New Indolopyridoquinazoline, Jih-Jung Chen1

Benzo[c]phenanthridines and Cytotoxic Hui-Yu Fang1

Chang-Yih Duh2
Constituents from Zanthoxylum integrifoliolum Ih-Sheng Chen3

Or
igi
na
l AbStK6st
Pa łermined łhrough specłral analyses. Among łhe isolałes, 7,8-
pe de- hydro-l-mełhoxyrułaecarpine, norcheleryłhrine,
oxycheleryłhr- ine, dihydrocheryłhrinylacełaldehyde, ð-
Yhree new alfialoids, 7,8-dehydro-l-mełhoxyrułaecarpine, iso- acełonyldihydrochelery- łhrine, l-hydroxyrułaecarpine, y-
decarine, and 8-demełhyloxycheleryłhrine, łogełher wiłh six- fagarine, sfiimmianine, (–)-ma- łairesinol, and canłhin-ð-one

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łeen finown compounds, norcheleryłhrine, oxycheleryłhrine, exhibiłed cyłołoxiciłies (ED50 val- ues < 4 µg/mL) againsł P-
decarine, dihydrocheryłhrinylacełaldehyde, ð- 388 or HY-S9 cell lines in vitro.
acełonyldihydro-
cheleryłhrine, rułaecarpine, l-hydroxyrułaecarpine, y-
fagarine, "ey woKdS
sfiimmianine, (–)-małairesinol, (–)-isoarcłigenin, (+)-epipinore- Zant7oxylum integrifoliolum · Rułaceae · rooł barfi · 7,8-
sinol, d-sesamin, lupeol, canłhin-ð-one, and dehydro- l-mełhoxyrułaecarpine · isodecarine · 8-
arnołłianamide demełhyloxychelery- łhrine · cyłołoxiciły
have been isolałed from łhe rooł barfi of Zant7oxylum

IfltKodUstEofl integrifoliolum has been found ło be one of łhe acłive

470 species. In- vesłigałion on chloroform-soluble fracłion of łhe
Zant7oxylum integrifoliolum (Merr.) Merr. (Rułaceae) is an rooł barfi of łhis species has led ło łhe isolałion of łhree
ever- green łree disłribułed in norłhern Philippine and on new alfialoids, 7,8-dehy- dro-l-mełhoxyrułaecarpine (7),
Lanyu Island in Yaiwan [l]. Iłs barfi was used by Ya-Mei isodecarine (€), and 8-demełhyl- oxycheleryłhrine (3), łogełher
aborigines as a folfi medicine ło łreał snafie-biłes and has wiłh sixłeen finown compounds (4
been a useful source for anłiplałeleł agenłs such as avicine – 79). Yhis paper describes łhe słrucłural elucidałion of 7–3
pseudocyanide and chelery- łhrine [S]. Isobułylamides, and łhe cyłołoxic acłiviłies of łhe isolałes.
benzo[c]phenanłhridines, quinolines, indolopyridoquinazoline,
and łriłerpenoids were łhe characłer- isłic consłiłuenłs of łhis
planł (fruił, barfi, rooł wood, and leaves) according ło M6teKE6lS 6fld MethodS
previous reporłs [3], [4], [5], [ð], [7], [8], [9], [l0]. Yhe
chemical consłiłuenłs and biological acłiviłies of iłs rooł GefleK6l expeKEmeflt6l pKosedUKeS
barfi have never been słudied. In our conłinuing słudies All melłing poinłs were dełermined on a Yanaco micro-
on łhe cyłołoxic consłiłuenłs of Formosan planłs, over l000 melłing poinł apparałus and were uncorrecłed. IR specłra
species have been screened for in vitro cyłołoxiciły ło dałe, (KBr) were łafien on a Perfiin Elmer sysłem S000 FY-IR
and Z. specłromełer. UV specłra
AffElE6tEofl
l Craduałe Insłiłułe of Pharmaceułical Yechnology, Yajen Insłiłułe of Yechnology, Pingłung, Yaiwan, R.O.C.
S Insłiłułe of Marine Resources, Nałional Sun Yał-sen Universiły, Kaohsiung, Yaiwan, R.O.C.
3 School of Pharmacy, Kaohsiung Medical Universiły, Kaohsiung, Yaiwan, R.O.C.
CoKKeSpofldeflse
Dr. J. J. Chen · Deparłmenł of Pharmacy · Yajen Insłiłułe of Yechnology · Pingłung · Yaiwan 907 · R.O.C. · Fax: +88ð-8-7ðS-5308 · E-mail:
Dr. I. S. Chen · School of Pharmacy · Kaohsiung Medical Universiły · Kaohsiung · Yaiwan 807 · R.O.C. · Fax: +88ð-7-3Sl-0ð83 · E-mail:
ReseEved July 7, S004 · Assepted November lð, S004
BEblEogK6phy
Planła Med S005; 7l: 470–475 · © Ceorg Yhieme Verlag KC Słułłgarł · New Yorfi DOI l0.l055/s-S005-8ð4l44
ISSN 003S-0943
 Or
igi
na
l
Pa
pe

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471

were obłained on a Jasco UV-S40 specłrophołomełer in EłOH. Republic of China.

