11a-Ethoxy-b-boswellic Acid and Nizwanone, a New Boswellic AcidDerivative and a New Triterpene, Respectively, from Boswellia sacra

by Ahmed Al-Harrasi*a), Liaqat Alia), Najeeb Ur Rehmana), Javid Hussaina), Hidayat Hussain*a),Ahmed Al-Rawahia), and Tania Shamim Rizvib)

a) Department of Biological Sciences and Chemistry, University of Nizwa, P.O. Box 33, Postal Code 616,Birkat Al Mauz, Nizwa, Sultanate of Oman (phone: þ968-92828487; fax: þ968-25446289;

e-mail: aharrasi@unizwa.edu.om; Hidayat110@gmail.com)b) International Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry,

University of Karachi, Karachi-75270, Pakistan

A new boswellic acid derivative, 11a-ethoxy-b-boswellic acid (EBA; 1) and a new ursane-typetriterpene, named nizwanone (2), were isolated from Omani frankincense Boswellia sacra Flueck.together with two known compounds papyriogenin B and rigidenol. The structures of 1 and 2 wereelucidated by detailed spectroscopic analysis using 1H- and 13C-NMR, 1H,1H-COSY, HMQC, HMBC,and HR-EI-MS techniques. The relative configurations of 1 and 2 were assigned by comparative analysisof the NMR spectral data with those of known analogs together with NOESY experiments. Structures ofknown compounds were identified by comparison with the reported data.

Introduction. – Frankincense is the gum resin obtained from incisions in the trunksof trees of the Boswellia genus of the family Burseraceae. Boswellia sacra Flueck. isnative to the southern part of Oman, while a closely related species, B. carteri, alsogrows in Yemen and Northern Somalia [1]. The properties of B. sacra have beenexploited in the traditional medicines of Oman where the frankincense of B. sacra isused against dental infections, as a tonic for the digestive system, and to treat stomachaches [2] [3]. The resin of B. scara is also used to relief joint pain caused by arthritis,muscle pain [2] [3], and for the treatment of colds, cough, fever, and asthma [2 –4].Boswellic acids (BAs), a group of pentacyclic triterpenoids, are mainly isolated fromthe gum resin of Boswellia spp. Their bioactivities against inflammation [5], arthritis[6], ulcerative colitis [7], chronic colitis [8], asthma [9], and hepatitis [10] are welldocumented. In addition, they have attracted new interest recently due to theiranticancer activities and ability to induce apoptosis [11]. BAs have been reported toinhibit growth and induce apoptosis in brain tumors [12], malignant glioma cells [13],colon cancer cells [14], and leukemia cells [15]. The biological activities of BAs and thetraditional uses of B. sacra prompted us to investigate its phytochemistry. In an earlierstudy, we reported essential oils from B. sacra [16]. To obtain the minor compounds, theresin of B. sacra was again collected and extracted with CH2Cl2/MeOH 1 : 1. The resinfraction was subjected to different chromatographic procedures including recyclingHPLC, resulting in the isolation and structure elucidation of 11a-ethoxy-b-boswellicacid (EBA; 1), a new boswellic acid derivative, along with one new ursane-typetriterpenoid named nizwanone (2).

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� 2013 Verlag Helvetica Chimica Acta AG, Z�rich

Results and Discussion. – Repeated chromatography of resin fractions of B. sacraresulted in the isolation of two new compounds, 1 and 2 (Fig. 1).

The IR spectrum of 1 displayed absorptions for a OH (3450 cm�1), an acid C¼O(1700 cm�1), and a C¼C bond (1655 cm�1) [17]. The 13C-NMR spectra of compound 1(Table) contained 32 signals, the multiplicities of which were determined by DEPTexperiments, and they were assigned to eight CH, eight Me, nine CH2 groups, and sevenquaternary C-atoms.

