Integrated synthesis of 3,4-carbazoquinone alkaloids N-Me-carbazoquinocin A, B and D–F

Carbazole alkaloids carbazoquinocin A–F possessing a 1-alkyl-2-methyl-3,4-ortho-carabazoquinone framework were isolated from the microorganism Streptomyces violaceus 2448-SVT2 in 1995. Furthermore, they were found to exhibit strong inhibitory activity against lipid peroxidation. Herein, we report the integrated synthesis of N-Me-analogues of 5 members of the carbazoquinocin family of natural products, namely, N-Me-carbazoquinocin A, B and D–F. We employed an acid-catalyzed, intramolecular benzannulation of indole-appended Z-enoate propargylic alcohols, which was developed earlier in our laboratory, for the construction of the required carbazole framework. All five natural products were obtained in an overall yield of 50–60%, starting from a commercially available indole.


Introduction
Carbazoles (dibenzopyrroles) are nitrogen-based tricyclic frameworks, which are essential in medicinal chemistry 1 and materials chemistry. 2They are also part of many structurally complex bioactive natural products and drug molecules. 3mong the many structurally diverse carbazole natural products, a 1-alkyl-2-methyl-carbazole framework has attracted special attention as it has been found to be present in different families of bioactive carbazole natural products such as lipocarbazoles, carbazoquinocins, and carazostatin. 4In 1995, the research group of Seto isolated six carbazoquinocins A-F, from the microorganism Streptomyces violaceus 2448-SVT2 (Fig. 1). 5 These natural products share a common 1-alkyl-2-methyl-3,4ortho-carbazoquinone framework and have been found to exhibit strong inhibitory activity against lipid peroxidation. 6everal research groups have reported the total synthesis of some of these novel alkaloids. 7For example, the total synthesis of carbazoquinocins A and D was achieved by the research group of Ogasawara in 1996.7a Alternatively, Hibino et al. reported the total synthesis of carbazoquinocins B-F.7b In 2003, Wulff and coworkers reported the total synthesis of carbazoquinocin C. 7c Recently, in 2022, we developed an approach for the total synthesis of the 1-alkyl-2-methyl-3,4-carbazoquinone natural product N-Me-carbazoquinocin C together with N-Mecarazostatin and N-Me-lipocarbazole A4. 7d This approach employs a Brønsted acid-catalyzed intramolecular benzannulation of C-3-tethered indole-propargylic alcohols (1) (Scheme 1A) as the key step for the construction of a carbazole unit (2).In continuation, we envisioned an opportunity to extend this methodology for the total synthesis of other members of the carbazoquinocin family, i.e., carbazoquinocin A, B, and D-F (3-7) (Fig. 1).According to our retrosynthetic analysis (Scheme 1B), butyraldehyde present in 2 can be converted into the respective alkyl chain (as in 8) required for carbazoquinocin A, B and D-F through the Wittig olenation reaction using suitable phosphorous ylides.Further, oxidation at the C-3 and C-4 positions of the resultant carbazoles 8 to generate the natural products carbazoquinocin A, B and D-F (3-7) can be achieved through C-3-methoxylation, followed by selenium promoted oxidations.
