Patent Application: US-31460394-A

Abstract:
withajardins a - d are new class of withanolides isolated from the leaves of deprea orinocensis and characterized by having a bicyclic c - 17β system and hydroxyl group at c - 14 and c - 17 . the bicyclic system consists of a lactone and a homocyclic ring with six carbon atoms unlike the acnistins which have a five - membered homocyclic ring . the two classes of compounds were differentiated and their structures were identified by a series of hmqc , hmbc and finally x - ray diffraction experiments . withajardins and acnistins would seem to evolve along quite dissimilar biosynthetic pathways although both types of compound show a marked immunosupressive effect on in vitro cultures of human lymphocytes .

Description:
the present molecules , which have an α - β unsaturated system in the ring a , are particularly interesting due to their application as immunomodulatory agents as demonstrated by the in vitro expermients described below . these substance could have applications as tools to study the cellular and molecular phenomena that occur during the immune response . pharmacologically they could be used as immunomodulators of the immune response . at low concentrations they stimulate mitogen or antigen - induced lymphocyte proliferation whereas at higher concentrations they exhibit inhibitory activity , suggesting that a low doses they could be used as immunopotentiator in immunodeficiencies such as aids , and at high doses they could be used in immunosupression for organ transplantation or autoimmune diseases . the withajardins are a group of steroid lactones with a withanolide skeleton which have a bicyclic 17 - α side chain . they have been isolated from the leaves of deprea orinocensis and their structure was established by 2d - nmr , hreims and x - ray diffraction analysis . withajardin a was the major product and exhibited interesting immunomodulatory activity in in vitro assays on human lymphocytes . a dose of 0 . 01 μg / ml potentiated 3 h !- thymidine uptake , an effect confined almost exclusively to t lymphocytes . these results were further corroborated by treatment with ppd and one - way expenmenus . after treatment with withajardin a cellular viability was still close to the 100 % level , thus ruling out possible cytoxicity . withajardin a , b , c , and d have similar immunosuppressive activity at concentrations of approximately 1 μg / ml or greater . the withanolides are a group of steroid lactones which have been isolated from several genera of solanaceae 1 . 2 and recently have also been found in a soft coral 3 . several of these substances have displayed various types of biological activity , such as cytotoxic 4 , anticancer 5 , immunosuppresive 6 , anti - inflammatory 7 , and hepatoprotective 8 properties . the withanolides are characterized chemically by a lactone chain at c - 17 and different oxidations , mainly in the a , b and e rings 2 . however , biogenetic transformations can produce very modified compounds such as the physalins 9 , trechonolide 10 , prejaborol 11 , withamelins 12 and acnistins 13 . in the course of a biological screening we studied the species dunalia solanaceae 14 , finding acnistins a and e . new withajardins a , b , c and d , with a new skeleton related to that of the acnistins , were found from the leaves of deprea orinocensis . withajardins can have limited structural variability in the a / b ring system without loss of immunomodulatory activity . in particular , the substituent r at c4 can be hydroxy , lower alkoxy , lower acyloxy or lower alkyl . in general , lower alkoxy will include straight or branched c 1 - 6 - alkoxy , lower acyloxy will include c 1 - 6 - acyloxy and lower alkyl will include c 1 - 6 - alkyl , with the smallest groups being preferred over the larger . preferred compounds will be those wherein r is hydroxy or acetyl . other preferred compounds include those wherein r is methyl or methoxy or ethoxy . esterification of compounds with oh at c4 proceeds in the cold and is selective for the equatorial 4β - oh . typically , acetic anhydride , with a catalytic amount of pyridine , in tetrahydrofuran , at about 0 ° c . is effective . the structures of acnistins a and e have recently been studies 14 by 2d - nmr . starting the analysis of the hmbc spectrum at the lactone carbonyl , easily identified by its shifts at 178 . 92 and 179 . 