipolarophile (III) and a suitable dipole (IV, nitrile oxide), which can afford the targeted molecules (prototype-A).¹¹ The β-carboline-allied dipolarophiles (III) in turn may be obtained from 1-formyl pyrido[3,4-b]indole derivatives (V). Apart from this, 1-formyl pyrido[3,4-b]indole (V) could also generate 1,3-dipoles, which upon reaction with a suitable dipolarophile may generate the C-3 substituted isoxazole derivatives. It is evident that these approaches have broad scope and advantages over the classical approach as they could yield the products with a high degree of diversity. In this context, we engineered a modified approach, where a versatile precursor in the form of 1-formyl-9H-β-carboline (an alkaloid known as Kumujian C)¹² was developed.^12f We successfully demonstrated the applicability of this synthon for the synthesis of canthin-6-one, harmicine and homofascaplysin mimics by using Morita–Baylis–Hillman (MBH) chemistry.¹³ More recently, we were able to fabricate β-carbolines-conjugated imidazo[1,2-a]pyridines and γ-lactones by using this template.¹⁴ Additionally, we could generate β-carboline D-ring fused frameworks by the application of intramolecular cycloaddition reactions.¹⁵ Notably, other research groups also effectively used this building block for the synthesis of β-carboline-containing natural products, catalysts and anticancer agents.¹⁶

Fig. 2 Proposed retrosynthetic pathways for the construction of prototype A.

An overview of the literature reflected that the cycloaddition of nitrile oxides with dipolarophiles is the best route to access isoxazole derivatives.¹⁷ However, thermal cycloaddition offers poor regioselectivity; therefore, various metal-assisted (Cu/Ru/Cr) approaches have been discovered to increase the efficacy of this reaction and to generate high chemo- and regioselectivity.¹⁸ Apart from the metal-assisted protocols, a few metal-free approaches using reagents like hypervalent iodine, Et₃N, DBU, NHCs and NaOCl/Et₃N have been developed.¹⁹ Due to the explosive nature and reactivity issues associated with hypervalent iodine reagents, we aimed to develop a cost-effective and benign approach to serve this purpose.²⁰ Since during recent years, much emphasis has been given to the development of sustainable approaches, we therefore directed our efforts towards engineering a metal-free strategy. A deep review of the literature revealed that there was only one report available towards the synthesis of β-carboline-conjugated isoxazoles; however, this was at the C-5 position with only three analogues.²¹ Additionally, this Pd-assisted approach was only able to produce the products in low yields under refluxing conditions. More importantly, a general approach dedicated to the synthesis of trisubstituted isoxazole-tethered β-carboline-based molecular hybrids has so far remained undeveloped. Therefore, a metal-free, simple, convenient, robust and cost-effective approach was developed and detailed studies were performed to achieve the synthesis of desired prototypes, which are presented herein.

Results and discussion

To achieve the synthesis of the designed prototypes, initially 1-formyl-9H-β-carbolines (7–8) and its N-substituted derivatives (10a–c) were prepared from L-tryptophan ester (1–2) by using a recently reported scale-up procedure, as illustrated in Scheme 1.^12f In the first phase of study, it was envisaged achieving the synthesis of C-5 substituted isoxazolyl-based molecular hybrids via a 1,3-dipolar cycloaddition approach. Accordingly, β-carboline substituted dipolarophiles (11a–d) were generated in a facile manner via a Wittig reaction of 1-formyl pyrido[3,4-b]indole derivatives (7, 10a–c) with triethyl phosphonoacetate in anhydrous THF.²² Interestingly, the products were obtained in excellent yields (89–97%) and could be collected by simple filtration. The nitrile oxides (A–C) were generated in situ to avoid the dimerization from the corresponding hydroxymoyl chlorides in a two-step process from aromatic carbaldehydes.²³ Next, the optimization studies were initiated with (E)-methyl 1-(3-ethoxy-3-oxoprop-1-en-1-yl)-9-methyl-9H-pyrido[3,4-b]indole-3-carboxylate (11a) as the dipolarophile with nitrile oxide A in the presence of Et₃N in anhydrous THF (Scheme 2).

Scheme 1 Synthesis of 1-formyl-9H-pyrido[3,4-b]indoles and N-substituted derivatives.

Scheme 2 Synthesis of β-carboline–isoxazole- (12) and β-carboline-pyrazole-based molecular hybrids (14).

To attain a high regioselectivity and to avoid the competitive dimerization of nitrile oxide, the cycloaddition reaction was performed at −78 °C under dilute conditions with the slow addition of Et₃N to form hydroxymoyl chlorides (A). It was pleasing to note that the nitrile oxides A reacted smoothly with 11a in a regioselective manner to yield the anticipated isoxazoline derivative (11aA) as a single diastereomer. The crystallisation/trituration of the crude product with MeOH afforded the analytically pure product in 92% yield. The optimisation studies for the cycloaddition reaction were also investigated with DABCO and DBU in THF, but offered the product 11aA in only low yields. Interestingly, it was observed that the isoxazoline product (11aA) was stable at low temperature but was prompt to undergo slow aerial oxidation at room temperature, which alleviated our objective. The further oxidation of isoxazoline product (11aA) was achieved in anhydrous THF : DMF (4 : 1) in the presence of KMnO₄. The oxidation process was very fast and exothermic when the reaction was performed in anhydrous DMF and required only 25 min for completion (80% yield, Table 1, ESI†) but work-up was slightly tedious. However, when THF was used as a solvent, the reaction was completed in 6 h, but simple filtration of the reaction contents through a celite bed, followed by evaporation of the solvent afforded the pure product (84%). Therefore, to take advantage of both, we performed the reaction in a 4 : 1 mixture of anhydrous THF and DMF (Table 1,† entry 3), and it yielded the product in a better yield (90%) in 40 min. With these optimized conditions in hand, we further investigated the scope of the reaction with various β-carboline-containing dipolarophiles (11a–c) with different nitrile oxides (A–C). To our pleasure, all the substrates responded positively towards the cycloaddition reaction and smoothly furnished the desired β-carboline-substituted isoxazoline derivatives (11aA–aC, 11bA–bC, 11cA–cC and 11dA–dC). The isoxazoline products (11) were isolated and purified, but immediately subjected to further oxidation with KMnO₄ to produce the corresponding β-carboline-conjugated isoxazole derivatives (12aA–aC, 12bA–bC, 12cA–cC and 12dA–dC) in good to excellent yields (76–92%) (Fig. 4), as illustrated in Scheme 2. Encouraged by this success, we planned to expand the scope of strategy for the synthesis of β-carboline and pyrazole-based molecular hybrids (14), as presented in Scheme 2. However, when 11a was treated with nitrile imine (D), a complex mixture of products was obtained. The major product was isolated in a 16% yield and on the basis of the spectroscopic data its structure was confirmed to be an oxidized product (14). We could not isolate compound 13 from the reaction mixture. Though, we attempted to optimize the reaction conditions (Table 2, see ESI†) by varying the solvent and base to obtain the product in a better yield, we did not have much success. It was also observed that nitrile imines were potentially allergic to the skin and were an irritant to the eyes, which halted our efforts in this direction.