EI- mass specłra were recorded on a VC Biołech Quałłro
50SS spec- łromełer. HR-EI-mass specłra were recorded on
a JEOL JMX-HX ll0 mass specłromełer. NMR specłra,
including COSY and NOESY experimenłs, were recorded on a
Varian Uniły 400 and Varian In- ova 500 specłromełer ał
400 or 500 MHz, wiłh chemical shifłs are given in ppm (6)
wiłh YMS as inłernal słandard. Silica gel (ð0 – S30, S30 –
400 mesh) (Mercfi) was used for CC, and silica gel ð0 F-
S54 (Mercfi) for YLC and preparałive YLC. Opłical roła-
łions were measured using a Jasco DIP-370 polarimełer in
CHCl3.

Pl6flt m6teKE6l
Yhe rooł barfi of Z. integrifoliolum was collecłed from Lanyu
Is- land, Yaiłung Counły, Yaiwan, in July S00S and idenłified
by Dr.
I. S. Chen. A voucher specimen (Chen 55S8) was
deposiłed in łhe herbarium of łhe School of Pharmacy,
Kaohsiung Medical Universiły, Kaohsiung, Yaiwan,
Chen J-J et al. New Indolopyridoquinazoline, Benzo[c]phenanthridines … Planta Med 2005; 71: 470 – 475
ExtK6stEofl 6fld ESol6tEofl
Yhe dried rooł barfi (l.ð fig) was exłracłed wiłh cold
MeOH and łhe exłracł concenłrałed under reduced
pressure. Yhe MeOH ex- łracł (lðl g), when parłiłioned
bełween HSO-CHCl3 (l : l), afford- ed a CHCl3-soluble
fracłion (fr. A, ð0 g). Fr. A (ð0 g) was chroma-
łographed on silica gel (S.ð fig), elułing wiłh n-hexane,
gradually increasing łhe polariły wiłh EłOAc and MeOH ło
give l7 frs: fr. Al (ð000 mL, n-hexane), fr. AS – A3
(each ð000 mL, n-hexane- EłOAc, l0 : l), fr. A4 – A5
(each 3000 mL, n-hexane-EłOAc, 5 : l), fr. Að – A8 (each
mg) (Rf = 0.79). Fr. Að – ð (lSS mg) was furłher purified by
pre- parałive YLC (n-hexane-EłOAc, l0 : l) ło obłain 77
(S3 mg) (Rf = 0.3l). Fr. Að – 7 (S0S mg) was furłher
purified by prepara- łive YLC (CHSClS-EłOAc, 30 : l) ło obłain
70 (ð.5 mg) (Rf = 0.S9). Fr. Að – 8 (l73 mg) was furłher
purified by preparałive YLC (CHCl3-EłOAc, S5 : l) ło obłain 7
(l.4 mg) (Rf = 0.34). Fr. A8 (4.l
g) was chromałographed on silica gel (l3S g) elułing wiłh n-
hex- ane-EłOAc (4 : l) ło give lS frs (each 500 mL, fr. A8 – l
– fr. A8 – lS). Fr. A8 – 4 (l88 mg) was furłher purified by
preparałive YLC (n-hexane-EłOAc, 5 : 3) ło obłain € (l.S mg)
(Rf = 0.5S). Fr. A8 – 5 (l34 mg) was furłher purified by
preparałive YLC (n-hexane- EłOAc, 5 : 3) ło obłain ð (5.S
mg) (Rf = 0.50) and 73 (4.5 mg) (Rf = 0.l7). Fr. A8 – ð
(l55 mg) was furłher purified by prepara- łive YLC (CHCl3-
Or MeOH, S0 : l) ło obłain 74 (3.l mg) (Rf = 0.ðl) and 75 (3.7
igi mg) (Rf = 0.3S). Fr. Al0 (3.l g) was chromałographed on silica
na gel (l05 g) elułing wiłh CHSClS-acełone (l0 : l) ło give 8
l 934 cm-l (OCHSO); EI-MS: m/s (rel. inł.) = 349 (M+, l00), 334
frs (each 400 mL, fr. Al0 – l – fr. Al0 – 8). Fr. Al0 – S (88
Pa mg) was furłher purified by preparałive YLC (CHCl3-acełone,
pe S0 : l) ło ob- łain 7€ (7.3 mg) (Rf = 0.5ð). Fr. Al0 – 3 (l0S
mg) was furłher pur- ified by preparałive YLC (CHCl3-MeOH,
35 : l) ło obłain 79 (4.S mg) (Rf = 0.38). Fr. All (3.4 g) was
chromałographed on silica gel (ll8 g) elułing wiłh CHSClS-
acełone (l0 : l) ło give l0 frs (each 500 mL, fr. All – l – fr.
All – l0). Fr. All – S (llS mg) was furłher purified by
preparałive YLC (CHSClS-acełone, l0 : l) ło ob- łain 7 (S.S mg)
(Rf = 0.89). Fr. All – 4 (98 mg) was furłher puri- fied by
preparałive YLC (CHSClS-acełone, l0 : l) ło obłain 77 (8.3
mg) (Rf = 0.ðl). Fr. All – ð (l45 mg) was furłher purified by
pre-
parałive YLC (CHSClS-acełone, 5 : l) ło obłain 78 (8.3 mg)
472
(Rf = 0.ð3). Fr. Al3 (S.85 g) was chromałographed on silica gel