09 ppm , respectively . in this way , sequentially following the two and three - bond correlations , the chemical shifts of the bicyclic side - chain could be unambiguously assigned as could the rest of the steroid system . furthermore , the structure of acnistin d was confirmed by x - ray diffraction analysis , enabling its absolute configuration to be established . the application of these bidimensional techniques to acnistin e ( with a structure earlier determined by x - ray diffraction 13 ) and to 3 - methoxy - acnistin e provided conclusive proof of the validity of the assignments . the chemical shifts of carbon atoms of the three acnistins were then compared with those found for withajardins a - d ( fig2 ) and from these data it would appear that a homocyclic five - membered side - chain ring was present as in the acnistins . nonetheless , some discrepancies were observed for the shifts of the atoms c - 20 , c - 22 , c - 24 , c - 25 , c - 27 and c - 28 , in particular ( table 1 ). hmbc 15 and hmqc 16 experiments on the four withajardins enabled their structures to be established conclusively and an x - ray diffraction analysis of the withajardin c established its absolute configuration . it was thus ascertained that the withajardins have a new carbon skeleton with a homocyclic six membered side chain ring . withajardin c ( 4 ), c 30 h 40 o 9 ( table ii ) has an α , β - unsaturated carbonyl in which the olefin protons form part of an amx system as can be deduced from the 1 h -- 1 h cosy spectrum since a doublet is seen at 7 . 00 ppm ( j = 6 . 0 hz and 9 . 8 hz ), a doublet at 6 . 25 ( j = 9 . 8 hz ) and another one at 4 . 26 ppm ( j = 6 . 0 hz ); the presence of an aceinte and an epoxide were deduced from the singlet at d 2 . 06 ( 3h ) and d 3 . 24 ( 1h ) ( table iii ). the dept spectrum of withajardin c ( 4 ) table i ) indicates three carbonyls at 201 . 10 , 178 . 37 and 170 . 1 ppm ( the latter corresponding to the acetate ), five oxygenated carbons , two of which are attributed to an epoxide ( 60 . 91 ) s and ( 60 . 73 ) d , five methyls ( one the acetate methyl ) and an oxygenated methine among other signals . hmqc established that the oxygenated methine corresponds to c - 22 which is shown in 1 h nmr as a doublet shifted to b 4 . 62 ( j = 1 . 7hz ) and in 13 c nmr as a doublet at 77 . 76 17 , 18 . using a combination of hmqc and hmbc ( table iv ) and starting from the lactone carbonyl at 178 . 37 three bond correlations could be observed with hj - 22 , h - 27 and h - 21ax ; the latter is also coupled with c - 17 and an oxygenated quaternary carbon at 70 . 37 ppm which was assigned to c - 24 since this is also connected by two and three bonds to h - 23ax and h - 28 . the latter correlation is very significant as it places a hydroxyl γ to the lactone carbonyl and so the homocyclic system closure is the side chain could be established between c - 21 and c - 25 and not between c - 21 adn c - 24 as in the acnistins . this fact was confirmed beyond possibility of doubt by the two and three bond correlations observed for c - 20 , c - 24 , c - 25 and those for h - 27 and h - 28 . these observations also hold good for the side chain of the withajardins a , b and d ( table iv ). the hydroxylations sites were also easy to deduce by hmbc ; one was established at c - 14 by correlation with me - 18 and another at c - 17 by connection with the c - 18 , c - 20 and c - 21 protons . the acetate group was positioned at c - 4 given the correlations of the acetate methyl with c - 4 and those of the h - 4 with the carbonyl at c - 4 . similarly , the epoxide could be sited between c - 5 , c - 6 . in the same way , connectivities between the other atoms in the molecule could be established ( table iv ). the relative configurations was deduced from the analysis of the roesy spectrum of withajardins a - d in which effects between hj - 4 and h - 6 , h - 8 and h - 19 and h - 23ax and h - 18 among others could be appreciated ( table v ). the absolute configuration of withajardin c was established by means of x - ray diffraction ; a perspective view of the structure is shown in fig3 . the central ring system is formed by the fusion of one five - membered and three six - membered rings characterized by two trans - connected rings b / c and c / d and the quasi cis connected a / b rings , four axial substitutes , two methyl groups at c - 10 and c - 13 ( β - position ) and two hydroxy substituents at c - 14 and c - 17 ( α - oriented ); the protonated carbons