Indian Journal of Chemistry Vol. 418, May 2002, pp. 1088-1092

Note

Novel diterpenoid and aliphatic compounds from Thuja orientalis

B K Mehta*, Vibha Nagar, Satish Shitut, Sanjay Nagar & Sanjay Sharma

School of Studies in Chemistry, Vikram University, Ujjain 456 0 I 0, Indi a

Received 9 August 1999; accepted (revised) 20 Jun e 2001

Two diterpenoids of labdane group and five long chain aliphatic compounds have been identified as 8( 17), 13-labdiene-16,15-0Iidc-IS-ate, S( 17), 13-labdiene-15, 16-olide- 18-ate , 8-hexyl-tricosane, octacosane-IO-ol-21-ene , decanyl-II-hydroxy-tritriacontanoate, pentyl triacontanoate and nonacosane on the basis of spectroscopic and chemical evidences.

Thuja orientalis is used in Chinese medicinal system, due to its anticancer and antihaemorrhagic activities. I.3 It contains bioflavonoids, terpenoids, fatty acids and cytotoxic principles.4 - 'o Our previous chemical examination on this plant" had led to the isolation of pimaric and isopimaric acids. Further investigation of hexane extract has now afforded two new diterpenoids of labdane group and four new aliphatic compounds. A hydrocarbon nonacosane has also been isolated. Herein we report the details of isolation and structural determination of each compound.

Results and Discussion The compounds were isolated by repeated column

chromatography on alumina gr.III and purity was checked by GC-MS. The structures of the compounds were established on the basis of ElMS, 300, 400 MHz IH NMR, and 100 MHz \3C NMR (DEPT I,II,III) spectral data. H-H COSY, 2 D-NMR spectral analysis confirmed the structure of compounds 1 and 2.

Compound 1: It was analysed for C21H3004 (methanol), M+ 346. The mass spectrum showed a

significant loss of 60 and 15 a. m.u . from the molecular ion, due to loss of acetic acid (C2H40 2) and angular methyl group (-CH3). The carbomethoxy fragment ion was detected at mJz 59. This suggested that 1 was an ester with an angular methyl group usually obtained in diterpenoids .1 2.13 It was further supported by the presence of absorption at 1750-1720 cm-I due to carbonyl group of ester and unsaturated ketone in five membered ring (Y-lactone)' 4.'5 and a singlet in 400 MHz NMR at 8 3.61 for three protons. The bands at 1250, 3079 and 780 cm-I were due to an olefinic group and exocyclic double bond respectively. The IH NMR was typical of labdane group of diterpene, indicating the presence of two methy I singlets at 8 0.52 and l.1 8. The exocyclic double bond and methylene group in five membered ring resonated at 8 7.11 and 4.79 as triplet and singlet in agreement with that of exocyclic, ~-unsaturated-ylactone NMR of 2-butenolide. '6

A series of spin decoupling experiments confirmed the assignments of chemical shifts in the compound 1 (Table I). On irradiation at 8 2.44 (4H, m) the dd pattern at 8 2.15 (H-6, J=9 and 6 Hz) collapsed to a singlet whil irradiation at 8 2.15 resulted into a doublet at 8 2.44. The base peak at mJz 121 and abundant fragment at mJz 189 confirmed its terpenic nature and position of exocyclic methylene group in ring B?I.22 Further, UV spectrum showed end absorption at 210 nm suggesting that the double bond and carbonyl group in lactone ring were not in conjugation . The \3C NMR and DEPT spectra of 1 showed 21 carbon atoms shared between three methyls, ni ne methylenes (two of which were olefinic) and six quarternary carbon atoms with one each attached to an oxygen atom (L 77 .60 ppm) and double bond (134.88 ppm) (Table I) . The exocyclic double bond revealed signals at 106.65 ppm, while olefinic group resonated at 145.73 and 147.48 ppm indicated labdane group of diterpene.

A 20 COSY (H-H and H-C) NMR spectra of 1 has shown a contour plot as in and connectivities were confirmed CTable II). The 20 NMR showed connectivities at 8 7.11 and 4.79 while the 4.79 peak has connectivity at 8 2.44 and 2.l5 confirming the position of exocyclic double bond at C-8 (Table II) .

