CHEMISTRY OF NATURAL PRODUCTS
DISSERTATrON SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF Muittv of $I)tlos(opt)p IN CHEMISTRY
BY SYBD MOHmUD AHMED
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 1993 DEDICATED
TO
MY
PARENTS DS2580 PHONt . (05 71) 400515 DEPARTMENT OF CHEMISTRY ALJGARH MUSLIM UNIVERSITY A L I G A R H —20:^ 002 Dalel ..lllA^
CERTIFICATE
This is to certify that the work described in the
d\ssertd.t\on entitled ' Chemistry of Natural Vxocxicts ' is the original work of Mr. Syed MQhmuc Ahmed and is suitable for submission for the award of M. Phil, degree in Chemistry.
L-l) IT , J, Ahmad) (Prof. M. Ilyas) o-supervisoi Supervisor ACKNOWLEDGEMENT
Words merely can not suffice my expression of gratitude to Professor Asif Zaman whose sagacious and invalu
able guidance was instrumental in the completion of this dissertation. I am highly indebted to Professor N. Islam,
Chairman and Professor A. Aziz Khan, ex-chairman. Department of Chemistry, for providing me necessary research facilities.
It gives me an immense pleasure to record my deep sense of gratitude to Supervisor, Professor M. Ilyas, under v
I humbly acknowledge my great indebtedness to
Professor K. M. Shamsuddin, Chairman, Department of Applied
Chemistry, Z.H. College of Engineering and Technology, for his eminent guidance whenever it was needed.
I tender my grateful thanks to co-supervisor. Dr. J.
Ahmed, for his useful advice and sincere encouragement during the entire tenture of the work.
I should not fail to express my highly indebtedness to lab. colleagues - Mr. Rafiuddin Ahmad Khan, Omair Zuberi and Ashraf Musharraf for rendering their generous help and sincere encouragement. I do not know how to extend my grateful thanks to my friends especially S. Muzaffar All, S. Shaida and Jamal
Feeroz for their cheerful companionship at all times.
My thanks are also due to Laboratory Staff Members for their kind cooperation.
I also wish to record my sincere thanks to Mr. Syed
Masahab All who very ably typed this manuscript.
Lastly, Financial assistance from the CCRUM, New
Delhi, is greatfully acknowledged.
.^
(S. MOHMUD AHMED) CONTENTS
PAGE NO,
INTRODUCTION
THEORETICAL 2-10
DISCUSSION 11 - 46
4. EXPERIMENTAL 47 - 53
BIBLIOGRAPHY 54 - 56 INTRODUCTION INTRODUCTION
Some work on Bridelia monoica v;as done earlier in
this laboratory but for various reasons could not be completed and was, therefore, taken up a fresh. This led to
the isolation, in small amounts, of a ferric positive compound. The compound v^as identified on the basis of work which is reported in this thesis as a glycoside of tamarexitin.
Another plant Gleditsia assamica was collected some years previously but could not be investigated. Present work led to the isolation of an aromatic ester identified as terephthalic acid methylester. Further work on a
'terpene' isolated from the plant is in progress. THEORETICAL spectroscopic Identification of Flavonoid Compounds:
Ultraviolet Spectroscopy:
The u.v. spectra of different flavonoids are very characteristic and along with colour reactions have been used extensively to distinguish various groups of this class of compounds. The absorption maxima of flavones have been correlated to the presence of cinnamoyi and a benzoyl chromophore, the former giving rise to the high wave length band at 320 to 380 nm and the latter to the low wave length band at 240 to 270 nm. On the basis of this generalisation important deductions have been made about the location of substituents in the two rings.
r=^=^^ +
(I (II) rr^^ .-^^
(III (IV) Substitution in the B ring, specially at 4' stabilises the cinnamoyl chrornophore resulting in a batho- chromic shift of band I whereas substitution in the A ring has a similar effect on the position of band II. Compounds having a free hydroxyl at 5 position absorb at higher wave length and methylation of this hydroxyl brings about a hypsochromic shift of 10 to 15 nm of both maxima. The presence of hydroxyl at this position is established by measuring the spectrum in presence of anhydrous aluminium chloride. Hydroxyl groups at 7,4' are more acidic than others and a bathrochromic shift of band I or II on addition of fused sodium acetate is a good indication of the presence of OH groups at these positions (1).