Fig. 3 X-ray crystallographic analysis of 18bC.

Fig. 4 Structures of various β-carboline- (C-5)isoxazole- and (C-3)isoxazole-based molecular hybrids synthesized during the present study.

Recently, Chauhan and co-workers demonstrated that β-carboline-based chalcones display potent cytotoxic properties against human breast cancer MCF-7 cell lines, with an IC₅₀ up to 2.25 μM.^16d Their findings stimulated us to explore these products as a template (as dipolarophiles) for the synthesis of new β-carboline–isoxazole-based molecular hybrids for our anticancer project. Accordingly, the synthesis of β-carboline-based chalcones (16a–c) was achieved via the Claisen–Schmidt condensation of the 1-formyl-pyrido[3,4-b]indole derivatives (7 and 10b) with different acetophenones (15a–c) having electron donating as well as electron withdrawing substituents, as depicted in Scheme 3. Interestingly, these dipolarophiles (β-carboline-based chalcones, 16a–c) also reacted smoothly with various nitrile oxides (A–C) in the presence of Et₃N in anhydrous THF at −78 °C to yield the desired isoxazoline derivatives (17aA–aC, 17bA–bC and 17cA–cC) in 81–93% yields. Interestingly, under these metal-free conditions, complete regioselectivity was obtained, which was further unambiguously assigned on the basis of the single crystal X-ray crystallographic analysis of 18bC (Fig. 3).²⁴ The β-carboline-linked isoxazoline derivatives (17aA–aC, 17bA–bC and 17cA–cC) were subsequently oxidized to the corresponding isoxazole derivatives (18aA–aC, 18bA–bC and 18cA–cC) with KMnO₄ within 0.5–3 h and afforded the products (Fig. 4) with high purity in good to excellent yields (60–92%). It is interesting to mention here that no column chromatographic purification was required at any step and that trituration/washing with MeOH furnished the analytically pure product.

Scheme 3 Synthesis of β-carboline–isoxazole molecular hybrids (18) using β-carboline based chalcones as template.

After the successful synthesis of β-carboline-(C-5)isoxazole-based molecular hybrids (12 and 18) from β-carboline-containing dipolarophiles (11 and 16), we directed our efforts to construct β-carboline-(C-3)isoxazole-based molecular hybrids (Scheme 4). It was envisaged that a cycloaddition reaction between a β-carboline-linked 1,3-dipole and a suitable external dipolarophile may fulfil our objective. Accordingly, ethyl 1-formyl-9-methyl-9H-pyrido[3,4-b]indole-3-carboxylate (10c) was treated with NH₂OH·HCl to obtain the corresponding oxime (19), which was subsequently reacted with N-chlorosuccinimide (NCS) in anhydrous DMF to yield the corresponding hydroxymoyl chloride (20). Furthermore, the β-carboline-tethered nitrile oxide was generated in situ in the presence of Et₃N at −78 °C and reacted with different dipolarophiles (X–Z), which smoothly furnished the desired molecular hybrids (20X–Z) in excellent yields (86–93%), as depicted in Scheme 4. Interestingly, this modified approach also afforded these novel β-carboline-conjugated isoxazole derivatives with complete regioselectivity, as confirmed by spectroscopic analysis. It is anticipated that low temperature, secondary interactions with the carbonyl functionality and steric limitation offered by the β-carboline ring are responsible for the high regioselectivity. Gratifyingly, both the approaches yielded the products with high purity and furthermore, no column chromatographic purification was required at any step.

Scheme 4 Synthesis of β-carboline-(C-3)isoxazole-based molecular hybrids (20X–Z) using β-carboline-linked dipoles as a template.

Conclusions

In conclusion, we successfully developed a simple, efficient and easy-to-execute metal-free protocol for the synthesis of β-carboline- (C-5)isoxazole- and (C-3)isoxazole-based molecular hybrids via a 1,3-dipolar cycloaddition approach. Unfortunately, the developed strategy was not found to be suitable for the synthesis of β-carboline-(C-5)pyrazole-conjugated derivatives. More importantly, all the products were afforded in high yield and no column chromatographic purification was required at any stage (except for the pyrazole derivative), and analytically pure products were obtained by a simple filtration of the reaction mixture or by recrystallization of the product with MeOH. In brief, this strategy offers an alternative to the possible classical approach and has several advantages, such as operational simplicity, easy purification procedure, wide functional group compatibility and, importantly, unlimited diversity can be introduced in these interesting molecular architectures. The anticancer evaluation and lead optimisation studies are underway and will be described in due course.

Experimental

General section

All chemicals and reagents were purchased from Sigma Aldrich, Acros, Avera Synthesis, Spectrochem Pvt. Ltd. and used without further purification. Commercially available anhydrous solvents (THF, DMF, toluene, MeOH and CH₂Cl₂; Spectrochem) were utilized in the reactions. Thin-layer chromatography (TLC) was performed using pre-coated aluminium plates purchased from E. Merck (silica gel 60 PF254, 0.25 mm). Column chromatography was performed using Spectrochem silica gel (60–120 mesh). Melting points were determined in open capillary tubes on a Precision Digital melting point apparatus (LABCO make) containing silicon oil and the results are uncorrected. IR spectra were recorded using an Agilent FTIR spectrophotometer. ¹H NMR and ¹³C NMR spectra were recorded either on an Avance III Bruker or JEOL JNM-ECS spectrometer at operating frequencies of 200/400 MHz (¹H) or 100 MHz (¹³C) as indicated in the individual spectrum, using TMS as an internal standard. The MS spectra were recorded on a Xevo G2-SQ Tof (Water, USA) or Thermo Finnigan LCQ Advantage Ion Trap Mass Spectrometer. Elemental analyses were performed on a Carlo-Erba 108 or an Elementar Vario EL III microanalyzer. The room temperature varied between 25 °C and 40 °C. The multiplicities in the ¹H-NMR spectra are presented as s for singlet, d for doublet, dd for doublet of doublet, t for triplet and m for multiplet.

Experimental section

General procedure for the synthesis of β-carboline-substituted isoxazoline derivatives (11 and 17) as exemplified for 11aA. To a stirred solution of β-carboline-linked dipolarophile 11a (0.20 g, 0.59 mmol) and hydroxymoyl chloride, A (0.14 g, 0.87 mmol) in dry THF (5 mL), Et₃N (0.12 mL, 0.87 mmol) was added dropwise at −78 °C and the contents stirred for 1 h at the same temperature. Thereafter, the reaction mixture was allowed to stir for an additional 4 h at room temperature. On completion of the reaction as monitored by TLC, THF was removed under reduced pressure to yield an off-white solid product, which was triturated twice with dry MeOH (2 × 5 mL). The solid product was finally washed with diethyl ether and filtered through a sintered funnel and dried under high vacuum to obtain an analytically pure white solid product, 11aA (0.25 g, 92%, R_f = 0.35 (hexane/EtOAc, 80 : 20, v/v)).