NOTES 1089

Table I _IH NMR and I3C NMR spectral data of compound 1

Carbon No. IH NMR (8, ppm) 13 C NMR (ppm)

8 Multiplicities J values 8 -C 8 -CH 8 - CH2

1.05

1.55

2.2

m

m

m

m

12 Hz

9 Hz

II Hz

9 Hz

38.68

19.94

38.23

44.0

56.16

I

2

3

4

5

6

7

8

9

10

II

12

13

14

15

16

17

18

19

20

21

1.55

2. 15

2.44

dd 9~Hz 24.66

1.27

1.9

1.82

7.11

4.79

4.59,4.90

1.18

3.6 1

0.52

m

dd

m

m

dd

s

s

These data are based upon H-H and H-C COSY NMR

These results are also confirmed by ID nOe experiment. Thus the compound has been identified as 8(17), 13-labdine-16, 15 olide-18-ate and being reported first time by us.

Compound 2: It was analysed for C21H300 4 (methanol) , M+346 and identified as an isomer of compound 1 with similar carbon skeleton of labdane type of diterpene. JR, PMR and CMR spectra were almost identical with slight change in the position and intensity of some peaks. The UV spectrum in

Table II - H-H COSY connectivities

Proton (8) Correlated proton (8)

7.11 4.79

4.90 1.82, 1.55

4.79

4.59

2.44

2.15

1.9

1.82

1.55

1.45

2.44,2.15

1.55

2.15,1.90,1.82

1.82, 1.55 , 1.05

1.05

1.54, 1.52, 1.18, 1.05

1.05

1.05

II Hz 39.25

134.88

9&6 Hz 56.36

40.29

9 Hz 21.88

9Hz 26.23

147.48

1.5 Hz 145.73

177.60

1.8 Hz 69.96

2&2 Hz 106.00

38.6

174.25

56.36

12.58

chloroform has shown absorption bands at A max 234 and 272 nm, indicating the presence of a conjugated system, which could only possible when the position of carbonyl group was at C-lS. Thus the compound was identified as 8(17), 13-labdiene-lS, 16-olide-18-ate, being reported first time by us.

Compound 3: White crystals, C29H60 (methanol), M+ 408. The IR spectrum showed saturated paraffinic nature. 18.19 A triplet (1=12 Hz) in the IH NMR spectrum, for nine methyl protons at 8 0.90 indicated

H;JC-(CH2113-CH2-9H-CH2-(CH21s-CH3

9H2 (yH214 CH3

3

OH I

H;JC-(CH21'3-CH2-CH-CH2-(CH21a-CH2-CH=CH-CH2-(CH214-CH3

4

o OH II I

H;JC-(CH21a-CHco-C-CH2-(CH21'9-CH2-CH-CHz-(CH21a-CH3

o II

5

H;JC-(CH212rCH2-C-D-CH2-(CH213-CH3 H;JC-(CH2121- CH3

6 7

1090 INDIAN J. CI-IEM ., SEC B, MA Y 2002

its branched chain nature. A doublet at 8 1.10 (1=7 Hz) for 6 protons was assigned to the methylene protons adjacent to methine proton. All the remaining methylene protons merged into a single peak at 8 1.20 fo r 44 protons. The methine proton at 8 1.56 appeared as unresolved triplet coupl ed with the base peak in the lTiass spectrum at mlz 83 indicated the pos ition of branching at C_8.17.20 Thus the compound was identi fied as 8-hexyl tricosane and bei ng reported first time by us.

Compound 4: White fl akes, C28H560 (benzene), M+ 408, monoacetate, m.p. 80 °C. The IR spectrum showed bands at 3400 and 1630 cm·1 for hydroxyl group and double bond respectively . The lH NMR showed triplet (1=8 Hz) for six terminal methyl protons at 8 1.08. A broad singlet at 8 1.40 for 40 protons indicated the presence of 20 methylene groups. The hydroxyl proton resonated at 8 1.68 as singlet (020 exchangeable) .

The methine proton attached to the hydroxyl group resonated at 8 3.44 as multiplet. The olefinic protons resonated at 8 4.80 (1= 10Hz) and 4.84 (1=8 Hz) as multiplets. The presence of rel atively abundant fragments in its mass spectrum at mlz 297 and 157 formed by allylic cleavage and a-cleavage indicated the position of double bond and hydroxyl group at C-21 and C-I0 respectively . The 13C NMR spectrum was consistent with the proposed structure. The assignments were made on the basis of known chemical shifts for olefinic and alcoholic groups.

The olefinic carbon resonated at 123.45 ppm, while the carbon adjacent to hydroxyl group resonated at 71 .99 ppm. The end methyl and methylene carbons resonated at 14.03, 25.63 and 25.32 ppm. The remaining methylenes resonated at 29.30 and 29.66 ppm21 .23. Thus the compound was identified as octacosane-lO- 01-21-ene.