Next, the regioselective electrophilic bromination of carbazole 12a with NBS in CHCl 3 at rt gave the corresponding 3bromocarbazole (13a) (90%) within 6 min reaction time.Next, we employed a two-step strategy for the efficient conversion of bromine in 13a into a hydroxyl group.Initially, heating a DMF suspension of 13a, NaOMe and CuI at 115 °C gave 3-methoxy carbazole (14a) in 90% yield.Subsequent treatment of 14a with BBr 3 in CH 2 Cl 2 at 0 °C to rt for 14 h resulted in the formation of the phenol product (15a) in 82% yield via deprotection of the methoxy group.Finally, the synthesis of N-Me-carbazoquinocin B 3 was achieved by employing the (PhSeO) 2 O-promoted oxidation of 15a.Accordingly, heating (50 °C) a solution of 15a and (PhSeO) 2 O in THF gave N-Me-carbazoquinocin B 3 in 95% yield.Aer successfully completing the total synthesis of N-Me-carbazoquinocin B 3, we extended this strategy for the synthesis of N-Me-carbazoquinocin D 4 by employing the carbazole-butyraldehyde 2 as the starting point (Scheme 2, R = Me).According to our retrosynthetic analysis (Scheme 1B), the only difference between carbazoquinocin B 3 and carbazoquinocin D 4 is the incorporation of an iso-propyl group (R = H) vs. sec-butyl group (R = Me) in aldehyde 2. Therefore, olenation of aldehyde 2 using sec-butylidene phosphorous ylide 9b (generated from (sec-butyl)PPh 3 Br 8 10b and n BuLi at 0 °C) afforded Z-olen 11b (R = Me) in 76% yield.The geometry of the olen in 11b was identied by using the correlations observed between ]CMe and ]C-H in the corresponding NOESY NMR spectrum.Further, by operating the same sequence of ve functional group transformations, as depicted in Scheme 2 (R = Me), i.e., hydrogenation (12b, 91%)-bromination (13b, 92%)-methoxylation (14b, 90%)-demethylation (15b, 91%)-oxidation (4, 89%), we efficiently converted olen 11b into N-Mecarbazoquinocin D 4, with an overall yield of 61% for ve steps.Subsequently, we envisioned the extension of this methodology for the total synthesis of N-Me-carbazoquinocin E 5 and F 6 (Scheme 3).The required alkyl chains at the C 1position present in compounds 5 and 6 could be achieved by incorporating an iso-butyl group (n = 0) and iso-pentyl group (n = 1) in butyraldehyde 2 via Wittig olenation reaction, respectively.Wittig olenation of aldehyde 2 using iso-butylidene phosphorous ylide 9c (ref.Finally, we also extended this strategy for the total synthesis of N-Me-carbazoquinocin A 7, which possesses a 3-methylpentyl alkyl chain at the C 1 -position of the carbazoquinone (Scheme 4).We envisioned carbazole ester (16) as a suitable starting material for this purpose.a-Methylation (for sec-methyl) followed by conversion of the carboxylate group into an ethyl group generates the required 3-methylpentyl chain at the C 1 -position.Accordingly, the LDA-mediated enolate formation of ester 16 followed by quenching with MeI gave a-methyl ester (17) in 59% yield.The reduction of 17 with LiAlH 4 followed by oxidation of the resultant primary alcohol (18) with Dess-Martin periodinane (DMP) gave aldehyde (19) in 84% overall yield for two steps.

Conclusions
In conclusion, we developed an efficient and practically viable synthetic approach for the unied synthesis of 3,4carbazoquinone-based carbazole alkaloids N-Mecarbazoquinocins A, B and D-F.Our earlier methodology, an acid-catalyzed, intramolecular benzannulation of indoleappended Z-enoate propargylic alcohols, was employed for the construction of the required carbazole framework.All ve natural products were obtained in an overall yield of 50-60%, starting from the commercially available indole.

General information
All solvents were distilled prior to use and anhydrous solvents were prepared according to the standard drying procedures.All non-aqueous reactions were carried out under an atmosphere of nitrogen in ame-dried glassware.Commercially available chemicals were purchased from Sigma-Aldrich, Alfa Aesar and Spectrochem Pvt.Ltd and were used as received without further purication.Infrared (IR) spectra were recorded on a JASCO 4100 FT-IR spectrometer. 1 H NMR spectra were measured on a Bruker AVANCE 400 MHz or Bruker AVANCE 500 MHz spectrometer.Chemical shis are reported in ppm relative to solvent signals. 13C NMR spectra were recorded on a Bruker AVANCE 100 MHz or Bruker AVANCE 125 MHz spectrometer with complete proton decoupling.Chemical shis are reported in ppm from the residual solvent as an internal standard [CDCl 3 d = 7.26 ppm for 1 H, d = 77.16ppm for 13 C or calibrated to tetramethylsilane (d = 0.00)].The following abbreviations are used to indicate multiplicities: s-singlet; d-doublet; t-triplet; qquartet; quint-quintet; sext-sextet; sept-septet; m-multiplet; dd-doublet of doublet; dt-doublet of triplet; dq-doublet of quartet; td-triplet of doublet; tt-triplet of triplet; dq-doublet of quartet; br-broad; J-coupling constant in Hz.The coupling constant J (Hz) was rounded to one decimal place for all compounds.When a coupling pattern can be assigned as a combination of multiplicities, the above-mentioned abbreviations were combined to describe the observed patterns (i.e., dt, doublet of triplets).Mass spectra were recorded by the electrospray ionization (ESI) method on a Q-TOF Micro with a lock spray source.For thin layer chromatography (TLC) analysis throughout this work, E-Merck precoated TLC plates (silica gel 60 F254 grade, 0.25 mm) were used and visualized using a UV lamp (366 or 254 nm) or by using of one of the following visualization reagents: PMA: 1 g phosphomolybdic acid/10 mL ethanol; KMnO 4 : 0.15 g potassium permanganate, 1 g K 2 CO 3 / 20 mL water.Acme (India) silica gel (100-200 mesh) was used for column chromatography.
