Nitration of C₁₃, C₁₈ and C₂₃ Carbazole Alkaloids using Ceric Ammonium Nitrate (CAN)

 

Mumu Chakraborty

Department of Chemistry, Govt. Girls’ General Degree College, 7, Mayur Bhanj Road, Kolkata – 700023, West Bengal, India

*Corresponding Author E-mail: mumu_chak@yahoo.com

In course of our study on the nitration of carbazole alkaloids isolated from Murraya koenigii, we have observed that there is no report of the nitration of pyranocarbazole alkaloids with ceric ammonium nitrate in the literature. So we were interested to study the nitration of pyranocarbazole alkaloids girinimbine, koenidine,   koenimbine and mahanimbine isolated from M. koenigii. Interestingly selective mononitation occurred in girinimbine and koenidine, whereas koenimbine and mahanimbine afforded dinitro derivatives  as major products.  Nitration of glycozolidine with ceric ammonium nitrate on the other hand furnished a dimeric diniro compound. The mechanism of formation of new nitro pyranocarbazole alkaloids  in these reactions  will be discussed.

 

 

 

INTRODUCTION:

Carbazole alkaloids isolated from plant sources e.g. Murraya koenigii, Momordica charantia Linn   Roots were found to possess potent anti-microbial activity^1,2, anti-oxidant properties^3,4 and hyperglycemic activities⁵. Some carbazole linked propeonones synthesized by Unnisa et al. showed anti-TB activity⁶. Herbal gels containing Murraya koyenigii leaves extract was shown to possess anti-bacterial activity⁷. The plant also possess significant anti-stress potential against a variety of biochemical and physiological perturbations during stress⁸. Murraya koenigii leaves at a dose of 100mg/kg produces significant cognitive enhancing potential in albino rats⁹.

 

In recent times, nitration of carbazole alkaloids has become a challenging problem. Nagarajan et al. ¹⁰ had shown that urea nitrate–acetic acid combination is a mild, selective and efficient reagent for nitration of carbazoles. Another efficient method is Olah’s method of nitration¹¹, in which a mixture of copper nitrate trihydrate and acetic anhydride is used as the reagent. Silica supported copper(II) nitrate [Cu(NO₃)₂.3H₂O] reagent was employed for the nitration of aromatic compounds¹². Application of ceric ammonium nitrate (CAN) for nitration of carbazole alkaloids was done by Chakraborty et al ¹³. In our course of investigation, application of the same reagent on girinimbine 2¹⁴ and koenidine 4¹⁵, C₁₈ carbazole alkaloids isolated from Murraya koenigii, resulted substitution of one nitro group either in the aromatic ring or at the olefinic double bond in the pyran ring system. Surprisingly, koenimbine 3¹⁶, another C₁₈ carbazole alkaloid from Murraya koenigiiafforded two dinitro derivatives andmahanimbine 5¹⁷, a C₂₃ carbazole alkaloid also from Murraya koenigii, on reaction with CAN gave a dinitro derivative 5a along with cyclisation of its side chain.

Glycozolidine 1, a C₁₃ carbazole alkaloid isolated from Glycosmis arborea ¹⁸ and Glycosmis pentaphylla, on treatment with CAN afforded a novel dimeric dinitro compound 1a along with three mono-nitro derivatives viz. 8-nitro 1b, 5-nitro 1c and 7-nitroglycozolidine 1d. Structures of all the compounds were assigned on the basis of ¹H and ¹³C NMR spectral data analysis.