(95 g) elułing wiłh CHSClS-MeOH (30 : l) ło give ð frs (each
300 mL, fr. Al3 – l – fr. Al3 – ð). Fr. Al3 – S (lS9 mg) was
furłher pur- ified by preparałive YLC (CHCl3-MeOH, 30 : l) ło
obłain 4 (3.ð mg) (Rf = 0.87). Fr. Al3 – 5 (l4ð mg) was
furłher purified by pre- parałive YLC (CHCl3-MeOH, S0 : l)
ło obłain 5 (4.S mg) (Rf = 0.5S). Fr. Al3 – ð (l0ð mg) was
furłher purified by prepara- łive YLC (CHCl3-MeOH, S5 : l) ło
obłain 3 (4.S mg) (Rf = 0.33). Fr. Al4 (3.8 g) was
chromałographed on silica gel (lS4 g) elułing wiłh CHCl3-
MeOH (l0 : l) ło give 7 frs (each l50 mL, fr. Al4 – l – fr. Al4
– 7). Fr. Al4 – S (98 mg) was furłher purified by preparałive
YLC (CHCl3-acełone, l0 : l) ło obłain 8 (S.5 mg) (Rf = 0.8S).
ISol6teS
Chen J-J et al. New Indolopyridoquinazoline, Benzo[c]phenanthridines … Planta Med 2005; 71: 470 – 475
3000 mL, n-hexane-EłOAc, l : l), fr. A9 – Al0 (each ð000 mL,
n-hexane-EłOAc, l : S), fr. All – AlS (each 5000 mL, EłOAc),
fr. Al3 – Al4 (9000 mL, EłOAc-MeOH, l0 : l), fr. Al5 (9000
mL, EłOAc-MeOH, 5 : l), fr. Alð (5000 mL, EłOAc-MeOH,
l : l), fr. Al7 (5000 mL, MeOH). Fr. Að (S.3 g) was
chromało- graphed on silica gel (78 g) elułing wiłh n-
hexane-EłOAc (l0 : l) ło give 9 frs (each 400 mL, fr. Að – l –
fr. Að – 9). Fr. Að – 4 (l74 mg) was furłher purified by
preparałive YLC (CHSClS-EłOAc, 30 : l) ło obłain 9 (ð.5 mg) (Rf
= 0.48). Fr. Að – 5 (l58 mg) was furłher pur- ified by
preparałive YLC (n-hexane-EłOAc, l : l) ło obłain 7ð (5.7
l034, 94S cm–l (OCHSO); EI-MS: m/s (rel. inł.) = 3l9 (M+,
l00), 304 (35), S7ð (S7), S34 (S3), S04 (8), l37 (l4), lS9 (ll),
77(l0);
HR-EI-MS: m/s found: 3l9.084S [M]+, calcd. for Cl9Hl3NO4
: 3l9.0845. lH- NMR (CDCl3, 500 MHz): 6 = 4.0ð (3H, s, OMe-
8),
5.95 (lH, br s, OH-7), ð.l4 (SH, s, OCHSO), 7.S8 (lH, s, H-l), 7.59
(lH, d, J = 9.0 Hz, H-9), 7.8ð (lH, d, J = 9.0 Hz, H-lS), 8.3ð (lH, d,
J = 9.0 Hz, H-l0), 8.37 (lH, d, J = 9.0 Hz, H-ll), 8.7S (lH,
s, H-4), 9.7l (lH, s, H-ð).
8-Demet7yloxyc7eleryt7rine (3): Colorless needles from CHCl3-
MeOH, m. p. SS4 – SSð °C. UV (EłOH): Zmax (log s) = S07
(4.Sl), S40 (4.S4), S80 (sh, 4.S4), S87 (4.33), 3S7 (sh,
3.8l), 375 (sh,
3.55); (KOH) S09 (4.S3), S5l (4.Sl), 30S (4.33), 340 (sh, 3.8l),
38S nm (sh, 3.58); IR: vmax = 3S7ð (OH), lð44 (C = O), l03ð,
(4ð), 3S0 (S3), 3l9 (4S), S75 (S5), l90 (33), lð3 (SS), 95 (3l),
55
(37); HR-EI-MS: m/s found: 349.0950 [M] +, calcd. for
CS0Hl5NO5 : 349.095l; lH-NMR (CDCl3, 400 MHz): 6 = 3.9l
(3H, s, N-Me), 4.0l (3H, s, OMe-7), 5.98 (lH, br s, OH-8), ð.ll
(SH, s,
OCHSO), 7.l7 (lH, s, H-l), 7.40 (lH, d, J = 8.5 Hz, H-9), 7.55 (lH,
Downloaded by: University of Pittsburgh. Copyrighted material.
s, H-4), 7.5ð (lH, d, J = 8.5 Hz, H-lS), 7.98 (lH, d, J = 8.5
Hz, H- l0), 8.0l (lH, d, J = 8.5 Hz, H-ll).
Di7ydroc7eryt7rinylacetalde7yde (7): [a]DS5: –lSS° (c 0.ll, CHCl
3 ).
6-Acetonyldi7ydroc7eleryt7rine (8): [a]S5
D : –l3l° (c 0.l0, CHCl 3)
{lił. [a]DS3: –l35° [ll]}.
(–)-Matairesinol (73): [a]DS5: –34.5° (c 0.l3, CHCl ) {lił. [a]S0: –
3
33.3° [lS]}. D
(–)-Isoarctigenin (74): [a]DS5: –S7.9° (c 0.ll, CHCl
3 ) {lił. [a]S5: –
S7.ð° [l3]}. D
(+)-epi-Pinoresinol (75): [a]S5 SS
D : + llS° (c 0.lS, CHCl3 ) {lił. [a] :
D
+ l03° [l4]}.
d-Sesamin (7ð): [a]S5 ) {lił. [a]S0: + ð8.S°
D : + ð7.5° (c 0.lS, CHCl 3
[l5]}. D
Lupeol (77): [a]S5: + S8.l° (c 0.l5, CHCl ) {lił. [a]SS: + S8.3° [7]}.
7,8-De7ydro-l-met7oxyrutaecarpine (7): Yellowish needles from
CHCl3-MeOH, m. p. S87 – S89 °C. UV (EłOH): Zmax (log s) = Sl0
(4.ð4), S48 (sh, 4.48), S85 (4.S7), 3Sð (4.4S), 345 (4.39), D 3 D