In flavanones absence of the cinnamoyl chrornophore has the effect of suppressing the high wave length band, which is either totally absent, or present only as an inflection. The spectra of isoflavones are also marked by the absence of high wave length band. Thus it is difficult to distinguish between flavanones and isoflavones with the help of u.v. alone.
Infrared Spectroscopy:
The i.r. spectrum of flavanone shows the carbonyl absorption at 1680 cm , the standard value for aromatic ketones. The shift of the carbonyl band to 1620 cm in
5-OH flavanones is largely due to electron donation by the orthohydroxyl group, coupled with chelation. Consequently methylation of 5-OH produces only a small hypsochromic shift of>^10 cm , A similar shift towards long wave length of 4'
substituted flavanone is, however, attributed to inter
molecular hydrogen bonding (2)- The i.r, spectrum of
flavone shows the carbonyl band at 1660 cm owing to conju gation with the olefinic double bond. Introduction of a hydroxyl at 5 position does not alter the band position
appreciably. The i.r. spectra of isoflavones are similar to
those of flavones. The aromatic region is not of any great usefulness as no reliable prediction can be made on the basis of absorption bands in this region.
Nuclear Magnetic Resonance Spectroscopy:
The n.m.r. spectrum provides unambiguous evidence for many structural features met with in flavonoids but progress in this field was hampered at first by the sparing solubility of flavonoids in solvents normally used for measurement of n.m.r. spectra. The derivatives of flavonoids such as methyl ether and acetates though soluble in these solvents are not ideally suited due to interference from signals of methoxyl and acetoxyl groups, which also effect the signals of the adjoining protons. All these difficulties have been overcome by the use of trimethyl silvl derivative flavonoids by Mabry et al (3). The trimethyl si-lyl derivatives can be convenienCly prepared by treatment of the compound with hexamethyl disilazane and trimethyl silyl chloride in pyridine and the spectrum is then measured in carbontetrachloride with tetramethyl silane as external or internal reference.
The n.m.r. spectra of flavonoids can also be rationalised on the basis of the resonance structures referred to in connection with u.v. spectroscopy. It is therefore 5 clear that chemical shifts and coupling patterns of the aromatic protons will be markedly influenced by the nature of the heterocyclic ring and the location of sub- stituents. The nature of this ring can be determined at the very outset by the position of the signal for the olefinic proton. Thus the 2 proton of isoflavones is more deshielded than the 3 proton of flavones and the respective values are
1.7 T and 3.2 T. In flavanones the signal of the olefinic proton is replaced by the absorption upfield of cis and trans coupled 2 and 3 protons. In flavonols the 3-OH gives rise to a signal at 0.6T and it can therefore, be easi]y differentiated from the more strongly chelated 5-OH {-3T) and other phenolic hydroxyl functions. A further important feature is that the presence of the 3-hydroxyl results in reducing deshielding of the 5-OH which now absorbs at -2.5T. Flavanones:
The most detailed and systematic studies of the 22 23 n.m.r. spectra of flavonoids are due to Massicot, '.
Batterham (4), Mabry (3), Henrick (5) and Clark - Lewis et al (6,), These studies have simplified the task of determining the substitution pattern of flavonoids with the help of n.m.r. spectroscopy. An obvious advantage of this technique in its application to flavanones is that they can be easily distinguished not only from flavones and isofla- vones but also from the isomeric chalkones which in view of the extreme case of the isomerization process, is often not possible with other methods.
The pholroglucinol substitution pattern of ring A of naturally occurring flavanones can be established by the presence of an AB quartet arising from the meta coupled
( J = 3 cps ) 6 and 8 protons. The signals for these two protons appear at 4.18, 3.98 respectively, upfield from the aromatic protons of B ring and so there is no over-lapping from signals of other protons. In the absence of a hydroxyl at 5 position the signal for the proton at this position appears at 2.3T. This deshielding of the C-5 hydrogen compared to the remaining protons of this ring is due to its being adjacent to the carboayl group. The absence of a substituent at C-5 also brings about a change in the chemical shifts and the coupling patterns of the remaining protons of this ring. The signals of 6 and 8 protons are both shifted downfield by about 0.4 and the AB doublet pattern is now obscured.