 

 

 

 

 

 

Figure 1:  Nitration of girinimbine 2, koenimbine 3, koenidine 4 and mahanimbine 5

 

 

 

 

 

 

 

RESULTS AND DISCUSSIONS:

Girinimbine 2 dissolved in acetonitrile, on treatment with equimolar amount of ceric ammonium nitrate resulted in the isolation of two mono-nitro derivatives. In one nitration occurred at the pyran ring system, which was indicated by the absence of the characteristic mutually coupled doublets at δ6.93 and 5.79 with coupling constant J=9.6Hz in girinimbine. In the second product, the aromatic proton singlet at δ 7.71 was absent. Structures of the products were confirmed to be 2’-nitrogirinimbine 2a and 4-nitrogirinimbine 2b by detailed ¹H and ¹³C NMR spectral data analysis summarized in Table 1 and 2 respectively. Koenidine 4 on similar reaction afforded the same C-4 substituted mono-nitro derivative, which was suggested by the absence of the aromatic singlet at δ7.45 in the PMR spectra of koenidine. Structure of the product was confirmed to be 4-nitrokoenidine 4a by detailed ¹H and ¹³C NMR spectral data analysis. Again koenimbine 3 dissolved in acetonitrile, on reaction with equimolar amount of CAN resulted in the isolation of two di-nitro derivatives. In both the products nitration occurred at the pyran ring system as well as in the aromatic ring, resulting in the formation of 4,2’-dinitrokoenimbine 3a and 7,2’-dinitrokoenimbine 3b and this was again indicated by the absence of the characteristic mutually coupled doublets at δ 6.60 and 5.69 with coupling constant J=9.6Hz in koenimbine. From the above facts it is clear that C-4 and C-2’ positions of pyranocarbazole alkaloids are favored for nitro group substitution.   Nitration of mahanimbine 5 was quite interesting. Mahanimbine 5 on treatment with CAN resulted in the isolation of a di-nitro derivative. One substitution occurred at aromatic A-ring and the other substitution occurred at the olefinic double bond present in the C₅H₉ residue in mahanimbine followed by cyclisation of the side chain. Presence of an aromatic singlet at δ 7.50 in the ¹H NMR spectrum of the product suggested that C-4 is unsubstituted unlike in most of the nitro derivatives of girinimbine, koenimbine and koenidine. Multiplicity and coupling constant of the remaining three aromatic signals clearly indicated that substitution occurred at C-6. Presence of the quaternary carbon at 61.4 ppm in the ¹³C NMR spectrum and comparison of the spectral data of the product with that of murrayazolinine ¹⁹, a compound isolated from Murraya koenigii,confirmed that the second nitro substitution occurred at C-7’. Finally structure of the product was confirmed to be 5a by detailed ¹H and ¹³C NMR spectral data analysis summarized in Table 1 and 2 respectively.  

 

 

 

 

 

 

Table-1: ¹H-NMR spectroscopic data (600 MHz, CDCl₃) for compounds 2a, 2b, 3a, 3b, 4a and 5a

Position	2a	2b	4a	5a	3a	3b
4	7.87, s	-	-	7.50, s	-	7.53
5	7.93, d (J=7.8Hz)	7.79,d (J=7.8Hz)	7.25, s	8.37, d (J=1.8Hz)	7.73	7.88
6	7.23, m	7.39, m	-	-	-	-
7	7.38, m	7.39, m	-	8.09, m	7.13, m	-
8	7.46, d (J=7.8Hz)	7.19, m	6.85, s	7.89, d (J=8.4Hz)	7.52	8.08,s
9	8.37, s	8.15, s	8.14, s	-	8.38, s	8.48, s
10	2.33, s	2.30, s	2.31, s	2.33, s	2.28, s	2.34, s
1’	8.15, s	6.62,d (J=10.2Hz)	6.58, d (J=10.2 Hz)	3.31, m	8.05,s	8.03, s
2’	-	5.83, d(J=10.2Hz)	5.80, d (J=10.2 Hz)	1.67, m 0.19, m	-	-
4’	1.81, s	1.51, s	1.49, s	2.40, m 2.03, m	1.50, s	1.62, s
5’	1.81, s	1.51, s	1.49, s	1.65, m	1.50, s	1.62, s
6’	-	-	-	1.95, m	-	-
8’	-	-	-	1.56, s	-	-
9’	-	-	-	1.34, s	-	-
10’	-	-	-	1.48, s	-	-
6-OCH3					3.99,s	4.07,s

 

 

 

 

 

 