c - 8 and c - 9 are α and β positioned , respectively . rings a and b adopted a half - chair conformation and the c ring is chair conformed . the five - membered d - ring adopts a conformation between half - chair and envelope . this moity is linked to another ring system with three six - membered rings ( c - 20 , c - 21 , c - 25 , c - 26 , o - 5 , c - 22 ; c - 20 , c - 21 , c - 25 , c - 24 , c - 23 , c - 22 , c - 23 , c - 24 , c - 25 , c - 26 , o - 5 ) all of which adopt a boat conformation ; the protonated c - 20 is β oriented and the molecule has several asymmetric centres with the following configurations : c - 4 s , c - 8 r , c - 9 s , c - 10 r , c - 13 r , c - 14 r , c - 17 s , c - 22 r , c - 24 r , c - 25 r . the molecules are linked together in the crystal by hyrimgen hands between hydroxy groups and the water molecule . all hydroxy h atoms act as donors in one intermolecular h - bond and two intramolecular h - bonds . on the other hand , the water molecule acts as both donor and acceptor . the geometry of the h - bonds is described below : the structure was solved by direct methods sir88 19 and refined by full - matrix least - square methods with anisotropic thermal parameters for non - h atoms . all h - atoms were found in a fourier difference map and were includes as isotropic contributors and refined . a weighting scheme was selected to prevent dependence in wδ 2 f & gt ; vs & lt ;| fu |& gt ; and & lt ; sinθ / λ . after several cycles of weighted mixed refinement , the final r and rw values were 3 . 5 and 4 . 0 , respectively . the final difference synthesis showed the residual electron density as no greater than 25 e å - 3 ; number of variables 525 , ratio of freedom 4 . 98 , degree of freedom 2094 . the absolute configuration was determined with bijvoet differences with δfo & gt ; 0 . 15 add with the least experimental error fo & gt ; 10e ( fo ). 37 more relevant bijvoet pairs from the raw data ( hkl and - h - k - l alternately measured intensities with no extra care on recentering , scan speed , etc ) gave the following results : r1 = σ δfo - δfe !/ n = 0 . 316 and r2 = σ δl - δlc !/ σ δlo != 0 . 879 for the right enantiomer model , the r1 and r2 values for the wrong enantiomer were 0 . 476 and 1 . 273 , respectively , thus the absolute configuration was established for the molecule . withajardin b ( 3 ) c 28 h 38 o 8 , is a very similar compound to withajardin c , which can be deduced from the similarity of their 1 ii and 13 c nmr spectra ( tables i and iii ), with only a few differences in the chemical shift , chiefly of h - 4 and c - 2 , c - 5 . moreover the 1 h and 13 c nmr spectra of withajardin b do not have the characteristic signals of the acetate group which in withajardin c was placed at c - 4 by hmbc ; therefore , withajardin b would seem to be 4 - hydroxy - 4 - deacetoxy - withajardin c . this is supported by the correlation of 4 - oh with c - 3 , c - 4 and c - 5 ( table iv ). to check out this theory , withajardin b was cold - acetylated and formed a monoacetate which proved identical to withajardin c in its ir and nmr spectra . withajardins a and d ( 1 and 2 ) seem also to be a very similar pair of compounds and are distinguished from the other two withajardins only by the presence of a double bond instead of an epoxide . sup . 17 , the rest of the molecule being the same . the position of the double bond between c - 5 , c - 6 was established by means of hmbc since two and three - bond correlations of c - 5 with h - 3 , h - 4 , h - 8 and h - 19 could be discerned ( table iv ). the β configuration of the substituent at c - 4 in both withanolides is similar to that of withajardins b and c and could be deduced by the chemical shift of me - 19 to a relatively low field 17 , 18 and by the presence of a roesy effect between h - 4 and h - 6 . the roesy effect between me - 27 and me - 28 was no clearly visible due to the fact that both groups have a very similar chemical shift . withajardin a is a withanolide with a new nucleus of natural products , isolated from leaves and stems of deprea orinocensis h . b . k . its structure was defined by the displacements of the carbonyl group of the lactone in 13 c nmr , found at 177 . 21 ppm which is a field lower than usual for withanolides . withajardin a : white powder ; hrms m / z ( rel int %) 448 . 2238 ( c 28 h 32 o 5 ) ( 25 ), 450 . 2574 ( m + - 2h 2 o ) ( 25 ), 432 . 2396 , ( 35 ), 417 . 2125 ( 15 ), 280 . 