Ethyl 4-(3-(methoxycarbonyl)-9-methyl-9H-pyrido[3,4-b]indol-1-yl)-3-phenyl-4,5-dihydroisoxazole-5-carboxylate (11aA). Yield: 92% (0.25 g from 0.20 g) as a white solid; mp 140–141 °C; R_f = 0.35 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1705 (CO₂CH₃ and CO₂CH₂CH₃), 1619 (C=N–O), 1261 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 1.17 (t, J = 7.2 Hz, 3H), 3.98 (s, 3H), 4.19 (q, J = 7.2 Hz, 2H), 4.35 (s, 3H), 6.56 (q, J = 4.8 Hz, 1H), 7.85 (d, J = 4.4 Hz, 1H), 7.37 (t, J = 7.2 Hz, 1H), 7.40–7.53 (m, 3H), 7.56 (d, J = 8.0 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.93 (d, J = 2.4 Hz, 2H), 8.18 (d, J = 7.6 Hz, 1H), 8.84 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.1, 29.8, 32.7, 52.5, 54.3, 62.2, 84.3, 110.2, 111.4, 118.4, 121.1, 121.3, 121.7, 127.9, 128.6, 130.4, 135.7, 136.9, 131.4, 141.1, 142.8, 156.6, 166.1, 169.7; MS (ES): m/z (%) = 458.2 (100) [M + 1]⁺; C₂₆H₂₃N₃O₅ (457.1638): calcd for C 68.26, H 5.07, N 9.19; found C 68.33, H 5.12, N 9.26.

General procedure for the synthesis of β-carboline-(C-4)isoxazoles-based molecular hybrids (12 and 18), as exemplified by compound 12aA. To a stirred solution of 11aA (0.20 g, 0.44 mmol) in anhydrous THF : DMF (4 : 1, v/v) (10 mL), KMnO₄ (0.60 g) was added portion-wise at room temperature and the reaction mixture stirred for an additional 30 min at room temperature. On completion of the reaction as monitored by TLC, the contents were filtered through a celite bed under vacuum and the bed was washed three times with chloroform. The collected organic layers were combined and concentrated under vacuum to yield a dull white solid product, which was washed twice with anhydrous diethyl ether and then air dried under vacuum to obtain the analytically pure white solid product, 12aA (0.16 g, 80%, R_f = 0.30 (hexane/EtOAc, 80 : 20, v/v)).

Ethyl 5-(3-(methoxycarbonyl)-9-methyl-9H-pyrido[3,4-b]indol-1-yl)-3-phenylisoxazole-4-carboxylate (12aA). Yield: 80% (0.16 g from 0.20 g) as a white solid; mp 132–133 °C. R_f = 0.30 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1703 (CO₂CH₃ and CO₂CH₂CH₃), 1617 (C=N–O), 1260 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 0.73 (t, J = 7.2 Hz, 3H), 3.68 (s, 3H), 3.98 (q, J = 7.2 Hz, 2H), 4.06 (s, 3H), 7.45–7.55 (m, 5H), 7.67–7.74 (m, 1H), 7.82 (t, J = 7.2 Hz, 2H), 8.29 (d, J = 7.8.0 Hz, 1H), 9.01 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 13.3, 31.2, 52.9, 60.9, 110.0, 118.9, 120.9, 121.4, 121.9, 127.6, 127.8, 128.3, 128.5, 129.1, 129.4, 129.7, 130.2, 130.8, 137.2, 142.9, 160.9, 162.7, 166.0, 170.8 ppm; MS (ES): m/z (%) = 456.1 (100) [M + 1]⁺; C₂₆H₂₁N₃O₅ (455.1481): calcd for C 68.56, H 4.65, N 9.23; found C 68.64, H 4.68, N 9.31.

Ethyl 3-(4-chlorophenyl)-5-(3-(methoxycarbonyl)-9-methyl-9H-pyrido[3,4-b]indol-1-yl)isoxazole-4-carboxylate (12aB). Yield: 85% (0.34 g from 0.40 g) as a white solid; mp 168–169 °C; R_f = 0.40 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1711 (CO₂CH₂CH₃), 1721 (CO₂CH₃), 1611 (C=N–O), 1262 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 0.71 (t, J = 7.1 Hz, 3H), 3.68 (s, 3H), 3.98 (q, J = 7.1 Hz, 2H), 4.10 (s, 3H), 7.45 (t, J = 7.7 Hz, 1H), 7.48–7.52 (m, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.71–7.75 (m, 1H), 7.78–7.82 (m, 2H), 8.29 (d, J = 7.9 Hz, 1H), 9.06 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 13.4, 31.4, 53.1, 61.2, 110.1, 113.1, 119.1, 121.0, 121.6, 122.1, 126.1, 128.7, 129.9, 130.9, 136.7, 137.3, 142.9, 160.8, 161.9, 166.1, 171.3 ppm; MS (ES): m/z (%) = 490.2 (100) [M + 1]⁺; C₂₆H₂₀ClN₃O₅ (489.1091): calcd for C 68.74, H 4.11, N 8.58; found C 68.85, H 4.16, N 8.70.

Ethyl 5-(3-(methoxycarbonyl)-9-methyl-9H-pyrido[3,4-b]indol-1-yl)-3-(p-tolyl)isoxazole-4-carboxylate (12aC). Yield: 82% (0.26 g from 0.32 g) as a white solid; mp 157–158 °C; R_f = 0.35 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1728 (CO₂CH₃ and CO₂CH₂CH₃), 1610 (C=N–O), 1263 (Ar-C–O)_isoxazole; ¹H NMR (200 MHz, CDCl₃) δ = 1.19 (t, J = 7.1 Hz, 3H), 2.40 (s, 3H), 3.98 (s, 3H), 4.20 (q, J = 7.1 Hz, 2H), 4.34 (s, 3H), 7.24–7.57 (m, 5H), 7.82 (d, J = 8.2 Hz, 2H), 8.18 (d, J = 7.8 Hz, 1H), 8.83 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.1, 21.6, 32.5, 52.4, 61.2, 110.2, 118.3, 121.0, 121.4, 121.7, 126.0, 127.8, 129.2, 129.4, 130.8, 135.4, 137.0, 137.3, 140.6, 142.8, 156.5, 166.4, 169.9 ppm; MS (ES): m/z (%) = 470.1 (100) [M + 1]⁺, 492.1 (33) [M + 23]⁺; C₂₇H₂₃N₃O₅ (469.1638): calcd for C 69.07, H 4.94, N 8.95; found C 69.19, H 5.00, N 9.06.