3ð4
(4.4ð), 385 nm (4.4S); IR: vmax = 3SðS (br, NH), lð87
cm–l (C = O); EI-MS: m/s (rel. inł.) = 3l5 (M+, l00), Sl4
(97), 300
(S0), S84 (3l), S7S (lS), S5ð (l0), SSl (8), l5S (l5), lS3 (l5),
ll5
(l0); HR-EI-MS: m/s found: 3l5.l009 [M]+, calcd.
for Cl9Hl3N3OS : 3l5.l005; lH-NMR (CDCl3, 400 MHz): 6 =
4.0ð (3H, s, OMe-l), 7.S7 (lH, dd, J = 8.0, l.S Hz, H-S), 7.34
(lH, łd, J = 8.0,
l.S Hz, H-l0), 7.4S (lH, ł, J = 8.0 Hz, H-3), 7.4ð (lH, łd, J =
8.0, l.S
Hz, H-ll), 7.53 (lH, dd, J = 8.0, l.S Hz, H-lS), 7.57 (lH, d, J =
8.0
Hz, H-8), 8.0S (lH, dd, J = 8.0, l.S Hz, H-9), 8.09 (lH, dd, J =
8.0,
l.S Hz, H-4), 8.7ð (lH, d, J = 8.0 Hz, H-7), l0.37 (lH, br s, NH,
ex-
changeable wiłh DSO).
Isodecarine (€): Colorless needles from CHCl3-MeOH, m. p. SS5
– SS7 °C. UV (EłOH): Zmax (log s) = S43 (4.54), S57 (sh,
4.53), S75 (4.59), 3S0 (sh, 4.0l), 380 nm (sh, 3.35); IR:
vmax = 3387 (OH),
in łriplicałe. Afłer łhree days of incubałion, P-388 cells
were en- umerałed wiłh MYY.
Yo measure łhe cyłołoxic acłiviłies of purified compounds
againsł HY-S9 cells, each cell line was iniłiałed ał l000 cells/
well in 9ð-well microłiłer plałes. Eighł concenłrałions
(łripli- całe) of łesł compounds encompassing a lS8-fold
range were added ło each cell line. HY-S9 cells were
enumerałed using MYY afłer exposure ło łesł compounds for ð
days, respecłively. Fifły µL of l mg/mL MYY were added ło
each well, and plałes were incu- bałed ał 37 °C for a
furłher 5 h. Formazan crysłals were redis- solved in DMSO
(Mercfi) for l0 min wiłh shafiing, and łhe plałe was read
immediałely on a microłiłer plałe reader (Dynałech) ał a
wavelengłh of 540 nm. Yhe ED50 was defined as łhe
concen- łrałion of łhe łesł compound resulłing in a 50 %
reducłion of ab- sorbance compared ło unłreałed cells in
łhe MYY assay. Yhe as- says above repeałed łhree łimes.
CytotoxEsEty 6SS6y
P-388 cells were fiindly provided by Prof. J. M. Pezzuło, of
łhe Universiły of Illinois ał Chicago; HY-S9 (human colon
carcinoma) was purchased from American Yype Culłure
Collecłion.
P-388 cells were culłured in Fisher's medium
supplemenłed wiłh l0 % heał-inacłivałed (5ð °C for 30 min)
fełal calf serum (FCS). HY-S9 cells were mainłained in
Rosewell Parfi Memorial Insłiłułe (RPMI) lð40 medium
conłaining l0 % heał-inacłivałed FCS. All cell lines were
mainłained in an incubałor ał 37 °C in hu- midified air
conłaining 5 % COS.
Yhe cyłołoxic acłiviłies of compounds againsł P-388 and HY-
S9 were assayed by a modificałion of łhe MYY [3-(4,5-
dimełhylłhia- zole-S-yl)-S,5-diphenylłełrazolium
bromide]colorimełric mełh- od [lð]. For P-388 cells, S00 µL
culłures were esłablished ał l500 cells/well in 9ð-well łissue
culłure plałes (Falcon). Compounds were dispensed ło
esłablished culłures ał eighł concenłrałions
7.S7 (dd, J = 8.0, l.S Hz), 7.4S (ł, J = 8.0 Hz), 8.09 (dd, J = 8.0, l.S
Hz) due ło H-S, H-3, and H-4, respec- łively; four mułually
coupling prołons ał 6 = 8.0S (dd, J = 8.0, l.S Hz), 7.34 (łd, J =
8.0, l.S Hz), 7.4ð (łd, J = 8.0, l.S Hz), 7.53
(dd, J = 8.0, l.S Hz) due ło H-9, H-l0, H-ll, and H-lS, respecłive-
ly. In addiłion, łhe presence of a mełhoxy and an NH group
was revealed by łhe signals ał 6 = 4.0ð (3H, s, OMe-l) and 6 =
l0.37 (lH, br s, exchangeable wiłh DSO), respecłively. According
ło łhe above dała, łhe słrucłure of 7 was elucidałed as 7,8-
dehydro-l- mełhoxyrułaecarpine, which was furłher confirmed
by lH-lH COSY and NOESY experimenłs (Fig. 7).
Isodecarine (€) was isolałed as colorless needles. Yhe HR-EI-MS
of € gave an [M]+ ion ał m/s = 3l9.084S (calcd. 3l9.0845), consis-
Fig. 1 Significant NOESY
correlations of 1.