Compound 5: White crystals, C43H860 3 (benzene), M+ 650, monoacetate, mp 95°C. The IR spectrum showed bands at 3480 and 1740 cm·1 for hydroxyl and ester groups respectively. The lH NMR showed a triplet at 8 0.80 for methyl groups and a broad peak for 70 protons resonated at 8 1.20 was due to methylene protons. The methylene protons of the ester group resonated as a di storted triplet at 8 3.66. The presence of fragments at mlz 509,171 and 141 indicated the position of hydroxyl group at C-ll. An abundant fragment at mlz 200 formed by McLafferty rearrangement indicated the position of efter group. This structure was further confirmed by 13C NMR

spectrum. The carboxyl carbon of ester appeared at 171.66 ppm. The end methyl groups appeared at 21.94 and 14.08 ppm. The rest of the methylenes resonated at 29. 19 and 29.37 ppm. Thus the compound was iden tified as dccanyl-II-hydroxy-tri tri acontanoate.

Compound 6: White crystals, C35H700 2 (methanol) , M+ 522. Strong absorpt ion in IR at 1740 cm-1 was ass igned to the ester group . lH MR showed a triplet for six methyl protons at 8 0.96. A broad singlet at 8 1.24 for 60 protons was due to 30 methylenes. The methylene protons adjacent to the ester group resonated at 8 3.68 and 2.38 as triplets (1=18 and 14 Hz) respectively. The fragments at m/z 451,115 (a-cleavage), 465,128 W-cleavage), 130 (Mclafferty rearrangement), 407 confirmed the position of ester group at C-6. Thus the compound was charaterised as pentyl triacontanoate.

Compound 7: White crystals, C29H60 (methano l), M+ 408. The IR, lH NMR and mass spectra showed it to be nonacosane. Further confirmation was done by comparing its mp and IR spectrum with the reported values. 24.25

Experimental Section Mps are uncorrected. c.c. was carried out on

alumina gr.IlI and TLC on silica gel G. The hi gh resolution 10 and 20 COSY experiments were carried out on Brucker WM 300 MHz spectrometer with the following conditions: for H-H COSY -20 spectral width 2345-2 Hz, acquisit ion time 0.2 18 sec., relaxation delay 1-500 sec., pulse width 89.6 degrees, temperature 25°C, number of repetitions 16, number of increment · 256 and total acquisition time 2 hr, l3C NMR spectra were recorded on Brucker WM 100 MHz spectrometer. In some cases I H NMR were recorded on 400 MHz WM Brucker spectrometer. IR spectra were taken in KBr pellets and mass spectra on Jeol JMS 0 300 mass spectrometer. The leaves were collected from the garden of our Chemistry Department in university campus and nearby places during the month July - Sept. and the taxonomic identification of the plant was done by the authorities of School of Studies in Botany, Vikram University, Ujjain.

Isolation of compounds 5.0 kg shade dried powdered leaves were extracted

with n-hexane for 64 hI' in soxhlet extractor and solvent was removed under reduced pressure by rotary film evaporator to yield a dark green mass.

NOTES 1091

8(17), 13-labdiene-16,15-olide-18-atel: Isolated from benzene eluate and crystallized from methanol, m.p.85°C (C2I H300 4), UV Amax (chloroform):210 nm; IR (KBr) : 3079, 2960, 2930, 2880, 2850, 1750-1720, 1650, 1470, 1460, 1450, 1414, 1390, 1350, 1250, 1230, 1200, 1170, 1160, 1130, 1100-10 I 0, 990, 950, 780, 750, 680, 450 and 430 cm· l . IH NMR and l3C NMR (Table I) ElMS mlz (rel.int.,%): M+ 346(5.0), 331 (13.0), 314(10.0), 286(48.0), 271 (20.0), 257(2.0), 189(11.9), 161(15.0), 121(100), 107(21.0) and 81 (25 .0) etc.

8(17), 13-Labdiene-15, 16-olide-18-ate 2 : Isolated from benzene eluate and crystallized from methanol, m.p. 92°C, C21H3004. IR, PMR, CMR and mass spectra were almost similar to compound 1. UV(chloroform): 234 and 272 nm.

8-Hexyltricosane 3: Isolated from hexane eluate and crystallised from methanol, m.p. 88°C, C29H60; IR (KBr) : 2960, 2920, 2860, 1420-1410 and 735-720 cm· l ; IH NMR (400 MHz, CDCI3, 8) : 0.90 (t, 9H, J = 12 Hz, 3 - CH3), 1.10 (d, 6H, J=7 Hz, 3-CH2), 1.20 (bs, 44H, 22-CH2) and 1.56(t, IH, -CH); EIMS(70 eV) mlz (re I. int.,%): M+ 408(2), 298(7), 279 (2), 265 (I), 257 (1), 251 (1), 223 (1), 209 (1), 195 (1), 181 (1), 167 (1), 156 (10), 125 (18), III (33),97 (60) and 83 (100) etc.

