Identification and synthesis of trinorcadalene phytoalexins formed by hibiscus cannabinus in honour of professor G. H. Neil towers 75th birthday

Article abstract of DOI:10.1016/S0031-9422(98)00183-6

Two trinorcadalene phytoalexins, hibiscanal (2,8-dihydroxy-4,7-dimethoxy-6-methyl-1-naph-thaldehyde) and o-hibiscanone (3,8-dimethyl-1,2-naphthoquinone), were isolated and identified from stem stele of kenaf (Hibiscus cannabinus) inoculated with the funga

Full text of DOI:10.1016/S0031-9422(98)00183-6

Phytochemistry\ Vol[ 38\ No[ 1\ pp[ 320Ð339\ 0887
Þ 0887 Elsevier Science Ltd[ All rights reserved
Printed in Great Britain
Pergamon
\
9920Ð8311:87:,Ðsee front matter
PII] S9920Ð8311"87#99072Ð5
IDENTIFICATION AND SYNTHESIS OF TRINORCADALENE
PHYTOALEXINS FORMED BY HIBISCUS CANNABINUS
IN HONOUR OF PROFESSOR G[ H[ NEIL TOWERS 64TH BIRTHDAY
ALOIS A[ BELL\ꢀ ROBERT D[ STIPANOVIC\$ JIUXU ZHANG\$ MARSHALL E[ MACE$
and JOSEPH H[ REIBENSPIES%
$USDA\ ARS\ Southern Crops Research Laboratory\ 1654 F+B Road\ College Station\ TX 66734\ U[S[A[
%Department of Chemistry\ Texas A+M University\ College Station\ TX 66732\ U[S[A[
"Received 29 October 0886^ revised 4 February 0887#
Key Word Index*Hibiscus cannabinus^ Gossypium species^ Malvaceae^ kenaf^ cotton^ Ver!
ticillium dahliae^ Fusarium oxysporum^ wilt diseases^ phytoalexins^ disease resistance^ terpenoids^
trinorcadalenes[
Abstract*Two trinorcadalene phytoalexins\ hibiscanal "1\7!dihydroxy!3\6!dimethoxy!5!methyl!0!naph!
thaldehyde# and o!hibiscanone "2\7!dimethyl!0\1!naphthoquinone#\ were isolated and identi_ed from stem stele
of kenaf "Hibiscus cannabinus# inoculated with the fungal pathogen Verticillium dahliae[ o!Hibiscanone also
was synthesized[ The ED49 values of hibiscanal and o!hibiscanone against V[ dahliae were 14[72 and 0[07 mg:ml\
respectively[ o!Hibiscanone killed all propagules of V[ dahliae and Fusarium oxysporum f[ sp[ vasinfectum at
7 mg:ml and 01 mg:ml\ respectively[ o!Hibiscanone is more toxic to V[ dahliae than desoxyhemigossypol\ the
most toxic phytoalexin known in cotton "Gossypium species#\ or mansonone C "2\7!dimethyl!4!isopropyl!0\1!
naphthoquinone# which has been isolated from other malvaceous species[ The implications of these _ndings
for the genetic engineering of cotton phytoalexins is discussed[ Þ 0887 Elsevier Science Ltd[ All rights reserved
INTRODUCTION
infected xylem vessels more rapidly than do sus!
ceptible cultivars ð2\ 6Ð09Ł[ Accumulation of the
phytoalexins is correlated closely with induction of
the genes coding for 2!hydroxy!2!methylglutaryl CoA
reductase "HMGR# and d!cadinene synthase "CADS#
ð7\ 00Ð02Ł[ In the most resistant cultivars\ maximum
accumulation of mRNA from these genes is observed
by 01 hr after inoculation\ whereas accumulation is
delayed by 13 to 37 hr in susceptible cultivars[ Most
modern cultivars of Upland cotton that have been
developed for resistance to Verticillium wilt exhibit
rapid accumulation of terpenoid aldehydes and rapid
induction of HMGR and CADS genes\ apparently
due to transfer of these characters from wilt!resistant
G[ barbadense cultivars ð7Ł[ It is unlikely that this
character can be improved much further because
maximum accumulation of mRNA for HMGR and
CADS occurs by 01 hr after inoculation\ which is even
before the conidia of V[ dahliae normally germinate[
Likewise\ the induction of these genes is so sensitive
in the resistant cultivars that phytoalexin synthesis is
triggered by dead as well as by live conidia of the
fungus ð2\ 03Ł[
A number of phytoalexins "antifungal antibiotics syn!
thesized by plants in response to microbial infections#
have been isolated and identi_ed from cotton plants
"Gossypium species\ tribe Gossypieae\ family Mal!
vaceae# infected with Verticillium dahliae Kleb[ The
most prominent phytoalexins in vascular tissues of
cultivated Upland cotton "Gossypium hirsutum L[# are
the cadalene terpenoids hemigossypol "HG# and
desoxyhemigossypol "dHG# which are accompanied
by relatively low concentrations of their 5!methyl
ethers\ hemigossypol!5!methyl ether "MHG# and
desoxyhemigossypol!5!methyl ether "dMHG# ð0Ð2Ł
"Scheme 0#[ High concentrations of the methyl ethers
are found in cultivated Pima and Egyptian cottons
"Gossypium barbadense L[# and in several wild Gos!
sypium species\ which may contain more than 49) of
the phytoalexins as the methyl ethers ð3\ 4Ł[ Raimondal
"1!methoxy!hemigossypol#^ occurs uniquely in the
wild species Gossypium raimondii Ulbr[ ð5Ł[
Cotton cultivars that are resistant to Verticillium
wilt and Fusarium wilt accumulate phytoalexins in
Further progress in improving the phytoalexin
response of cotton might be achieved by increasing
the potency of the phytoalexins produced[ The phyto!
