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Spectral Assignments and Reference DataReceived: 10 June 2008 Revised: 9 March 2009 Accepted: 14 March 2009 Published online in Wiley Interscience: 28 April 2009

(www.interscience.com) DOI 10.1002/mrc.2439

Nepetanal and nepetanoate: A new diterpenealdehyde and a benzene derivative ester fromNepeta juncea†

Javid Hussain,a∗ Nargis Jamila,a Farman Ullah Khan,a Krishna PrasadDevkota,b M. Raza Shahc and Saeed Anward

One new tricyclic clerodane type diterpene aldehyde nepetanal (1) and one new benzene derivative nepetanoate (2) have beenisolated from a plant Nepeta juncea together with two known compounds oleanolic acid (3) and ursolic acid (4). The structuresof the isolated compounds were elucidated by means of modern spectroscopic techniques and comparison with literature data.Copyright c© 2009 John Wiley & Sons, Ltd.

Keywords: NMR; 1D/2D NMR; Nepeta juncea; labiatae; nepetanal; nepetanoate

Introduction

Nepeta, also called Glechoma and Cataria, gets its name afterthe ancient Italian city of Nepi[1], and is a multi-regional genuscomprising about 300 species. These are widely spread in Asiaand Europe with the greatest diversity and richness of speciesin Southwestern Asia, especially in Iran (about 67 species)and the Western Himalayas including Hindukosh (58 speciesin Pakistan).[2 – 5] Members of the genus Nepeta are sub-shrubs,perennial or annual herbs, monoecious or dioecious and usuallyaromatic in nature.[6 – 7]

Literature survey reveals that members of the genus Nepetaare rich in fatty acid, flavones, flavone glycosides, coumarins,steroids, iridoid glycosides, monoterpenic lactones, eudesmanesesquiterpenes, abietane diterpenoids, carbohydrates etc.[7 – 8]

However, no phytochemical investigation has so far beingconducted on Nepeta juncea, which inspired us to work on thisplant. The present work has led to the isolation of one newclerodane type diterpene aldehyde nepetanal (1) and one newbenzene derivative nepetanoate (2) (Fig. 1) along with two knownconstituents namely oleanolic acid and ursolic acid which havebeen isolated for the first time from N. juncea.

Results and Discussion

The CHCl3 fraction obtained from MeOH extract of N. junceawas chromatographed on silica gel. The elution was carried outwith a gradient of increasing polarity of hexane, chloroform, andmethanol, which afforded a new clerodane type diterpenoid 1.The structure of 1 was elucidated by using modern spectroscopictools including 1D- and 2D NMR.

The EI-MS of (1) showed [M]+ at m/z 334 [Correction made hereafter initial online publication] and fragment ions at m/z 55, 81,135, 152, 177, 220, 249 and 286 indicating the diterpenoid skeletonwith hydroxyl group and peak at m/z 81 was due to the presenceof furan ring with an alkyl chain.[8] The HR-EI-MS of 1 provided[M]+ peak at m/z 334.1990 [Correction made here after initial

online publication] indicating the molecular formula C20H30O4

(calculated for C20H30O4; 334.987) [Correction made here afterinitial online publication]. IR spectrum of 1 indicated the presenceof hydroxyl (3470 cm−1), furan (1510, 875 cm−1), and aldehyde(1720 cm−1) functionalities.

The 1H-NMR (400 MHz, CDCl3) spectrum of 1 exhibited signalsfor two tertiary methyl groups at δ 0.95 and 0.94 (3H, s) and onesecondary methyl group at δ 0.83 (d, J = 7.3 Hz). A proton at δ

4.76 (dd, J = 13.8, 5.4 Hz) was assigned to the geminal protonof a secondary OH group. Typical high frequency signals in the1H-NMR spectrum of 1 at δ 6.25, 7.15 and 7.30 were attributed toH-14, H-15 and H-16, respectively, suggesting the presence of aβ-substituted furan ring.[8]