High-resolution mass spectrometric (HR-MS) and 13C-NMR data (Table) of 1provided the molecular formula C32H52O4, indicating seven indices of hydrogendeficiency (IHD), five of which were attributed to the five rings of the pentacyclictriterpene skeleton. One equivalent was assigned to a COOH C¼O group (d(C) 182.9),and the last equivalent was assigned to a C¼C bond, being consistent with that of a D12-ursane skeleton, in accordance with its 13C-NMR resonances (d(C) 125.1 (C(12)) and142.9 (C(13))). The appearance of a vinylic H-atom signal at d(H) 5.27 (H�C(12)) as abroad doublet and the appearance of H�C(18) signal as a doublet at d(H) 1.32 (J¼13.6), in addition to the two Me group signals (Me(29) and Me(30)), each as a doubletat d(H) 0.78 (d, J¼5.6, 3 H) and 0.85 (d, J¼5.6, 3 H), respectively, and five Me singletsat d(H) 0.77, 0.91, 1.00, 1.06, and 1.33 unambiguously confirmed the presence of a D12-ursane skeleton [18]. The location of the COOH group was found to be C(4) based onthe downfield shift of C(4) signal at d(C) 47.5, similar as in reference compounds[18] [19]. The 13C-NMR signal for an O-bearing CH C-atom at d(C) 70.6 was assignedto C(3) on biosynthetic [18] [20] and analogy grounds [18] [21], as well as 1H,1H-COSY,HMQC, and HMB correlations (Fig. 2).

The latter revealed cross-peaks between Me H-atom signal (Me(23)) at d(H) 1.33(s), and those of the CH-O C-atom (C(3)) at d(C) 70.6, the quaternary C-atom (C(4))at d(C) 47.5, and the CH C-atom (C(5)) at d(C) 49.0. In addition, strong HMBCsbetween the signals of COOH C-atom (C(24)) at d(C) 182.9 and the Me H-atoms(Me(23)) at d(H) 1.33. These established connectivities supported the substitutionpattern of ring A. The 1H- and 13C-NMR spectra of 1 were similar to those of b-boswellic acid [22] with a difference observed for C(11): a CH2 group of C(11) (d(C)23.4; d(H) 1.92 (m) and 1.62 (m)) in b-boswellic acid was replaced by RO�CH (d(C)75.8 (d); d(H) 3.91 (dd, J¼3.0, 9.6)) in 1. Additionally, the 1H-NMR spectrum of 1showed typical signals for an EtO group [23] at d(H) 3.34 – 3.38, 3.56 –3.60 (m, each

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Fig. 1. Structures of EBA (1) and nizwanone (2) isolated from B. sacra

1 H), and 1.11 (t, J¼7.2 Hz, 3 H). The position of the EtO group at C(11) wasconfirmed from HMBC of CH2(31) to C(11). These data suggested that 1 was an 11a-ethoxy analog of b-boswellic acid, namely 11a-ethoxy-b-boswellic acid (EBA).

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Table. 1H- and 13C-NMR Dataa)b) of Compound 1 and 2

Position 1 2

d(H) d(C) d(H) d(C)

1 1.29 –1.34 (m, Ha), 33.8 1.39–1.44 (m, Ha), 33.11.44 –1.48 (m, Hb) 1.48–1.51 (m, Hb)

2 1.93 –1.99 (m, 2 H) 26.6 2.09–2.12 (m, Ha), 32.8– – 2.13–2.18 (m, Hb)

3 4.06–4.10 (m, 1 H) 70.6 – 218.34 – 47.5 1.22–1.26 (m, 1 H) 42.25 1.44 –1.49 (m, 1 H) 49.0 1.45–1.49 (m, 1 H) 48.96 1.63 –1.67 (m, Ha), 19.5 1.64–1.68 (m, Ha), 18.3

1.75 –1.80 (m, Hb) 1.73–1.77 (m, Hb)7 1.92 –1.96 (m, 2 H) 34.9 3.39 (br. s, 1 H) 76.28 – 38.7 – 33.79 1.77 (d, J¼9.6, 1 H) 50.8 1.70–1.74 (m, 1 H) 59.1

10 – 42.9 – 36.911 3.91 (dd, J¼3.0, 9.6, 1 H) 75.8 2.09–2.12 (m, Ha), 23.3

– 2.15–2.19 (m, Hb)12 5.27 (d, J¼2.4, 1 H) 125.1 5.11 (br. s, 1 H) 124.213 – 142.9 – 139.614 – 42.2 – 40.215 1.53 –1.56 (m, 2 H) 26.4 1.50–1.54 (m, 2 H) 26.616 1.95 –1.99 (m, 2 H) 27.9 1.91–1.95 (m, 2 H) 25.317 – 33.7 – 37.418 1.32 (d, J¼13.6, 1 H) 58.6 1.35 (d, J¼13.1, 1 H) 47.519 0.83–0.86 (m, 1 H) 39.5 0.85–0.89 (m, 1 H) 39.720 0.86–0.90 (m, 1 H) 39.4 0.86–0.90 (m, 1 H) 39.621 1.23 –1.26 (m, Ha), 31.1 1.25–1.29 (m, Ha), 31.3