The chemical shifts of ring B protons again vary with the substitution pattern. The simplest case is that of
4' substituted compounds where the splitting pattern is that of 1,4 disubstituted benzenes, i.e. A'A, B'B. The two independent AB doublets are clearly seen in the spectrum of liquiritigenin (3). In 3', 4' disubstituted flavanones the chemical shifts of 2', 6' and 5' protons are almost identical and two to three overlapping signals are usually observed.
The methine and methylene protons of the hetero cyclic ring give rise to an ABX type of splitting pattern with a downfield quartet from the methine proton and the quartets of cis and trans coupled methylene protons upfield.
Flavones:
The important difference in the n.m.r. of flavones is that now there is substantial deshielding of the 2'-6' protons due to conjugation of the B ring with the carbonyl group. In 4' substituted flavones the doublet from 2'-5' protons mores downfield whereas the position of the doublet from 3',5' protons remains approximately the same as in flavanones. In 3',4', disubstituted flavones the 5' proton gives rise to a doublet at 3.15 showing ortho coupling whereas the signals from 2',6' protons give rise to two or three almost coincident peaks at 2.65. The system,therefore, approximates to an AA'B coupling pattern. In flavonols the
3 hydroxyl causes some alteration in the position of the signals from B ring protons but the effect is not very marked. These differences are clearly brought out by a comparison of the n.m.r. spectra of apigenin and liquiri- tigenin.
Isoflavones:
The main difference in the n.m.r. spectra of flavones and isoflavones derives from the fact that in the latter, as also in flavanones, the B ring is no longer conjugated with the carbonyl function and, therefore, no deshielding effects is working on the protons of this ring.
The AB quartet of the B ring protons in 4' substituted isoflavones has .therefore, the same appearance and field position as in flavanones. The signals from 2',5',6' protons in 3'-4' substituted compounds again merge into 2 or
3 peaks centred at about 3.08. The n.m.r. spectra of 2', 4' substituted flavones and isoflavones having unsubstituted 6 and 8 protons present a somev7hat different picture. The AB quartet of the 6,8 protons is now somewhat distorted due to overlapping with signals from the protons of the B ring. The 6' proton gives rise to a doublet at lowfield centred at 2.7 and the 3',5' protons give rise to a doublet and a quartet respectively which is superimposed on the signal from the 8 proton.
Mass Spectroscopy:
The mass spectra of oxygenated heterocycles have been the subject of detailed studies by Pelter(21), Reed
(7 ), Barnes et al (:8 ). According to Pelter (9 ) the mass spectral fragmentation pattern of these compounds is sensitive to variation in the oxygenation pattern which makes it difficult to formulate a general break down pattern for different members of this class of compounds. Thus for example, the retrodiene fragmentation of flavone (III) itself gives rise to a peak due to the species (IV) which is
80% of the molecular ion peak in intensity whereas in more highly oxygenated compounds its intensity is only 15-187c, of the molecular ion peak. 10
,.^^="N^0 R.D.A, -f-
0 ^H B'
(V) (VI) (VII)
Substitution pattern in ring A and B can be detected
by examining m/z value of A 'a B ",for example 5,7-dihydroxy-
flavone' gives the same B ' fragment (m/z 102) but produces
A • ion 32 m.u. higher that is at m/z 152 instead of m/z
120, thus indicating two additional oxygen atoms in ring A.
Similarly, m/z value can pinpoint substitution pattern in
ring B, leuteolin 3', 4'-dimethyl ether gives B * ion at
•Ti/z 162 which clearly indicaten the presence of two methyl groups in the molecule.