The 13C-NMR spectrum (BB and DEPT) corroborated thepresence of three methyl, five methylenes, eight methines andfour quaternary carbons. The 13C-NMR chemical shift of C-19 wasobserved at δ 18.8; C-20 at δ 18.1, while C-17 at δ 17.7. These valuesrevealed the trans configuration at the A/B ring junction of 1.[9]

In the HMBC experiment of 1, the proton at δ 4.76 (H-3) showedcorrelations to the carbons at δ 174.1 (C-18), 45.1 (C-4), 45.5 (C-5),and 27.2 (C-2) and proton at δ 3.46 (H-4) showed correlations tothe carbons at δ 174.1 (C-18), 45.5 (C-5), and 27.2 (C-2). In addition,

∗ Correspondence to: Javid Hussain, Department of Chemistry, Kohat Universityof Science and Technology, Kohat, NWFP, Pakistan.E-mail: javidhej@yahoo.com

† This article was published online on 28 April 2009. Errors were subsequentlyidentified. This notice is included in the online and print versions to indicatethat both have been corrected; 18 May 2009.

a Department of Chemistry, Kohat University of Science and Technology, Kohat,NWFP, Pakistan

b Institute of Forestry, Tribhuvan University, Pokhara Campus, P. O. Box: 43,Pokhara, Kaski, Nepal

c ICCS, HEJ Reserch institute of Chemistry, Karachi University, Karachi, Pakistan

d Islamia College University, Peshawar, NWFP, Pakistan

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J. Hussain et al.

HH

H

HO O

O

CH3

O

H

CHO

HO

OH

1

1′′

2′′

3′′

1′

2′3′

4′

5′6′

21

545 6 7

8

12

3

910

11

12 13

14

17

18

19

20 H

Figure 1. Structure of compounds 1 and 2.

δ 0.95 of CH3-19 was correlated to the carbons at δ 45.13 (C-4),76.19 (C-6), and 45.30 (C-10), which established the presence ofan aldehyde group at C-4. The AB pattern centered at δ 1.50 and2.05 could be assigned to CH2-11, the HMBC of which showedcorrelations to the carbons at δ 45.3 (C-10), 41.1 (C-9), 31.3 (C-8),and 28.14 (C-12), confirming the attachment of alkyl chain at C-9.

The relative stereochemistry of 1 was determined by comparisonof the spectral data of 1 with those recorded for other known clero-dane diterpenoides[6,7,10] as well as from the NOE spectrum. TheCH3-19 showed NOE correlation with H-3, H-4, and H-6, whereasCH3-17 showed similar correlation with H-6. This result is consis-tent with a cis-relationship between these groups. H-10 however,did not exhibit NOE correlation either with CH3-19 or CH3-20signals, suggesting transfused A/B rings of decalin system of 1.[11]

From these observations and from the comparison ofspectral data with the literature, the compound 1 was de-termined to be (−) 3α,6α-dihydroxy-15,16-epoxy-5β ,8β ,9β-methyl,10α – cleroda-13,14-dien-18-al, commonly named asnepetanal.

Compound 2 was obtained as a colorless solid from chloroformfraction of MeOH extract when eluted with chloroform: hexane(1.5 : 8.5). The EI-MS exhibited the molecular ion peak at m/z 292and base peak at m/z 120. Molecular formula of 2 was determinedthrough HR-EI-MS as C18H28O3 (calculated m/z 292.3389, observedm/z 292.3387). In addition to the molecular ion peak, otherprominent peaks were appeared at m/z 57, 71, 107 and 120.