ꢀAuthor to whom correspondence should be addressed[
320

321
A[ A[ BELL et al[
Scheme 0[ Terpenoid phytoalexins of Gossypium[
Table 0[ Rf values of o!hibiscanone and hibiscanal in di}er!
alexins MHG and dMHG\ and raimondal\ are only
about one!half as toxic as the nonmethylated parent
compounds to several plant and human pathogens
ð4\ 04Ð07Ł[ Accordingly\ the evolutionarily advanced
Gossypium species\ G[ hirsutum\ G[ arboreum L[\ and
G[ herbaceum L[ ð08Ł\ have evolved regulatory genes
to restrict the methylation of desHG and\ thus\
decrease the percentage of the phytoalexins that are
methylated ð4\ 19Ł[ Further\ when genes were intro!
duced into Upland cotton from other Gossypium spec!
ies to increase the percentages of the less potent phyto!
alexins MHG and dMHG or raimondal among total
phytoalexins\ there was a concomitant decrease in
resistance to Verticillium wilt in each of four family
ent solvent systems
Solvent Systems
o!Hibiscanone Hibiscanal
Cyclohexane ] EtOAc "59 ] 39# 9[38
9[09
9[92
9[07
Toluene ] MeOH "84 ] 4#
Hexane ] Me1CO "59 ] 39#
CHCl2 ] Me1CO ] HCO1H
"74 ] 04 ] 0#
9[42
9[42
9[58
9[22
Column chromatography was used to isolate the
lines[ Thus\ the low potency of cotton phytoalexins inhibitory compounds demonstrated by the TLC plate
appears to limit their e}ectiveness against wilt patho! bioassay[ Antifungal activity was found in column
gens[
fractions 2 and 4[ Chromatography of these fractions
Kenaf "Hibiscus cannabinus L[#\ an annual _ber on TLC plates and crystallization of the eluted com!
crop used for the manufacture of newsprint\ might be pounds were used to purify the inhibitory compounds\
the source of genes that could be used to improve which were identi_ed as o!hibiscanone and hibiscanal[
the potency of cotton phytoalexins[ This species\ like Rf values of these compounds on TLC plates in vari!
cotton\ belongs to the Malvaceae family[ It was the ous solvents is shown in Table 0[
most resistant of any dicotyledonous species to iso!
On developed TLC plates\ hibiscanal appeared as a
lates of Verticillium dahliae from 06 species belonging yellow spot\ and o!hibiscanone as a yellow!orange
to 00 plant families in Russia ð10Ł[ Four kenaf cultivars spot[ A dark!blue color appeared when the com!
also showed high levels of resistance to both defol! pounds were sprayed with ferric chloride!potassium
iating and nondefoliating strains of V[ dahliae isolated ferricyanide[ With phloroglucinol spray\ hibiscanal
from cotton in the United States ð11Ł[ In this paper initially gave a brown color that turned to orange
we report the isolation and biological activity of two after several minutes[
trinorcadalene phytoalexins induced by V[ dahliae
infection in stele tissues of kenaf plants[ A synthesis
is reported for the more potent phytoalexin\ o!hib!
Identi_cation of o!hibiscanone
iscanone[
o!Hibiscanone was identi_ed as 2\7!dimethyl!0\1!
naphthoquinone "Scheme 1# by x!ray crystallography[
Its H! and 02C!NMR and mass spectra are given in
the Experimental Section and it was synthesized as
shown in Fig[ 0[
0
RESULTS
Isolation of phytoalexins from kenaf
TLC plate bioassays of the crude extracts of kenaf
stem steles inoculated with V[ dahliae clearly showed
two spots inhibitory to V[ dahliae and Fusarium oxy!
Identi_cation of hibiscanal
Hibiscanal has the structure shown in Scheme 2[ It
sporum Schlecht f[ sp[ vasinfectum Atk[ Sny[ + Hans[ was obtained as orange crystals\ mp 114Ð115>\ from
"F[o[v[#[ One spot was extremely inhibitory to both acetone\ and gave a parent peak in the mass spectrum
pathogens[ No antifungal activities were observed at m:z 151 "6[5)#[ High resolution mass measurement
from extracts of healthy stem stele[
gave the formula C03H03O4 "Calcd[ 151[9730^ Found

Identi_cation and synthesis of trinorcadalene phytoalexins formed by Hibiscus cannabinus
322
Scheme 1[ Comparison of o!hibiscanone structure with those of related terpenoids in other species of the Malvaceae[
Fig[ 0[ Synthesis of o!hibiscanone "see Experimental section for reagents and product _elds#[
151[9735^ Di}[ 9[4 ppm#[ A fragment ion at m:z 133 chelated ortho!hydroxy proton appeared at d 01[66[
indicated the loss of H1O which is characteristic of Two aromatic methoxyl groups "d 2[78 and 3[91# and
sesquiterpenoid aldehydes found in other members of a methyl group "d 1[21# were also evident[ Aromatic
the Malvaceae "e[g[\ hemigossypol ð0Ł "Scheme 0# and proton singlets were observed at d 5[61 and 6[46[ The
raimondal ð5Ł "Scheme 2# from Gossypium#[ This frag! 0H!0H COSY spectrum showed a correlation between
mentation requires the presence of an aldehyde group the methyl group "d 1[21# and the aromatic proton at
at C!0 and hydroxyl groups at C!1 and C!7[ Hibiscanal d 6[46[ This establishes that this aromatic proton is
also gave a signi_cant peak at m:z 159 "8)# which adjacent to the methyl group[
was shown by high resolution mass measurement to be
The 02C!NMR spectrum "Table 1# showed 03 car!
C03H01O4 "Calcd[ 159[95736^ Found 159[95736^ Di}[ bon atoms[ An inverse 0H!detected heteronuclear mul!
9[90 ppm#[
tiple quantum coherence "HMQC# spectrum estab!
