-

Indian Journal (It"C h<:mistry Vol. 3 ~B. D<:<.:<:lllhn 1'1'1'1. lip . 135'1- 1303

New protolimonoids and limonoids: Part I-Isolation, structure elucidation of new protolimonoids and limonoid from the root wood of

Chisocheton paniculatus Hiern (Meliaceae)

R D Yadav , J C S Kataky* & R K Mathur

Organic Chemi stry Divi sion , Regional Research Laboratory, Jorhat 785 006 , Assam. Indi a

Received 3 Augus/ 1998; accepted (revised) 14 April 1999

Five new protolimonoids paniculatin B, paniculatin C, panicualtin D, paniculatin G, paniculatin H, a new Iimonoid arunachalin and known compound paniculatin including ~-sitostero l have been isolated from the mature root wood of Chi­soche/o/l I'({/licu/a/us Hi ern (Fam. Meli aceae). Structures of the new compounds have been estab li shed by spectral data and of the known compounds by comparing with au thent ic compounds

In our continuing studies I on Meliaceae of the North­

Easte rn region of Indi a we have investigated the root wood of Ch is()cheroll paniculatus , a member of Me­liac.eae from thi s region . The plant mate rial was col­lected from the tropical evergreen forest of Namsai, Chessa from Aru nachal Pradesh, India.

Limonoids have attracted much attention because of the marked insect antifeedant and growth regulat­ing activity. Antifungal acti vity of azadirachtin and re lated limonoids iso lated from the neem tree and also o f few limonoids iso lated from the fruits of Chi­socheron pOllicufarus Hiern2

-4 More recently, li­

monoids have shown anticarc inogenic and antitumou­rogenic acti v ili es~atl. More than 400 limonoids and

proto limono id s ha ve been iso lated and characterized

from nalural sources and in a lmost a ll cases correct u­orientati on I al C-7 is depicted . However two early

examples of ~ -ori entat i o n have also been reported in the I ile rature),6 . We report he re the isolation, identifi­

cati on of known paniculatin 3, ~-s itos terol and structure e luc idation of new compounds arunachal in 1, paniculatin B 4 , paniculatin-C 5, panicualtin-D 6, panicul alin-G and paniculatin-H.

Paniculalin 3 and ~ -s itostero l were identified by a compari son o f their mp, IR and IH-NMR data with the reported va lues I, as well as by compari son with authentic samp les. The "CNMR spectra l data for new compounds also a llowed unambi guous ass ignment of the signals for most of the c~rbon atoms.

Arunachalin 1, C1oH .. 40 5 (HRMS ), showed pos iti ve Liberman-Buchard react ion and ye llow colouration with tetranitromethane (TNM) but failed to give Ehr-

H

1 : 7,8-fl-epoxide-9,11-ene fl-5itosterol 2 : 7,8~-epoxide

o

o R,

""OCOCH3 ""

OCOCH3

3

R10

4 : R1 = H, R2 = Ac, R, = A 5 : R1 = H, R2(a-)= Ac R3 = C 6 : R1 = H, R2(a-)= Ac R, = B

¢:" lich colour test. The compound showed absorption maxima in its IR spectrum fo r cyc\ohexanone ( 171 2 em-I) and hydroxy l (3600-3200 em-I) functions . A

less intense ion peak at mlz 483 (4%) [M+-Ht in its mass spectrum showed thermal instability of the

1360 INDIAN J CHEM, SEC B, DECEMBER 1999

molecule. Fragment ions at mlz 325 (6%) and mlz 271 (10%), corresponding to the loss of [M+ -side chain-2Ht, and [M+-side chain-C6H6 (part of ring 0 ) -CH2t respec ti ve ly, are characteristic of lanostane type of triterpene alcohols having a monounsaturated skele ton8

. High resoluti on IH NMR spectrum of 1 showed five quaternary methyl s at 8 0 .88, 1.02, 1.02,

1.05 , 1.05 and two epoxy methyl s at 8 1.32

ppm. Presence of a six proton s ing let at 8 1.32 ppm and a doublet at 2.86 ppm sugges ts for an (y, -oxirane ring in the s ide chain which is also supported by an ion peak at mlz 71 (20%) in the mass spectrum. The

presence of a broad one-proton si ng le t at 8 5.32 pPI" and a doublet at 2.52 (J=2.5Hz) exchangeable with 0 20 , just ified the presence of a hydroxyl function at C_2 17. The NMR spectrum of 1 (ef Table I) is very much similar to that of cneorin-NP38 27, except that 1

ex hibited one additional double bond at ~l)( I I ) which is

supported by the presence of a doublet for one proton

at 8 5.36 ppm lO . These observati ons lead us to pro­

pose the st ructure fo r compound 1 wit h 7(8)-P ­

epoxide , the most sought precursor of p -o riented C-7 oxygen function , proclaiming the poss ib le ex istence

of perfec t p-oriented function at C-7 in the limonoid series5

.6 of natura l products.