11
DISCUSSION
Bridella Monoica:
Bridelia monoica belongs to the natural order
Euphorblaceae which comprises about thirteen species. These are listed in the diagram below alongwith reported chemical constituents of some species. c ro 12 n ^ o CQ E
.CM CO m
1 •H 1 •W ->2 o 1- U!- ! pq CO •^ ca en 0 E ->.ON a PQ cfl CO < •^ 00 •u w pq o < ca c Pi -^r^ • o o CQ E CO U u ->vo o c o m E M w CD -rJ^in m M 0 4-» 0) pa u CO c -» ^ a « -> rH pa' CM a o c o )-i CO O -o c cc o OiN •r-t 00 •r-l 0 Xro U (U •V O CO -o •r4 0!N x; QJ 0) ••-1 U >-^ a -d X! en QJ •r-l •1-1 •f-l 0 a r-l C/) en c en CO 0 0 E - 0) o 3 CO CO 00 1 c I 0 X. •d 1 1 1 O a o ro en m m O E u o <^ I )- 0 00 CO a c C VD 14 0) 0) 0 CC c c S 0 0 03 1—1 1 }—1 1 c m en c 1 1 •H QJ c o c C c OJ 0 0 •o 0 •r^ X 1 •a •r-l 1 -0 o m 1 U3 ro 1 x: 1 0 1 in 4.) (N C c c CNJ 0) 03 0) E 1- •H i -o >^ -0 n TH 1 ^j 1 a 1 in r-l o in T—• >^ u CM X a^ 0 ON o i r-l 1 1-1 K) 03 -v T-i iJ TH iJ 1 >^ G TH 03 x: 0 1 0 1 c ON c CN 15 Bridelia monoica stems, cut into small pieces were extracted first with petroleum ether and then with MeOH. Chromotography of the methenol extract yielded a glycoside, m.p. 204°C,^labelled as M-1. Because of practical difficulties the mass spectrum of M-1 could not so far be obtained but other spectral data is consistent with its identification as the flavonoid glycoside (I) . OCH. OH (I) The flavonoid nature of the compound is evident from its uv spectrum which shows a band at 256 nm with a shoulder at 265 and another at 352 nm. Bearing in mind that the high wave length absorption of flavones is at 304-350 nm and of flavonols between 253-385 nm, the compound could be either 1'3 a flavone or a flavonol. If the flavonoid structure is assumed, the presence of a shoulder at 265 nm is indication of either mono or dioxygenation of ring B. The oxygenation pattern shown in (2) is the only one that accords with the nmr spectrum of the compound which shows besides aromatic and glycosidic protons a methoxy methyl at 3.9 ppm. H 0 0 (2) The arori- ic region of the spectrum shows a total of 5 protons, 2 of which appear as a pair of doublets at (^.^ and 6.2 ppm with J = 2.2 Hz. These values are typical of the C-6 and _C-8 hydrogens in flavones. The most deshielded proton gives rise to a double doublet at 7.8 [J= 8.8+1.8Hz]. The chemical shift and multiplicity suggest that it is the 6' proten in a 3',4' substituted flavone or flavonol. This assignment is forced bv the metacouoled doublets referred to 17 earlier which are possible only if the C-5 position is substitued so that the downfield double doublet cannot be the signal of the C-5 hydroden. The other deshielded proton of ring B ie C-2'H is coupled only to C-6'H and its doublet is visible at 7.5 ppm. These two values correspond closely to the values reported (18) for the 2',6' protons in 3,4 dioxygenated flavonols. It is noteworthy that the chemical shift values for 2',6' hydrogens in 3',4' substituted flavones are much higher 7.2-7.3 ppm. The third proton of ring B ie C-5'H is not conjugated with the pyrone carbonyl and besides is ortho to an oxygen. Its doublet, therefore, appears at higher fieldand the chemical shift 7.0 ppm is close to the values reported in literature e.g 6.80 in Querecetin (19) . The nmr data discussed above establishes the substitution of the flavone moiety of M-1 but leaves open the question of the position of the OMe group and the point of attachment of the sugar residue. In order to establish this, one has to