ReSUltS 6fld DESsUSSEofl

7,8-Dehydro-l-mełhoxyrułaecarpine (7) was isolałed as

yellow- ish needles. Yhe EI-MS afforded łhe posiłive ion
[M]+ ał m/s = 3l5, implying a molecular formula of
Cl9Hl3N3OS, which was confirmed by łhe HR-EI-MS. Yhe UV
absorpłions ał Sl0, S48 sh, S85, 3Sð, 345, 3ð4, and 385 nm
were similar ło łhose of 7,8-de- hydrorułaecarpine [l7], and
suggesłed łhe presence of a 7,8-de-
hydroindolopyridoquinazoline nucleus. Yhe presence of NH
and carbonyl groups in łhe molecule of 7 was revealed by
łhe bands ał 3SðS and lð87 cm-l, respecłively, in łhe IR
specłrum. Yhe lH- NMR specłrum of 7 showed łhe presence
of nine aromałic pro- łons including łwo ort7o-coupling
prołons ał 6 = 8.7ð and 7.57 (J = 8.0 Hz) due ło H-7 and
H-8, respecłively; łhree mułually coupling prołons ał 6 =

Chen J-J et al. New Indolopyridoquinazoline, Benzo[c]phenanthridines … Planta Med 2005; 71: 470 – 475
łenł wiłh a molecular formula of Cl9Hl3NO4. Yhe UV
absorpłions of € ał S43, S57 sh, S75, 3S0 sh, and
380 sh nm were similar ło łhose of norcheleryłhrine
[l8] and suggesłed łhe presence of a S,3,7,8-
łełraoxygenałed benzo[c]phenanłhridine sfielełon. Yhe IR
specłrum of € showed łhe hydroxy band ał 3387 cm–l
and łhe mełhylenedioxy bands ał l034, 94S cm–l. Yhe
l
H-NMR spec- łrum of € was similar ło łhał of
norcheleryłhrine (4), also isolałed in łhis słudy, excepł
łhał OH-7 [6 = 5.95 (lH, br s)]of € replaced OMe-7 [6
= 4.l3 (3H, s)] of norcheleryłhrine (4). According ło łhe
above dała, łhe słrucłure of € was elucidałed as 7-
hydroxy- 8-mełhoxy-S,3-
mełhylenedioxybenzo[c]phenanłhridine, which was furłher
confirmed by lH-lH COSY and NOESY experimenłs (Fig.
€). Yhis is łhe firsł reporł of łhe occurrence of € in a
nałural source, alłhough ił has been synłhesized and
named isodecarine by Yolfiachev eł al. [l9].
8-O-Demełhyloxycheleryłhrine (3) was isolałed as
colorless nee- dles. Iłs molecular formula was
esłablished as CS0Hl5NO5 by EI- MS ([M]+, m/s = 349)
and HR-EI-MS specłromełry (found: 349.0950, calcd.:
revealed by łhe band ał 3S7ð cm–l in łhe IR specłrum of 3,
which was confirmed by łhe signal ał 6 = 5.98 (br s, DSO
ex- changeable, OH-8) in łhe lH-NMR specłrum. Yhe IR of 3
also showed łhe conjugałed carbonyl absorpłion ał lð44 cm–l
and łhe mełhylenedioxy bands ał l03ð, 934 cm–l. Yhe lH-
NMR spec- łrum of 3 was similar ło łhał of 5 [S0] excepł
łhał OH-8 (6 = 5.98) of 3 replaced OMe-8 (6 = 3.99) of
5. According ło łhe above dała, łhe słrucłure of 3 was
elucidałed as 8-demełhyloxy- cheleryłhrine, which was
furłher confirmed by lH-lH COSY and NOESY experimenłs
(Fig. 3). Yhis is łhe firsł reporł of łhe occur- rence of 3 in a
nałural source, alłhough ił has been synłhesized by
Ishifiawa eł al. [Sl].
Fig. 2 Significant
NOESY correlations
of 2. Or
igi
na
l
Pa
pe

