Our phytochemical research on Jatropha species

Jatropha is an important genus of the family Euphorbiaceae. The plants of this genus contain complex chemical constituents which are structurally impressive. The medicinal properties of the plants and their various constituents are valuable. We carried out phytochemical investigation on three Jatropha species, viz., Jatropha gossypifolia , Jatropha curcas and Jatropha multifida . We isolated several constituents: diterpenes, lignans, coumarinolignans and other compounds. Some of the isolates possess significant antibacterial and anticancer properties. Here, we discuss our phytochemical research on Jatropha species informing of the isolation, structures and bioactivity of their chemical constituents


Introduction
Jatropha is a genus of flowering plants in the family Euphorbiaceae.The name "Jatropha" is derived from the Greek word "Jatros" (meaning "physician") and "trophe"(meaning "food").Since ancient time, the plants of this genus have been used in ethno-medicine.The genus comprises around 170 species of shrubs and small trees.The plants are naturally distributed mainly in the tropical and sub-tropical regions of the Americas and Africa but they are now cultivated around the world.The species have attracted much attention in recent years due to their impressive medicinal properties, such as, antimicrobial, antidiabetic, anti-inflammatory, antimalarial, antioxidant, anti-HIV and anticancer activities.Other potential benefits include their applications for bio-diesel production, insecticide preparation and plastic formulation.[3][4][5][6][7][8][9][10][11] We conducted phytochemical research on three Jatropha species: Jatropha gossypifolia, Jatropha curcas and Jatropha multifida.The plant materials were collected from the local areas.Our investigation resulted in the isolation of various novel constituents.The biological activities of some of these constituents were also examined.Here, we briefly review our phytochemical studies on Jatropha species.

Discussion
We successfully isolated and characterized several diterpenes, lignans, coumarinolignans and other constituents from three investigated Jatropha species.The structures of the new compounds were settled by extensive studies of their spectroscopic data.The X-ray crystallographic analysis of some of the complex compounds were also performed.The known compounds were characterized by comparison of their spectral properties with those reported in the literature.The transformations and syntheses of various constituents have been carried out.

Diterpenes
Diterpenes are the major isolated constituents of the Jatropha species.Thirty diterpenes have been obtained from these species.Fourteen of these compounds are new.These thirty compounds are listed in Table 1, with their source(s) and references to our group's work.

Jatropha multifida Known
From Jatropha gossypifolia two macrocyclic diterpenes, jatrophenone (1) 12 and citlalitrione (15) 20 were isolated.The first compound is a novel diterpene while the other was isolated from the species for the first time.The structure of jatrophenone (1) was established mainly by detailed studies of its 1D and 2D NMR spectra.2D NMR spectra clearly showed that A ring of the molecule is saturated and it contains an acetoxy group (β-configuration) at C-3 [δ 2.09 in the 1 H NMR spectrum and δ 170.8, 21.3 in the 13  Jatrophenone (1) was found to exhibit significant antibacterial activity against Staphylococcus aureus; the activity was comparable to that of penicillin G. 12 Citlalitrione (15) and related diterpenes were reported earlier from other Jatropha species.These compounds are considered as the useful taxonomic markers within the genus. 20Both the compounds 1 and 15 were isolated from Jatropha multifida. 13,14,16,18nvestigation on Jatropha curcas yielded a large number of diterpenes (Table 1). 15,21Four of these constituents, 2-5, are new compounds.Two of them, 2 and 3, are of lathyrane type while the other two, 4 and 5 are of podocarpane type.From careful analysis of the spectral data compound 2 was characterized as 15-Oacetyl-15-epi-4(E)-jatrogrossidentadione and 4 and 5 as 3β-acetoxy-12-methoxy-13-methyl-podocarpa-8,11,13-trien-7-one and 3β,12-dihydroxy-13-methylpodocarpa-8,10,13-triene respectively.Compound 3 is an epoxylathyrane diterpene related to 2. 15 The molecule 2 contains a cyclopropane moiety, a hydroxyl, an acetoxy and five methyl groups.All the signals for the protons and carbons in the 1 H and 13 C NMR spectra respectively were assigned from 2D NMR ( 1 H -1 H COSY, NOESY, HSQC and HMBC) and APT experiments.The NOESY experiment clearly supported the placement of the acetoxy group in the epimeric form at C-15.Both the compounds 2 and 3 possess a similar general structure.However, the compound 3 contains an epoxide ring at C-5, C-6 (instead of a double bond at C-4, C-5).The other difference is that the latter contains a tetrasubstituted double bond at C-14, C-15 with an acetoxy group at C-14.The trans E configuration of the C-14 -C-15 double bond was established from a NOESY experiment.