unambigously determine the positions of free hydroxyl groups in the molecule. Treatment of M-1 with ethanolic hydrochloric acid on water bath cleaved the glycosidic linkage and supplied the aglycon m.p. 260'~'c. 18 OH MeO OMe OH OH 0 OH 0 Isorhamnetin Rhamnetin 295^0 m.p. 306OC OMe OH 0 Tamarexitin 259®C As seen from the above data the m.p. of the aglycon of M-1 is almost identical with that of tamarexitin and differs widely from those of the other two tetrahydroxy flavonols—rhamnetin and isorhamnetin. M-1 is thus a glycoside of tamarexitin but the point of attachment of the sug?r residue has still to be established. This can be 19 done with some certainty on the basis of characteristic shifts in the uv spectrum of the glycoside on addition of NaOAc and AlCl^. Thus while addition of NaOMe to the methanol solution of polyhydroxy flavonols ionises all the phenolic OH groups, addition of NaOAc ionises only the more acidic 3,7 and 4' OH groups. lonisation of the 7-OH group affects only the benzoylchromophore and hence produces a large bathochromic shift in the position of band II. Since the 4' position is methylated, the compound having been identified by the m.p, of its aglycon as tamarexitin glycoside, any bathochromic shift observed on addition of NaOAc can only be due to the presence of hydroxyl at 7 and/or 3 position of the flavone nucleus. Comparison of the uv spectra of the glycoside in MeOH and MeOH + NaOAc shows that addition of NaOAc results in merger of the shoulder at 265 nm with the main peak to produce a sharp high intensity band at 275 nm. NaOAC addition thus produces a bathochromic shift of 9.5 nm. Such a large shift is possible only if the C-7 OH group is free in M-1. The sugar residue is, therefore, attached to either the C-5, C-3 or C-3' hydroxyl, '!easurement of the uv spectrum of the compound in MeOH in presence of AlCl^ has been extensively used in locating the position of OH groups in the flavone molecule. AlCl,, forms chelate complexes with 0-dihydroxyphenols but these 20 complexes are not acid stable and the bathochromic shift produced in the MeOH spectra of such phenols on addition of AlCl^ is nullified by addition of HCl. As against this flavones having hydroxyls at C-5 or C-3 produce acid stable chelate complexes with the participation of the C-4 carbonyl group. This AlCl^ induced shift in such compounds persists even in presence of HCl. A further significant point is that whereas compounds in which both C-5 and C-3 OH groups are free give large bathochromic shift with AlCl-,. Compounds in which the C-3 OH group is substituted give a comparatively smaller shifts. This is highlighted in table 1. Bathochromic shifts in the position of band I on addition of AlCl-, in compounds in which both 5 and 3 OH groups are free and compounds in which only the C-5 OH is free (20). Compound No Position Position Batho of Band I of Band I chromic in MeOH in MeOH - shift AICI3/ AICI3 - HCl 54=1 Galanoin 359 413 54 21 58=2 Kaempferol 367 424 57 OH HO OH 0 59=3 Kaempferol 364 424 60 7-0-neohesperidoside rhamnoglucosyl-0 OH 0 61=4 Kaempferol 367 423 56 4'-methylether OCH. HO .0. OH OH 0 22 64=5 Herbacetin 377 435 58 S-methylether OH OH 0 65=6 Quercetin 370 428 58 OH OH 0 66=7 Quercetin 372 426 54 7-0-rhamnoside OH rhamnosyl - 0 OH 0 23 76=8 Rhamnetin 371 423 52 OH H3CO OH 0 77=9 Isorhamnetin 370 428 58 OH OH 0 80=10 Tamarixetin 369 427 58 7-0-neohespericloside OH OCH- rhamnoglucosyl- OH 0 2A 81=11 Tamarexitin 367 423 56 7-0-ruti noside