Paniculatin-B 4 . It was obtained as crystalline solid by repeated crysta lli sat ion from a mixture of

be nzene-eth yl ace tate (70:30) and through PTLC. The molecular fo rmula C.12 H:;OO C, was obtained fro m HRMS uata. The ElMS data at 70 eV showed frag­ment ion peaks a t Il1Iz 497 [M+-H10-CH1t , 372 (59) [M +-s idc c il a in f sh owi ng two distinct parts in the structure pe rtaining to a charac teri st ic c leavage in the apo- tiru ca l,lIIe side chain ') . The compound showed

Tahle I_I 'c NMR ( 100 M Hz) and IH NMR (400 MHz) data for compounds 1,4, 5 and 6 (CDCIJ ) •

-'

:i (,

7

I)

10 /I I::> i\ 1--1 IS 16 17 IX I I)

2() ::>1

2') ,\ 1)

C() \h· CO\I l'

I iS H

1.1)0 uti

::>.70 1.')5 ~ . 7~

,'i .Y, ::>02

::>.::> 1 2.7 1 ::> . 10 OX~

OX8

5.-' 2

\00 ::>~()

1 .. \2 I .. \ ~ I.()) 1{J5 I .0::>

4

iSe iSH

33.04 1.90 22.64 I .R5 77.')9 4.66 36.0--1 41 .74 2. 72 2347 1.95 72.0::1 4.06 4--1 .,'() --I I A--I 2.52 37. --1 8 16.17 1. 85 32.H7 2.00 46.59

162.27 11 942 549 -' --1 .92 2. 20 52.56 2 04 20.3() 090 19. 11 0.92

--1 5.32 'n43 4.66 31.25 1.70 77.22 3.0() 67 .58 2X'i 57 .--1 5 27.77 13 2 ::>U)7 U2 2--1 . ' ) I .OH 2.'1.50 1.08 1') .60 085 21.35 2.0X 17().lJ6

5 (,

iSC iS H iSC iSH

33 .12 um 33 . I S 1.90 22.68 1. 80 22.72 1. 85 78.05 3.92 78.07 3.93 36.08 36. 12 4 1.78 2.68 41.81 2.70 23.55 1.90 23.56 1.90 72. 35 4.66 72. 18 4.67 44. 36 44.3 8 41.62 2.55 41.55 2.55 3744 37.57 16. 34 I ~ O 16.21 I.S5 34.03 2.00 33 . I S 2.00 46.60 46.57

162.29 162. 14 119.66 5.30 119.56 548 34.72 2.25 34.9 1 2.25 52.14 2.05 52.67 2.04 21.67 0.85 21.73 0.87 15.10 0.90 15.10 0.90 3S .72 44.72 70.02 3.44 96.69 5.32 36.26 30.07 1.70 86.--14 75.53 4.54 64.36 2.90 7 S. 05 3. 16 73.94 73. 59 28.3H 1. 2H 27 .79 1.28 23. ')7 1.2H 27.53 1.28 27. 50 0.90 26.66 1.08 27. 82 0 ')3 2642 1.0R 19.13 1.30 19.85 1.30 21.36 2 . m~ 2 1.36 2. 10 170')2 170.S7

YADAV et al.: NEW PROTOLIMONOIDS & L1MONOIDS: PART I 136 1

pos iti ve Li hc rlllan-Buchard reaction and a yellow co lourati on with TNM, but failed to gi ve Ehrlich col­nul' tes t. Thc IR spectrum showed absorption bands at ~)~) -:n ()o . I nt,. 1240 and 870 cm·1

, indicati ve of hydroxy l group. acetoxy group and a double bond . Hi gh reso luti on I H NMR spectrum of 4 showed seven Illethyl groups at 8 0.8). 0.90, 0.92, 1.08, 1.08, 1.32 and 1 .~2 ppm and one acetoxy group at 8 2.08, one ole fini c pro tons at 8 ).49. two oxymethine protons at 8 .+ .66 and ~.90 ppm and one di stinct epoxy methine prolUn at 8 2.8) ppm (c( Table I) . Comparing these da ta with scvc ral known molecules like meli anol, Illc li ;.ln one. structure 4 is suggested for paniculatin-B