O OR
In the new molecule 4(E)-jatrogrossidentadione acetate (7) 16 the acetoxy group (δ 2.05, 3H, s, in the 1 H NMR spectrum and δ 170.0, 22.1 in the 13 C NMR spectrum) was placed at C-6 because a comparision of the 1 H NMR spectra of this compound and of 4(E)-jatrogrossidentadione (26) showed that Me-17 (δ 1.75 for 7 and 1.48 for 26) appeared at a downfield region in 7. The 13 C NMR spectra (C-6: δ 84.4 for 7 and 73.5 for 26) also supported this.The X-ray crystallographic analysis of 7 was accomplished. 16The structure of 8 17 is similar to that of 7; the only difference is that in the former the hydroxy group at C-15 is with α-configuration while in the latter with β-configuration. 17ultifidone (9) 18 another constituent of Jatropha multifida, is structurally interesting as it possesses a six membered A ring in contrast to cyclopentane ring generally found in lathyrane-type diterpenes.The A ring contains two carbonyl groups at C-1 and C-4 and a trisubstituted double bond at C-2 -C-3 .The macrocyclic B ring was found to possess a dihydrofuran ring.The E-configuration of the double bond present in the furan ring was suggested from the 13 C NMR spectrum of the molecule.The sructure of 9 was confirmed from X-ray crystallographic analysis. 18ultifidone (9) was examined for in vitro cytotoxic activity against four different cancerous cell lines: THP-1 (human acute monocytic leukaemia), HL-60 (human promyelocytic leukaemia), A-375 (human malignant melanoma) and A-549 (human lung carcinoma) using etoposide as the positive control.The compound 9 showed significant decrease in cell viability in all the tested cell lines in a concentration dependent manner. 18-O-Acetyljapodragrone (10), 14 a new constituent of Jatropha multifida, contains a tetrahydrofuran moiety in the ring B but it contains no cyclopropane ring.The acetyl group (δ 2.09, 3H, s in the 1 H NMR spectrum) at C-15 is in the α-configuration.The compound is the acetyl derivative of japodagrone (29) 14 which contains a hydroxy group at C-15.The 13 C NMR spectrum suggested the placement of this acetoxy group at C-15 as this carbon (δ 88.9) showed a downfield shift compared to the corresponding carbon (δ 82.4) of japodagrone.In the 1 H NMR spectrum H-1 showed a downfield shift (δ 7.30 in 10 and 6.80 in 29).2D NMR spectra also supported the structure of 10. 14 In other new constituent, a lathyrane type diterpene 11, 14 a trisubstituted double bond is situated at C-5-C-6 position instead of C-4-C-5 position and the other at C-1-C-2.The structure is related to that of 4Ejatrogrossidentadione (26). 17The compound contains a hydroxy group at C-15.From the NOESY experiment H-4 and OH-15 was suggested to be α and β oriented respectively. 14ltifidanol (12) 13 and multifidenol (13) 13 two new diterpenes of Jatropha multifida, possess cyclopentanol A ring (instead of cyclopentanone).The structures of these two compounds are closely related.In multifidanol (12) C-1, C-2-bond is saturated while this bond is unsaturated in multifidenol (13).Both the compounds contain other two hydroxyls and a keto group.On the basis of 1D and 2D NMR spectral correlations these two hydroxyls were placed at C-6 and C-15 positions while the keto group at C-14.All the three -OH groups were found to have α -configuration. 13ultifidanol (12) and multifidenol (13) were evaluated for cyctotoxic activity (in vitro) against different cancerous cell lines using doxorubicin as the positive control.Both the compounds exhibited promising activity against some of these cell lines.It was observed that the absence of the double bond at C-1, C-2 enhanced the activity of multifidanol (12) against A-549 and MCF-7cell lines but the presence of a double bond at this position increased the activity of multifidenol (13) against Neuro-2aHeLa and MDA-231 cell lines. 13he antibacterial activity of 12 and 13 was also tested against some bacterial organisms using neomycin as the positive control.Compound 12 showed impressive activity against Bacillus subtilis and Escherichia coli but compound 13 showed selective activity against Staphylococcus aureus. 13om Jatropha multifida another novel diterpene, multidione ( 14) 19 was isolated.The structure of the compound was settled from 1D and 2D NMR spectra.The spectra indicated that multidione possess a 2,4disubstituted phenolic moiety containing a methyl group at C-2 and a long side chain at C-4.The side chain was found to have four methyl groups, two carbonyl groups and a cyclopropane ring which were properly placed in the molecule by spectral correlations.The relative stereochemistry of the side chain was established from NOESY experiment and interproton or heteronuclear coupling constants.Ring B of the lathyrane skeleton has been cleaved to produce the side chain.Multidione ( 14) has possibly been derived biogenetically from a related diterpene. 19long with the new diterpenes several known diterpenes were also isolated for the first time from Jatropha multifida.These compounds included jatropholones A (23) 13,14 and B (24), 14 4-(E)-jatrogrossidentadione (26), 14,16,17 15-epi-4-(E)-jatrogrossidentadione ( 27), 14,[16][17][18] jatrophone (28), [16][17][18] japodagrone (29) 13,14 and jatrothrin (30). 13jatrophone (28) was reported earlier to exhibit promising antileukemic activity against P-388 lymphocytic leukemia and also cytotoxicity against KB cell culture. 22