OCH- rhamnoglucosyl -0 OH 0 82=12 Morin 370 419 49 OH 0 90=13 Patuletin 371 427 56 OH OH 0 15 55=14 Galangin 340{sh) 393 53 3-methyl ether r^^^^ 60=15 Kaempferol 3-0- 350 400 50 Robinoside 7-0- Rhamnoside (Robin) rhamnosyl 0-rhamnogalactosyl 67=16 Quericitin 362 405 43 3-0-galactoside OH 0-galactosyl 26 68=17 Quericitrin 350 AOl 51 OH 0 - rhamnosyl OH 0 69=18 Rutin 359 402 43 O-rhamnoglucosyl 70=19 Quercetin 3,7-0- 355 402 47 Diglucoside OH OH 0-glucosyl 27 71=20 Qucrcetin 3-0-glucoside 358 404 46 7-0-rhamnoside OH rhamnosyl -0 0-glucosyl OH 0 72=21 Quercitin 3-0-glucoside 358 404 46 7-0-rutinoside OH rhamnoglucosyl-O O-glucosyl OH 0 73=22 Quercetin 3-methylether 358 402 44 OH OH 28 lh=2?) Quercetin 3-methylether 349 400 51 4'-0-glucoside 7-0-Di- glucoside OH 0-glucosyl glucoglucosyl - 0 OH 0 78=25 Isorhamnetin 357 403 46 3-0-galactosicie OCH. OH HO O-galactosyl OH 0 79=26 Isorhamnetin 356 399 43 3-0-rut:inoside OCH- OH O-rhamnoglucosyl OH 0 29 84=27 Penduletin 340 396(sh) 56 ,0H CB3O 0 CH3O OCH- OH 0 85=28 Pendulin 330 403{sh) 73 0-glucosyl 87=29 Jaceidin 351 411(sh) 60 OCH. OH 30 88=30 Jacein 352 A07(sh) 55 OCH- OH glycosyl -0- OCH OH 0 31 Comparison of the uv spectra of Isorhamnetin-3-0- rutinoside and tamarexitin-7-O-neohesperidoside with that of M-1 OCH, OH OH OCH, rhamnoglucosyl-O 0-rhamnoglucosyl OH 0 MeOH 254,256(sh),305(sh),356 nm MeOH 255,269(sh),369 nm MeOH 268,278(sh),300(sh) MeOH 266, 301{sh), AlCl 369(sh), 402 nm AICI3 360 (sh), 429 nm OH OCH- OH 0 R=H or gl MeOH 230, 256.5, 265(5h), 352 nm MeOH 226, 269.5, 295(sh), 359, 404.5 nm AlCl 32 is crucial in identifying the position of sugar residue in M-1. The comparison reveals that the position of the high V7ave length band in M-1 uv spectrum in MeOH/AlCl- A05.5 nm is closer to that in isorhamnetin-3-0 - gl. than in tamarexitin-7-O-gl, This is further supported by the fact that in Morin which also has free OH groups at both 5 and 3 positions the wave length band in the MeOH/AlClo spectrum is at 421 nm. It follows that in M-1 the residue is located at C-3 and hence it must have structure (I). For identification of the sugar residue of M-1, the hydrolystate was extracted with ethyacetate after addition of water. The aquous layer was concentrated and used for paper chromatography. The chromatogram was run with n-BuOH-AcOH-water in the ratio of 4:1:5. Arabinose, galactose, rhamnose, glucose and xylose were taken for comparison. The sugar residue of M-1 was identified in this way as L-arabinose. The ethylacetate layer was worked up to yield the aglycone identified as tamarexitin on the basis of its melting point and nnr spectrum which was similar to that of the glycoside M-1 except for the absent of the sugar moietv in the .iiidfield resion. 33 The structure of M-1 can ,therefore,be defined as the S-arabinoside of tamarexitin. OMe 0-arabinosyl OH 0 34 SpectriiM, Orgl Grapli ELI CO SLi51 im ^^,Vz' { y ./"N. ft I \ llllinuIMIIillMKIIIHKtllMIIIIIMIlllllHKMIPUXIIItlMlirUKtMltH'XMIHMirMnllltnilliniMIt IM111M M M11111MIM1111M11111 • 11 n^^ml^ml^ian Tinki I WW 21,1 33,08 ns/div 5S0,9 M ; HeOH Speed ; Fast • •4. i \l 1 Sn-iiict :I ?s)i:S30i S Me ; 02-96-94 line ;ll;22 a« • X 35 Spectnyi, Orgl Grapli ELI CO SL151 2.581 I I ,ri- y 3 k- ."in?- ji I ! a ••^r t^ssi^ias •J 1 U V 18,3 38,99 iiM/diy oi,8 Sasple ; :i-i(HaOH8) Speeil ; Fast at9 ; 82-S6-M IiM8 ;ll:40as /105 36 Sp^ctniH. OFII Graph ELICO SL15i 33.09 iiH/diy see.8 Sp89d ; Fast i r>^j.< =)-!