1439 ( c 19 h 20 o 2 ) ( 10 ), 238 . 1316 ( c 17 h 18 o ) ( 90 ), 125 . 1468 ( c 7 h 9 o 2 ) ( 60 ), 55 . 00993 ( c 4 h 7 ) ( 100 ). in vitro studies have shown that withajardin a has a dual effect on the proliferation of human peripheral blood lymphocytes induced by mitogens ( pha , con a or pwm ), alloantigens or microbial antigens ( purified protein derivative , ppd ). at low doses ( 0 . 01 μg / ml ) there is an increased in 3 h - thymidine incorporation , while at high concentrations ( 10 μg / ml ) the proliferative response is completely inhibited . mononuclear cells obtained from healthy individuals were cultured for 72 h with either 1 μg / ml of pha , 5 μg / ml of cona or 10 μg / ml of pwm . cultures of lymphocytes obtained from tuberculin positive healthy subjects were stimulated with 10 μ / ml of ppd and incubated for 120 h . six to eight hours before harvesting the cultures were pulsed with 0 . 5 μci / well of 3 h - tdr . kinetics studies showed that withajardin a added during the first 24 h of culture is able to induce either stimulation or suppression of lymphocyte proliferation depending on the concentration used . removal of withajardin a by washing and replacement with medium suggest that if withajardin a is present for 24 h or more its effects are irreversible . cell viability , as determined by tryptan blue exclusion , was always over 90 % at 72 h of culture in the presence of withajardin a up to 10 μg / ml . withajardin a was able to inhibit il - 2 and il - 4 production by pha stimulated lymphocytes in a dose dependant way . the above discussion established that withajardins have a bicyclic lactone side chain with a six - membered homocycle which is different from the bicyclic system of the acnistins which consists of a five - membered homocycle , although the class of carbon atoms on 13 c nmr is exactly the same for both types of substances . there are other differences , too , between withajardins and acnistins : the first have an extra hydroxyl at c - 14 and the configuration of the side chain is 17β . the fact that acnistins have 17α configuration is abnormal in the withanolide field but has been accounted for by a biogenetic mechanism of dehydration - rehydroxylation 22 similar to that postulated 23 for the formation of 14 α - oh . the compounds obtained from deprea orinocensis and dunalia solanaceae suggest the existence of very specific enzymatic biogenetic mechanisms since , starting from a common precursor , only one type of substances forms in each species , which has been demostrated in our case by means of intensive phytochemical screening of both species . in no instance have withajardins and acnistins been encountered together . the biogenesis of all of these compounds may be explained as owing to a mechanism which requires prior oxidation of the me - 21 ( very possible in glycoside form ) followed by an - oh electrophilic attack on c - 24 or c - 25 depending on the species , and then a shift of the duble bond on the oxidized me - 21 . this mechanism follows a different path from that for the formation of the withametelins 2 in which an electrophilic attack on 24 , 25 - epoxy - withanolide takes place from the 21 - ch 2 oh . paradoxically , and in spite of a withanolide which does have 21 - ch 2 oh having been obtained from datura metel 12 ( whence the term withametelins ), no other type of substance with a bicyclic system equal to that of the withajardins or acnistins has been reported to date from this species . physalin b , a type of substance obtained from other solanaceae species such as physalis or witheringia 2 has also been found in d . orinocensis thus indicating an extra oxidative mechanism in this species at the me - 18 level . despite the different positions of the hydroxy groups in relation to the lactone carbonyl group in acnistins and withajardins , the chemical shift in this latter is very similar in 13 c nmr in both types of compounds ( table i ), although it is different from that of normal withanolides in which it appears at 166 ppm . in the case of acnistins this shift could be accounted for neighbouring β - hydroxylation . however in the withajardins there would seem to be other factors which notably influence the shift of the lactone carbonyl since the hydroxyl at c - 24 in theory should exercise a effect on it , shifting it further upfield than normal . such an effect may be cancelled out by the formation of h - bonding between the hydroxyls of c - 24 , c - 17 , and c - 14 the presence of which has been firmly established , as seen above , by x - ray diffraction of withajardin c ; in the same way , the generation of a bicyclic system with two six - membered rings apparently reduces the tension on the carbonyl which does not occur in the case of the acnistins . the chemical shift of me - 28 , further upfield than expected for a hydroxy geminal group can be accounted for by it being situated in the unshielded zone of the lactone carbonyl . studies both with acnistins and with withajardins are under way to determine their immunomodulatory activity . 