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349.095l). Yhe UV absorpłions of 3 ał S40, S80 sh, S87,
3S7 sh, and 375 sh nm were similar ło łhose of
oxychelery- łhrine (5) [S0], also isolałed in łhis słudy,
and łhe occurrence of a bałhochromic shifł in alfialine
Fig. 3 Significant
solułion suggesłed łhe presence of a phenolic S,3,7,8-
NOESY correlations
łełraoxygenałed benzo[c]phenanłhridin-ð- one sfielełon. of 3.
Yhe presence of a hydroxy group in łhe molecule was
Yhe finown isolałes including five benzo[c]phenanłhridine values rang- ing from l.l5 ło l.80 µg/mL againsł łhe P-388 cell
alfia- loids, norcheleryłhrine (4) [SS], oxycheleryłhrine (5) [S0], line. Sfiimmia- nine (7€) exhibiłed łhe mosł połenł cyłołoxiciły,
decar- ine (ð) [SS], dihydrocheryłhrinylacełaldehyde (7) wiłh an ED50 va- lue = 0.l5 µg/mL againsł łhe HY-S9 cell line.
[S3], ð-aceło- nyldihydrocheleryłhrine (8) [S3], łwo
indolopyridoquinazoline alfialoids, rułaecarpine (9) [9], l-
hydroxyrułaecarpine (70) [9], łwo furoquinoline alfialoids, y- 473
fagarine (77) [SS], sfiimmianine (7€) [SS], four lignans, (-)-
małairesinol (73) [S4], (–)-isoarcłigen-
in (74) [l3], (+)-epipinoresinol (75) [S5], d-sesamin (7ð) [9], a
łri- łerpenoid, lupeol (77) [7], canłhin-ð-one (78) [Sð],
arnołłiana- mide (79) [SS], which were readily idenłified
by comparison of physical and specłroscopic dała (UV, IR, lH-
NMR, [a]D, and mass specłromełry dała) wiłh corresponding
aułhenłic samples or lił- erałure values.

Or Yhe cyłołoxic effecłs of łhe isolałes from łhe rooł barfi of

igi Formosan
na Z. integrifoliolum were łesłed in vitro againsł P-388 and HY-
l S9 cell lines. Yhe cyłołoxiciły dała are shown in Yable 7. Yhe
Pa clinically ap- plied anłicancer agenł, miłhramycin, was used as
pe łhe reference compound. An ED50 value of ≤ 4.0 µg/ml is
considered ło represenł significanł cyłołoxiciły [S7]. 7,8-
Dehydro-l-mełhoxyrułaecarpine (7), norcheleryłhrine (4),
oxycheleryłhrine (5), dihydrocheryłhri- nylacełaldehyde (7), ð-
acełonyldihydrocheleryłhrine (8), l-hydro- xyrułaecarpine (70),
y-fagarine (77), sfiimmianine (7€), (–)-małair- esinol (73), and
canłhin-ð-one (78) exhibiłed cyłołoxiciły (ED50 values < 4
µg/mL) againsł łhe P-388 cell line. Dihydrocheryłhrinyl-
acełaldehyde (7), (–)-małairesinol (73), and canłhin-ð-one
(78) are łhe mosł cyłołoxic among łhe isolałes, wiłh ED50

Chen J-J et al. New Indolopyridoquinazoline, Benzo[c]phenanthridines … Planta Med 2005; 71: 470 – 475
AsflflowledgemefltS l993; 4ð: llð5 – 73
3
Ysai IL, Lin WY, Huang MW, Chen YL, Chen IS. N-
Isobułylamides and bułyrolacłone from łhe fruiłs of
Yhis worfi was supporłed by granłs from łhe Nałional
Zant7oxylum integrifoliolum. Helv Chim Acła S00l; 84: 830 – 3
Science Council of łhe Republic of China (NSC 9S-S3S0- 4
Chen IS, Chen YL, Lin WY, Ysai IL, Chen YC. Isobułylamides
B-lS7-004) and łhe Yajen Insłiłułe of Yechnology from łhe fruił of Zant7oxylum integrifoliolum. Phyłochemisłry l999;
(Yajen-9S 04ð). 5S: 357 – ð0

Downloaded by: University of Pittsburgh. Copyrighted material.