' 1 R ' I C7 f ' S6 \\' Itllr ort entl'c - oxyge n uncti on' . . Paniculatin-C 5. It was al so obtained as a crystal­

line solid hy rcpeated chromatography (benzene-ethyl acetate. t, (j :20 ) and rec rys talli sa ti on fro m ethanol. The Illolec ul ar formu la C 12 H"20 () was obtained from HRMS and the cll ill pound is a non furanoid one (ncg ati vc I-: hrli ch tes t ). The ElM S data at 70 eV showed fragment ion peaks at mlz 5 14 due to [M+­H20r. ThL' IR spec trum showed absorption bands at ~) 3 ~-~ ~ 7 {) . 172) . i 2.+8 CIll ·

1 , indicati ve of hydroxy l

gn)up ,lIlel ace toxy group. respecti ve ly. The IH NMR \ pcc tru m or 5 showed seven meth yl groups at 8 0.85, () l) (). ()90 . () (n. 1.28. 1.18 and 1.30 ppm, one ace­toxy group at 8 2 .m~. ll ne olefini c protons at 85.30 pp m (c( Tahle I) . Pcrforming the experiments liC DEPT. COSY . the complete structure fo r paniculatin­C is ass igned as 5 .

Panicula tin-D 6. It '-'vas obtained as a crystalline so l id by repeated crys ta II izat ion fro m methanol . The molecular formul a C ,2 H,20 7 was obtained from HR MS data. The compound is a tetrahydroxy non­furanoid apo- tiruca ll anc de ri vati ve in which the ep­ox ide rin g is opened in a cis-manner. Thi s has an a­oriented oxyge n functi on (OCOCH1) at C-7 similar to paniculatin -C 5 already described above. The IR spectrum shuwed the presence of hydroxy l fun ction ( 3697- 342~ CIll·'). and an acetyl group ( 1727, 1244 C Ill ·

I) . The ElMS da ta at 70 eV showed fragment ion peaks at Illh ~n [M+-side chaint and 175 [side

chain- I r correspondin g to two di stinct parts in the structure of molecul ar mass mlz 548. The fragment ion at mlz 372 [M +-s ide chain t is more stable and furth er degrades to mlz 354 [M+-side chain-H20t. That paniculatin-D lac;ked epox ide in the side chain is revealed by the absence of a doublet at 8 2.85 ppm (H-24). However, presence of a long and sharp singlet

at 8 2.10 ppm corresponds to a-oriented C-7 oxygen function (OCOCH~) similar to that in paniculatin-C S. The presence of a triplet at 84.54 and a broad singlet at 8 3.16 ppm is attributed to two hydroxy functions at C-23 and C-24 respecti vely (cf Table I). The po­siti on of other hydroxy function at C-21 as that in arunachal in-l is registered by a broad singlet at 8 5.32 ppm. The two cis-hydroxy functions at C-23 and C-24 are confirmed by the formati on of acetonide. By comparing the data with those of panicul atin-C 5, the complete structure for panicul atin-D is ass igned as 6 .

Paniculatin-G. It was obtained as crystalline plates from eth anol. The IR spectrum of the com­pound resembles the spectrum of panicul atin-B 4. The IH NMR of panicul atin-G is also similar to that of paniculatin-B 4 except that the former di splays almost all signals down fi eld . The I3C NMR differs only in terms of relati ve intensities of the signals. Inciden­tally the compound di ssolves in chl oroform and when chl oroform is allowed to evaporate at room tempera­ture in a few days, a gum is obtained which renders a mi crocrystalline product like that of paniculatin-B with comparable melting point when crystallized in ethanol. This reveal s at once that panicul atin-B and panicul atin-G are conformational isomers.

Paniculatin-H. It was also obtained as crystalline so lid but is very unstable in solvent like chloroform. There is no signal for epox ide or six-membered het­erocyc li c ring in its IH NMR spectrum. The I3C NMR spectrum indicates that it is an epimeric mixture and consi sts of an unsaturated fi ve-membered heterocy­cli c ring in the side chain similar to paniculatin-D.

Experimental Section General. Melting points were determined in open

capill aries on a Buchi oil heated (Model 510) appa­ratus and are uncorrected. Optical rotations were taken on an Optical ac ti vity polarimeter (Model AA 1000). UV spectra were recorded in MeOH on a Hitachi 320 and Jasco UV-Vis 7800 spectropho­tometers, IR on a Perkin-Elmer 983 spectrometer ( Vll tlX in cm-I), and NMR spectra on a Varian EM-390,

Brucker wrvt -'+OO spec trometer using TMS as internal refe rence (chemi ca l shifts in 8, ppm). Mass spectra were reco rded on Jeol-300 or INCOS 50 GC/M S/DS mass spectrometer. Thin Layer Ch romatography (TLC) and preparati ve TLC were performed on Si ge l-G (E. Merck) and Co lumn Chromatography was performed 0 11 Si gel (B D H) of 60-1 20 mesh size .