General procedure I (GP-I): Wittig olenation with primary alkyl-based phosphonium salts and t BuOK base. 9,4d RPPh 3 X (2.0 equiv.)and anhydrous THF (8 mL) were added to a ame-dried round-bottom ask under an N 2 atmosphere.Aer the mixture was cooled to 0 °C, t BuOK (1.8 equiv.) was solubilized in anhydrous THF (4 mL) and added dropwise to the reaction mixture over a period of 5 min.The resulting mixture was warmed to room temperature and stirred for 1 h.Then the reaction mixture was cooled back 0 °C, and a solution of aldehyde 2 (1.0 equiv.) in anhydrous THF (5 mL) was added dropwise.The nal reaction mixture was stirred at room temperature until the TLC showed the complete consumption of the aldehyde.Aer completion, saturated NH 4 Cl solution (6 mL) was added to quench the reaction at 0 °C.Next, diethyl ether (5 mL) and water (5 mL) were added.The color of the mixture changed from pale-yellow to white.Aer separation of the layers, the residual compound from the aqueous layer was extracted with EtOAc (3 × 5 mL).The combined organic layers were dried over anhydrous Na 2 SO 4 , ltered, and concentrated in vacuo.Purication of the crude product using silica gel column chromatography (9 : 1 hexanes/EtOAc) provided the corresponding alkene.
General procedure II (GP-II): Wittig olenation with secondary alkyl-based phosphonium salts and n-BuLi base. 9RPPh 3 X (2.0 equiv.)and anhydrous THF (8 mL) were added to a ame-dried round-bottom ask under an N 2 atmosphere.Aer the mixture was cooled to 0 °C, n-BuLi (3.0 equiv., 1.6 M in hexane) was solubilized in anhydrous THF (4 mL) and added dropwise to the reaction mixture over a period of 5 min.The resulting mixture was warmed to room temperature and stirred for 1 h.Then the reaction mixture was cooled back 0 °C, and a solution of aldehyde 2 (1.0 equiv.) in anhydrous THF (5 mL) was added dropwise.The nal reaction mixture was stirred at room temperature until TLC showed the complete consumption of the aldehyde.Aer completion, saturated NH 4 Cl solution (6 mL) was added to quench the reaction at 0 °C.Next, diethyl ether (5 mL) and water (5 mL) were added.The colour of the mixture changed from pale yellow to white.Aer separation of the layers, the residual compound from the aqueous layer was extracted with EtOAc (3 × 5 mL).The combined organic layers were dried over anhydrous Na 2 SO 4 , ltered, and concentrated in vacuo.Purication of the crude product using silica gel column chromatography (9 : 1 hexanes/EtOAc) provided the corresponding alkene.
General procedure III (GP-III): hydrogenation of internal alkenes using Pd/C.Pd/C (10 w/w%) was added to a well-stirred solution of internal alkenes (1.0 equiv.) in EtOAc (10 mL).The resulting reaction mixture was stirred under hydrogen (1 atm) atmosphere for an appropriate time at room temperature.Aer completion, the reaction mixture was ltered through a Celite® pad by using EtOAc (20 mL).The ltrate was concentrated in vacuo.Purication of crude product using a silica gel column chromatography (hexanes/EtOAc) provided the desired reduced product.