_A.,^ 3"7 37 Spscinn, 05jl teh ELKO SU5i U I ! ^ 3 A I 'Jl U V J i Jt3 isr^i^SMSiJSS'^iiisiini 13 ii;i/cliy •J J J I W MT3 ; 92-%-'?4 hue i ""'J. ^ i J^. V ;^ a - 1 ~ ^K^^^^ 38 SpecipiiM, Opgl Grapli ELI CO SL151 /i,sa I'. 39,88 iiM/ai 589,8 Saspia ; ;i-l(flaOSo-ia) Is? : HeOH Spsed : Fast ilnalyii ; Mmu """ Sats : 02-86-94 Ii»8 li2;25 ps •^Ax 39 I/) U) 6e hi (•/-) BDNViilWSNVbi 40 (•/.) 3DHViilW^NVdi 0) >> V/1 Q 'in J W 1 ' 1 1 i(r ^1 J ^ ;'. '^' y 0 ! —1 1 o > n u r^ ' u x9 0 1 < y T o ^ ^ / S / .V , " 0 0) rV' r ^n 1 I I I I J- V >> ^ Ji t'l ^-^ en -— -d -J ^^ A M _ o 2. d;d^. > . tA. x9 ^ u > V u."' 6 o ;; , 1 MM ri 1 -, I' I I i 1 1 ' 1 ^e 1 i 43 GLEDITSIA ASSAMICA Gleditsia assamica belongs to the family of Leguminosae. Its stem pieces were extracted first with petroleuiii ether and then methanol. Work up of the methanol extract yielded white crystallized material m.p. 148 C. The ir spectrum of the compound showed a carbonyl band at 1720 alongwith bands at 1500, 1435 cm" character istic of an aromatic compound. The uv spectrum showed maximum at 265 alongwith weak absorption at 340 nm. The nmr spectrum of the compound was very simple with just two singlets, one at 3.95 and the other at 8.2 ppm. The singlets at higher field obviously arise from aromatic methoxyl, the one at lower yield must then be allotted to identical aromatic protons. These features are compatible only v^7ith identification of the compound as terephthalic acid methyl ester. COOCH. COOCH. 44 l;28:5l t^m-L.*!* t.-«x«MJ« ! \ k •J 1 ,1 A n •" -I ,' ' /diu .^' \ a^«",™ •». _.. - -— X j*a-.«ib.'iii<>i;.i'iiii.;iu..a ^uai ^"""'" ' I .i" i"'" "....I, ii,,„i ,1. .7\-,| - -A 1 J. 33;3,:] 13,8eiw'diy 383,.!: jnahs'J- t ; ^MmA Data ; i7-95-M Ine ;li;43 m 45 3 o o o O o o o o en CO 1^ iC in •*r (1 CM ("/») 33NViilWSNVyi ii o 1 • J X (O 1 O 1 1 UJ 8 to 1 i ( ") , 1 1 46 III 1 , , ^^ •o 1 1 ^' CL LI 1 1 X X 1 o r, 1 1 2 X •:> :c _l '^ I- ' • • u 7 i 1 j ZL UJ UJ H- -i 1 i 1 1 ^1 Q I J H- 3 S n t/) • • Q > vr 1 - t( o) 1 i cc u • • -.j: X 1- ^ I 47 EXPERIMENTAL The melting points were taken using a Kofler block and are uncorrected - IR spectra were recorded in KBr/Nujol on a PYE UNICAM SP3-100 Spectrophotometer. UV spectra in methanol solutions were measured on ELICO SL 151 spectro photometer . H-NMR spectra were obtained from I.I.T., Ne w Delhi using TMS as internal standard in DMSO-d, and CDCl^. Column chromatography was carried over SISCO silica gel (60-120 mesh) and TLC over MERCK Kieselgel (60 G). Iodine and alcoholic ferric chloride were used for visualisation of TLC plates. AZA'^mO yr \ 48 BRIDELIA MONOICA Extraction: The stem-wood (10 kg) of the plant was chopped into small pieces and exhaustively extracted in a soxhlet, first with petrol to remove the fatty material, and then with methanol. The methanol extract after removal of the solvent under reduced pressure provided a black resinous mass. This black resinous mass was refluxed with petroleum ether and ethyl acetate respectively. The ethyl acetate fraction which contained a mixture of several compounds as revealed by TLC examination was subjected to column chromatography over silica gel. Elution of the column with a mixture of ethyl acetate-benzene (90:10) afforded a glycosidic compound labelled as M-1. The compound gave a dark blue colour with alcoholic ferric chloride solution. M-1 (Tararexitin-3-O-arabinoside): It was repeatedly crystallised from ethanol- petroleum ether as yellowish crystals, yield 200 mg, m.p. 204°C. 49 Spectral Data of M-1: UV (MeOH)X : 230, 