Ethyl 5-(9-ethyl-3-(methoxycarbonyl)-9H-pyrido[3,4-b]indol-1-yl)-3-phenylisoxazole-4-carboxylate (12bA). Yield: 85% (0.34 g from 0.40 g) as an off-white solid; mp 150–151 °C; R_f = 0.35 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1710 (CO₂CH₂CH₃), 1725 (CO₂CH₃) 1617 (C=N–O), 1263 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.55 (t, J = 7.1 Hz, 3H), 1.16 (t, J = 7.0 Hz, 3H), 3.89 (q, J = 7.1 Hz, 2H), 3.94 (s, 3H), 4.05–4.19 (m, 2H), 7.46 (t, J = 7.4 Hz, 1H), 7.60 (t, J = 7.1 Hz, 3H), 7.76 (t, J = 7.7 Hz, 1H), 7.83–7.95 (m, 3H), 8.60 (d, J = 7.9 Hz, 1H), 9.22 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 13.5, 14.1, 39.6, 53.0, 61.0, 110.3, 113.0, 119.1, 121.4, 121.5, 122.1, 127.7, 128.4, 129.0, 129.5, 129.8, 130.4, 131.1, 136.4, 137.2, 142.0, 160.7, 162.9, 166.1, 171.4 ppm; MS (ES): m/z (%) = 470.2 (100) [M + 1]⁺; C₂₇H₂₃N₃O₅ (469.1638): calcd for C 69.07, H 4.94, N 8.95; found C 69.19, H 5.01, N 9.02.

Ethyl 3-(4-chlorophenyl)-5-(9-ethyl-3-(methoxycarbonyl)-9H-pyrido[3,4-b]indol-1-yl)isoxazole-4-carboxylate (12bB). Yield: 78% (0.29 g from 0.40 g) as an off-white solid; mp 171–172 °C; R_f = 0.45 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1704 (CO₂CH₂CH₃), 1721 (CO₂CH₃), 1620 (C=N–O), 1262 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.51 (t, J = 7.1 Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H), 4.18 (q, J = 7.1 Hz, 2H), 4.28–4.34 (m, 2H), 4.40 (s, 3H), 7.41 (t, J = 7.5 Hz, 1H), 7.50–7.60 (m, 2H), 7.74 (t, J = 7.6 Hz, 1H), 7.81–7.90 (m, 2H), 7.94 (d, J = 6.4 Hz, 1H), 8.50 (d, J = 7.8 Hz, 1H), 9.01 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 13.5, 14.1, 39.7, 53.0, 61.1, 110.3, 112.9, 119.1, 121.4, 121.6, 122.2, 126.2, 128.8, 129.9, 130.1, 131.2, 136.4, 137.2, 142.0, 160.8, 162.0, 166.1, 171.7 ppm; MS (ES): m/z (%) = 504.2 (100) [M + 1]⁺; C₂₇H₂₂ClN₃O₅ (503.1248): calcd for C 64.35, H 4.40, N 8.34; found C 64.44, H 4.43, N 8.40.

Ethyl 5-(9-ethyl-3-(methoxycarbonyl)-9H-pyrido[3,4-b]indol-1-yl)-3-(p-tolyl)isoxazole-4-carboxylate (12bC). Yield: 83% (0.33 g from 0.40 g) as an off-white solid; mp 147–148 °C; R_f = 0.40 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1720 (CO₂CH₃), 1702 (CO₂CH₂CH₃), 1626 (C=N–O), 1259 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 0.71 (t, J = 7.1 Hz, 3H), 1.30 (t, J = 7.2 Hz, 3H), 2.45 (s, 3H), 3.97 (q, J = 7.1 Hz, 2H), 4.06 (s, 3H), 4.07–4.13 (m, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.44 (t, J = 7.5 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.69–7.73 (m, 3H), 8.29 (d, J = 7.8 Hz, 1H), 9.06 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 13.5, 14.1, 21.6, 39.6, 53.0, 61.0, 110.3, 113.0, 119.1, 121.3, 121.5, 122.1, 124.6, 129.2, 129.4, 129.8, 131.1, 136.4, 137.1, 140.6, 142.0, 160.9, 162.9, 166.2, 171.2 ppm; MS (ES): m/z (%) = 484.2 (100) [M + 1]⁺; C₂₈H₂₅N₃O₅ (483.1794): calcd. for C 69.55, H 5.21, N 8.69; found C 69.66, H 5.25, N 8.77.

Ethyl 5-(3-(ethoxycarbonyl)-9-methyl-9H-pyrido[3,4-b]indol-1-yl)-3-phenylisoxazole-4-carboxylate (12cA). Yield: 80% (0.16 g from 0.20 g) as a white solid; mp 153–154 °C; R_f = 0.40 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1703 (CO₂CH₂CH₃), 1719 (CO₂CH₃), 1616 (C=N–O), 1260 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 1.14 (t, J = 7.1 Hz, 3H), 1.29 (t, J = 7.1 Hz, 3H), 4.13–4.19 (m, 2H), 4.31 (q, J = 7.1 Hz, 2H), 4.39 (s, 3H), 7.41 (t, J = 7.5 Hz, 1H), 7.50–7.60 (m, 3H), 7.74 (t, J = 7.3 Hz, 1H), 7.81–7.90 (m, 2H), 7.94 (d, J = 6.4 Hz, 1H), 8.50 (d, J = 7.8 Hz, 1H), 9.01 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.1, 14.5, 32.4, 54.3, 61.3, 62.1, 110.2, 118.2, 121.0, 121.3, 121.7, 127.9, 128.6, 128.9, 129.1, 130.3, 130.8, 135.7, 136.9, 137.1, 142.8, 156.5, 165.9, 169.7 ppm; MS (ES): m/z (%) = 470.2 (100) [M + 1]⁺; C₂₇H₂₃N₃O₅ (469.1638): calcd for C 69.07, H 4.94, N 8.95; found C 69.20, H 5.00, N 9.06.

Ethyl 3-(4-chlorophenyl)-5-(3-(ethoxycarbonyl)-9-methyl-9H-pyrido[3,4-b]indol-1-yl)isoxazole-4-carboxylate (12cB). Yield: 92% (0.34 g from 0.37 g) as a white solid; mp 130–131 °C; R_f = 0.50 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1702 (CO₂CH₂CH₃), 1717 (CO₂CH₃), 1611 (C=N–O), 1260 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 0.73 (t, J = 7.2 Hz, 3H), 1.48 (t, J = 7.2 Hz, 3H), 3.69 (s, 3H), 3.99 (q, J = 7.2 Hz, 2H), 4.54 (q, J = 7.2 Hz, 2H), 7.42–7.54 (m, 4H), 7.71 (t, J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 2H), 8.28 (d, J = 8.0 Hz, 1H), 9.02 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 13.8, 14.6, 31.4, 61.2, 62.0, 110.1, 113.0, 119.0, 121.0, 121.5, 122.1, 126.1, 128.7, 128.9, 129.9, 130.9, 136.7, 137.3, 137.6, 142.9, 160.9, 161.9, 165.5, 171.3 ppm; MS (ES): m/z (%) = 504.2 (100) [M + 1]⁺; C₂₇H₂₂ClN₃O₅ (503.1248): calcd for C 64.35, H 4.40, N 8.34; found C 64.47, H 4.44, N 8.42.

