Trimeric and Dimeric Carbazole Alkaloids from Murraya microphylla

Seventeen new carbazole alkaloid derivatives, including a trimeric carbazole racemate, (±)-microphyltrine A (1), 15 dimeric carbazole racemates, (±)-microphyldines A–O (2–16), and a C-6–C-3″-methyl-linked dimeric carbazole, microphyldine P (17), were isolated from the leaves and stems of Murraya microphylla (Merr. et Chun) Swingle. The structures of the new compounds were elucidated on the basis of HRESIMS and NMR data analysis. The optically pure isomers of these isolated carbazole alkaloids were obtained by chiral HPLC separation and their absolute configurations were determined by electronic circular dichroism (ECD) data analysis.


Introduction
Carbazole alkaloids, one type of bioactive constituents from the Murraya genus, have been demonstrated to possess anti-inflammatory, antitumor, antimicrobial, antioxidant, and antidiabetic properties [1]. Many of the biologically active carbazole alkaloids have been isolated from four closely related genera, Clausena, Glycosmis, Murraya, and Micromelum of the family Rutaceae [2][3][4]. Murraya microphylla (Merr. et Chun) Swing (M. microphylla) is a shrub distributed in the thickets of sandy areas or coastal regions in the Hainan Province of China [5]. Previous chemical investigations have confirmed that M. microphylla contains abundant carbazole alkaloids [6,7].
The 13 C nmR data (Table 3) showed 37 carbon resonances, comprising 30 olefinic, six methyl, and one methoxy carbons. The above data, coupled with information from the literature [4,14,15] and 2D nmR analysis, indicated a dimeric carbazole skeleton of 2, and the two carbazole units were deduced as koenigine [12] and murrayamine A [16] moieties, respectively. The HMBC correlations from H-5 to C-8 indicated that the two units were linked by a C-8-C-6 bond ( Figure S12, Supporting Information). Therefore, the planar structure of 2 was assigned as shown ( Figure 1).
Compound 2 could also be a racemate owing to the disappeared specific rotation and Cotton effects in the ECD spectrum, thus, it was then separated by a chiral HPLC to give the enantiomers 2a and 2b for almost equal quantity, which possess the opposite ECD curves and specific rotations. Similar to 1, the ECD spectrum of 2a exhibited sequential negative and positive Cotton effects at 252 nm and 224 nm, indicating an (R a ) configuration for (−)-microphyldine A (2a) [14] and, accordingly, the configuration of (+)-microphyldine A (2b) was defined as (S a ). Furthermore, the ECD spectra of (R a )-and (S a )-2 were calculated and compared with the experimental spectra to support the results of ECD exciton coupling ( Figure 4).
(±)-Microphyldine B (3) Tables 2 and 3) of 3 showed close resemblance to those of 2, except that one of the phenyl singlets in 3 was missing, but an additional methoxy signal was observed at δ H 3.99 (3H, s). After 2D nmR analysis ( Figure S19, Supporting Information), the two units were deduced to be both koenigine units [12]. The deficiency of H-8 and H-5 signals suggested 3 to be a C-8-C-5 -linked dimeric carbazole. Thus, the planar structure of 3 was assigned as shown ( Figure 1).
a Assignments were based on HSQC and HMBC experiments. b Measured in 500 MHz, and others in 400 MH measured in CDCl3 and others were measured in acetone-d6.