Lignans
From Jatropha gossypifolia several lignans have been isolated (Table 2; Figure 1).These lignans are dibenzyl and arylnaphthalide types.Some of the molecules contain butyrolactone and butyrolactol moieties.Both trans (E)-and cis (Z)-configurations of the olefinic double bonds present in the molecules are observed.They also possess (S) as well as (R) stereochemistry.Jatrophan (31) is the first lignan reported from the genus Jatropha. 23,24Its structure was deduced from its spectral data and X-ray crystallographic analysis.The compound contains a trans (E)-olefinic double bond.On the other hand, gadain (32), another new lignan constituent of the same plant contains a cis (Z)-olefinic double bond. 24In the 1 H NMR spectra the olefinic proton of jatrophan (31) appeared at δ 7.54 (1H, d, J =1.6 Hz) while that of gadain (32) at δ 6.59 (1H, d, J =1.6 Hz).An interesting cis-trans isomerisation of gadain (32) was observed when its NMR spectra in CDCl 3 were studied.This transformation was assumed to be catalyzed by the usual trace of HCl present in CDCl 3. In fact, when 32 was kept in HCl (1.0 M) for 72 h it changed to its trans (E) isomer.The structure as well as stereochemistry [(S)] of gadain was confirmed from its synthesis from jatrophan (31) (Scheme 1).The latter was demethylated with BBr 3 to produce a dihydoxy derivative which on treatement with bromochloromethane afforded the trans (E)-isomer (37) of gadain (32).This isomeric compound yielded gadain (32) by UV irradiation. 24Isogadain (37) was also isolated later from Jatropha gossypifolia. 29Various chemical conversions and synthetic studies of jatrophan (31) and gadain (32) were performed (Scheme 1). 24,35,36On treatment with DDQ the former yielded retrochinensin (43) while the latter produced justicidin E (44), both the products are naturally occurring aryl naphthalide lignans.Another natural aryl naphthalide lignan, justicidin B (45) was also prepared from jatrophan (31) by NBS treatment. 24 total synthesis of jatrophan in racemic form started with the Stobbe condensation of piperonal (46) with dimethyl succinate followed by methylation to yield a diester (48) (Scheme 2).A second Stobbe condensation of this diester with veratraldehyde and subsequent Bouveault-Blanc reduction generated (±)-jatrophan. 35nother butyrolactone lignan, gossypifan (36), structurally related to jatrophan (31), was isolated from the aerial parts of Jatropha gossypifolia.The compound also possesses an olefinic trans (E)-double bond and (S)stereoconfiguration.Two aromatic rings with their functionalities have been interchanged in 36. 28wo dibenzyl lignans (without having butyrolactone moiety), prasanthaline (33) 25 and gossypiline (38) 30 are also the constituents of Jatropha gossypifolia.The occurrence of dihydroprasanthaline (34) in the plant was also reported. 26The structures of the compounds, 33 and 38 (established from the spectral data) are related.The difference is that prasanthaline (33) contains one methylenedioxy and two methoxy groups while gossypiline (38) contains two methylenedioxy groups.However, the stereochemistry of these two compounds are opposite; compound 33 bears (R)-configuration but 38 (S)-configuration.Scheme 1.Chemical conversions of jatrophan (31) and gadain (32). 24,35,36asanthaline (33) 25 and gossypiline (38) 30 were prepared from the natural lignans, suchilactone (50) and isogadain (37) respectively (Scheme 3) by reduction with lithium aluminium hydride followed by acetylation with acetic anhydride and pyridine.Thus the structures (along with the stereochemistry) of the two compounds were confirmed.© ARKAT USA, Inc Scheme 3. Preparation of prasanthaline (33) and gossypiline (38). 25,30emical investigation on the same plant, Jatropha gossypifolia, resulted in the isolation of two aryl naphthalide lignans, 2,3-bis(hydroxymethyl)-6,7-methylenedioxy-1-(3´,4´-dimethoxyphenyl)naphthalene ( 35) 27 and 4´-O-demethyl retrochinensin (41). 