Ethyl 5-(3-(ethoxycarbonyl)-9-methyl-9H-pyrido[3,4-b]indol-1-yl)-3-(p-tolyl)isoxazole-4-carboxylate (12cC). Yield: 83% (0.33 g from 0.40 g) as a white solid; mp 137–138 °C; R_f = 0.45 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1704 (CO₂CH₂CH₃), 1620 (C=N–O), 1262 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 1.19 (t, J = 7.1 Hz, 3H), 1.44 (t, J = 7.2 Hz, 3H), 2.40 (s, 3H), 4.20 (q, J = 7.1 Hz, 2H), 4.37 (s, 3H), 4.44 (q, J = 7.2 Hz, 2H), 7.24 (s, 2H), 7.38 (t, J = 7.5 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.81 (d, J = 8.1 Hz, 2H), 8.20 (d, J = 7.8 Hz, 1H), 8.85 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 13.6, 14.0, 21.1, 31.9, 53.7, 60.7, 61.6, 83.6, 109.7, 117.6, 120.5, 120.6, 121.1, 125.4, 127.2, 128.7, 128.9, 130.1, 135.0, 136.4, 136.5, 140.2, 142.2, 155.9, 165.2, 169.3 ppm; MS (ES): m/z (%) = 484.1 (100) [M + 1]⁺; C₂₈H₂₅N₃O₅ (483.1794): calcd for C 69.55, H 5.21, N 8.69; found C 69.66, H 5.26, N 8.74.

Ethyl 5-(3-(methoxycarbonyl)-9H-pyrido[3,4-b]indol-1-yl)-3-phenylisoxazole-4-carboxylate (12dA). Yield: 81% (0.26 g from 0.32 g) as an off-white solid; mp 201–202 °C. R_f = 0.30 (hexane/EtOAc, 70 : 30, v/v); IR (KBr): ν_max = 3379 (N–H), 1705 (CO₂CH₃ and CO₂CH₂CH₃), 1623 (C=N–O), 1269 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.96 (t, J = 7.1 Hz, 3H), 3.95 (s, 3H), 4.25 (q, J = 7.1 Hz, 2H), 7.40 (t, J = 7.4 Hz, 1H), 7.62 (d, J = 1.9 Hz, 3H), 7.68 (t, J = 7.5 Hz, 1H), 7.79–7.82 (m, 3H), 8.52 (d, J = 7.9 Hz, 1H), 9.14 (s, 1H), 12.38 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.0, 52.5, 62.6, 111.6, 112.6, 118.7, 121.6, 121.8, 127.6, 128.2, 128.9, 129.7, 130.5, 131.2, 134.4, 137.7, 141.2, 161.2, 163.6, 165.8, 167.5 ppm; MS (ES): m/z (%) = 442.1 (100) [M + 1]⁺; C₂₅H₁₉N₃O₅ (441.1325): calcd for C 68.02, H 4.34, N 9.52; found C 68.14, H 4.39, N 9.61.

Ethyl 5-(3-(methoxycarbonyl)-9H-pyrido[3,4-b]indol-1-yl)-3-(p-tolyl)isoxazole-4-carboxylate (12dC). Yield: 76% (0.15 g from 0.20 g) as an off-white solid; mp 217–218 °C; R_f = 0.35 (hexane/EtOAc, 70 : 30, v/v); IR (KBr): ν_max = 1697 (CO₂CH₃ and CO₂CH₂CH₃), 1621 (C=N–O), 1264 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 0.98 (t, J = 7.1 Hz, 3H), 2.42 (s, 3H), 3.95 (s, 3H), 4.26 (q, J = 7.1 Hz, 2H), 7.38–7.43 (m, 3H), 7.67–7.71 (m, 3H), 7.80 (d, J = 8.2 Hz, 1H), 8.52 (d, J = 7.9 Hz, 1H), 9.14 (s, 1H), 12.40 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃ + DMSO-d₆) δ = 12.2, 20.0, 50.9, 60.2, 111.9, 117.5, 119.6, 120.4, 123.4, 126.7, 127.2, 128.1, 129.5, 133.3, 135.2, 139.0, 140.8, 159.5, 161.0, 164.1, 166.5 ppm; MS (ES): m/z (%) = 456.2 (100) [M + 1]⁺; C₂₆H₂₁N₃O₅ (455.1481): calcd for C 68.56, H 4.65, N 9.23; found C 68.68, H 4.70, N 9.30.

Experimental procedure for the synthesis of β-carboline-pyrazole-based molecular hybrid (14). A stirred solution of 11b (0.20 g, 0.57 mmol), hydrazonyl chloride,^23b D (0.65 g, 2.84 mmol) and DIPEA (0.29 mL, 1.70 mmol) in dry toluene (10 mL) was heated at 110 °C for 72 h. Afterwards, toluene was evaporated under vacuum and the crude mass was extracted with ethyl acetate (3 × 10 mL). The organic layer was further washed with NaHCO₃ solution (10 mL) and brine (10 mL). Thereafter, the crude product was purified through silica gel (230–400 mesh) column chromatography using ethylacetate and hexane as a eluent to yield the white solid product, 14 (0.05 g, 16%, R_f = 0.40 (hexane/EtOAc, 70 : 30, v/v)). We wish to mention that even after 3 days the reaction was not complete and 5–6 products were visible in the crude mixture on TLC analysis. We purified the major product from the reaction mixture, which was analysed as 14 (in situ oxidized product).

Methyl 1-(4-(ethoxycarbonyl)-1,3-diphenyl-1H-pyrazol-5-yl)-9-ethyl-9H-pyrido[3,4-b]indole-3-carboxylate (14). Yield: 16% (0.05 g from 0.20 g) as a white solid; mp 170–171 °C; R_f = 0.40 (hexane/EtOAc, 70 : 30, v/v); IR (KBr): ν_max = 1703 (CO₂CH₂CH₃), 1718 (CO₂CH₃); ¹H NMR (400 MHz, CDCl₃) δ = 0.56 (t, J = 7.2 Hz, 3H), 1.17 (t, J = 7.2 Hz, 3H), 3.77–3.89 (m, 3H), 4.08 (s, 3H), 4.10–4.16 (m, 1H), 7.17 (d, J = 7.3 Hz, 3H), 7.38 (t, J = 7.5 Hz, 1H), 7.43–7.49 (m, 6H), 7.64 (t, J = 7.5 Hz, 1H), 7.85 (d, J = 7.9 Hz, 2H), 8.23 (d, J = 7.8 Hz, 1H), 8.97 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 13.0, 14.4, 43.6, 51.8, 61.2, 109.4, 122.4, 122.6, 122.7, 127.6, 128.1, 128.2, 128.3, 128.8, 128.9, 135.5, 137.3, 139.2, 140.0, 141.3, 141.9, 144.3, 160.8, 164.9 ppm; MS (ES): m/z (%) = 545.0 (100) [M + 1]⁺; C₃₃H₂₈N₄O₄ (544.2111): calcd for C 72.78, H 5.18, N 10.29; found C 72.90, H 5.22, N 10.36.