General procedure IV (GP-IV): bromination of carbazoles using N-bromosuccinimide (NBS).N-Bromosuccinimide (1.0 equiv.) was added a solution of carbazole derivative (1.0 equiv.) in chloroform (CHCl 3 , 10 mL) at room temperature under a nitrogen atmosphere.The reaction mixture was stirred at the same temperature for 6 min until TLC showed the complete consumption of the starting material.Aer completion, water (10 mL) was added.The residual compound from the aqueous layer was extracted with CH 2 Cl 2 (3 × 5 mL).The combined organic layers were dried over anhydrous Na 2 SO 4 , ltered, and concentrated in vacuo.Purication of the crude product using silica gel column chromatography (hexanes/EtOAc) provided the desired 3-bromocarbazole derivative.
General procedure V (GP-V): methoxylation using NaOMe.A 15 mL Schlenk tube equipped with a magnetic stirrer was evacuated, and then backlled with nitrogen gas.This process was repeated three times.Next, 2 mL of anhydrous MeOH was added, and the reaction vessel was cooled to 0 °C.To this stirring MeOH solvent, sodium metal (54 equiv.) was carefully added in portions under a positive nitrogen pressure to form an ∼2.7 M solution of sodium methoxide (NaOMe) in MeOH.Aer the complete dissolution of Na in MeOH, the solution became thick and light yellowish.Next, the corresponding 3-bromocarbazole (1.0 equiv.)dissolved in DMF (1.4 mL) and CuI (4.0 equiv.)were added to this freshly prepared NaOMe solution under a nitrogen atmosphere.The resulting reaction mixture was transferred to a preheated oil bath and stirred at 115 °C for 15 h.Aer the complete consumption of the starting material, as indicated by TLC, the crude reaction mixture was ltered through a short plug of Celite® and washed with EtOAc.The ltrate was sequentially washed with saturated NH 4 Cl solution (5 mL), water (10 mL) and brine (5 mL), dried over anhydrous Na 2 SO 4 and concentrated in vacuo.Purication of the crude product using a silica gel column chromatography (9 : 1 hexanes/EtOAc) provided the desired 3-methoxycarbazole derivatives.
General procedure VI (GP-VI): BBr 3 -mediated deprotection of the methoxy group to phenol.A 1 M solution of boron tribromide (BBr 3 ) in CH 2 Cl 2 (3.0 equiv.) was added dropwise to a stirred solution of the corresponding 3-methoxycarbazole derivative (1.0 equiv.) in anhydrous CH 2 Cl 2 (4 mL) at 0 °C.The reaction mixture was allowed to warm up to room temperature and further stirred for the appropriate time.Aer completion of the reaction, the reaction mixture was quenched with H 2 O (5 mL).The organic layer was separated, and the residual compound from the aqueous layer was extracted with EtOAc (3 × 5 mL).The combined organic layers were washed with H 2 O, brine and dried (Na 2 SO 4 ) and concentrated in vacuo.Purication of the crude product using silica gel column chromatography (4 : 1 hexane/ EtOAc) provided the desired 3-hydroxycarbazole derivative.
General procedure VII (GP-VII): synthesis of N-Me carbazoquinocin natural products.(PhSeO) 2 O (2.0 equiv.) was added to a solution of N-methyl-2-alkyl-3-hydroxy carbazole (1.0 equiv.) in THF (4 mL) at rt under a nitrogen atmosphere.The reaction mixture was stirred at 50 °C for 30 min.Aer cooling to room temperature, the mixture was quenched with water, and the residual compound from the aqueous layer was extracted with EtOAc (3 × 5 mL).The combined organic layers were washed with H 2 O, brine and dried (Na 2 SO 4 ) and concentrated in vacuo.Purication of the crude product using silica gel column chromatography (hexane/EtOAc) provided the desired N-methylcarbazoquinocin natural products.
General procedure VIII (GP-VIII): preparation of required Wittig salts. 8Triphenylphosphine (1.0 mmol) was placed in an oven-dried Schlenk tube and dissolved in 2 mL toluene at room temperature under an N 2 -atmosphere.To this reaction mixture, the required alkyl bromide (1.1 mmol) was added.A white precipitate was formed aer stirring and reuxing at 110 °C for 48 h using an oil bath.The reaction mixture was cooled to room temperature, and the phosphonium salt was recrystallized from petroleum ether and ethyl acetate solvent mixture.The salt was separated, washed with Et 2 O, and dried in vacuo to give the corresponding phosphonium bromide.

Fig. 1
Fig. 1 Structures of carbazoquinocin A-F natural products.