1.33 –1.37 (m, Hb) 1.35–1.39 (m, Hb)22 1.24 –1.27 (m, Ha), 41.4 1.23–1.27 (m, Ha), 41.6

1.37 –1.41 (m, Hb) 1.36–1.40 (m, Hb)23 1.33 (s, 3 H) 24.6 1.06 (s, 3 H) 23.424 – 182.9 0.98 (s, 3 H) 28.825 1.00 (s, 3 H) 14.4 0.76 (s, 3 H) 15.526 1.06 (s, 3 H) 18.2 0.78 (s, 3 H) 16.927 0.91 (s, 3 H) 22.5 1.54 (s, 3 H) 22.328 0.77 (s) 28.7 0.83 (s, 3 H) 28.329 0.78 (d, J¼5.6, 3 H) 17.4 0.95 (d, J¼5.6, 3 H) 15.430 0.85 (d, J¼5.6, 3 H), 21.3 0.94 (d, J¼5.6, 3 H) 21.431 3.34–3.38 (m, 1 H), 62.1 – –

3.56–3.60 (m, 1 H)32 1.11 (t, J¼7.2, 3 H) 15.9 – –

a) Bruker-DRX-600 NMR spectrometer; recorded in CDCl3. b) Assignments accomplished by DEPT,1H, 1H-COSY, HMQC, and HMBC experiments.

The relative configuration of 1 was established by means of NOESY (Fig. 2). Thecross-peaks H�C(3)/Hb�C(2), Hb�C(2)/Hb�C(1), H�C(11)/Me(25), and H�C(11)/Me(26) confirmed the b-axial orientation of H-atoms at C(3) and C(11). The axialorientation of Hb�C(3) was further confirmed from the chemical shift of C(3) in 1(d(C) 70.6; d(H) 4.06– 4.10) with b-boswellic acid (d(C) 70.8; d(H) 4.08) [22]. TheO�CH H-atom (H�C(11)) resonated at d(H) 3.91 (dd, J¼3.0, 9.6, 1 H), showing Jvalues consistent with an axial orientation [23]. Finally, the assigned structure was fullyconfirmed with 1H,1H-COSY, HMQC, HMBC, and NOESY data (Fig. 2). From theabove evidence, compound 1 was assigned as 11a-ethoxy-b-boswellic acid (EBA; 1).

Compound 2 was isolated as a white solid, and its molecular formula wasestablished as C30H48O2 by FD-MS. The 1H-NMR spectrum (Table) of 2 displayedsignals of six tertiary Me (d(H) 1.54 (s, Me(27)), 1.06 (s, Me(23)), 0.98 (s, Me(24)), 0.83(s, Me(28)), 0.78 (s, Me(26)), and 0.76 (s, Me(25))) and two secondary Me groups(d(H) 0.95 (d, J¼6.6, Me(29)) and 0.94 (d, J¼6.6, Me(30))), and this was furtherconfirmed by the 13C-NMR spectrum (d(C) 28.3 (C(28)), 23.4 (C(23)), 22.3 (C(27)),21.4 (C(30)), 15.4 (C(29)), 16.9 (C(26)), 15.5 (C(25)), and 28.8 (C(24)).

The 1H-NMR spectrum also showed a signal of an O-bearing CH group at d(H)3.39 (br. s, H�C(7)) and another arising from trisubstituted olefin (d(H) 5.11 (br. s,H�C(12)). The 1H- and 13C-NMR (DEPT) spectra of 2 displayed signals of six tertiaryMe, two secondary Me, eight CH2, seven CH (one O-bearing) groups, and sevenquarternary (one sp2 hybridized) C-atoms, indicating that compound 2 was apentacyclic ursane-type triterpenoid with a trisubstituted C¼C bond (d(C) 139.6 and124.2) and one O-bearing functional group. This functional group was proposed to be aOH group, corresponding to an IR absorption at 3410 cm�1 [24]. The IR spectrumshowed a strong ketonic C¼O absorption band at 1728 cm�1, and the 13C-NMRspectrum exhibited the C(3)¼O resonance at d(C) 218.3, which showed correlations inthe HMBC spectrum (Fig. 3) with Me(23) and Me(24) H-atom resonances, confirming

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Fig. 2. Key 1H,1H-COSY (——) , HMB (H!C), and NOESY (H$H) correlations of 1

the position of the ketone group. The structure was assembled by analysis of thecorrelation spectra (1H,1H-COSY, HMQC) and HMBC experiments.