1 h nmr spectra were recorded on a bruker am400 spectrometer at 400 mhz and 13 c nmr at 100 mhz ; chemical shift are given in parts per million ( δ ) in relation to the solvent peaks . ir spectra were taken on a perkin - elmer model 1600 spectrometer . low - resolution mass spectra were run on a hewlett - packard 5995 and hrms on a vg micromass zab - 2f , e1 , 70 ev for withajardins a , b and d and 15 ev for withajardin c . column chromatography was carried out on silica gel ( merck 0 . 2 - 0 . 5 nun ) and tlc on silica gel plates , 0 . 25 mm , with visualization on oleum , heated to 120 ° c . the nmr data are given in tables i , iii , iv and v . extraction and isolation procedures . deprea orinocensis leaves ( 1 . 2 kg ) collected in el jardin , colombia in july 1991 were extractred by soxhlet with hexane and then meoh . the meoh extract was evaporated almost to dryness , diluted with water and re - extracted with hexane ( 4 × 11 ) and etoac ( 6 l ). the etoac extracts were chromatographed on silica gel with hexane - etoac ( 4 : 1 - 1 : 8 ) mixtures and 180 fractions , of 100 ml each were collected . withajardin d ( 60 mg ) and withajardin c ( 180 mg ) were obtained from the less polar fractions and withajardins a ( 1 . 2 g ) and b ( 800 mg ) appeared after repeated column chromatography and / or crystallization on meoh . the withajardins were detected by tlc due to the colour developed ( blue or red ) when the plates were sprayed and heated . withajardin a ( 1 ): white powder ; hrms m / z ( rel int . %) 448 . 2238 ( c 28 h 32 o 5 ) ( 25 ), 450 . 2574 ( m + - 2h 2 o ) ( 25 ), 432 . 2396 ( 35 ), 417 . 2125 ( 15 ), 280 . 1439 ( c 19 h 20 o 2 ) ( 10 ), 238 . 1316 ( c 17 h 18 o ) ( 90 ), 125 . 1468 ( c 7 h 9 o 2 ) ( 60 ), 55 . 00993 ( c 4 h 7 ) ( 100 ). withajardin b ( 3 ): white powder ; lrms m / z ( rel int . %) 487 ( m + - me ) ( 7 ), 450 ( 15 ), 432 ( 60 ), 399 ( 15 ), 249 ( 40 ), 238 ( 37 ), 171 ( 65 ), 125 ( 30 ), 107 ( 90 ), 91 ( 100 ), 55 ( 80 ). withajardin c ( 4 ): crystallzied from etoh ; hrms m / z ( rel int . %) 545 . 2747 ( m + + h + , c 30 h 41 o 9 ) ( 1 ), 484 . 2450 ( c 28 h 36 o 7 ) ( 1 ), 254 , 1263 ( c 17 h 18 o 2 ) ( 42 ), 238 . 1247 ( c 13 h 18 o 4 ) ( 29 ), 173 . 0982 ( c 12 h 13 o ) ( 39 ), 124 . 0545 ( c 7 h 8 o 2 ) ( 93 ), 55 . 0995 ( c 4 h 7 ) ( 100 ). crystal data . formula c 30 h 39 o 9 . h 2 o , molecular weight 545 , 648 , calculated density 1 . 2971 g . cm - 1 , μ = 7 . 428 cm - 1 , z = 4 . the compound crystallizes in the space group p212121 . a suitable crystal of 0 . 35 × 0 . 30 × 0 . 20 mm . was used for data collection . cell dimensions were determined by least - square from setting 38 reflections with 10 0 & lt ; θ & lt ; 40 0 : a = 27 . 934 ( 3 ), b = 12 . 344 , c = 8 . 1032 ( 2 ) å . the data were collected on a philips pw 1100 diffractometer with graphite monochromated cukα radiation . a total of 2753 independent reflections were measured and 2619 were considered as observed when i & gt ; 2σ ( i ), and were used for the structure determination and refinement . two reference reflections were checked every 90 treflections and they showed no intensity variation . the intensities measurements was performed up θ = 65 0 , ω / 2θ scan technique , scan speed 0 . 050 s . g - 1 , scan width 1 . 