1362 INDIAN J CHEM, SEC B, DECEMBER 1999

Plant material. The cuttings of main root of Chi­s(lcheron , )(I/lieu/o/IIS Hi ern were collected from Na msa i <lnd Chessa reserve forest of Jtanagar, Arun­ach,11 Pratksh. India during November 1990. Identifi­cat ion W,I S made by Forest Research institute, Chessa, Itanagar. Government of India from the Herbarium spec ies maintained in the Institute.

Extraction and isolation. The dried root wood (850 g) was powdered and extracted exhaustively with re fiu xing pet. ether (60-80°C) in a Soxhlet appa­ratu s for I ~ hr. The ex tract on concentration under redu ced press ure gave 39.41 g as a gummy material. This gum was furth er reflux ed in pet. ether (40-60 DC)

for a period of 6 hI' and was allowed to stand for 7 days. Art er filtrati on a so lid was obtained which was repeat edl y \-vashed with pet. ether (40-60 "C) to give 8 0 t!. of ;1 so lid. The so lid on further crystallisation from pel. et her gave 4.9 g of powdered material (S) ,Inc! the wash ing and filtrat e gave 2.8 g of a gummy material (K). The filtrat e and washing from above were combined and concentrated under reduced pres­sure to give ,In oil 00.9 g. limonoid fraction) . The oil was ~ ubi ec t e d to ex haustive chromatography (TLC, PTLC. Coillmn ) to yield the reported compounds (TLC profile s). The so lid S (4.9 g) from above was ~ uhkcled t() coluilln chromatography using C6H6 with IIlcreasin g <lmoullt of EtOAc as e lu ent to give the fract ions. The fracti ons were grouped on the basis of the ir TLC profile s in to fi ve maj or fractions (A-E) fro m whi ch the prese nt compounds were isolated. Also the gUlllmy pmduct K was subjected to similar colllilln chromatography using C6H6 with increasing allluunt of I-:tOAc as eluent to give the fractions F and G.

P<lnicul<ltil1 J. the reported limonoid was isolated frolll th e () il ()hclined after trituration of the gummy 111,ll eri<l1 with pet. et her. as a pure compound (PTLC, TLC. co lullln chroillatography) and identified by c\llllp<lring wilh ~In authenti c sample (TLC. IR. mmp) .

Fr,ll' ti on A (60 mg. 7YIc Cr,HJ25 % EtOAc) which showed <I sill gle spot on TLC was purified by PTLC (C'!-Ir,. x:i) al1d cry~ tallised from a mixture of C6H6 and iV1 eOH (00: 10 ) to give white plates (30 mg) and

characterised as arunachal in 1 by comparison of the IH NMR spectra or cneorin-NP1R 2 , a known com­pound 7 Fract ion B (70 mg, 75 % C6HJ25 % EtOAc) gave ~-sitosterol (SO mg) on crystallisation from EtOH after PTLC (C6H6, x5) and identified by direct comparison with an authentic sample. Fraction C

(830 mg, 70% C6HJ30% EtOAc) showed one major and few minor spots on TLC and crystallised from MeOH and the crystallised product was purified by PTLC (C6H6-EtOAc, 80:20, x5) to furnish pure paniculatin-B 4, (280 mg) . Fraction 0 (960 mg, 70%C6HJ30% EtOAc) yielded panicuiatin-C 5 after column chromatography (C6H6-EtOAc, 80:20) and crystallisation from EtOH as pure product (790 mg). Fraction E (360 mg, EtOAc) showed a single spot on TLC (C6H6) and purified by repeated crystallisation from EtOH and MeOH to furnish the pure crystalline panicualtin-D 6 (280 mg) . Fraction F (330 mg, 80% C6HJ20% EtOAc) yielded paniculatin-G after purifi­cation through PTLC (C6H6: EtOAc, 80:20 , x5) as pure solid (180 mg) which was then crystallised from EtOH to yield the pure compound. Fraction G (300 mg, 70%Cr,HJ30% EtOAc) furnished paniculatin-H as a pure crystalline compound (250 mg) on crystalli­sation from MeOH.