33In the 1 H NMR spectrum of 35 two hydroxyls (δ 3.05) appeared as a broad signal.The methylene protons at C-2 and C-3 resonated at δ 4.55 (2H, s) and 4.85 (2H, s) indicating their association with the hydroxyls as -CH 2 OH.The 13 C NMR spectral data (δ 60.71, C-2 ; 65.25, C-3) also supported this.The substitution pattern of the molecule 35 was confirmed from the analysis of its mass spectrum.The spectral data of 41 revealed that its sructure is similar to that of retrochinensin (43) but it contains a hydroxyl group at C-4' instead of a methoxy group present in the latter.The compound 35 was synthesised from jatrophan (31) (Scheme 4) by oxidative cyclization with DDQ followed by reduction with lithium aluminium hydride.o novel lignans, jatrodien (39) 31 and gossypidien (40) 32 each containing two olefinic trans (E)-double bonds, were isolated from Jatropha gossypifolia.The 1 H NMR spectrum of 39 showed the appearance of two deshielded olefinic protons at C-7 and C-7' at δ 7.76 and 7.72 (1H each, s).On the other hand, in the 1 H NMR spectrum of 40 the two deshielded olefinic protons appeared at δ 7.82 (s 2H, H-7 and H-7') The compound 40 is symmetrical and the 13 C NMR spectrum revealed the signals for only 11 carbons present in the half of the molecule.The total syntheses of 39 and 40 involving Stobbe condensation were also accomplished.These two compounds represent the intermediates in Haworth's biosynthetic scheme for the formation of lignans. 31,32atrolactol (42) 34 is an another new lignan of Jatropha gossypifolia.The 1 H and 13 C NMR spectral data suggested that the compound is structurally related to jatrophan (31).They possess two 1,3,4-trisubstituted aromatic rings, a trisubstituted olefinic double bond, one methylenedioxy and two methoxy groups.The difference is that jatrophan contains a lactone ring while jatrolactol a lactol group at C-9.The H-9 in jatrolactol (42) appeared at δ 5.32 in its 1 H NMR spectrum and C-9 at δ 108.4 in the 13 C NMR spectrum.The βconfiguration of the hydroxyl at C-9 was settled from its 2D NMR spectra.The compound on oxidation with Fétizon's reagent yielded jatrophan (31) and thus its structure was confirmed. 34
Structures of the coumarinolignans of Jatropha gossypifolia and of cleomiscosin A diacetate (59).
Venkatasin (52) is the first acetylated coumarinolignan obtained from nature. 37The structure of the compound was established from its 1 H and 13 C NMR spectral data.The compound contains a hydroxyl group (phenolic) at C-4´and an acetoxy group at C-9´.A direct comparison of the 1 H NMR spectrum of venkatasin (52) with that of authentic cleomiscosin A (58) showed that the signals for H 2 -9' of 52 resonated at δ 4.42 -4.28 (m) but the signals for these two protons of 58 appeared at δ 3.81 (1H, dd, J =13.0, 2.0 Hz and 3.56 ( 1H, dd, J =13.0, 3.0 Hz).This observation suggested to place the acetoxy group (δ2.04, 3H, s in the 1 H NMR spectrum and δ 20.62 in the 13 C NMR spectrum) at C-9' of venkatasin (52).The acetylated product of 52 was found to be identical to cleomiscosin A diacetate (59).The regioselective acetylation of the hydroxyl group (alcoholic) at C-9´ of cleomiscosin A (58) using NaHSO 4 •SiO 2 catalyst 42 afforded venkatasin (52), thus confirming the structure of the latter as 9´-O-acetyl cleomiscosin A. Venkatasin (52) was also directly synthesised 43  Scheme 5. Interconversions of coumarinolignans of Jatropha species. 42,43ther two cleomiscosin A derivatives, jatrorins A (53) and B (54) were isolated from Jatropha gossypifolia.Jatrorin A (53) is 6-O-demethyl derivative while jatrorin B (54) is 6-O-demethyl-4´-O-methyl derivative of cleomiscosin A (58).The structures of these two compounds, 53 and 54 were clearly derived from detailed analysis of their 1 H and 13 C NMR spectra. 38rom the whole plant of Jatropha gossypifolia two new propacin analogues, jatrocins A (55) and B (56) were also isolated.Jatrocin A was characterized as 6-O-demethylpropacin and jatrocin B as 5´-methoxypropacin. 397][18] The compound is known to possess cytotoxic and anti-HIV properties.It also exhibits immunomodulatory and liver protective activities. 44