The 0H!NMR spectrum of hibiscanal "Table 0# lished the attachment of protons to the speci_c car!
showed a low _eld aldehyde proton at d 09[05^ and a bons in the naphthalene ring as shown in Table 1[

323
A[ A[ BELL et al[
Scheme 2[ Comparison of hibiscanal structure with those of related terpenoids in other species of the Malvaceae[
Table 1[ 0H!NMR and 02C!NMR chemical shifts of hibiscanal\ raimondal\ and 1\3!
dihydroxyacetophenone
Raimondal 1\3!Dihydroxyacetophenone
Hꢀ
C
C
C
C(
d
d
d
d
0
009[7
055[0
83[8
000[3
055[0
030[8
023[2
006[2
016[9
031[7
033[2
004[9
014[9
088[9
05[4
002[8
054[0
092[4
054[0
097[5
022[8
1
2
3
4
5
6
7
8
5[61s
6[46q
060[1
004[2
029[8
038[2
037[3
014[1
008[4
089[7
05[5
09
00
01
09[08s
1[21d
02
16[6
03
19[0
04
19[0
C3!OCH2
C6!OCH2
C1!OH
3[91s
2[08s
01[66s
48[3
46[2
59[8
ꢀsꢁsinglet\ qꢁquartet "9Ð7 Hz#^ dꢁdoublet "9[7 Hz#
The speci_c point of attachment on the naphthalene unique chemical shift of the signal at d 83[8 indicated it
ring of the protons and most of the groups was estab! was located between two carbons attached to oxygen
lished by an inverse heteronuclear multiple bond con! moieties[ The proton attached to this carbon "d 5[61#
nectivity "HMBC# experiment "Fig[ 1#[ Thus\ the alde! showed long!range correlations to signals at d 009[8
hyde showed long!range correlations with the signal "C!0# and d 008[3\ and to two oxygenated atoms d
at d 009[8 and d 014[1[ This established positions C!0 054[7 and d 060[1[ The HMBC experiment showed
and C!8\ respectively\ in the naphthalene ring[ The that the signal at d 060[1 is attached to an O!CH2

Identi_cation and synthesis of trinorcadalene phytoalexins formed by Hibiscus cannabinus
324
Fig[ 1[ HMBC correlation of hibiscanal[
group and thus the signal d 054[7 must be C!1 and the
signal at d 008[3 must be C!09[
The shifts for carbons −0 to −3 of hibiscanal are
in general agreement with C!0\ C!1\ C!2\ and C!3 in
1\3!dihydroxyacetophenone ð12Ł "Table 1#[ Sub!
stituents on the second naphthalene ring were estab!
lished as follows[ The HMBC experiment showed the
proton at d 6[46 was correlated to the signals at d
014[1 "C!8# and d 038[2 "C!OCH2# and thus meta to
these carbons[ Thus\ this proton is attached to either
C!4 or C!6[ The protons on the aromatic methyl group
showed long range coupling to carbons at chemical
shifts of d 029[8\ 004[1 "C!H# and 038[2 "C!OCH2#[
Thus\ the aromatic methyl group must be attached to
the carbon at d 029[8 with the carbons at d 004[1
and 038[2 located on either side[ The remaining ring
carbon "i[e[\ C!7# which showed no long!range cor!
relation was assigned to the signal at d 037[3[ The
chemical shift of C!4\ C!5\ C!6\ and C!7 in hibiscanal
are very similar to those in raimondal "Table 1^ Fig[
2# and thus are assigned as indicated[ Oxygen sub!
stitution in this ring is also in agreement with that of
o!hibiscanone[
Fig[ 2[ Toxicity of hibiscanal to Verticillium dahliae and
Fusarium oxysporum f[ sp[ vasinfectum[ Values for each con!
centration are means of 5 replications from each of 1Ð3
experiments[
Toxicities of phytoalexins to fun`al patho`ens
Both hibiscanal and o!hibiscanone are toxic to the
fungal wilt pathogens V[ dahliae and F[o[v[ "Figs 2
and 3# Verticillium dahliae is more sensitive than F[o[v[
to both compounds[ Hibiscanal had ED49 values of
14[7 mg:ml and 25[4 mg:ml against V[ dahliae and
F[o[v[\ respectively[ o!Hibiscanone had signi_cantly
lower ED49 values toward these organisms "0[1 mg:ml
for V[ dahliae and 0[0 mg:ml for F[o[v[#[ o!Hibiscanone
killed all propagules of V[ dahliae and F[o[v[ at 7
and 01 mg:ml\ respectively[ As shown in Fig[ 4\ o!
hibiscanone was twice as toxic to V[ dahliae as man!
sonone C which di}ers only by the addition of an
isopropyl group at C!4 "Scheme 1#[
Fig[ 3[ Toxicity of o!hibiscanone to Verticillium dahliae and
Fusarium oxysporum f[ sp[ vasinfectum[ Values for each con!
centration are means of 5 replications from each of 1Ð3
experiments[ Propagules of V[ dahliae and F[o[v[ were killed
at 7 and 01 mg:ml\ respectively[
sels beginning about 13 hr after inoculation and
accumulate to toxic levels by 61 hr after inoculation[
They do not accumulate in H1O!inoculated plants[
DISCUSSION
Hibiscanal and o!hibiscanone function as phyto! Because they are synthesized in response to stress and
alexins synthesized by kenaf cells in response to infec! accumulate to fungitoxic levels they are phytoalexins[
tion by fungal wilt pathogens[ In separate studies "Bell
We propose that the pre_x hibiscan! be used to
and Stipanovic\ unpublished# we have shown that name the trinorcadalenes of Hibiscus cannabinus to
these compounds are secreted into infected xylem ves! distinguish them from the hibiscones "cadinenes# and

325
A[ A[ BELL et al[
all propagules at 7 mg:ml[ Thus\ o!hibiscanone is more
toxic than any of the known phytoalexins in the Mal!
vaceae[ If the biosynthetic pathway\ key enzymes and
gene control of o!hibiscanone can be deciphered\ it
may be possible using recombinant DNA technology
to transfer the necessary genes from kenaf to cotton
in order to allow synthesis of the trinorcadalene
phytoalexins and improve the resistance of cotton to
wilt pathogens[
EXPERIMENTAL
General
Mass spectra were obtained on a double!focusing
VG 69Ð149 EHF spectrometer with a VG 00Ð149 data
system by direct probe using electron impact to gen!