Octacosane-l0-o1-21-ene 4: Isolated from benzene eluate and recrystallised from benzene, m.p.100°C; IR (KBr): 3400, 2960, 2920, 2840, 1630, 1470, and 735-725 cm· l ; IH NMR (400 MHz, CDCb): 81.08 (t, 6H, J=8 Hz, 2CH3), 1.40(bs, 40H, 20x-CH2), 1.68 (s, 1 H, -OH,D20 exchangeable) 1.70 (t, 4H, 2x-CH2)' 3.44 (m, IH, -CH), 4.80 (m, IH, J=10 Hz, =CH) and 4.84 (m, IH, J=8 Hz, =CH); ElMS (70 eV); mlz (reI. int., %) : M+ 408(30), 380 (17), 337 (12), 323 (5), 297 (57), 281 (7), 269 (50), 195 (30), 187 (31), 181 (41), 167 (35), 157 (57), 153 (51), 138 (12), 126 (22), III (40), 96 (68) and 85 (100) etc. CMR (100 MHz, CDCl3): 123.45, 71.99, 37.48, 31.87, 29.66, 29.30, 25.63,25.32 and 14.03 ppm, monoacetate, m.p. 80°C.

Decanyl-ll-hydroxy tritriacontanoate 5: Isolated from benzene: methanol (7:3) eluate, recrystallised from benzene.m.p. 80°C; IR (KBr): 3480, 2960, 2920, 2860,1740,1470,1170,740, and 730 cm· l ; IH NMR (400 MHz, CDCI3): 8 0.80 (t, 6H, 2-CH3), 1.20 (bs, 70H, 35-CH2)' 1.06 (t, 4H, 2-CH2)' 2.02 (m, 2H, -CH2), 2.02 (m, IH, -OH) and 3.66 (t, 3H, -CH and -O-CH2); ElMS, (70 eV): mlz (reI. int.%) (M+) 650(8), 622 (5), 594 (5), 566 (5), 509 (15), 475 (22), 454(1), 277 (15), 239 (10), 200 (20),185 (16), 182

(20), 171 (15),141 (17), 110 (90), 71 (78), 57 (100); CMR (100 MHz, CDCb): 171.66, 84.99, 64.35 , 34.41, 29.72, 29.37,' 29.28, 29.19, 25.05, 21.94 and 14.08 ppm, mono acetate : m.p. 95°C.

Pentyl triacontanoate 6: Isolated from ethanol eluate and recrystallised from methanol, m.p. 71°C; IR (KBr): 2920, 2860,1740,1440,1390,1190,1070, 730-720 cm· l ; IH NMR (400 MHz, CDCb): 80.96 (t, 6H, 2-CH3), 1.24 (bs, 60H, 30-CH2), 2.38 (t, 2H, J=18 Hz, -CO-CH2) and 3.68 (t, 2H, J=14 Hz, -O-CH2); ElMS (70 eV) mlz (reI. int,%): M+ 522 (10), 489 (10), 480 (5), 465 (60), 451 (20), 438 (20), 420 (25), 407 (14),392 (50), 365 (22), 252 (12), 250 (12), 238 (10), 208 (25), 190 (15), 180 (25), 168 (45), 153 (70), 142 (50), 130 (90), 128 (50), 126 (20), 11 0 (35), 96 (70), 71 (13), 68 (80), 57 (100) and 56 (89).

Nonacosane 7: Isolated from hexane: benzene (9: 1, v/v) eluate and recrystallised from methanol, m.p. 95°C; IR (KBr): 2960, 2949, 2860, 1480 and 725-720 cm· l ; IH NMR (400 MHz, CDCl3,): 80.90 (t, 6H, 2-CH3) and 1.28 (bs, 54H, 27-CH2), ElMS (70 eV) mlz (reI. int,%) : M+ 408(3), 278 (2), 250 (3), 236 (10),223 (4),209 (4), 195 (7), 181 (2), 167 (18), 156 (28), 139 (10), 111 (30), 97 (70) and 83 (100) etc.

Acknowledgement Thanks are due to MPCOST, Bhopal for financial

assistance and RSIC, CDRI, Lucknow and RSIC, lIT, Mumbai for spectral data.

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