Other constituents
Besides diterpenes, lignans and coumarinolignans some other constituents were also isolated from the investigated Jatropha species.
Three deoxypreussomerins, palmarumycins JC 1 (60), JC 2 (61) and CP 1 (62), were isolated from a collection of the stem of Jatropha curcas (Table 1). 45The first two compounds, 60 and 61 are new deoxypreussomerins while 62 is a known compound.All these compounds were characterized from spectral evidence.The 1 H NMR spectrum of JC 1 showed the signals at δ 3.56 (1H, dd, J = 4.5, 3.0 Hz), 3.64 (1H, d, J = 4.5 Hz) and 5.47 (1H, d, J = 3.0 Hz) corresponding to three oxymethine protons.The 13 C NMR spectrum also revealed the signals of three oxymethine carbons (δ 53.3, 50.5 and 60.4).The 1 H-1 H COSY and HMBC experiments suggested the presence of a hydroxy group at C-1 and an epoxide ring at C-2, C-3.The βconfigurations of HO-1, H-2 and H-3 were settled by comparision of the spectral data and optical property of JC-1 with those of related compounds.The X-ray crystallographic analysis of 60 was also performed. 45The hydroxyl group at C-1 of JC-1 was oxidised to a keto group in JC-2 (61).The 1 H NMR spectrum of JC-2 showed the signal of a chelated -OH group (δ 12.27, 1H, brs).The spectrum also indicated that C-2 was not oxygenated but C-3 is oxygenated.A hydroxyl group was placed at this position.The β-configuration of this hydroxyl group was suggested from the observation that JC-2 showed almost opposite optical rotation to that of the compound of similar system having α-OH group at C-3.The isolation of these compounds, 60, 61 and 62 in reasonable quantities indicated their presence as constituents and excluded their occurrence in any endophytic fungus present in the plant.These compounds were examined to possess impressive antibacterial activity against the organism Staphylococcus aureus.Their activity was comparable to that of the standard compound, penicillin G. 45 From the roots of Jatropha gossypifolia the phenolic compounds, tetradecyl-(E)-ferulate (63), ferulic acid (64) and fraxetin (65) were obtained (Figure 3). 46The first compound (63) is a new natural product and the other two compounds (64 and 65) were reported for the first time from the species.][15] A new minor imidazole derivative, 4-butyl-2-chloro-5-formyl-1H-imidazole (66) was obtained from Jatropha curcas. 47The molecule is structurally impressive as it is associated with various functionalities.Three functionalities (-Cl, -Bu and -CHO) were placed at C-2, C-4 and C-5 respectively on the basis of its 1D and 2D NMR spectral data.The compound was found to possess significant antibacterial activity.This imidazole derivative was also isolated from Jatropha multifida. 14atropha curcas also yielded 2-methoxyanthraquinone (67), scopoletin (68), 3-O-(Z)-coumaroyl oleanolic acid (69) and tomentin (70), 15 and Jatropha multifida yielded pictolinarigenin (71) 16,17 and fraxidin (72).13

Conclusion
In the present article we have described our phytochemical investigation on three Jatropha species viz., Jatropha gossypifolia.Jatropha curcas and Jatropha multifida which resulted in the isolation of a large number of their chemical constituents.These constituents are of different types: diterpenes, lignans, coumarinolignans, flavones, coumarins, simple phenolics etc.Some of the compounds possess novel interesting molecular structures.Various chemical transformations and synthetic studies on several constituents were performed.Antibacterial and anticancer activities of some of the isolates are impressive.

Figure 3 .
Figure 3. Structures of miscellaneous constituents of Jatropha species.

Table 2 .
Lignans isolated from Jatropha gossypifoliaa a All the lignans are new compounds.
from cleomiscosin A diacetate (59) by applying NH 4 OAc as the catalyst for selective deprotection of aromatic acetate group (Scheme 5).