erate ions[ 0H! and 02C!NMR were obtained on a
Bruker ARX!299 spectrometer in CDCl2^ all carbon
assignments were con_rmed by heteronuclear multiple
quantum coherence "HMQC# and heteronuclear mul!
tiple bond connectivity "HMBC# experiments[
Fig[ 4[ Comparison of the toxicity of o!hibiscanone and
mansonone C to Verticillium dahliae isolate V33[
hibisquinones "cadalenes# isolated from heartwood of
other Hibiscus species ð13\ 14Ł[ As shown in Schemes
1 and 2\ the compounds from kenaf apparently di}er
Patho`ens
Verticillium dahliae Kleb[ strains V!33 and V!65
from other terpenes found in malvaceous plants ð13Ð "cotton defoliating isolates# and PH and TS1 "cotton
17Ł because the isopropyl group\ normally present in nondefoliating isolates#\ were cultured on PDA for 6
the cadalene ring\ is absent from the naphthalene ring day at 14>[ Conidia were harvested in sterile distilled
of the trinorcadalenes[ In other species of the Mal! H1O and _ltered through Whatman No[ 3 _lter paper
vaceae the terpenes apparently are derived from a to remove mycelia[ Spores were washed twice by cen!
cadinene\ for example d!cadinene in Gossypium ð00\ trifugation\ resuspended in H1O\ and adjusted to ca[
01Ł\ and the entire carbon structure is maintained dur! 096 spores:ml "A[949 at 599 nm for V[ dahliae ð6Ł#[ An
ing synthesis of the biologically active terpenoids[ isolate of Fusarium oxysporum Schlecht f[ sp[ vas!
Kenaf apparently produces a unique enzyme that infectum Atk[ Sny[ + Hans[ "F[o[v[^ American Type
allows removal of the isopropyl carbons from the Culture Collection 35533# from cotton in California
cadinene ring\ or even before ring closure[ Precursor was cultured and prepared as described for V[ dahliae[
feeding studies are being used to test this possibility[
Several observations suggest that o!hibiscanone and
hibiscanal may be responsible for the high level of
Growth and inoculation of kenaf
resistance of kenaf to wilt pathogens[ The ability to
Kenaf\ cv[ Everglade\ plants were grown in the _eld
synthesize trinorcadalenes appears to be unique to at College Station\ Texas[ When the stems were 3Ð4
kenaf among species of the Malvaceae[ o!Hibiscanone month old\ they were cut into 19 cm sections\ and the
is more toxic to V[ dahliae than is mansonone C "Fig[ bark "cortical tissue# tissue was removed to expose the
4#\ which is produced by other species of the Mal! cambial tissue of the stele[ These stele sections were
vaceae ð16\ 17Ł[ Yet\ these compounds are identical in immediately dipped into a mixture of conidial sus!
structure "Scheme 1#\ except for the isopropyl group[ pensions prepared from the four V[ dahliae strains\
This suggests that the removal of the isopropyl group and were incubated over moist towels in plastic stor!
has improved the toxicity of the terpenoid o!naph! age containers placed in large plastic bags without
thoquinones to wilt pathogens[ Dumas et al ð18Ł found sealing to assure aeration[ The containers were kept
that the mansonones A\ C\ D\ E\ F\ and G also varied in darkness at room temperature for 61 hr[ Non!
signi_cantly in toxicity to the fungal wilt pathogen inoculated plant steles served as the control[
Ceratocystis ulmi due to small di}erences in the struc!
ture of these terpenoid o!quinones[ o!Hibiscanone
also is considerably more toxic to V[ dahliae than
Extraction of phytoalexins
dHG\ the most potent phytoalexin produced by
The inoculated stele sections were cut into ca[ 1 cm
Gossypium species against wilt pathogens ð11Ł[ dHG pieces and put into a 1 l ~ask with Me1CO "4 ml:g
has an ED49 value of 2[4 mg:ml against V[ dahliae and fresh tissue# and shaken for about 4 min[ Then\ the
kills all propagules at 04 mg:ml[ In comparison\ o! extract was _ltered\ using a Whatman No[ 31 _lter
hibiscanone has an ED49 value of 0[07 mg:ml and kills paper to remove plant tissue[ This extraction was per!

Identi_cation and synthesis of trinorcadalene phytoalexins formed by Hibiscus cannabinus
326
formed twice[ The Me1CO was evaporated from the Characterization of o!hibiscanone[
extract in a rotary evaporator at 29>[ The remaining
o!Hibiscanone was obtained as red crystals from
Me1CO]hexane\ mp 034Ð035>[ UV] lEtOHmax nm "e#
aqueous suspension was mixed with an equal volume
of EtOAc in a separatory funnel\ and the H1O phase
was discarded[ The EtOAc extract was kept in a
refrigerator for further use[ The extract from non!
inoculated kenaf stele tissue was used as control[
192 "11\549#\ 142 "16\599#\ 244 "1\699#\ 308 "2\099#^
KBr
max
IR] n
cm−0 0569\ 0546^ MS] m:z ")# 077 "0[4\
ðMŁ¦¦1#\ 075 "1[2\ ðMŁ¦#\ 048 "06#\ 047 "099\ ðMŁ¦!
CO#\ 046 "05#\ 029 "11#\ 018 "25#\ 017 "13#\ 016 "00#\
004 "43#^ 0H!NMR "CDCl2#] d 0[87 "2H\ bs\ C2!CH2#\
1[59 "2H\ s\ C7!CH2#\ 6[94 "0H\ d\ Jꢁ6[5 Hz\ C4!H#\
6[03 "0H\ bs\ C3!H#\ 6[04 "0H\ d\ Jꢁ6[5 Hz\ C6!H#\
6[28 "0H\ t\ Jꢁ6[5 Hz\ C5!H#^ 02C!NMR "CDCl2#] d
04[2 "C2!CH2#\ 11[7 "C7!CH2#\ 016[7 "C4#\ 017[0 "C8#\
022[5 "C6#\ 023[6 "C5#\ 024[7 "C2#\ 025[2 "C09#\ 031[5
"C3#\ 034[2 "C7#\ 070[9 "C0#\ 070[3 "C1#[
Puri_cation of phytoalexins by column chromato`raphy
The extract was chromatographed on a 27×1[4 cm
column of SilicAR CC!3 "Mallinckrodt\ Inc[#[ The
concentrated extract was dried onto a few grams of
the Si gel and then placed at the top of the column
which was eluted with 49 ml of each 9\ 4\ 09\ 04\ 19\
29\ 39 and 099 percent Me1CO in cyclohexane[ 49 ml
fractions were collected separately in ~asks and dried
on a rotary evaporator[ Antifungal activities of each
fraction were tested using the TLC bioassay[
X!ray crystallo`raphic identi_cation of o!hibiscanone
o!Hibiscanone crystallized in the monoclinic space
Ä
group P10:n with cell constants aꢁ8[954 "3#A\
Ä
Ä
bꢁ09[786 "5#A\ cꢁ07[791 "8# A[ The data were cal!