The 13C-NMR signals in the BB spectrum of compound 2 wereresolved through DEPT experiments into one methyl, four methine,ten metylene and three quaternary carbons. The high frequencyquaternary carbon at δ 173.0 (C-1) revealed the presence of acarbonyl group. In the 1H-NMR spectrum (CDCl3, 400 MHz), a pairof triplets integrating each for two protons resonated at δ 4.23(J1′′′ , 2′′ = 7.1 Hz) and δ 2.85 (J2′′ , 1′′ = 7.1 Hz) were assigned tomethylene protons of 1′′ and 2′′ respectively. The 1H and 13C- NMRchemical shifts of CH2-1′′ (δ 4.23 and 64.8) revealed its attachmentto an oxygen atom. The appearance of two doublets at δ 7.07and 6.75 (J = 8.3 Hz) each for two protons indicated a parasubstitution pattern. The signal at δ 130.1 was assigned to C-2′,C-6′ while C-3′ and C-5′ appeared at δ 115.3. The quaternary signalat δ 138.1 was assigned to C-1′.[8] The chemical shift value at δ

154.2 of a quaternary signal revealed its attachment to a hydroxylgroup and was assigned to C-4′. The 1H and 13C NMR data forcompound 2 are presented in Table 2. All chemical shifts wereconfirmed through 1H- NMR, 13C- NMR, HMBC, HMQC and COSYtechniques and comparison with the reported data of relatedcompounds.[9] The discussion so far led us to assign compound2 as 3-(4-hydroxyphenyl) propyl nonanoate commonly named asnepetanoate. Oleanoic acid and ursolic acid were also isolated

from the chloroform fraction of the crude extract and identified bycomparison with the literature data.[12 – 15]

Experimental Section

General

Column chromatography (CC) was done using silica gel, 70–230mesh. Flash chromatography was carried out using silica gel230–400 mesh. Thin layer chromatography (TLC) was performedwith precoated silica gel G-25-UV254 plates and detection wasdone at 254 nm, and by ceric sulphate in 10% H2SO4 solution.Silica gel (E. Merck, 230–400 mesh) was used for CC. The IRand UV Spectra were recorded on a Jasco-320-A and Hitachi-UV-240 spectrophotometers, respectively. Optical rotations weremeasured on a Jasco-DIP-360-digital polarimeter using a 10-cmcell-tube. Mass spectra (EI- and HR-EI-MS) were measured inan electron impact mode on Finnigan MAT 12 or MAT 312spectrophotometers; ions are presented in m/z (%).

The NMR spectra were obtained on a Bruker AMX 400spectrometer operating at 400.1 MHz for 1H and 100.6 MHz for13C. The one-dimensional 1H- and 13C-NMR spectra were acquiredunder standard conditions on a 5-mm DUAL -1H/13C direct probe.The digital resolution of 1H-NMR was 0.73 Hz per point, number oftransients (NS) 128; the digital resolution of 13C-NMR was 1.84 Hzper point, NS 25 000. Compounds 1–4 were dissolved in CDCl3 atabout 10 mg ml−1 each and transferred into a 5-mm NMR tube.Chemical shifts (δ ppm) were measured with accuracy of 0.01 and0.5 ppm, respectively. Internal lock and tetramethylsilane (TMS)were used as internal reference. The two-dimensional experimentswere acquired and processed with XWIN-NMR software providedby Bruker. COSY 45◦ spectra SF 400.03 MHz, NS 32, P1 5.70 µs,P2 2.70 µs, DE 10 µs; for the NOESY experiments SF 300.133 MHz,NS 64, DE 305 µs, F1 2903.69 Hz, F2 2903.69 Hz; for the HMQCspectra NS 32, F1 1688.52 Hz, F2 3071.71 Hz; for the HMBC spectraNS 128, F1 3212.30 Hz, F2 23 236.21 Hz. All pulse sequences arestandard in the Bruker XWIN-NMR software. For all experiments,the temperature was maintained at 300 K.

Plant material

Whole parts of Nepeta juncea were collected from Parachinar,Kurrum Agency, Pakistan in April 2005 and identified byMohammad Siraj Assistant Professor, Government Post GraduateCollege (Jehan Zeb), Swat, Pakistan.

A Voucher Specimen (No. KU. 005) was deposited in theHerbarium of the college.