60 . with the same measurement time for both backgrounds as for the peak . the data were corrected for lorentz and polarization effects , but not for absorption . the atomic scattering factors and the anomalous dispersion corrections were taken from the literature 24 . all calculations performed on a vax 6410 and the programs from reference 20 , 21 and several local programs . withajardin d ( 2 ): white powder ; lrms m / z ( rel int . %) 450 ( m + -- hoac -- h 2 o ) ( 15 ), 432 ( 16 ), 249 ( 24 ), 238 ( 68 ), 223 ( 34 ), 197 ( 27 ), 171 ( 65 ), 125 ( 32 ), 109 ( 43 ), 69 ( 76 ), 55 ( 100 ). the present invention is further described by reference to the following , illustrative examples . withajardin a (&# 34 ; wo &# 34 ;) stimulates at low concentrations and inhibits at high concentrations the induction of lymphocyte ( pbl ) proliferation by the mitogens pha , con a and pwm peripheral blood lymphocytes were obtained and cultured by standard methods . the cells were incubated with phytohemagglutinin (&# 34 ; pha &# 34 ;), concanavalin a (&# 34 ; con a &# 34 ;) and pokeweed mitogen (&# 34 ; pwm &# 34 ;) at concentrations of 2 , 5 and 10 μg / ml , respectively , and without wo , &# 34 ; 0 ,&# 34 ; or with wo at concentrations of 0 . 001 , 0 . 01 , 0 . 1 , 1 and 10 μg / ml . proliferative activity of the cells was measured by standard assay of 3 h - thymidine is measured by incorporation . the results , expressed in counts per minute (&# 34 ; cpm &# 34 ;), are displayed in fig1 and showed that wo stimulates mitogen - induced proliferation at low concentrations and inhibits it at higher concentrations . pbls were obtained by standard methods from two patients suffering from tuberculosis infection ( i . e ., lymphocytes sensitized to the effects of ppd ). the cells were incubated without wo , &# 34 ; 0 ,&# 34 ; and with wo at concentrations of 0 . 001 , 0 . 01 , 0 . 1 , 1 and 10 μg / ml . proliferative activity of the cells was measured by standard assay of 3 h - thymidine is measured by incorporation . the results , displayed in fig2 as counts per minute (&# 34 ; cpm &# 34 ;) per cent of the control , showed that wo inhibits ppd - induced proliferation at the higher concentrations . withajardin a (&# 34 ; wo &# 34 ;) inhibits nk cell activation by pha in a dose - dependent manner nk cells were obtained and cultured by standard methods and incubated with pha in presence and absence of withajardin a (&# 34 ; wo &# 34 ;). nk cell activation was measured by standard cytotoxicity assay at a series of target ratios (&# 34 ; e : t &# 34 ; in fig3 .). the dose - dependent inhibition of pha - activation of nk cells is shown in fig3 . mixed lymphocyte culture were prepared and cultured by standard methods . the results for two samples , &# 34 ; a &# 34 ; and &# 34 ; b ,&# 34 ; are shown in fig4 . the a or b responder cells were stimulated by b or a cells , respectively , which had been pretreated with mitomycin c to block proliferation ( the pretreated cells are designated &# 34 ; bx &# 34 ; and &# 34 ; ax &# 34 ; in fig4 ). the inhibitory effect of wo is seen in the plot based on thymidine incorporation set out in fig4 . withajardin (&# 34 ; wo &# 34 ;) inhibits the generation of cytotoxic lymphocytes (&# 34 ; ctl &# 34 ;) in mixed lymphocyte culture (&# 34 ; mlc &# 34 ;) the effects of wo on ctl generation in mlc was determined as described above for pha - mediated nk cell activation in example 3 . as shown in fig5 wo inhibits ctl generation at the tested concentrations . experiments similar to those set out in examples 5 and 3 , with the results set out in fig6 showed that wo inhibits nk cell activity in a dose - dependent manner . experiments similar to those set out in examples 3 , 5 and 6 were performed with the results set out in fig7 and showed that wo inhibits ctl activity in a dose - dependent manner . withajardin (&# 34 ; wo &# 34 ;) inhibits nitrate production by macrophage cells in absence and the presence of lps (&# 34 ;+ lps &# 34 ;) macrophage cells were cultured by standard methods in the absence and presence of lps ( lipopolysaccharide ) and the absence or presence at various concentrations of wo . the results , shown in fig8 demonstrate that wo dose - dependently inhibits nitrate production in the presence and absence of lps . shows that withajardin a (&# 34 ; wo &# 34 ;) inhibits expression of the interleukin - 2 receptor (&# 34 ; il - 2r &# 34 ;) in pha - stimulated cells il - 2 - receptor expression was measured by standard methods in control and pha - stimulated cells . the cells were tested for viability , as well . the results are shown in fig9 . withajardin a (&# 34 ; wo &# 34 ;) inhibits il - 2 and il - 4 production . cells were obtained and cultured by standard methods for il - 2 and il - 4 assay and incubated in the absence and in the presence of various concentrations of wo . il - 2 and il - 4 production were measured by standard elisa using a commercially available kit . as shown in fig1 , wo inhibits the production of both interleukins in a dose - dependent manner . table 1__________________________________________________________________________ . sup . 