culated on a Nicolet R2m:v X!ray defractometer with
Ä
MoKÃ"9[609962 A# radiation for 3[9>²19²49[9>
ðw"Wycko}# scans\ −09²2²09\ 9²k²01\ 9²l\
²11Ł at 082>K[ Scan width\ on v\ for the data col!
TLC bioasssays for phytoalexins
Test samples were spotted on Baker Silica Gel lection was 9[59>\ with a variable scan rate of 0[49 to
G:HR TLC plates that were developed with solvent 03[54> min−0[ A total of 0782 unique re~ections\ with
system 3 "see below#[ The plates were dried at room =I= r1[9o I\ were used for structure solution[ The
temp[ in a fume hood\ and then hot PDA medium was structure was solved by direct Methods ð29Ł[ Full
sprayed on the plates and allowed to solidify[ Spore matrix least!squares anisotropic re_nement for all
suspension "096 spores:ml# of V[ dahliae isolate V!33 non!hydrogen atoms yielded Rꢁ9[964\ wRꢁ9[967
or F[o[v[ then was sprayed on the TLC plate[ Plates and Sꢁ1[07 at convergence[ The _nal di}erence Four!
were incubated over moist towels in glass trays cov! ier map was featureless with rhomaxꢁ9[28 and
ered by thin plastic wrap[ They were kept in the dark rhominꢁ−9[28 e A−2[ Full details of the X!ray analysis
Ä
at room temp[ "11Ð14># for 1 days[ Plates then were will be provided upon request[
observed for fungal growth and inhibitory zones[
Characterization of hibiscanal
Hibiscanal was obtained as red crystals from
TLC puri_cation of phytoalexins
Me1CO]hexane\ mp 114Ð115>[ MS] m:z ")# 151 "6[5#\
159 "8#\ 134 "14#\ 133 "099\ ðMŁ¦!H1O#\ 118 "23\ 133!
TLC plates "19×19 cm# were prepared with Baker
EtOH
CH2#\ 102 "03#\ 190 "04\ 118!CO#^ UV] l
nm "e#
max
Silica Gel G:HR "9[4 mm#[ Developing solvents were]
system 0\ cyclohexane!EtOAc "59 ] 39#^ system 1\ hex!
ane!Me1CO "54 ] 24#^ system 2\ toluene!MeOH "84 ] 4#^
and system 3\ CHCl2!Me1CO!HCO1H "73 ] 04 ] 0#[
Inhibitory compounds were located by visible colors
or ~uorescences under UV "254 nm#[ Zones containing
inhibitory compounds were scraped from plates with
391 "9[7949#\ 237 "4\499#\ 174 "06\399#^ 0H!NMR and
02C!NMR spectra are given in Table 1[
Biolo`ical activity of phytoalexins
The toxicities of compounds inhibitory to V[ dahliae
a razor blade immediately after development[ Com! "V!33# and F[o[v[ were quantitatively determined using
pounds were eluted from silica gel with 39) Me1CO a direct turbidimetric bioassay[ The bu}ered nutrient
in cyclohexane\ dried and dissolved in EtOAc[ solution "pH 5[2# was composed of 049 mM Na!K!Pi
Developing solvent systems 0Ð3 were used successively bu}er
containing
29 mM
NaNO2\
0 mM
until antifungal compounds were pure[ Compounds MgSO3[6H1O and 004 mM D!glucose[ Phytoalexins
were detected by their visible colors\ ~uorescence were dissolved in DMSO[ The solutions were diluted
under 254 nm UV\ and reaction with phloroglucinol with nutrient solution to give a _nal concentration of
reagent "4) phloroglucinol in EtOH]conc[ HCL\ 0:0\ 1) DMSO[ The toxicities of the compounds to V[
freshly mixed# and FeCl2!K2Fe"CN#5 reagent "a 0 ] 0 dahliae "V!33# and F[o[v[ were assayed by adding vari!
mixture of 0) FeCl2 and 0) K2Fe"CN#5#[ All iso! ous concentrations of the phytoalexins to conidia "095
lation procedures were modi_cations of methods used spores:ml# in nutrient solution[ The assay mixtures
for cotton phytoalexins ð0Ł[
"199 ml:well# were incubated in a 85!well cluster plate[

327
A[ A[ BELL et al[
Each concentration of a compound was replicated X5 "CDCl2#] d 05[1 "CH2#\ 15[3 "C2#\ 22[2 "C1#\ 23[0 "C3#\
for each experiment[ Plates were incubated for 39 hr 44[2 "OCH2#\ 098[8 "C4?#\ 015[4 "C2?#\ 015[4 "C5?#\
at 14> in darkness\ and optical densities were read 029[7 "C1?#\ 021[7 "C0?#\ 045[0 "C3?#\ 079[1 "C0#[
directly on a Molecular Devices Precision Microplate
Reader at 449 nm[ Each experiment was repeated 1Ð3
times[ Assay medium with 1) DMSO served as the
Preparation of 6!methoxy!5!methyl!0!tetralone
control[
3!"3!Methoxy!2!methylphenyl# butyric acid "11 g#
was mixed with polyphosphoric acid "029 g# under N1
and heated at 74> for 5 hr[ The mixture was cooled\
carefully diluted with ice and H1O "199 ml# and
extracted with Et1O "499 ml#[ The Et1O soln was
Preparation of 2!"3!methoxy!2!methylbenzoyl#!pro!