Arunachalin J : Crystalline solid (C6H6-MeOH, plates), mp 216-217"C; [aJ"'o -12" (c 2.0 CHCb) ; UY: A lllax 2 16, 219 and 221 nm; IR: V max 3600-3200, 1712, 1450, 1370, I 090, and 810 cm-I; I H NMR: see Table 1; ElMS (70 eY) rnlz 483 [M+- I t 455,325 (6) [M+-side chain-2Ht, 271 ( 10) [M+-side chain-C6H6-CH2t, 109 , 69 ( 100) ; calcd . for C}oH-l-lOs.

Paniculatin 3: Crystalline solid (MeOH), mp 185-186 "c (Iitl , mp 186-188"C); [a] IS!) 118" (c 0.5) (Iitl [a] 25D 119" (c 0.5»; UY: A max 220 nm (£ 10,000 ); IR: V lllax 1750, 1675, 1505 and 875 cm-I; EIMS (70 eY) : mlz [M +] 494 corresponding to C}OH }S06.

Paniculatin-B 4 : Crystalline solid (MeOH), mp 24 I-243°C ; [a] 25D- I.609°(c 2.0, CHCI}); UV: AntlX 203 nm ; IR : V lila x 3535-3300, 1728, 1385, 1370, 1240, I 180, 1060, and 870 cm-I; I H NMR and IJC NMR : see Table I ; ElMS (70 eY): rnlz [M+-H20t 512,372 (59) [M+-side chaint ; Caled for C:nHS00 6.

Paniculatin-C 5 : Crysta lline solid (MeOH); mp 200-202"C ; [a] lS !) -2 .59° (c 4.0, CHCI ,); UV : Alllax

205 nm ; IR : V lllax 3533 , 3371, 1725. 1382 , 1372, 1248, 1175,1072,824 and 732 cm-I

; IH NMR and I' C NMR : see Table i : ElMS (70 eY) rnlz [M+-H20t 514 ( 17.9), 475 ( 14.9) , 396 (10.8) 373 ( 17 .0)[M+-side chain+ I r, .172 (40.9)f M+-side chaint, 354 ( 18 .7), 160 (:i I ) I side chainr: Ceded for C}2 H5l0 6

Panicuiatill-D 6 : Crystalline solid (MeOl-f) , mp 148- 15 1"C: 10:.1 -' \) - 2 . .')9" (c 4.0, CHCI}); UV : 1"111<1 X

205 11m: lR VI1 1:1\ 3697-3424, 1727. 1460, 1375, 12'+4. 1095. X72. and 807 cm-I; IH NMR and 11C

~.

Y ADAVelal. : NEW PROTOLIMONO IDS & L1MONO IDS: PART I 1363

NM R see Table I; ElMS (70 eV): m/z [M+] 548 ( 179 ) .470 ( 14.9).373 ( 12.6)[M+-side chai n+lt, 372 (26.5)[M+-s ide chain t, 354 ( 13.1 ), 175 ( 14.1 ) [s ide chail1 - 1 t: 160 (3 1) [side chain t ; Ca lcd. for C ;2 H,20 7'

Paniculatin-G Cry, talline so lid (EtOH , plates), mp 220-222"C; [CX] 2S j) -2 .59" (c 4.0, CHCl l); UV : /,,,,,,, 205 11m ; IR : V illa" 3697-3424, 1727, 1460, 137.\ 1244. 1095.872, and 8 10 cm' l; ' H NMR and 11C NMR: see Table I: ElMS (70 eV): mlz [M+ - H20r, 5 12 ( 179). 373 (6.7) , 372 (2 1.2) [M +-side cha int. 1'(;7 ( 10. 1), 175 ( 10.0) , 173 ( 12.0), 160 ( I -U\). 159 ( 19.0). 135 (20 .0) , 83 ( 100); Calcd. for C ,2 i-h ,O(, .

Paniculatin-H : Crys talline so lid (MeOH), mp 122-12'i"C; ' H MR al1d I'C NM R are complex , ElM S (70 eV) m/z ' M+-HlW 6 14, 576, 5 18, 502 , 470,464, -+'i4 . 426. 372. 330_ 2 19 ( I O(J): un stab le and diffi cult to characterize.

Acknowledgement We thank the Direc tor Regional Research Labora­

tory . .f orh at for prov iuing research fac ilities . One of us (ROY) thank s CS IR , New Delhi for financial as-

sistance, RSIC (C DRI,Lucknow) for major analytical data.

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355 1. ~ It o T, Tamaillura T & M :lI~a mllIO T . Li/Jids. II . 1976.434. SI Nakan ishi T, I nad~, A. Ni ~ hi M, Miki T. Hino R & Pujiwara

T, Chelll istr." Lett. 1976. 6lJ . 10 Lavic D. J:lin M K & Kir~on 1..1 rhl'llI Soc (C)' 1971. 17S15.