Methyl 1-(4-(3,4-dimethoxybenzoyl)-3-phenylisoxazol-5-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (18aA). Yield: 72% (0.18 g from 0.25 g) as an off-white solid; mp 154–155 °C; R_f = 0.20 (hexane/EtOAc, 70 : 30, v/v); IR (KBr): ν_max = 3360 (N–H), 1659 (CO), 1710 (CO₂CH₃), 1621 (C=N–O), 1259 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 3.68 (s, 3H), 3.75 (s, 3H), 3.79 (s, 3H), 6.87 (t, J = 7.6 Hz, 1H), 7.39 (s, 2H), 7.53–7.60 (m, 4H), 7.69 (s, 3H), 7.89 (s, 1H), 8.47 (d, J = 6.6 Hz, 1H), 8.94 (s, 1H), 12.47 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 50.1, 53.9, 54.0, 108.7, 109.1, 111.7, 114.3, 116.6, 119.0, 119.3, 120.2, 123.3, 126.1, 127.3, 127.7, 128.7, 129.2, 132.1, 134.7, 140.4, 147.0, 151.8, 159.4, 163.5, 164.6, 186.3 ppm; MS (ES): m/z (%) = 534.1 (100) [M + 1]⁺; C₃₁H₂₃N₃O₆ (533.1587): calcd for C 69.79, H 4.35, N 7.88; found C 69.92, H 4.40, N 8.00.

Methyl 1-(3-(4-chlorophenyl)-4-(3,4-dimethoxybenzoyl)isoxazol-5-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (18aB). Yield: 60% (0.15 g from 0.25 g) as a white solid; mp 197–198 °C; R_f = 0.30 (hexane/EtOAc, 60 : 40, v/v); IR (KBr): ν_max = 3472 (N–H), 1720 (CO₂CH₃), 1672 (CO), 1619 (C=N–O), 1262 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 3.94 (s, 3H), 4.03 (s, 3H), 4.11 (s, 3H), 7.13 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.6 Hz, 2H), 7.34–7.39 (m, 1H), 7.57 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 3.9 Hz, 2H), 7.78 (d, J = 1.7 Hz, 1H), 8.18 (d, J = 7.9 Hz, 1H), 8.85 (s, 1H), 9.01 (d, J = 7.1 Hz, 1H), 9.70 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 52.7, 56.1, 56.2, 110.1, 110.6, 111.0, 112.5, 118.0, 121.2, 121.8, 126.5, 126.8, 128.3, 128.7, 129.0, 129.2, 129.5, 130.5, 135.1, 136.6, 136.7, 140.9, 149.5, 154.7, 157.4, 166.3, 193.8 ppm; MS (ES): m/z (%) = 568.1 (100) [M + 1]⁺; C₃₁H₂₂ClN₃O₆ (567.1197): calcd for C 65.55, H 3.90, N 7.40; found C 65.67, H 3.94, N 7.49.

Methyl 1-(4-(3,4-dimethoxybenzoyl)-3-(p-tolyl)isoxazol-5-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (18aC). Yield: 70% (0.14 g from 0.20 g) as a white solid; mp 173–174 °C; R_f = 0.25 (hexane/EtOAc, 60 : 40, v/v); IR (KBr): ν_max = 3457 (N–H),1722 (CO₂CH₃), 1669 (CO), 1617 (C=N–O), 1256 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 2.31 (s, 3H), 3.69 (s, 3H), 3.85 (s, 3H), 3.97 (s, 3H), 7.10 (d, J = 8.6 Hz, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.51 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 1.4 Hz, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 9.03 (s, 1H), 12.21 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 21.5, 52.6, 56.2, 56.3, 110.6, 111.1, 112.4, 117.8, 121.1, 121.2, 121.8, 125.4, 126.5, 127.4, 128.5, 129.6, 130.4, 135.0, 136.7, 140.8, 141.1, 141.5, 149.5, 154.5, 158.2, 166.4, 194.0 ppm; MS (ES): m/z (%) = 548.1 (100) [M + 1]⁺; C₃₂H₂₅N₃O₆ (547.1743): calcd for C 70.19, H 4.60, N 7.67; found C 70.31, H 4.65, N 7.76.

Methyl 1-(4-(4-chlorobenzoyl)-3-phenylisoxazol-5-yl)-9-ethyl-9H-pyrido[3,4-b]indole-3-carboxylate (18bA). Yield: 85% (0.17 g from 0.20 g) as an off-white solid; mp 159–160 °C; R_f = 0.35 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1719 (CO₂CH₃), 1667 (CO), 1615 (C=N–O), 1248 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 1.36 (t, J = 7.0 Hz, 3H), 3.80 (s, 3H), 4.47 (q, J = 7.0 Hz, 2H), 7.27 (d, J = 7.8 Hz, 2H), 7.42–7.51 (m, 4H), 7.66 (d, J = 5.9 Hz, 2H), 7.74 (t, J = 8.1 Hz, 3H), 7.88 (d, J = 7.8 Hz, 1H), 8.52 (d, J = 7.5 Hz, 1H), 9.00 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.4, 40.4, 52.3, 110.6, 118.8, 119.0, 121.4, 121.6, 122.0, 127.7, 128.5, 128.7, 129.0, 129.9, 130.5, 131.2, 132.0, 135.7, 136.3, 136.9, 140.0, 142.3, 165.8, 169.0, 188.5 ppm; MS (ES): m/z (%) = 536.1 (100) [M + 1]⁺; C₃₁H₂₂ClN₃O₄ (535.1299): calcd for C 69.47, H 4.14, N 7.84; found C 69.60, H 4.20, N 7.91.