pionic acid
Succinic anhydride "009 g# and o!methylanisole washed with H1O and placed over dry Na1SO3[ The
"011 g# were dissolved in nitrobenzene "499 ml# under solvent was evaporated\ and the crude product was
dry conditions and cooled to −04> in an ice!salt bath[ distilled "bp\ 037Ð041>:1[4 mm Hg#[ The product
EtOH
max
Dry AlCl2 "183 g# was added to the stirred soln over a "06[2 g# solidi_ed\ mp 33Ð34>^ UV] l
nm "e# 195
period of 0 hr[ The soln was warmed to 9> over 2 hr\ "06\999# 114 "19\299#\ 151 "01\499# 219 "3\399#^ IR]
and then slowly to room temp[ with continued stirring nKmBaxr cm−0 0563\ 0529\ 0455\ 0384^ 0H!NMR "CDCl2#]
for 39 hr[ The reaction mixture was poured onto 0[1 kg d 1[95 "1H\ quin\ C2#\ 1[10 "2H\ s\ CH2#\ 1[46 "1H\ t\
of ice and 225 ml of 5N HCl[ The white ppt[ was Jꢁ5[1 Hz\ C1#\ 1[71 "1H\ t\ Jꢁ5[0 Hz\ C3#\ 2[71 "2H\
removed by _ltration and set aside[ The _ltrate was s\ OCH2#\ 5[87 "0H\ s\ C4#\ 6[30 "0H\ s\ C7#^ 02C!NMR
extracted with Et1O "799 ml#\ and the organic phase "CDCl2#] d 05[4 "CH2#\ 12[5 "C2#\ 17[6 "C3#\ 27[7 "C1#\
was washed with 09) NaOH "299 ml#[ The alkaline 44[3 "OCH2#\ 095[6 "C7#\ 029[7 "C4#\ 020[2 "C8#\ 022[6
solution was acidi_ed with 5N HCl[ The ppt[ was "C5#\ 026 "C09#\ 045[4 "C6#\ 087[1 "C0#[
removed by _ltration and combined with the ppt[ from
above[ After washing with H1O\ the ppt[ was re~uxed
Preparation of 0\1!Dihydro!5!methoxy!3\6!dimethyl!
2 hr with 19) NH3OH "3[4 l#[ The soln was decol!
naphthalene
orized with activated carbon and _ltered[ The _ltered
soln was cooled in ice and acidi_ed with HCl[ The
6!Methoxy!5!methyl!0!tetralone "06[0 g# was dis!
ppt[ was _ltered and washed with H1O to provide solved in 0 l of Et1O under N1[ A 2M solution of
084 g of white crystals] mps 049[4Ð041>\ lit[ 041> ð20Ł^ methyl magnesium bromide "020 ml# was added drop!
0H!NMR "CDCl2# d 1[66 "1H\ t\ Jꢁ5[5 Hz\ C1#\ 1[11 wise over 0 hr[ The soln was re~uxed for 1 hr\ and then
"2H\ s\ CH2#\ 2[13 "1H\ t\ Jꢁ5[5 Hz\ C2#\ 2[57 "2H\ s\ cooled[ An aq[ soln of NH3Cl "12 g in 53 ml H1O# was
OCH2#\ 5[72 "0H\ d\ Jꢁ7[5 Hz\ C4?#\ 6[66 "0H\ d\ added dropwise[ The Et1O soln was separated\ washed
Jꢁ1[4 Hz\ C1?#\ 6[70 "0H\ dd\ Jꢁ7[5 Hz\ 1[4 Hz\ C5?#^ with H1O and evaporated to dryness[ The crude prod!
02C!NMR "CDCl2# d 05[1 "CH2#\ 17[1 "C1#\ 21[6 "C2#\ uct "08[97 g# was dissolved in C5H5 "699 ml# and a
44[1 "OCH2#\ 098[1 "C4?#\ 015[7 "C2?#\ 017[1 "C5?#\ catalytic amount of p!toluenesulfonic acid was added[
018[9 "C0?#\ 029[6 "C1?#\ 050[8 "C3?#\ 067[7 "C0#\ 085[6 The soln was re~uxed for 0 hr under N1 using a Dean!
"CO#[
Stark trap[ After cooling\ Et1O "299 ml# was added\
washed with 4) NaHCO2 soln"149 ml#\ dried over
Na1SO3\ and evaporated[ The crude product was dis!
tilled "bp\ 004>:9[34 mm Hg# to provide 02[5 g of a
Preparation of 0!"3!methoxy!2!methylphenyl#!butyric
acid
0
light yellow oil^ H!NMR "CDCl2#] d 1[97 "2H\ bq\
Zinc "67 g#\ HgCl "6[7 g#\ H1O "029 ml# and conc[ Jꢁ0[66 Hz\ Cl!CH2#\ 1[12 "1H\ m\ C3#\ 1[14 "2H\ bs\
HCl "3 ml# were mixed for 7 min[ The solution was C5!CH2#\ 1[57 "1H\ t\ Jꢁ7[1 Hz\ C3#\ 2[75 "2H\ s\
decanted and H1O "38 ml#\ conc[ HCl "003 ml#\ toluene OCH2#\ 4[73 "0H\ bt\ C1#\ 5[65 "0H\ s\ C7#\ 5[82 "0H\
"54 ml#\ and 2!"3!methoxy!2!methylbenzoyl#!pro! bs\ C4#^ 02C!NMR "CDCl2#] d 04[7 "C5!CH2#\ 08[3 "Cl!