 

 

 

 

Table-2: ¹³C-NMR spectroscopic data (150 MHz, CDCl₃) for compounds 2a, 2b, 4a and 5a

Position	2a	2b	4a	5a
1	102.4	108.6	107.2	107.4
2	145.3	149.3	149.4	157.2
3	118.5	119.7	112.3	112.4
4	120.4	143.9	142.8	119.9
4a	123.2	106.7	109.4	112.4
4b	117.9	112.1	111.9	138.9
5	125.0	126.0	103.3	118.9
6	125.2	121.1	147.9	132.7
7	119.5	120.5	144.5	115.2
8	110.9	110.7	94.0	109.1
8a	136.4	134.8	134.7	144.9
9a	139.3	139.6	134.7	143.1
10	15.8	10.7	10.9	15.4
1’	126.9	131.8	132.0	27.9
2’	151.0	116.3	116.3	21.6
3’	78.7	77.2	77.2	77.2
4’	25.7	29.7	27.5	35.8
5’	25.7	27.6	27.5	36.8
6’	-	-	-	48.2
7’	-	-	-	61.4
8’	-	-	-	29.7
9’	-	-	-	29.9
10’	-	-	-	23.4
6-OCH3	-	-	56.4	-
7-OCH3	-	-	56.0	-

 

Glycozolidine 1 dissolved in acetonitrile, on treatment with equimolar amount of ceric ammonium nitrate resulted in the isolation of four compounds. Presence of four aromatic methoxy and two aryl methyl groups in the ¹H NMR spectrum of 1a was indicative of its dimeric nature. FAB-MS showing m/z 570 (C₃₀H₂₆N₄O₈) was also supportive of the fact.It also showed the presence of six aromatic singlets which suggested that two more positions were substituted in each half of the dimer. The positions (C-1, C-5 and C-7) ortho to the methoxy groups are more nucleuphilic in nature. Absence of the upfield chemical shift in 1a at δ6.74 (H-1), present in 1,indicated that the linkage between the two moieties is present between H-1 and H-1’. The nitro group might be present at C-5 as well as C-7. Since ¹H NMR spectrum of 1a showed no splitting of the aromatic signals, the nitro groups must be present at C-7 and C-7’. Structures of the mono-nitro derivatives were determined by the coupling of aromatic proton signals in each case. ¹H NMR spectrum of compound 1b showed the presence of two aromatic singlets (at δ7.76 and 6.96) and two meta-coupled aromatic doublets at δ 7.77 and 7.81 with coupling constant J=2.4Hz, which suggested that in this compound the nitro group is at C-8 . ¹H NMR spectrum of compound 1c showed the presence of two aromatic singlets (at δ7.63 and 6.82) and two ortho-coupled aromatic doublets at δ 7.43 and 7.05 with coupling constant J=8.4Hz indicating position of the nitro group at C-5. Whereas four aromatic singlets at δ7.78, 6.87, 7.54 and 8.01 was present in the ¹H NMR spectrum of compound 1d, which is possible only when the nitro group is present at C-7. FAB-MS of all the three compounds showed m/z 286 (C₁₅H₁₄N₂O₄). Finally structures of the three compounds 1b, 1c and 1d were determined to be 8-nitro, 5-nitro and 7-nitroglycozolidine respectively by detailed ¹H and ¹³C NMR spectral data analysis summarized in Table 3 and 4 respectively.

 

Table-3: ¹H-NMR spectroscopic data (600 MHz, CDCl₃) for compounds 1a, 1b, 1c and 1d

 

 

 

Figure 2: Nitration of glycozolidine 1

 

 

Table-4: ¹³C-NMR spectroscopic data (150 MHz, CDCl₃) for compounds 1b, 1c and 1d

 

 

EXPERIMENTAL:

Transformation of girinimbine (2) into nitrogirinimbines 2a and 2b

213 mg of girinimbine 2 (0.0008mole)was dissolved in 30 ml of filtered acetonitrile. 456mg Ce(NH₄)₂(NO₃)₆ (0.0008 mole)was added to it. The reaction mixture was refluxed in a water bath for 15 minutes. Progress of the reaction was monitored by TLC. Solvent was evaporated under reduced pressure. The product was extracted with CHCl₃ using a separating funnel and washed with water (3 times). Then again it was concentrated under reduced pressure. TLC (Solvent system: Benzene: EtOAc = 9:1) showed the presence of multiple spots. The mixture of products was chromatographed on a column of silica gel (mesh size 60-120). Gradient elution was carried out with petroleum ether (bp 60-80ºC) followed by various mixtures of petroleum ether and benzene (3:1, 1:1, 1:3 and 100% benzene) and again various mixtures of benzene and chloroform (200 mL. each). Fractions giving similar spots were combined. The major product 2a (100 mg)was isolated as red crystalline solid with a 1:1 mixture of petroleum ether and benzene. The crystals were filtered and dried. Preparative TLC of the mother liquor with solvent system Benzene: EtOAc = 9:1 resulted in the isolation of compound 2b (40 mg) as an orange amorphous solid. EIMS showed (m/z) 308[M]⁺ for both the compounds (C₁₈H₁₆N₂O₃); ¹H NMR(Table-1) and ¹³C NMR (Table-2).

 

Transformation of koenimbine (3) into nitrokoenimbines 3a and 3b

70 mg of koenimbine 3 (0.00024mole)was dissolved in 20 ml of filtered acetonitrile. 133 mg Ce(NH₄)₂(NO₃)₆ (0.00024 mole)was added to it. The reaction mixture was stirred for one hour using a magnetic stirrer at room temperature. Progress of the reaction was monitored by TLC. Solvent was evaporated under reduced pressure. The products were extracted with CHCl₃ using a separating funnel and washed with water (3 times). Then again it was concentrated under reduced pressure. TLC (Solvent system: Benzene: EtOAc = 1:1) showed the presence of multiple spots. The mixture of products was chromatographed on a column of silica gel (mesh size 60-120). Gradient elution was carried out with petroleum ether (bp 60-80ºC) followed by various mixtures of petroleum ether and benzene (3:1, 1:1, 1:3 and 100% benzene) and again various mixtures of benzene and chloroform (200 mL. each). Fractions giving similar spots were combined. Preparative TLC of one fraction (eluted with benzene) with solvent system Benzene: EtOAc = 1:1 resulted in the isolation of compounds 3a (7 mg)and 3b (8 mg), both as yellow amorphous solids. ¹H NMR data of both the compounds are given in  Table-1.

 

Transformation of koenidine (4) into nitrokoenidine 4a

266 mg of koenidine 4 (0.00048mole)was dissolved in 40 ml of filtered acetonitrile. 266mg Ce(NH₄)₂(NO₃)₆ (0.00048mole)was added to it when the colour changed to dark green. The reaction mixture was refluxed in a water bath for a few minutes. Progress of the reaction was monitored by TLC. Solvent was evaporated under reduced pressure. The product was extracted with CHCl₃ using a separating funnel and washed with water (3 times). Then again it was concentrated under reduced pressure. TLC (Solvent system: 100% EtOAc) showed the presence of multiple spots. The mixture of products was chromatographed on a column of silica gel (mesh size 60-120). Gradient elution was carried out with petroleum ether (bp 60-80ºC) followed by various mixtures of petroleum ether and benzene (3:1, 1:1, 1:3 and 100% benzene) and again various mixtures of benzene and chloroform (200 mL. each). Fractions giving similar spots were combined. The major product 4a (110mg)was eluted with CHCl₃ as a black amorphous solid. FAB-MS showed (m/z) 368[M]⁺ (C₂₀H₂₀N₂O₅); ¹H NMR(Table-1) and ¹³C NMR (Table-2).