5
Chen IS, Chen YL, Chang YL, Yeng CM, Lin WY. Chemical
consłiłuenłs and biological acłiviłies of łhe fruił of Zant7oxylum
integrifoliolum.J Nał Prod l999; ðS: 833 – 7
RefeKeflseS ð
Liu SL, Ysai IL, Ishifiawa Y, Harayama Y, Chen IS. Bishordeninyl
łerpene alfialoids from Zant7oxylum integrifoliolum. J Chin Chem
l
Chang CE, Harłley YC. Rułaceae in Flora of Yaiwan. Snd Soc S000; 47: 57l–4
7
ediłion. Ediłor- ial Commiłłee of łhe Flora of Yaiwan, Yaipei, Ishii H, Chen IS, Afiaifie M, Ishifiawa Y, Lu SY. Słudies on łhe
Yaiwan: l993; Vol. 3: 5l0 – 44 chemical consłiłuenłs of Rułaceous planłs. XLIV. Yhe chemical
S
Ko FN, Hsiao C, Chen IS, Wu SJ, Yeng CM. Inhibiłion of consłiłuenłs of Xant7oxylum integrifoliolum (Merr.) Merr. (Fagara
collagen-in- duced plałeleł aggregałion and adhesion by a integrifoliola Merr.)
pseudocyanide deriva- łive of avicine isolałed from (l) Yhe chemical consłiłuenłs of łhe rooł wood. Yafiugafiu
Zant7oxylum integrifoliolum Merr. Bio- chem Pharmacol \asshi l98S; l0S: l8S – 95

Table 1 Cytotoxic effects of compounds isolated from the root bark of Zanthoxylum integrifoliolum against P-388 and HT-29 cell lines a

Compound ED50 [µg/mL]

P-388 HT-29

Mithramycinb 0.06 ± 0.01 0.08 ± 0.01

CHCl3-soluble fraction 14.1 ± 1.3 18.2 ± 1.6
7,8-Dehydro-1-methoxyrutaecarpine (1) 3.88 ± 0.42 8.54 ± 0.82
Isodecarine (2) 4.71 ± 0.56 10.3 ± 1.1
8-Demethyloxychelerythrine (3) 4.41 ± 0.52 6.17 ± 0.70
Norchelerythrine (4) 2.32 ± 0.23 6.56 ± 0.56
Oxychelerythrine (5) 3.25 ± 0.44 5.48 ± 0.46
Decarine (6) 4.27 ± 0.56 8.79 ± 0.73
Dihydrocherythrinylacetaldehyde (7) 1.15 ± 0.22 4.08 ± 0.34
6-Acetonyldihydrochelerythrine (8) 3.77 ± 0.45 5.39 ± 0.42
Rutaecarpine (9) 10.5 ± 0.9 33.6 ± 2.9
1-Hydroxyrutaecarpine (10) 3.72 ± 0.36 7.44 ± 0.86
y-Fagarine (11) 3.58 ± 0.39 22.5 ± 2.6
Skimmianine (12) 2.62 ± 0.28 0.15 ± 0.02
(–)-Matairesinol (13) 1.80 ± 0.16 4.68 ± 0.41
(–)-Isoarctigenin (14) 5.19 ± 0.65 4.33 ± 0.39
(–)-Epipinoresinol (15) 17.0 ± 1.9 5.45 ± 0.50
d-Sesamin (16) 25.0 ± 2.9 5.74 ± 0.55
Lupeol (17) 60.2 ± 8.2 70.2 ± 8.6
Canthin-6-one (18) 1.67 ± 0.12 6.24 ± 0.57
Arnottianamide (19) 7.28 ± 0.63 4.74 ± 0.41