Methyl 1-(4-(4-chlorobenzoyl)-3-(4-chlorophenyl)isoxazol-5-yl)-9-ethyl-9H-pyrido[3,4-b]indole-3-carboxylate (18bB). Yield: 90% (0.18 g from 0.20 g) as an off-white solid; mp 181–182 °C; R_f = 0.45 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1672 (CO), 1716 (CO₂CH₃), 1620 (C=N–O), 1263 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 1.37 (t, J = 6.9 Hz, 3H), 3.81 (s, 3H), 4.46 (q, J = 6.9 Hz, 2H), 7.25 (d, J = 8.3 Hz, 2H), 7.44 (t, J = 7.4 Hz, 1H), 7.59 (d, J = 8.3 Hz, 2H), 7.71 (d, J = 8.2 Hz, 4H), 7.77 (t, J = 7.4 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 8.52 (d, J = 7.8 Hz, 1H), 9.00 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.4, 40.4, 52.6, 110.6, 118.7, 118.8, 121.4, 121.7, 122.0, 126.1, 127.4, 128.7, 129.3, 129.8, 130.0, 131.1, 132.1, 135.7, 136.2, 136.8, 136.9, 140.1, 142.3, 161.2, 165.7, 169.1, 188.4 ppm; MS (ES): m/z (%) = 570.2 (100) [M + 1]⁺; C₃₁H₂₁Cl₂N₃O₄ (569.0909): calcd for C 65.27, H 3.71, N 7.37; found C 65.33, H 3.72, N 7.40.

Methyl 1-(4-(4-chlorobenzoyl)-3-(p-tolyl)isoxazol-5-yl)-9-ethyl-9H-pyrido[3,4-b]indole-3-carboxylate (18bC). Yield: 85% (0.17 g from 0.20 g) as an off-white solid; mp 189–190 °C; R_f = 0.40 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1672 (CO), 1716 (CO₂CH₃), 1622 (C=N–O), 1262 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 1.36 (t, J = 7.0 Hz, 3H), 2.35 (s, 3H), 3.97 (s, 3H), 4.48 (q, J = 7.0 Hz, 2H), 7.25–7.32 (m, 4H), 7.44 (t, J = 7.5 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.69–7.79 (m, 3H), 7.89 (d, J = 8.4 Hz, 1H), 8.52 (d, J = 7.7 Hz, 1H), 9.00 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.4, 40.4, 52.5, 110.6, 118.8, 119.0, 121.4, 121.6, 122.0, 124.7, 127.7, 128.3, 128.7, 129.7, 129.9, 131.2, 131.9, 135.8, 136.3, 136.9, 139.9, 140.8, 142.3, 162.0, 165.8, 168.8, 188.7 ppm; MS (ES): m/z (%) = 550.1 (100) [M + 1]⁺. C₃₃H₂₄ClN₃O₄ (549.1455): calcd for C 69.88, H 4.40, N 7.64; found C 70.01, H 4.45, N 7.73.

Methyl 9-ethyl-1-(4-(4-fluorobenzoyl)-3-phenylisoxazol-5-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (18cA). Yield: 90% (0.27 g from 0.30 g) as an off-white solid; mp 178–179 °C; R_f = 0.40 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1667 (CO), 1716 (CO₂CH₃), 1616 (C=N–O), 1269 (Ar-C–O)_isoxazole; ¹H NMR (200 MHz, CDCl₃) δ = 1.47 (t, J = 7.2 Hz, 3H), 3.85 (s, 3H), 4.41 (q, J = 7.2 Hz, 2H), 6.93 (t, J = 8.6 Hz, 2H), 7.38–7.45 (m, 4H), 7.59–7.70 (m, 4H), 7.84–7.91 (m, 2H), 8.22 (d, J = 7.8 Hz, 1H), 8.90 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.4, 40.4, 52.6, 110.6, 115.5, 115.7, 118.8, 119.2, 121.5, 121.6, 122.1, 127.7, 127.8, 128.5, 129.0, 129.9, 130.5, 131.9, 132.5, 132.6, 133.7, 136.4, 136.9, 142.3, 162.1, 164.7, 165.9, 167.2, 168.9, 188.2 ppm; MS (ES): m/z (%) = 520.2 (100) [M + 1]⁺; C₃₁H₂₂FN₃O₄ (519.1594): calcd for C 71.67, H 4.27, N 8.09; found C 71.76, H 4.31, N 8.16.

Methyl 1-(3-(4-chlorophenyl)-4-(4-fluorobenzoyl)isoxazol-5-yl)-9-ethyl-9H-pyrido[3,4-b]indole-3-carboxylate (18cB). Yield: 92% (0.23 g from 0.25 g) as an off-white solid; mp 183–184 °C; R_f = 0.50 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1665 (CO), 1712 (CO₂CH₃), 1615 (C=N–O), 1260 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 1.48 (t, J = 7.1 Hz, 3H), 3.86 (s, 3H), 4.42 (q, J = 7.1 Hz, 2H), 6.94 (t, J = 8.6 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.42 (t, J = 7.8 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.72 (t, J = 7.7 Hz, 1H), 7.85–7.89 (m, 2H), 8.22 (d, J = 7.8 Hz, 1H), 8.85 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.4, 40.4, 52.6, 110.6, 115.5, 115.7, 118.9, 121.4, 121.7, 122.1, 126.2, 127.4, 129.3, 129.8, 130.0, 132.0, 132.4, 132.5, 136.8, 136.9, 142.3, 161.2, 165.8, 169.0, 188.0 ppm; MS (ES): m/z (%) = 554.1 (100) [M + 1]⁺; C₃₁H₂₁ClFN₃O₄ (553.1205): calcd for C 67.21, H 3.82, N 7.59; found C 67.32, H 3.86, N 7.67.

Methyl 9-ethyl-1-(4-(4-fluorobenzoyl)-3-(p-tolyl)isoxazol-5-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (18cC). Yield: 80% (0.16 g from 0.20 g) as an off-white solid; mp 190–191 °C; R_f = 0.45 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1667 (CO), 1718 (CO₂CH₃), 1620 (C=N–O), 1265 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 1.47 (t, J = 7.2 Hz, 3H), 2.36 (s, 3H), 3.84 (s, 3H), 4.41 (q, J = 7.2 Hz, 2H), 6.94 (t, J = 8.6 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 7.5 Hz, 1H), 7.55 (d, J = 8.2 Hz, 3H), 7.71 (t, J = 7.5 Hz, 1H), 7.87–7.90 (m, 2H), 8.22 (d, J = 7.9 Hz, 1H), 8.86 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.4, 21.6, 40.3, 52.6, 110.6, 115.5, 115.7, 118.8, 121.4, 121.6, 122.1, 124.7, 127.8, 128.4, 129.7, 129.8, 131.9, 132.5, 132.6, 133.7, 136.9, 140.8, 142.3, 162.0, 164.7, 165.9, 167.2, 168.7, 188.3 ppm; MS (ES): m/z (%) = 534.1 (100) [M + 1]⁺; C₃₂H₂₄FN₃O₄ (533.1751): calcd for C 72.04, H 4.53, N 7.88; found C 72.13, H 4.57, N 7.94.