13 c nmr of withajardins a - d ( 1 - 4 ) and acnistins a ( 5 ) and e ( 6 ) withajardins acnistinscarbon a ( 1 ). sup . a d ( 2 ). sup . b b ( 3 ). sup . a c ( 4 ). sup . b a ( 5 ). sup . b e ( 6 ). sup . b__________________________________________________________________________ 1 203 . 23 s 202 . 39 s 201 . 64 s 200 . 61 s 202 . 36 s 202 . 37 s 2 127 . 40 d 130 . 83 d 131 . 28 d 133 . 73 d 129 . 87 d 133 . 08 d 3 146 . 15 d 140 . 00 d 145 . 06 d 139 . 70 d 143 . 56 d 141 . 31 d 4 67 . 38 d 70 . 20 d 68 . 59 d 71 . 32 d 31 . 00 t 70 . 27 d 5 138 . 09 s 134 . 33 s 63 . 01 s 60 . 91 s 62 . 14 s 64 . 02 s 6 128 . 75 d 133 . 16 d 59 . 22 d 60 . 73 d 62 . 89 d 61 . 69 d 7 24 . 38 t 25 . 01 t 24 . 91 t 25 . 75 t 32 . 76 t 30 . 92 t 8 35 . 77 d 36 . 15 d 33 . 32 d 35 . 98 d 30 . 74 d 30 . 72 d 9 35 . 86 d 36 . 32 d 36 . 81 d 37 . 41 d 43 . 85 d 43 . 14 d10 48 . 78 s 49 . 36 s 47 . 07 s 47 . 62 s 46 . 94 s 47 . 85 s11 21 . 17 t 21 . 51 t 19 . 33 t 20 . 87 t 22 . 69 t 21 . 52 t12 26 . 13 t 26 . 41 t 25 . 24 t 25 . 31 t 33 . 26 t 32 . 97 t13 49 . 95 s 50 . 67 s 49 . 95 s 50 . 60 s 48 . 54 s 46 . 80 s14 85 . 21 s 86 . 80 s 85 . 09 s 86 . 90 s 50 . 42 d 50 . 71 d15 32 . 15 t 33 . 70 t 32 . 11 t 33 . 52 t 23 . 50 t 23 . 96 t16 35 . 73 t 35 . 90 t 35 . 93 t 35 . 92 t 36 . 92 t 36 . 69 t17 84 . 26 s 85 . 09 s 84 . 18 s 85 . 09 s 85 . 43 s 85 . 07 s18 18 . 92 q 19 . 91 q 18 . 51 q 19 . 60 q 14 . 41 q 14 . 00 q19 21 . 85 q 21 . 36 q 16 . 10 q 15 . 98 q 14 . 20 q 16 . 57 q20 41 . 85 d 41 . 25 d 41 . 96 d 41 . 25 d 51 . 33 d 51 . 36 d21 26 . 00 t 27 . 12 t 26 . 00 t 27 . 09 t 37 . 09 t 37 . 13 t22 76 . 54 d 77 . 00 t 76 . 46 d 77 . 10 d 84 . 05 d 84 . 03 d23 40 . 93 t 41 . 67 t 40 . 83 t 41 . 67 t 41 . 20 t 41 . 22 t24 69 . 53 s 70 . 27 s 69 . 55 s 70 . 30 s 45 . 30 s 45 . 35 s25 47 . 28 s 47 . 80 s 47 . 27 s 47 . 91 s 76 . 63 s 76 . 70 s26 177 . 21 s 178 . 29 s 177 . 15 s 178 . 80 s 179 . 09 s 178 . 92 s27 14 . 47 q 14 . 44 s 14 . 46 q 14 . 52 q 25 . 55 q 25 . 56 q28 27 . 67 q 27 . 49 s 27 . 68 q 27 . 57 q 19 . 92 q 19 . 89 qmecoo 170 . 20 s 170 . 10 smecoo 21 . 61 q 19 . 91 s__________________________________________________________________________ . sup . a : recorded at 100 . 0 mhz , dmsod6 . . sup . b : recorded at 100 . 0 mhz , cdcl . sub . 3 . multiplicities were determine by dept experiments . table ii__________________________________________________________________________mass , ir spectra and mps . of withajardins 1 - 4 highest masscompound formula ions observed . sup . a ir , cm . sup .- 1 m . p ., ° c . __________________________________________________________________________withajardin a ( 1 ) c . sub . 28 o . sub . 38 o . sub . 7 450 . 2574 3300 , 1725 , 1660 225 , descomp . 450 . 2568 ( m . sup .+ -- h . sub . 2 o ) withajardin b ( 3 ) c . sub . 28 h . sub . 38 o . sub . 8 487 ( m . sup .+ -- me ) 3400 , 1730 , 1665 228 , descomp . withajardin c ( 4 ) c . sub . 30 h . sub . 40 o . sub . 9 545 . 2747 3450 , 1740 , 1725 , 1660 268 , descomp . 545 . 2769 ( m . sup .+ + h . sup .+) withajardin d ( 2 ) c . sub . 30 h . sub . 40 o . sub . 8 450 ( m . sup .+ -- acoh -- h . sub . 2 o ) 3350 , 1735 , 1725 , 1660 182 , descomp . __________________________________________________________________________ . sup . a : observed / calculated for specific formula . i . e ., 70 ev , except fo withajardin c , 15 ev table iii__________________________________________________________________________ . sup . 1 h nmr of withajardins a - d ( 1 - 4 ) h withajardin a ( 1 ). sup . a withajardin d ( 2 ). sup . b withajardin b ( 3 ). sup . a withajardin c ( 4 ). sup . b__________________________________________________________________________2 5 . 81 ( d , 4 . 3 ) 6 . 01 ( d , 10 . 0 ) 6 . 15 ( d , 9 . 8 ) 6 . 25 ( d , 9 . 8 ) 3 6 . 81 ( dd , 4 . 6 , 10 . 0 ) 6 . 69 ( dd , 4 . 7 , 10 . 0 ) 7 . 07 ( dd , 6 . 3 , 9 . 8 ) 7 . 00 ( dd , 6 . 0 , 9 . 8 ) 4 4 . 55 ( t , 4 . 5 ) 5 . 76 ( d , 4 . 7 ) 3 . 55 ( dd , 4 . 3 , 6 . 1 ) 4 . 62 ( d , 6 . 0 ) 6 6 . 82 ( d , 4 . 2 ) 6 . 09 ( d , 2 . 8 ) 3 . 24 ( brs , 4 . 5 ) 3 . 30 ( brs , 5 . 0 ) 7 - ax 2 . 09 ( dd , 3 . 8 , 18 . 6 ) 1 . 95 ( m ) 1 . 98 ( dt , 4 . 7 , 17 . 0 ) 2 . 5 ( d , 11 . 8 ) 7 - eq 1 . 21 ( d , 14 . 29 1 . 16 ( d , 13 . 6 ) 1 . 25 ( d , 13 . 7 ) 8 1 . 75 ( m ) 1 . 82 ( m ) 1 . 67 ( m ) 1 . 80 ( m ) 9 1 . 50 ( m ) 1 . 42 ( m ) 1 . 40 ( m ) 1 . 