In the HMBC spectrum, the correlations from H�C(7) to C(5), C(6), C(8), andC(9); and from H�C(12) to C(9), C(11), C(13), and C(14) indicated that OH groupand C¼C bond were located at C(7), and between C(12) and C(13), respectively. Therelative configuration of C(3) was confirmed from NOESY correlation of Me(27) toH�C(7). Consequently, the structure was established as 2, named nizwanone.

The two known compounds papyriogenin B and rigidenol were identified bycomparison with published data [25] [26].

The authors would like to thank The Oman Research Council (TRC) for the generous supportthrough the project ORG/CBS/10/002, and the University of Nizwa through the project (A109-10-UON/28/A & S/IF).

Experimental Part

General. Column chromatography (CC): silica gel (SiO2; Merck); with hexane/CH2Cl2 and MeOH/CH2Cl2 in varying concentrations. TLC: Precoated SiO2 60 F254 plates (E. Merck); visualization underUV at 254 and 366 nm, and by spraying with Ce(SO4)2 reagent. Recycling prep. HPLC: JAILC-9210NEXT; HPLC-grade solvents from Sigma�Aldrich by using AcOEt/hexane 1 : 1 system. IR Spectra:Bruker Tensor 27 FT-IR ; n in cm�1. 1H- and 13C-NMR Spectra: Bruker NMR spectrometers (at 600 (1H)and 150 MHz (13C)); d in ppm rel. to Me4Si as internal standard; J in Hz. EI- and HR-EI-MS: JEOL JMSHX 110 mass spectrometers; in m/z (rel. %).

Plant Material. The Hougari Regular (HR) frankincense samples of B. sacra were collected from thevarious locations in Oman and were also supplied by a trustful partner (Mr. Saleh Al-Amri). All thesesamples were authenticated by Dr. Mustafa Mansi (botanist), Department of Biological Sciences andChemistry, University of Nizwa, the Sultanate of Oman, and a voucher specimen (No: BSHR-01/2012)was deposited with the Herbarium of the Department of Biological Sciences and Chemistry.

Extraction and Isolation. The air-dried ground material (500 g) of Hougari Regular (HR) resin wasexhaustively extracted with 100% MeOH at r.t. The extract was evaporated to yield the residue (50 g).The MeOH extract (50 g) was subjected to CC (SiO2 (1000 g; 70 –230 mesh; Merck; hexane/CH2Cl2 up to100% CH2Cl2, and then by the gradient of MeOH (1–100%)) yielded ten fractions, Frs. 1–10. Fr. 6obtained with 2% MeOH/CH2Cl2 was also loaded directly on recycling HPLC, and by using 1 :1 solventsystem of AcOEt/hexane, the 11a-ethoxy-b-boswellic acid (1, 5.0 mg), was eluted as a UV-inactivecompound at a retention time of 34 min with 4 ml/min flow rate. Fr. 5 (42 g) of the first column wasloaded on SiO2 in order of increasing polarity (hexane, hexane/CH2Cl2, pure CH2Cl2, and CH2Cl2/

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Fig. 3. Key HMBCs (H!C) for nizwanone (2)

MeOH), to afford 12 subfractions, Frs. 5.1–5.12. Fr. 5.4 yielded nizwanone (2 ; 14.4 mg) after CC(CH2Cl2/hexane 4 :6). Fr. 4 gave papyriogenin B (5.0 mg) and rigidenol (6.0 mg) after CC (CH2Cl2/hexane 3.5 : 6.5).

11a-Ethoxy-b-boswellic Acid (¼ (3a,11a)-11-Ethoxy-3-hydroxyurs-12-en-24-oic Acid; 1): Whitesolid. [a]25

D ¼ þ6.0 (c¼0.06, in CH2Cl2). IR (KBr): 3450, 1700, 1655. 1H- and 13C-NMR (CDCl3): Table.EI-MS (2308): 500.3 (55, Mþ ), 454.3 (96), 262.2 (40), 218.1 (90), 203.1 (40), 133.1 (35), 95.1 (25), 55.0(20). HR-EI-MS: 500.3876 (Mþ , C32H52Oþ

4 ; calc. 500.3866).Nizwanone (¼ (7b)-7-Hydroxyurs-12-en-3-one ; 2) . White solid. [a]25

D ¼ �5.0 (c¼0.05, in CH2Cl2).IR (KBr): 3410, 2930, 1728, 1275, 1070. 1H- and 13C-NMR (CDCl3): Table. EI-MS (2308): 440.1 (34, Mþ ),272.2 (20), 218.1 (70), 207.1 (30), 135.1 (31), 95.1 (45), 55.0 (20). FD-MS: 440.1 (Mþ ).

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Received July 4, 2012

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