pionic acid "39 g# were combined[ The mixture was CH2#\ 12[4 "C2#\ 16[3 "C3#\ 44[5 "OCH2#\ 094[2 "C7#\
re~uxed for 19 hr during which conc[ HCl "21 ml# was 013[6 "C5#\ 013[7 "C1#\ 017[0 "C09#\ 018[6 "C4#\ 021[0
added X2 at 5 hr intervals[ The soln was cooled to "C0#\ 023[3 "C8#\ 045[2 "C6#[
room temp[\ H1O "029 ml# was added\ and the organic
layer was separated[ The H1O layer was extracted with
Preparation of 6!methoxy!0\5!dimethylnaphthalene
Et1O "X2\ 49 ml#[ The organic extracts were combined\
washed with H1O and placed over dry Na1SO3[ The
0\1!Dihydro!5!methoxy!3\6!dimethyl!naphthalene
solvent was evaporated\ and the product distilled "02[1 g# was dissolved in decalin "099 ml#[ Palladium
under vacuum "bp\ 056>:0mm Hg#[ The product on carbon "09)\ 1 g# was added and the mixture was
0
"11[5 g# solidi_ed\ mp 46[4Ð48>^ H!NMR "CDCl2#] d re~uxed for 07 hr under N1[ The catalyst was removed
0[82 "1H\ q\ Jꢁ6[4 Hz\ C2#\ 1[10 "2H\ s\ CH2#\ 1[26 by _ltration\ and most of the decalin was removed by
"1H\ d\ Jꢁ6[4 Hz\ C1#\ 1[48 "1H\ d\ Jꢁ6[4 Hz\ C3#\ distillation^ the product "00[3 g# was crystallized from
EtOH
2[70 "2H\ s\ OCH2#\ 5[64 "0H\ d\ Jꢁ7[8 Hz\ C4?#\ 5[85 cyclohexane\ mp 58Ð69>^ UV] l
nm "e# 120
max
"0H\ bs\ C1?#\ 5[86 "0H\ bd\ Jꢁ7[8 Hz\ C5?#^ 02C!NMR "35\499#\ 157 "2\299#\ 166 "2\699#\ 176 "2\099#\ 202

Identi_cation and synthesis of trinorcadalene phytoalexins formed by Hibiscus cannabinus
328
KBr
"0\099#\ 216 "0\499#^ IR] n
cm−0 0518\ 0488\ 0491\
1[ Stipanovic\ R[ D[\ Bell\ A[ A[ and Howell\ C[ R[\
Phytochemistry\ 0864\ 03\ 1750[
2[ Bell\ A[ A[\ in Challen`in` the Future] Proceedin`s
of the World Cotton Research Conference!0\ ed[
G[ A[ Constable and N[ W[ Forrester[ CSIRO\
Melbourne\ Australia\ 0884\ p[ 114[
3[ Bell\ A[ A[\ Stipanovic\ R[ D[\ O|Brien\ D[ H[ and
Fryxell\ P[ A[\ Phytochemistry\ 0867\ 06\ 0186[
4[ Bell\ A[ A[\ Stipanovic\ R[ D[\ Mace\ M[ E[ and
Kohel\ R[ J[\ Recent Advances in Phytochemistry\
0883\ 17\ 120[
5[ Stipanovic\ R[ D[\ Bell\ A[ A[ and O|Brien\ D[ H[\
Phytochemistry\ 0879\ 08\ 0624[
6[ Bell\ A[ A[\ Phytopatholo`y\ 0858\ 48\ 0008[
7[ Cui\ Y[\ Bell\ A[ A[\ Puckhaber\ L[\ Joost\ O[ and
Magill\ C[\ Molecular Plant!Microbe Interactions[
0887\ submitted[
max
0
0336^ H!NMR "Me1CO!d5#] d symbol] 1[21 "2H\ bs\
C5!CH2#\ 1[50 "2H\ bs\ Cl!CH2#\ 2[85 "2H\ s\ OCH2#\
6[07 "0H\ t\ Jꢁ6[9 Hz\ C2#\ 6[11 "0H\ bd\ C1#\ 6[12
"0H\ bs\ C7#\ 6[46 "0H\ d\ Jꢁ6[9 Hz\ C3#\ 6[48 "0H\
bs\ C4#^ 02C!NMR "Me1CO!d5#] d 05[7 "C5!CH2#\ 08[5
"Cl!CH2#^ 44[5 "OCH2#\ 091 "C7#\ 013[9 "C2#\ 015[0
"C3#\ 017[4 "C5#\ 018[8 "C0#\ 029[1 "C4#\ 022[3 "C09#\
022[4 "C8#\ 046[6 "C6#[
Preparation of 2\7!dimethyl!1!naphthol
6!Methoxy!0\5!dimethylnaphthalene "09 g# was dis!
solved in dry CH1Cl1 "019 ml#[ The soln was cooled to
−79> in a dry ice:Me1CO bath[ A 0 M solution of BBr2
in CH1Cl1 "59 ml# was added[ The soln was allowed to
warm slowly to room temp[ The soln was diluted with
Et1O "099 ml# and washed with 4) NaHCO2 soln
followed by H1O[ After drying over Na1SO3\ the sol!
8[ Mace\ M[ E[\ Physiolo`ical Plant Patholo`y\ 0874\
01\ 0[
vent was evaporated[ The naphthol "1[0 g# was recrys! 09[ Stipanovic\ R[ D[\ Mace\ M[ E[\ Altman\ D[ W[
tallized from hexane\ mp\ 89[4Ð80>^ UV] lEmtaOxH nm "e#
and Bell\ A[ A[\ in Biolo`ical Active Natural Prod!
ucts\ ACS Symposium Series Number 279\ ed[ H[
G[ Cutler[ American Chemical Society\ Wash!
ington\ D[C[\ 0877\ p[ 151[
120 "47\799#\ 160 "2\799#\ 170 "3\899#\ 181 "3\999#\ 207
KBr
max
"0\899#\ 220 "1\999#^ IR] n
cm−0 2241\ 0518\ 0421\
0339\ 0124^ 0H!NMR "Me1CO!d5#] d 1[26 "2H\ bs\ C2!
CH2#\ 1[41 "2H\ bs\ C7!CH2#\ 6[01 "0H\ t\ Jꢁ6[6 Hz\
C5#\ 6[05 "0H\ bd\ Jꢁ6[6 Hz\ C6#\ 6[29 "0H\ bs\ C0#\
6[43 "0H\ d\ Jꢁ6[6 Hz\ C4#\ 6[47 "0H\ bs\ C3#^ 02C!