57 ( m ) 11 - ax 1 . 89 ( m ) 1 . 90 ( m ) 1 . 90 ( m ) 11 - eq1 . 56 ( dd , 4 . 7 , 10 . 2 ) 1 . 54 ( m ) 1 . 64 ( dd , 4 . 5 , 11 . 0 ) 12 - ax1 . 81 ( m ) 1 . 78 ( m ) 1 . 80 ( m ) 1 . 90 ( m ) 12 - eq1 . 60 ( m ) 1 . 61 ( m ) 1 . 61 ( m ) 1 . 62 ( m ) 15 - β1 . 78 ( m ) 1 . 75 ( m ) 1 . 76 ( m ) 1 . 74 ( m ) 15 - α1 . 47 ( dd , 4 . 2 , 11 . 0 ) 1 . 46 ( m ) 1 . 45 ( m ) 1 . 47 ( dd , 4 . 3 , 13 . 0 ) 16 - β1 . 99 ( dq , 4 . 2 , 11 . 5 , 16 . 3 ) 1 . 89 ( m ) 2 . 15 ( m ) 2 . 15 ( m ) 16 - α1 . 77 ( m ) 2 . 05 ( m ) 1 . 82 ( m ) 18 0 . 84 ( s ) 0 . 89 ( s ) 0 . 79 ( s ) 0 . 86 ( s ) 19 1 . 33 ( s ) 1 . 39 ( s ) 1 . 30 ( s ) 1 . 41 ( s ) 20 2 . 24 ( m ) 2 . 25 ( m ) 2 . 28 ( m ) 2 . 25 ( m ) 21 - ax2 . 16 ( d , 13 . 4 ) 2 . 18 ( m ) 2 . 28 ( m ) 2 . 18 ( m ) 21 - eq1 . 40 ( dd , 1 . 9 , 11 . 6 ) 1 . 45 ( m ) 1 . 50 ( m ) 1 . 47 ( d , 12 . 2 ) 22 4 . 62 ( brs , 6 . 0 ) 4 . 61 ( brs , 6 . 0 ) 4 . 62 ( brs , 6 . 0 ) 4 . 62 ( d , 1 . 7 ) 23 - ax2 . 42 ( d , 14 . 4 ) 2 . 46 ( d , 15 . 0 ) 2 . 42 ( d , 14 . 6 ) 2 . 44 ( d , 13 . 7 ) 23 - eq1 . 85 ( m ) 1 . 85 ( m ) 1 . 82 ( m ) 1 . 87 ( m ) 27 0 . 97 ( s ) 1 . 09 ( s ) 0 . 97 ( s ) 1 . 11 ( s ) 28 0 . 98 ( s ) 1 . 09 ( s ) 0 . 99 ( s ) 1 . 13 ( s ) mecoo 2 . 08 ( s ) 2 . 05 ( s ) 4 - oh 5 . 43 ( d , 4 . 4 ) 5 . 58 ( d , 4 . 2 ) 14 - oh5 . 34 ( d , 6 . 3 ) 3 . 16 ( s ) 5 . 39 ( s ) 3 . 02 ( s ) 17 - oh6 . 43 ( t , 5 . 1 )) 6 . 18 ( s ) 6 . 84 ( s ) 6 . 18 ( s ) 24 - oh4 . 03 ( d , 7 . 2 ) 5 . 21 ( s ) 4 . 90 ( s ) 5 . 21 ( s ) __________________________________________________________________________ . sup . a dmsod . sub . 6 . . sup . b cdcl . sub . 3 . scalar coupling constants , were determined from homo2d - j and roesy spectroscopy . table iv__________________________________________________________________________three and two bond correlations ( hmbc experiment ) of withajardins a - d ( 1 - 4 ). sup . ac withajardin a ( 1 ). sup . b withajardin d ( 2 ). sup . c withajardin b ( 3 ). sup . b withajardin c ( 4 ). sup . c__________________________________________________________________________ 1 19 - me 3 , 19 - me 2 , 3 , 19 - me 19 - me 2 4 3 4 4 , 5 , 4 - oh 4 4 2 2 , 3 , 4 - oh 2 , 3 5 19 - me 3 , 4 , 19 - me 4 , 4 - oh , 19 - me 3 , 4 , 7 , 19 - me 6 8 7 6 6 8 6 6 9 19 - me 19 - me 7 , 19 - me 19 - me10 2 , 6 , 19 - me 2 , 4 , 6 , 19 - me 2 , 4 , 19 - me 2 , 4 , 19 - me12 18 - me 18 - me 18 - me13 14 - oh , 18 - me 14 - oh , 18 - me 14 - oh , 18 - me 18 - me14 18 - me 18 - me 18 - me15 8 , 14 - oh16 20 15 - β17 18 - me 17 - oh , 20 , 21 - ax 17 - oh , 21 - ax 18 - me , 21 - ax20 17 - oh 17 - oh , 21ax 17 - oh21 28 - me 27 - me 27 - me 27 - me23 28 - me 28 - me 28 - me 20 ,- 24 - oh24 23 - ax , 25 - oh , 27 - me 21 - ax , 23 , 24 - oh 23 - ax , 24 - oh , 28 - me 21 - eq , 21 - ax , 23 - ax , 28 - me25 28 - me 21 - ax , 28 - me 21 - ax , 23 - ax , 24 - oh , 27 - me 28 - me26 27 - me 21 - ax , 27 - me 12 - ax , 22 21 - ax , 22 , 27 - me28 24 - oh 24 - oh 24 - ohme -- coo -- 4 , me -- coo -- me -- coo -- 4 , me -- coo__________________________________________________________________________ table v__________________________________________________________________________roesy correlations for withajardinsproton withaiardin a ( 1 ) withaiardin d ( 2 ) withaiardin b ( 3 ) withaiardin c ( 4 ) __________________________________________________________________________h - 2 h - 3 h - 3 h - 3 h - 3h - 3 h - 2 , 4 - oh h - 2 , h - 4 , 4 - ohh - 4 h - 6 , 4 - oh h - 3 , h - 6 , 4 - ohh - 6 h - 4 , h - 7ax h - 3 h - 4 h - 3h - 8 19 - me 19 - me 19 - me 19 - meh - 11ax 19 - me 19 - me , h - 12eqh - 16α h - 21eq , h - 23eq h - 23eq h - 21eq h - 23eqh - 20 18 - me , h - 21eq h - 23eq 18 - me , h - 21eq h - 23eqh - 21ax h - 21eq , h - 22 h - 21eq , h - 22 , 24 - oh h - 22 , 17 - oh , 24 - oh h - 21eq , h - 22h - 21eq h - 16α , 27 - me h - 16α , 27 - me h - 16α , 27 - meh - 22 h - 20 , h - 23eq , h - 23ax h - 20 , h - 21ax , h - 21eq h - 20 , h23aq , h - 23ax h - 20 , h - 21ax , h - 21eqh - 23ax h - 16α , 24 - oh h - 22 , h - 23eq , 18 - me h - 16α , 24 - oh h - 21eq , h - 23eq , 18 - me24 - oh h - 21ax , h - 23ax , 17 - oh , 27 - me h - 21ax , h - 23ax , 17 - oh h - 21ax , h - 23ax h - 21ax , h - 23ax17 - oh 14 - oh , 24 - oh 14 - oh , 24 - oh4 - oh h - 3 , h - 4 h - 3 , h - 4__________________________________________________________________________ 1 . kirson i ., glotter e . j . nat . prod . 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