Compound 2 could also be a racemate owing to the disappeared specif Cotton effects in the ECD spectrum, thus, it was then separated by a chiral the enantiomers 2a and 2b for almost equal quantity, which possess the curves and specific rotations. Similar to 1, the ECD spectrum of 2a exhibi negative and positive Cotton effects at 252 nm and 224 nm, indicating an ( tion for (−)-microphyldine A (2a) [14] and, accordingly, the configuration phyldine A (2b) was defined as (Sa). Furthermore, the ECD spectra of (Ra)-a calculated and compared with the experimental spectra to support the resu citon coupling ( Figure 4)  Tables 2 and 3) of 3 showed clos to those of 2, except that one of the phenyl singlets in 3 was missing, but methoxy signal was observed at δH 3.99 (3H, s). After 2D NMR analysis (Fi porting Information), the two units were deduced to be both koenigine u deficiency of H-8 and H-5'' signals suggested 3 to be a C-8-C-5''-linked dim Thus, the planar structure of 3 was assigned as shown ( Figure 1). Similar to 2, compound 3 is also an atropisomeric racemate, and the fo HPLC resolution afforded 3a and 3b in a ratio of 1:1, and their absolute were established as (Ra) and (Sa), respectively, by comparison of their EC Supporting Information) and specific rotations with those of 2a and 2b [14] (±)-Microphyldine C (4)   Similar to 2, compound 3 is also an atropisomeric racemate, and the following chiral HPLC resolution afforded 3a and 3b in a ratio of 1:1, and their absolute configurations were established as (R a ) and (S a ), respectively, by comparison of their ECD ( Figure S18, Supporting Information) and specific rotations with those of 2a and 2b [14].     Tables 2 and 3) suggested that the structure of 4 resembled that of 3, except for the disappearance of one methoxy group in 4, and the replacement of an aromatic singlet in 3 by two aromatic doublets (δ H 7.00 (1H, d, J = 8.0 Hz, H-7 ), 7.31 (1H,d,J = 8.0 Hz,). This suggested that 4 is the demethoxy derivative of 3. Further 2D nmR analysis ( Figure S26, Supporting Information) deduced the structure of 4 as shown (Figure 1). The isolation of individual enantiomers (4a and 4b) was accomplished by a chiral HPLC separation and their absolute configurations were established as (R a ) and (S a ), respectively, by comparison of their ECD ( Figure S25, Supporting Information) and specific rotations with those of 2a and 2b [14].
(±)-Microphyldine D (5)  The HMBC correlations from H-5 to C-2 and C-7 and from H-6 to C-3 , C-7 , and C-8 suggested that the isopentenyl moiety is located at C-5 ( Figure 2). Compound 5 is also a pair of atropisomers, and the following chiral HPLC resolution afforded 5a and 5b in a ratio of 1:1, and their absolute configurations were established as (R a ) and (S a ), respectively, by comparison of their ECD ( Figure S32, Supporting Information) and specific rotations with those of 4a and 4b [14]. The experimental ECD spectra of 5a/5b are almost similar to those of 4a/4b, indicating that the C-3 configuration did not have much influence on the ECD curves, which was proofed by the calculated ECD data of the different configurations of C-3 . Thus, the C-3 configuration was undetermined in this paper.
(±)-Microphyldine F (7) was shown to have the same molecular formula as 2, according to its 13 C nmR data and the [M − H] − ion at m/z 585.2390 in the HRESIMS (calcd. for C 37 H 33 N 2 O 5 , 585.2389). Analysis of 1 H and 13 C nmR data (Tables 2 and 3) of 7 showed a close structural resemblance to 2, a dimeric carbazole formed by koenigine [12] and murrayamine A [16] units. The difference between them is that the linkage mode is shifted from C-8-C-6 in 2 to C-8-C-8 in 7, as deduced from the deficiency of H-8 and H-8 signals in 7. Accordingly, the structure of 7 was determined as shown ( Figure 1). A subsequent chiral HPLC isolation was performed to obtain the pure enantiomers of 7a and 7b in a ratio of 1:1. After ECD and specific rotation determination, the absolute configurations of 7a and 7b were established as (R a ) and (S a ), respectively, by comparison of their ECD ( Figure S45, Supporting Information) and specific rotations with those of 2a and 2b [14].
(±)-Microphyldine H (9) (Tables 2 and 3) analysis revealed that the structure of 9 is a dimeric carbazole formed by a koenigine [12] and an isomurrayafoline B [17,18] unit. The 2D nmR data ( Figure S60, Supporting Information) analysis indicated the linkage mode of 9 was C-8-C-1 due to the absence of H-8 and H-1 protons. Accordingly, the structure of 9 was determined as shown ( Figure 1). A subsequent chiral HPLC isolation was performed to obtain the pure enantiomers of 9a and 9b in a ratio of 1:1. After ECD and specific rotation determination, the absolute configurations of 9a and 9b were established as (R a ) and (S a ), respectively, by comparison of their ECD ( Figure S59, Supporting Information) and specific rotations with those of 2a and 2b [14].