 

Transformation of mahanimbine (5) into 5a

50 mg of mahanimbine 5 (0.00015mole)was dissolved in 20 ml of filtered acetonitrile. 83mg Ce(NH₄)₂(NO₃)₆ (0.00015 mole)was added to it. The reaction mixture was stirred overnight using a magnetic stirrer at room temperature. Progress of the reaction was monitored by TLC. Solvent was evaporated under reduced pressure. The product was extracted with CHCl₃ using a separating funnel and washed with water (3 times). Then again it was concentrated under reduced pressure. TLC (Solvent system: petroleum ether:benzene=1:1) showed the presence of multiple spots. The mixture of products was chromatographed on a column of silica gel (mesh size 60-120). Gradient elution was carried out with petroleum ether (bp 60-80ºC) followed by various mixtures of petroleum ether and benzene (3:1, 1:1, 1:3 and 100% benzene) and again various mixtures of benzene and chloroform (200 mL. each). Fractions giving similar spots were combined. The major product 5a (26 mg)was eluted with a 1:1 mixture of petroleum ether and benzene, as an orange amorphous solid. Molecular formula (C₂₃H₂₅N₃O₅); ¹H NMR (Table-1) and ¹³C NMR (Table-2).

 

Nitration of glycozolidine 1

50 mg of glycozolidine 1 (0.0002mole)was dissolved in 20 ml of filtered acetonitrile. 116mg Ce(NH₄)₂(NO₃)₆ (0.0002 mole)was added to it. The reaction mixture was stirred overnight at room temperature. Progress of the reaction was monitored by TLC. Solvent was evaporated under reduced pressure. The product was extracted with CHCl₃ using a separating funnel and washed with water (3 times). Then again it was concentrated under reduced pressure. TLC (Solvent system: Benzene: EtOAc = 9:1) showed the presence of multiple spots. The mixture of products was chromatographed on a column of silica gel (mesh size 60-120). Gradient elution was carried out with petroleum ether (bp 60-80ºC) followed by various mixtures of petroleum ether and benzene (3:1, 1:1, 1:3 and 100% benzene) and again various mixtures of benzene and chloroform (200 mL. each). Fractions giving similar spots were combined. The major product 1b (15 mg) and the dimeric compound 1a (7 mg)was eluted as orange amorphous solids with a 1:1 mixture of petroleum ether and benzene and with 100% benzene respectively. Again Preparative TLC of a fraction (eluted with a 1:3 mixture of petroleum ether and benzene) with solvent system Benzene: EtOAc = 9:1 resulted in the isolation of compounds 1c and 1d (10 mg each). FAB-MS showed m/z 286 [M]⁺ for 1b, 1c and 1d (C₁₅H₁₄N₂O₄) and m/z 570 [M]⁺ for 1a (C₃₀H₂₆N₄O₈)  ; ¹H NMR(Table-3) and ¹³C NMR (Table-4).

 

CONCLUSION:

Application of ceric ammonium nitrate (CAN) on C₁₃, C₁₈ and C₂₃ carbazole alkaloids isolated from Murraya koenigii and Glycosmis pentaphylla resulted in the isolation of a number of nitro derivatives of the carbazole alkaloids. In case of pyrano carbazole alkaloids (C₁₈), the major site of nitration was observed to be C-4. Some nitration also occurred at the pyran ring system. When mahanimbine (C₂₃ carbazole) was treated with CAN, Nitration occurred at the side chain with the formation of a new ring. On application of CAN at the C₁₃ alkaloid, nitration occurred mostly at C-5, C-7 and C-8 position along with the formation of an interesting dimmer.

 

ACKNOWLEDGEMENT:

I sincerely acknowledge my Ph.D. supervisor Dr.Sibabrata Mukhopadhyay, Former Scientist, IICB, Kolkata. Special thanks to Director of IICB for his keen interest in the work. Thanks are also due to Ajanta Mukherjee (Bose Institute, Kolkata) for references and Mr. R. Padmanaban for NMR data. Finally the authors are very grateful to ‘Council of Scientific and Industrial Research’ for financial support.

 

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Received on 21.02.2018         Modified on 20.03.2018

Accepted on 07.04.2018         © AJRC All right reserved