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Nepetanal and Nepetanoate

Table 1. The full assignments of protons and carbon signals of 1(CDCl3)

Position 1H 13C

1 1.54 ddd (14.0, 12.0, 5.5) 17.7

2 2.23 m 27.2

3 4.76 dd (13.8, 5.4) 77.2

4 3.46 d (6.2) 45.1

5 – 45.5

6 3.46 m 76.1

7 1.43 m (overlapped) 35.1

8 1.57 m 31.3

9 – 41.1

10 1.27 br.d (11.5) 45.3

11 1.50–2.05 m (overlapped) 38.4

12 2.15 m 28.1

13 – 125.1

14 6.25 br.s 110.6

15 7.15 d (1.5) 143.1

16 7.30 s 138.0

17 0.83 d (7.3) 17.7

18 9.92 s 174.1

Extraction, fractionation and isolation

The whole plant of N. juncea was dried in shade, chopped andground to coarse powder. The powdered plant (4 kg) was initiallyextracted with methanol (7 days × 3) at room temperature. Thecombined methanolic extract was evaporated under reducedpressure leaving behind a greenish, syrup residue (150 g). Usingseparating funnel, the methanol extract was partitioned applyingdifferent solvents i.e. hexane, chloroform, ethyl acetate, butanoland water (in order of increasing polarity) successively.

A portion of the CHCl3 extract (50 g) was chromatographed overa silica gel column. The elution was carried out with a gradient ofincreasing polarity of hexane, chloroform, and methanol. Thefractions showing similar TLC profiles were mixed and fourfractions (A–D) were obtained after compilation. These collectivefractions were individually subjected to repeated CC on silica gel.

Fraction D was obtained through elution with n-hexane-chloroform (1 : 1) and was subjected to CC with the solventsystem (n-hexane-chloroform). This fraction afforded one pure andnew compound 1, after eluting with n-hexane-chloroform (4 : 6).Compound 1 was further purified by washing and recrystallizedfrom methanol. Similarly compound 2 was obtained from fractionA and eluted with chloroform: hexane (1.5 : 8.5). The sub-fractionA was subjected to flash CC, eluted by acetone: chloroform (2 : 8)to afford compounds 3 and 4.

Nepetanal (1): Amorphous solid (CHCl3); [α]23D = −4.28

(c = 0.15, CHCl3). UV (MeOH): λmax = 214 (5.2) nm; IR (KBr):νmax (cm−1) = 3470, 1720, 1510, 870; EI-MS m/z = 334 [M]+, 55(36), 81 (100), 135 (30), 152 (23), 177 (8), 220 (5), 249 (3), 286.HR-EI-MS m/z = 334.1990 calculated for C20H30O4: 334.1987. 1Hand 13C-NMR (CDCl3) data see Table 1 [Correction made here afterinitial online publication].

Nepetanoate (2): Colorless solid (CHCl3); [α]23D = +2.81

(c = 0.15, CHCl3). UV (MeOH): λmax = 276 nm; IR (KBr):νmax (cm−1) = 3320, 1750 cm−1; EI-MS m/z = 292 [M]+, 57,71, 107, 120 (100), 135 (30), 152 (23), 177 (8), 220 (5), 249 (3), 286[Correction made here after initial online publication].

Table 2. The full assignments of protons and carbon signals of 2(CDCl3)

Position 1H 13C

1 – 173.0

2 1.89 t (6.4) 31.9

3 1.63 s 29.7

4 1.63 s 29.6

5 1.63 s 29.3

6 1.63 s 29.1

7 1.63 s 24.9

8 2.23 s 22.7

9 0.87 t (6.9) 14.0

1′ – 138.1

2′ 7.07 d (8.3) 130.1

3′ 6.75 d (8.3) 115.3

4′ – 154.2

HR-EI-MS m/z = 292.3387 calculated for C18H28O3. 292.3389.1H and 13C- NMR (CDCl3) data see Table 2 [Correction made hereafter initial online publication].

Acknowledgements

The authors wish to thank Higher Education Commission,Government of Pakistan, for providing financial support for thecurrent study under the National Research Program for Universities(NRPU).

References

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