a
An ED50 value of ≤ 4.0 µg/ml (for a pure compound) or ED50 ≤ 20.0 µg/mL (for a crude extract) is considered represent a significant cytotoxicity [23].
b
Mithramycin was used as a positive control.
8
Jen CM, Ysai IL, Horng DJ, Chen IS. Isolałion and słrucłure conjugałe addiłions. Enanłioselecłive synłhesis of bułyrolacłone
dełermina- łion of łwo new compounds from Zant7oxylum nałural pro- ducłs: (–)-enłerolacłone, (–)-arcłigenin, (–)-isoarcłigenin,
integrifoliolum. J Nał Prod l993; 5ð: S0l9 – Sl (–)-nephros- łeranic acid, and (–)-roccellaric acid. J Org Chem S00S; ð7:
9
Sheen WS, Ysai IL, Yeng CM, Ko FN, Chen IS. l738 – 45
l4
Indolopyridoquinazoline alfialoids wiłh anłiplałeleł aggregałion Nishibe S, Ysufiamoło H, Hisada S. Effecłs of O-mełhylałion and O-glu-
acłiviły from Zant7oxylum integrifoliolum. Planła Medica l99ð; cosylałion on carbon-l3 nuclear magnełic resonance chemical shifłs
ðS: l75 – ð of małairesinol, (+)-pinoresinol and (+)-epipinoresinol. Chem. Pharm.
l0
Liu SL, Ysai IL, Ishifiawa Y, Harayama Y, Chen IS. Bishordeninyl Bull. l984; 3S: 4ð53 – 7
l5
łerpene alfialoids from Zant7oxylum integrifoliolum. J Chin Chem Banerji A, Sarfiar M, Dałła R, Sengupła P, Abraham K. Amides from
Soc S000; 47: 57l–4 Piper brac7ystac7yum and Piper retrofractum. Phyłochemisłry S00S;
ll
Chaaib F, Queiroz EF, Ndjofio K, Diallo D, Hosłełłmann K. Anłifungal 59: 897 – 90l
lð
and anłioxidanł compounds from łhe rooł barfi of Fagara Mosmann Y. Rapid colorimełric assay for cellular growłh and
sant7oxyloides. survival: applicałion ło proliferałion and cyłołoxiciły assays. J
Planła Medica S003; ð9: 3lð – S0 Immunol Mełh l983; ð5: 55 – ð3
l7
lS
Cozler B, Arar C, Cozler Y, Hesse M. Isodaurinol, an Ifiuła A, Nafiamura Y, Urabe H. Indolopyridoquinazoline, furoquinoline
arylnaphłhalene lignan from Haplop7yllum cappadocicum. and canłhinone łype alfialoids from P7ellodendron amurense callus
Phyłochemisłry l99S; 3l: łissues. Phyłochemisłry l998; 48: S85 – 9l
S473–5
l3
Sibi MP, Liu P, Ji J, Hajra S, Chen J-X. Free-radical-mediałed
Chen J-J et al. New Indolopyridoquinazoline, Benzo[c]phenanthridines … Planta Med 2005; 71: 470 – 475
l8
Ceen CR, Mann IS, Mullane MV, McKillop A. A versałile
synłhesis of fully aromałic benzo[c]phenanłhridine alfialoids.
Yełrahedron l998; 54: 9875 – 94
l9
Yolfiachev ON, Savina AA, Sheichenfio VI, Prosfiudina VV.
On łhe mechanism of łhermolysis of łhe
benzo[c]phenanłhridine alfialoid cheleryłhrine. Pharm Chem
J l999; 33: 3S3 – 5
S0
Hanaofia M, Mołonishi Y, Mufiai C. Chemical łransformałion
of proło- berberines. Parł 9. A biomimełic synłhesis of
oxycheleryłhrine, dihy- drocheleryłhrine, and cheleryłhrine
from berberine. J Chem Soc Per- fiin Yrans l, l98ð: SS53 – ð
Sl
Ishifiawa Y, Yafiami A, Abe M, Chen IS, Harayama Y, Ishii
H. Concise synłhesis of łhe oxo derivałives of
benzo[c]phenanłhridine bases by N-deformylałed
cyclizałion based on Vilsmeier-Haacfi Reacłion. Chem
Pharm Bull l995; 43: 7ðð – 70
SS
Chen IS, Ysai IW, Yeng CM, Chen JJ, Chang YL, Ko FN, Lu MC,
Pezzuło JM. Pyranoquinoline alfialoids from Zant7oxylum
simulans. Phyłochemis- łry l997; 4ð: 5S5 – 9
S3
Decaudain N, Kunesch N, Poisson J. Alcaloïdes de Or
Zant7oxylum tsi7animposa. Phyłochemisłry l974; l3: 505 – 7 igi
S4
Chen IS, Lin YC, Ysai IL, Yeng CM, Ko FN, Ishifiawa Y, Ishii H.
na
Coumarins and anłi-plałeleł aggregałion consłiłuenłs from
Zant7oxylum sc7inifolium. Phyłochemisłry l995; 39: l09l – 7 l
S5
Rahman MMA, Dewicfi PM, Jacfison DE, Lucas JA. Lignans of Pa
Forsyt7ia intermedia. Phyłochemisłry l990; S9: l97l – 80 pe

Downloaded by: University of Pittsburgh. Copyrighted material.

Sð
Cain M, Campos O, Cuzman F, Coofi JM. Selenium dioxide
oxidałions in łhe indole area. Synłhesis of þ-carboline
alfialoids. J Am Chem Soc l983; l05: 907 – l3
S7
Ceran RI, Creenberg NH, Macdonald MM, Schumacher AM,
Abbołł BJ. Prołocols for screening chemical agenłs and nałural
producłs againsł animal łumors and ołher biological sysłems.
Cancer Chemołher Rep, Parł 3 l97S; 3: l03

475

Chen J-J et al. New Indolopyridoquinazoline, Benzo[c]phenanthridines … Planta Med 2005; 71: 470 – 475