(E)-Ethyl 1-((hydroxyimino)methyl)-9-methyl-9H-pyrido[3,4-b]indole-3-carboxylate (19). Yield: 98% (1.03 g from 1.00 g) as a light brown solid; mp 179–180 °C; R_f = 0.10 (hexane/EtOAc, 70 : 30, v/v); IR (KBr): ν_max = 1699 (CO₂CH₂CH₃), 3451 (NOH); ¹H NMR (400 MHz, CDCl₃) δ = 1.50 (t, J = 7.1 Hz, 3H), 4.07 (s, 3H), 4.25 (s, 1H), 4.53 (q, J = 7.1 Hz, 2H), 7.37 (t, J = 7.5 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 8.18 (d, J = 7.8 Hz, 1H), 8.84 (s, 1H), 8.85 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.5, 33.7, 62.1, 110.3, 118.6, 121.2, 121.9, 122.0, 130.2, 132.0, 133.8, 134.9, 137.6, 143.1, 148.6, 164.4 ppm; MS (ES): m/z (%) = 298.3 (100) [M + 1]⁺; C₁₆H₁₅N₃O₃ (297.1113): calcd for C 64.64, H 5.09, N 14.13; found C 64.70, H 5.11, N 14.17.

General procedure for the synthesis of β-carboline-substituted (C-3)isoxazole (20X–Z), as exemplified by compound 20X. To a stirred solution of 20 (0.40 g, 1.21 mmol) and methyl propiolate, X (0.14 mL, 1.51 mmol) in dry THF (8 mL), Et₃N (0.25 mL, 1.81 mmol) dissolved in THF (2 mL) was added dropwise at −78 °C and the contents stirred for 30 min at the same temperature. Thereafter, the reaction mixture was allowed to stir for an additional 1.5 h at room temperature. On completion of the reaction as monitored by TLC, THF was removed under reduced pressure to yield an off-white solid product, which was triturated twice with dry MeOH (10 mL). The product was finally washed with anhy. diethyl ether, filtered through a sintered funnel and dried under high vacuum to obtain an analytically pure white solid product, 20X (0.41 g, 89%, R_f = 0.40 (hexane/EtOAc, 80 : 20, v/v)).

Methyl 3-(3-(ethoxycarbonyl)-9-methyl-9H-pyrido[3,4-b]indol-1-yl)isoxazole-5-carboxylate (20X). Yield: 89% (0.41 g from 0.40 g) as a white solid; mp 187–188 °C; R_f = 0.40 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1741 (CO₂CH₃), 1717 (CO₂CH₂CH₃), 1630 (C=N–O), 1259 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 1.38 (t, J = 7.1 Hz, 3H), 3.74 (s, 3H), 3.99 (s, 3H), 4.42 (q, J = 7.1 Hz, 2H), 7.43 (s, 1H), 7.76 (s, 2H), 7.82 (s, 1H), 8.56 (s, 1H), 9.13 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 13.1, 31.6, 51.6, 59.8, 109.3, 110.1, 116.7, 119.3, 119.8, 120.4, 128.3, 128.9, 129.6, 135.0, 135.7, 141.7, 155.4, 158.6, 161.2, 163.5 ppm; MS (ES): m/z (%) = 380.1 (100) [M + 1]⁺; C₂₀H₁₇N₃O₅ (379.1168): calcd for C 63.32, H 4.52, N 11.08; found C 63.43, H 4.57, N 11.16.

Ethyl 3-(3-(ethoxycarbonyl)-9-methyl-9H-pyrido[3,4-b]indol-1-yl)isoxazole-5-carboxylate (20Y). Yield: 93% (0.44 g from 0.40 g) as a white solid; mp 160–161 °C; R_f = 0.45 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1754 (CO₂CH₃), 1716 (CO₂CH₂CH₃), 1627 (C=N–O), 1257 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, DMSO-d₆) δ = 1.39 (t, J = 6.8 Hz, 6H), 3.75 (s, 3H), 4.44 (q, J = 7.1 Hz, 4H), 7.44 (t, J = 7.1 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.0 Hz, 1H), 8.56 (d, J = 7.5 Hz, 1H), 9.13 (s, 1H) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ = 14.0, 14.4, 32.7, 61.1, 62.4, 111.1, 111.6, 118.4, 120.4, 121.2, 122.4, 129.8, 130.5, 136.2, 136.7, 142.9, 156.3, 160.1, 162.5, 164.9, 175.0 ppm; MS (ES): m/z (%) = 394.2 (100) [M + 1]⁺; C₂₁H₁₉N₃O₅ (393.1325): calcd for C 64.12, H 4.87, N 10.68; found C 64.23, H 4.90, N 10.74.

Ethyl 9-methyl-1-(5-phenylisoxazol-3-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (20Z). Yield: 86% (0.30 g from 0.30 g) as a white solid; mp 168–169 °C; R_f = 0.55 (hexane/EtOAc, 80 : 20, v/v); IR (KBr): ν_max = 1717 (CO₂CH₂CH₃), 1628 (C=N–O), 1260 (Ar-C–O)_isoxazole; ¹H NMR (400 MHz, CDCl₃) δ = 1.50 (t, J = 7.1 Hz, 3H), 3.96 (s, 3H), 4.55 (q, J = 7.1 Hz, 2H), 7.22 (s, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.49–7.57 (m, 4H), 7.70 (t, J = 7.4 Hz, 1H), 7.91–7.93 (dd, J₁ = 7.8 Hz, J₂ = 8.3 Hz, 2H), 8.27 (d, J = 7.8 Hz, 1H), 8.97 (s, 1H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ = 14.6, 32.2, 61.8, 101.9, 110.4, 118.0, 121.1, 121.2, 121.4, 121.8, 126.1, 127.4, 129.2, 129.5, 129.7, 130.5, 131.2, 132.1, 136.9, 137.4, 143.4, 162.7, 165.8, 170.2 ppm; MS (ES): m/z (%) = 398.1 (100) [M + 1]⁺; C₂₄H₁₉N₃O₃ (397.1426): calcd for C 72.53, H 4.82, N 10.57; found C 72.60, H 4.88, N 10.66.

Acknowledgements

D. S., N. D. and V. K. acknowledge Science and Engineering Research Board (SERB)-Department of Science and Technology (DST), Council of Scientific and Industrial Research (CSIR) and Ministry of Human Resource Development (MHRD), New Delhi, India for Junior and Senior Research Fellowships. V. S. and B. S. Kaith gratefully acknowledges the financial support in the form of research grant from CSIR (02(0202)/14/EMR-II), DST (CS-361/2011), and DST-FIST (CSI-228/2011) New Delhi (India). Central Facility, Indian Institute of Technology Ropar and Central Instrumental Facility, GJ University of Science and Technology, Hisar is gratefully acknowledged for recording MS, elemental analysis and NMR spectra reported in this paper. V. S. and co-workers gratefully acknowledges the motivation and guidance provided by Prof. Diwan S. Rawat, University of Delhi, Delhi.

Notes and references

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24. The single crystal XRD deposition number is CCDC 1485761.†.

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Footnote

† Electronic supplementary information (ESI) available: General information and NMR spectra (¹H and ¹³C) of new compounds. CCDC 1485761. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra15875g

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