(±)-Microphyldine I (10) was isolated as a brown amorphous powder. Its molecular formula was defined as C 26 H 20 N 2 O 2 via its 13 Tables 2 and 3) of the monomeric unit of 10 resembled those of 1-hydroxy-3-methylcarbazole [19], except for the absence of H-4 proton and a shift of the C-4 signal downfield to δ C 125.0, indicating that the two units are linked through C-4-C-4 . Compound 10 was a pair of atropisomer mixtures inferred from its almost zero specific rotation and weak ECD Cotton effects. The pure enantiomers of 10a and 10b were obtained by a chiral HPLC separation. The absolute configurations of 10a and 10b were defined as (R a ) and (S a ), respectively, from their experimental ECD spectra and computed ECD spectra using the TDDFT method at the B3LYP/6-311 + G(d) level ( Figure S66, Supporting Information).
(±)-Microphyldine J (11) (Tables 2 and 3) showed many similarities to those of murrayaquinone A [20], except for the replacement of a methoxy singlet in murrayaquinone A by a hydroxy singlet (δ H 8.80 (1H, br s)) in 11. The 2D nmR analysis, especially of HMBC correlation from OH-1 to C-9a, from CH 3 -3 to C-2, and from CH 3 -3 to C-4 proved the above deduction ( Figure S74, Supporting Information). Compound 11 was separated by a chiral HPLC to give the enantiomers of 11a and 11b. The absolute configurations of 11a and 11b were defined as (R a ) and (S a ), respectively, from their experimental ECD and computed ECD spectra ( Figure S73, Supporting Information).
(±)-Microphyldine K (12) (Table 4) of 12 were found to be similar to those of murrafoline D [21]. The apparent differences were the replacement of two aromatic signals in murrafoline D by two active hydrogen signals in 12, suggesting that 12 is a dihydroxy derivative of murrafoline D [21]. The two hydroxy groups were deduced to be located at C-6 and C-7 , respectively, via the HMBC correlation of one hydroxy proton (δ H 7.66 (1H, br s)) with C-5/C-7, and the other hydroxy proton (δ H 8.23 (1H, br s) with C-6 and C-8 ( Figure S80, Supporting Information). The optical inactivity of 12 indicated that it is a pair of enantiomer mixture, thus a chiral HPLC isolation was performed to obtain the pure enantiomers of 12a and 12b. The ECD spectra of 12a/12b were calculated using the TDDFT method at the B3LYP/6-311G(d) level to determine the absolute configuration. The computed ECD spectrum of (1 S)-12a matched the experimental curve for 12a ( Figure 5). Thus, the absolute configuration of 12a was defined as (1 S) and, accordingly, 12b was defined as (1 R).
(±)-Microphyldine L (13) Table 4) of 13 were closely comparable to those of microphyldine K (12), indicating that it also has a biscarbazole skeleton like 12. The difference between them is that 13 is a dimeric carbazole formed by koenigine [12] and murrayamine A [16] units, which were further deduced from the 2D nmR data. The linkage of these two units was determined to be via the C-1 -C-6 bond, supported by the HMBC correlations of H-2 and C-6 , and H-1 and C-5 /C-7 ( Figure S87, Supporting Information). Accordingly, the structure of 13 was determined as shown ( Figure 1). A subsequent chiral HPLC isolation was performed to obtain the pure enantiomers of 13a and 13b in a ratio of 1:1. The absolute configurations of 13a and 13b were defined as (1 S) and (1 R), respectively, by comparison of the experimental and calculated ECD spectra ( Figure S86, Supporting Information).