NMR "Me1CO!d5#] d 05[6 "C2!CH2#\ 08[3 "C7!CH2#\
095[0 "C0#\ 012[2 "C5#\ 015[1 "C4#\ 015[5 "C6#\ 016[5
"C2#\ 018[6 "C09#\ 029[3 "C3#\ 021[4 "C7#\ 022[6 "C8#\
044[1 "C1#[
00[ Alchanati\ I[\ Patel\ J[ A[ A[\ Liu\ J[\ Benedict\ C[
R[\ Stipanovic\ R[ D[\ Bell\ A[ A[\ Cui\ Y[ and
Magill\ C[ W[\ Phytochemistry[ 0886\ in press[
01[ Chen\ X[ Y[\ Chen\ Y[\ Heinstein\ P[ and Davis!
son\ V[ J[\ Archives of Biochemistry and Biophys!
ics\ 0885\ 213\ 144[
02[ Joost\ O[\ Bianchini\ G[\ Bell\ A[ A[\ Benedict\ C[
R[ and Magill\ C[ W[\ Molecular Plant!Microbe
Interactions\ 0884\ 7\ 779[
Preparation of 2\7!dimethyl!0\1!naphthoquinone "o!
hibiscanone#
03[ Bell\ A[ A[ and Stipanovic\ R[ D[\ Myco!
patholo`ia\ 0867\ 54\ 80[
04[ Mace\ M[ E[\ Stipanovic\ R[ D[ and Bell\ A[ A[\
Physiolo`ical Plant Patholo`y\ 0874\ 15\ 198[
05[ Mace\ M[ E[\ Stipanovic\ R[ D[ and Bell\ A[ A[\
Pesticide Biochemistry and Physiolo`y\ 0889\ 25\
68[
06[ Mace\ M[ E[\ Stipanovic\ R[ D[ and Bell\ A[ A[\
Natural Toxins\ 0882\ 0\ 183[
07[ Zhang\ J[\ Mace\ M[ E[\ Stipanovic\ R[ D[ and
Bell\ A[ A[\ Journal of Phytopatholo`y\ 0882\ 028\
136[
Benzene selenic anhydride "187 mg# was added to
dry THF "05[4 ml#[ A soln of 2\7!dimethyl!0!naphthol
"022 mg# in dry THF "7 ml# was added dropwise over
04 min[ The reaction was heated to 49> and held at
that temp[ for 0 hr[ After cooling\ Et1O "14 ml# was
added to the reaction[ The soln was washed twice with
brine "49) satd soln\ 19 ml# and dried over Na1SO3[
The solvent was evaporated\ and the product "096 mg#
was crystallized from C5H5:hexane\ mp 036Ð037>\
mixed 034Ð035>[
08[ Fryxell\ P[ A[\ The Natural History of the Cotton
Tribe[ Texas A+M University Press\ College
Station\ Texas\ 0868\ p[ 134[
19[ Kohel\ R[ J[ and Bell\ A[ A[\ Journal of Heredity[
0886\ in press[
10[ Idessis V[ F[\ in Cotton Wilt\ ed[ M[ V[ Mukh!
amedzhanov[ U[ S[ Department of Commerce\
National Technical Information Service\ Spr!
ing_eld\ Va[ 11040\ 0855 "translated 0861#\ p[ 50[
11[ Stipanovic\ R[ D[\ Puckhaber\ L[ S[ and Bell\ A[
A[\ in Synthesis and Chemistry of New and Poten!
tial A`rochemicals[ eds[ D[ R[ Baker\ J[ G[ Fenyes\
G[ S[ Basarab\ D[ A[ Hunt[ American Chemical
Society\ Washington\ D[C[\ 0886\ in press[
Acknowled`ements*We thank Dr[ Joseph Veech for
providing kenaf plants and Dr[ Ross Beier for mass
spectral measurements[ The R2m:V single crystal X!
ray di}ractometer and crystallographic computing
system in the Crystal and Molecular Structures Lab!
oratory at the Department of Chemistry\ Texas A+M
University was purchased from funds provided by the
National Science Foundation[ "CHE!7402162#[
REFERENCES
0[ Bell\ A[ A[\ Stipanovic\ R[ D[\ Howell\ C[ R[ and
Fryxell\ P[ A[\ Phytochemistry\ 0864\ 03\ 114[

339
A[ A[ BELL et al[
12[ Dhami\ K[ S[ and Stothers\ J[ B[\ Canadian Jour!
nal of Chemistry\ 0854\ 32\ 368Ð387[
13[ Ferreira\ M[ A[\ King\ T[ J[\ Ali\ S[ and Thomson\
R[ H[\ J[ C[ S[ Perkin I\ 0879\ 138[
V[\ Indian Journal of Chemistry\ 0872\ 11H\ 84[
18[ Dumas\ M[ T[\ Strunz\ G[ M[\ Hubbes\ M[ and
Jeng\ R[ S[\ European Journal of Forestry Path!
olo`y\ 0875\ 05\ 106[
14[ Ali\ S[\ Singh\ P[ and Thomson\ R[ H[\ J[ C[ S[
Perkin I\ 0879\ 146[
15[ Sankaram\ A[ V[ B[\ Reddy\ N[ S[ and Shoolery\
J[ N[\ Phytochemistry\ 0870\ 19\ 0766[
16[ Letcher\ R[ M[ and Shirley\ I[ M[\ Phytochemistry\
0881\ 20\ 3060[
17[ Neelakantan\ S[\ Rajagopalan\ V[ and Raman\ P[
29[ Sheldirck\ G[ M[ SHELXTL!Plus "rev[ 3[00# User
Manual[ Siemens Analytical X!ray Instruments
Inc[\ Madison\ WI\ 0881[
20[ Violland\ R[\ Violland!Duperret\ N[\ Pacheco\ H[\
Trouiller\ G[ and Leblane\ A[\ Chimie Thera!
peutique\ 0860\ 5\ 085[