256.5, 265(sh), 253 nr max ' ' (NaOMe)/- : 235.5, 273.5, 397.5 nm max (AlCljX : 226, 269.5, 295.5(sh), 404.4 nm. 3 max ' ' • ' (NaOAO^X max : 232,' 275.5,' 337(sh),' 388.5 nm. (NaOAc)X : 233, 274.5, 336.5(sh), 387 nm. ^ max ' ' ' after 10 mins. IR (Nuiol)•^ 'maV x : 3400-3500, 1650 cm~^ IH-NMR (DMS0-d6) : 3.85 (3H, S, -OCH^), 6.2 (IH,d, J=2.2Hz, C-8), 6.4 (lH,d,J=2.24Hz, C-6), 7.06 (lH,d,J=8.79, C-5') 7.4(lH,d,J=1.831 , C-2'') 7.7 (H, dd, J^ = 8.79 & J2 = 1.8, C-6'), 9.29 (IH, s, C-3'), 10.93 (lH,s,C-7), 12.53 (lH,s,C-5). Hydrolysis of H-1: 100 mg of the compound was refluxed with ethanol (10 ml) containing few drops of HCl on a water bath for about two hours. After concentration, the hydrolystate was extracted with ethyl ^acetate after dilution with water. 50 The ethylacetate layer was worked up to yield the aglycon labelled as M-2. M-2 (Tamarexitin): It was crystallised from methanol-petroleum ether as bright yellow crystals, yield 30 mg, m.p. 260 C. Spectral data: IR (KBr)V^ : 3500, 1655 cm"^ r max ' IH-NMR (DMSO-dg) : 3.84(3H,s,- OCH3), 6.19 (lH,d, J=2.197Hz, C-8) 6.42 (lH,d, J=2.198Hz, C-6), 7.09 (lH,d, J=7.97Hz, C-5'), 7.63 (2H, m, C-6' & C-2'), 9.35 (2H, m, C-3'& C-7) , 12.477 (IH, s, C-5). Identification of Sugar: The aquous layer obtained during hydrolysis of the glycosidic compound M-1 was concentrated and chromatographed in the descending mode on 'Whatman no.3' paper along with standard samples of L-arabinose, galactose, glucose, rhamnose and xylose. The chromatogram was run in n.BuOH - AcOH - water (4:1:5). The development of the chromaogram was made by spraying it with aniline phthalate (2g of 51 Phthalic acid + 1ml of Aniline in 200 ml of n.BuOH). The isolated sugar had the same Rf and colour as that of L-arabinose. 52 GLEDITSIA ASSAMICA Extraction: Stem wood (15 kg) of the plant v/as cut into small pieces and defatted three times by refluxing with petroleum ether to remove the fatty acids. The plant material was then extracted repeatedly by refluxing with methanol. TLC examination of the concentrated methanolic extract revealed it to be a mixture of several compounds, and hence it was subjected to column chromatography over silica gel. Chromatography of the methanolic extract: The extract was absorbed on silica gel and the slurry was dried. The powder so obtained was deposited on a column of silica gel and the column eluted with petroleum ether - benzene (80:20) yielded a solid labelled as M-3. M-3 (Dimethylterephthalate): It was repeatedly crystallized from petroleum ether- benzene in the form of white crystals, yield 250 mg, m.p. 148°C. 53 Spectral data: Uv (MeOH)X ,^ . oon 9/,n r.,. max : JoU, zq-U nm. IR (KBr/NujoDy^ : 1720, 1500, 1435, 1400 cm""^ IH - NMR ( CDCI3) : 3.96 (6H, S, -OCH3), 8.2 (4H, S). BIBLIOGRAPHY 54 BIBLIOGRAPHY 1. L. Jurd, The Chemistry of Flavonoid Compound (Edited by T.A. Giessman), 107 (1962) 2. H.L. Hegert and E.F. Kurth, J. Am. Chem. Sec, 75, 1622 (1953) 3. T.J. Mabry, J. Kagan and H. Rosier, Nuclear Magnetic Resonance, Analysis of Flavonoids, No.6418, University Texas, Austin, Texas (1964) 4. T.J. Batterhara and R.J. Highet, Aust. J. Chem., 17, 428 (1964) 5. C.A. Henrick and P.R. Jefferies, Aust. J. Chem., 17, 1934 (1964) 6. J.W. Clark Lewis, J.M. Jackman and T.M. Spotswood, Aust. J. Chem., 17, 632 (1964) 7. R.I. Reed and J.M. Wilson, J. Chem. Sec. , 5949 (1963) 8. C.S. Barnes and J.L. Occolowitz, Aust. J. Chem., 17, 975 (1964) 9. A. Pelter, P. 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