olecules 2021, 26, x FOR PEER REVIEW curve for 12a ( Figure 5). Thus, the absolute configuration of 12a was define accordingly, 12b was defined as (1'R).  (Table 4) of 13 were closely comparable to those of microph indicating that it also has a biscarbazole skeleton like 12. The difference be that 13 is a dimeric carbazole formed by koenigine [12] and murrayamine which were further deduced from the 2D NMR data. The linkage of these determined to be via the C-1'-C-6'' bond, supported by the HMBC correl and C-6'', and H-1' and C-5''/C-7'' ( Figure S87, Supporting Information). Ac structure of 13 was determined as shown ( Figure 1). A subsequent chiral H was performed to obtain the pure enantiomers of 13a and 13b in a ratio of 1:1 configurations of 13a and 13b were defined as (1'S) and (1'R), respectively, b of the experimental and calculated ECD spectra ( Figure S86, Supporting Inf  (Table 4) and 2D nmR data showed many similarities to those of 13, except for the replacement of a hydroxy singlet (δ H 7.14 (1H, br s)) in 13 by a methoxy singlet (δ H 3.64 (3H, s)) in 14. This suggested that 14 is a 7-methoxy derivative of 13, as deduced from the HMBC correlation of the methoxy protons with C-7 (δ C 149.6) ( Figure S94, Supporting Information). Hence, the structure of 14 was defined as shown ( Figure 1). The pure enantiomers of 14a and 14b were obtained by a chiral HPLC separation. A comparison of the experimental and calculated ECD spectra facilitated the assignment of the absolute configurations of 14a and 14b as (1 S) and (1 R), respectively ( Figure S93, Supporting Information).    (Table 4) and 2D nmR data showed many similarities to those of 13 and the apparent difference was the presence of a set of resonances for the isopentenyl group (δ H 1.54 (3H, s, H-10 ), 1.62 (3H, s, H-9 ), 2.15 (2H, m, H-6 ), 5.11 (1H, t, J = 6.0 Hz, H-7 ), δ C 18.1 (C-10 ), 23.9 (C-6 ), 26.2 (C-9 ), 125.7 (C-7 ), 132.3 (C-8 )) in 15. The HMBC correlations from H-5 to C-2 , C-6 , and C-7 and from H-6 to C-3 and C-5 suggested that the isopentenyl moiety is located at C-5 ( Figure 2). Hence, the planar structure of 15 was assigned as shown ( Figure 1). The following chiral HPLC resolution and ECD determination defined the absolute configurations of 15a and 15b as (1 S) and (1 R), respectively (Figure S100, Supporting Information).

General Experimental Procedures
UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu Co., Tokyo, Japan). Optical rotations were measured on a Rudolph Autopol IV automatic polarimeter (NJ, USA). ECD data were acquired on a J-810 CD spectrophotometer (JASCO, Japan). IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer (MA, USA). The nmR spectra were measured with a Bruker Plus-400 nmR spectrometer (Bruker Co., Switzerland) or a Varian INOVA-500 nmR spectrometer (Varian Co., USA), using acetone-d 6 or CDCl 3 as solvent, and the chemical shifts were referenced to the solvent residual peak. HRESIMS experiments were measured on a Waters Xevo G2 Q-TOF mass spectrometer (Waters Co., Milford, MA, USA). Column chromatography (CC) was performed on silica gel ( Qingdao Marine Chemical Co. Ltd.,Qingdao,China). Semipreparative HPLC was carried out using a ZORBAX Eclipse XDB-C18 column (10 mm × 250 mm, 5 µm) on an Agilent 1200 series LC instrument with a DAD detector (Agilent Technologies, Palo Alto, CA, USA). Preparative TLC and TLC analyses were carried out on the pre-coated silica gel GF254 plates (Qingdao Marine Chemical Co. Ltd., Qingdao, China). Spots were visualized under the UV lights (254 and 365 nm) or by heating after spraying with 2% vanillin-H 2 SO 4 solution. All the solvents used for isolation were of analytical grade and the solvents used for HPLC were of HPLC grade.

Plant Material
The dry leaves and stems of Murraya microphylla were collected from the Hainan Province, People's Republic of China, in July 2015. The plant material was identified by one of the authors (P.F. Tu). A voucher specimen (no. MM201507) has been deposited at the Herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine.