The first total syntheses of (±)-norphoebine, dehydrophoebine, oxophoebine, dehydrocrebanine, oxocrebanine and uthongine and their cytotoxicity against three human cancer cell lines

Article abstract of DOI:10.1080/10286020.2016.1177025

The first total syntheses of (±)-norphoebine, dehydrophoebine, oxophoebine, dehydrocrebanine, oxocrebanine and uthongine have been achieved. The crucial step involved the formation of ring C by a microwave-assisted direct biaryl coupling to produce the aporphine skeleton in high yields. The synthetic alkaloids were evaluated for their cytotoxicity against three human cancer cell lines MCF7, KB and NCI-H187. The results showed that uthongine was the best candidate of the series and it exhibited cytotoxicity against a human breast cancer MCF7 line with an IC₅₀?=?3.05?μM

Full text of DOI:10.1080/10286020.2016.1177025

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
The first total syntheses of (±)-norphoebine,
dehydrophoebine, oxophoebine,
dehydrocrebanine, oxocrebanine and uthongine
and their cytotoxicity against three human cancer
cell lines
Kanok-on Rayanil, Cholthicha Prempree & Surachai Nimgirawath
To cite this article: Kanok-on Rayanil, Cholthicha Prempree & Surachai Nimgirawath
(2016): The first total syntheses of (±)-norphoebine, dehydrophoebine, oxophoebine,
dehydrocrebanine, oxocrebanine and uthongine and their cytotoxicity against
three human cancer cell lines, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2016.1177025
To link to this article: http://dx.doi.org/10.1080/10286020.2016.1177025
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Date: 10 May 2016, At: 00:10

Journal of asian natural Products research, 2016
http://dx.doi.org/10.1080/10286020.2016.1177025
The first total syntheses of ( )-norphoebine,
dehydrophoebine, oxophoebine, dehydrocrebanine,
oxocrebanine and uthongine and their cytotoxicity against
three human cancer cell lines
Kanok-on Rayanil, Cholthicha Prempree and Surachai Nimgirawath
faculty of science, department of chemistry, silpakorn university, nakorn Pathom, thailand
ABSTRACT
ARTICLE HISTORY
received 17 July 2015
accepted 6 april 2016
The first total syntheses of ( )-norphoebine, dehydrophoebine,
oxophoebine, dehydrocrebanine, oxocrebanine and uthongine have
been achieved. The crucial step involved the formation of ring C by a
microwave-assisted direct biaryl coupling to produce the aporphine
skeleton in high yields. The synthetic alkaloids were evaluated for
their cytotoxicity against three human cancer cell lines MCF7, KB and
NCI-H187. The results showed that uthongine was the best candidate
of the series and it exhibited cytotoxicity against a human breast
cancer MCF7 line with an IC50 = 3.05 μM
KEYWORDS
oxophoebine; oxocrebanine;
uthongine; cytotoxic activity;
total syntheses
1. Introduction
e aporphine alkaloids possess a tetracyclic skeleton derived by bonding between rings
A and D of the benzylisoquinoline structure. More than 500 aporphine alkaloids have
been isolated from a large number of plant families and they play a crucial role in the field
of drug discovery research due to a broad range of pharmacological properties including
cytotoxic [1], antioxidant [2], antimalarial [3], antimicrobial [2,4], acetylcholinesterase
inhibiting [5], anti-HIV [6], antiparkinsonian [7] activities and anti-platelet aggregation
[8]. Unfortunately, natural aporphines occur in extremely minute quantities, making the
study of their biological activity an impossible undertaking. us, a number of syntheses
of aporphine alkaloids and their derivatives have been undertaken in order to study the
structure activity relationship and also to discover new drugs [5,7,9].
As part of our search for new anticancer drugs from natural sources, phoebine and
crebanine-type natural alkaloids have attracted our attention because several compounds
in these series showed promising anticancer activity. Oxophoebine has been identified as a
DNA topoisomerase inhibitor [10] and has been shown to have cytotoxicity against GLC-
82 and HCT cell lines [11]. Dehydrocrebanine was shown to have strong activity against
several cancer cell lines (HL-60, HUCCA-1, BC and MOLT-3) [4] whereas crebanine was
CONTACT Kanok-on rayanil
rayanil_k@su.ac.th
supplemental data for this article can be accessed http://dx.doi.org/10.1080/10286020.2016.1177025.
© 2016 informa uK limited, trading as taylor & francis Group

2
K.-o. RayaNIl eT al.
found to significantly inhibit the proliferation of human leukemic cells (HL-60, U937 and
K526), human fibrosarcoma cells (HT1080) and cervix cancer cell lines (KB-3-1 and KB-V1)
[12,13]. However, some alkaloids in the phoebine and crebanine series isolated from natural
sources still have not been evaluated for their cytotoxicity. In order to explore their struc-
ture activity relationship, we decided to undertake the first total syntheses of six natural
aporphine alkaloids; ( )-norphoebine, dehydrophoebine, oxophoebine, dehydrocrebanine,
oxocrebanine and uthongine and a non-natural aporphine alkaloid norcrebanine in which
the formation of ring C was achieved via a microwave-assisted direct biaryl coupling reac-
tion and the evaluation of cytotoxicity against three human cancer cell lines (MCF7, KB
and NCI-H187) was tested. Our investigation will be a preliminary study for the further
synthesis of aporphine alkaloid derivatives which will lead to the discovery of novel and
effective anticancer drugs.
2. Results and discussion
e syntheses of phoebine (1a), norphoebine (1a′), crebanine (1b) and 8,9-dimethoxy-1,
2-methylenedioxynoraporphine (norcrebanine, 1b′) (Scheme 1) were based on the con-
struction of ring C of the aporphine skeleton by palladium-catalyzed direct biaryl coupling
reaction under microwave radiation, recently developed by Chaudhary et al. [14]. Prior to
the report of Chaudhary et al., the formation of ring C of a number of aporphine alkaloids
was achieved by radical cyclization using a radical initiator and tributyltin hydride. However,
this methodology has limited applicability with low to moderate yields for concomitant
formation of the reduction products [15]. On the other hand, the microwave-assisted direct
biaryl coupling using palladium as a catalyst represents a promising route for this process
and has previously been applied successfully for several alkaloid syntheses [9]. erefore, we
decided to further apply this method for the synthesis of a number of aporphine alkaloids
with different oxygenation in order to study the scope and limitation of this process. e
isoquinolines (12) required for this crucial step were synthesized as follows. Condensation
of 2,3,4-trimethoxyphenethylamine (5) [16] with 6-bromo-3,4-methylenedioxyphenylacetic
acid (6) and 3,4-methylenedioxyphenethylamine (7) [17] with 6-bromo-2,3-dimethoxyphe-
nylacetyl chloride (8) [18] afforded, respectively, amides 9a and 9b which were converted
by a Bischler–Napieralski reaction to give dihydroisoquinolines 10a and 10b in good yields.
Sodium borohydride reduction then produced the tetrahydroisoquinolines 11a and 11b.
Subsequent protection of compounds 11a and 11b as methyl carbamate, trifluoacetamide
and formamide gave isoquinolines (12a, 12a′, 12b and 12b′) in reasonable yields. With the
desired precursors in hand, the palladium-catalyzed biaryl coupling of 12a, 12b and 12b′
under microwave irradiation at 850 W for 5 min afforded aporphines (13a, 13b and 13b′) in
81–96% yields. However, under the same reaction condition, compound 13a′ was obtained
in a low yield (19%). Several attempts to optimize the reaction conditions to improve the
yields were fruitless. e cause of this was not further investigated and remains obscure.
e structures of 13a and 13a′ were supported by the presence of singlets at δH 7.90 and
7.91 due to the proton at C-11 of the aporphine skeleton. Similarly, the H-11 signal of 13b′
exhibited a doublet at δH 7.86 (J = 8.7 Hz) whereas that of 13b was observed as two dou-
blets at δH 7.85 (J = 8.7 Hz) and 7.84 (J = 8.7 Hz) of the two conformers. Reduction of the
carbomethoxy group in 13a and the formyl group in 13b with lithium aluminum hydride
gave phoebine (1a, 84% yield) and crebanine (1b, 97% yield), respectively. On the other

JouRNal oF aSIaN NaTuRal PRoduCTS ReSeaRCH
3
Scheme 1. syntheses of phoebine (1a), norphoebine (1a′), crebanine (1b) and norcrebanine (1b′).
reagents and conditions: (a) reflux, xylene; (b) 10% nahco3, chcl3; (c) Pocl3, benzene, reflux; (d) naBh4,
etoh, reflux; (e) ch3co2cl, et3n, chcl3 (11a →12a); (f) (cf3co)2o, et3n, ch2cl2 (11a →12a′, 11b →12b′);
(g) formic acid, dcc, ch2cl2 (11b →12b); (h) Pd (oac)2, pivalic acid, di-tert-butyl(methyl) phosphonium
tetrafluoroborate, K2co3, dMa, microwave, 850 W, 5 min; (i) lialh4, reflux, thf (13a, 13b →1a, 1b); (j)
na2co3, reflux, 90% Meoh-h2o (13a′, 13b′→1a′, 1b′).

4
K.-o. RayaNIl eT al.
hand, removal of the trifluoroacetyl groups from 13a′ and 13b′ was achieved using aqueous
sodium carbonate to yield norphoebine (1a′) and norcrebanine (1b′) in excellent yields.
Dehydrophoebine (2a) and dehydrocrebanine (2b) were synthesized by treatment of
phoebine (1a) and crebanine (1b) with 10% palladium on carbon in acetonitrile under
reflux in 90% and 99% yields, respectively (Scheme 2). Oxidation of norphoebine (1a′)
and norcrebanine (1b′) with a saturated solution of Fremy’s salt at room temperature for
6 h gave oxophoebine (3a′) and oxocrebanine (3b′) in 81 and 86% yields (Scheme 3).
Finally, treatment of oxocrebanine with iodomethane gave uthongine (4) with a 91% yield
1
(Scheme 4). e H and 13C-NMR spectral data of synthetic ( )-norphoebine, dehydro-
phoebine, oxophoebine, dehydrocrebanine, oxocrebanine and uthongine were identical in
all respects with those previously reported for the natural alkaloids [19–23].
All synthetic compounds (1–4) were screened for their cytotoxicity against NCI-H187
(human small cell lung cancer), KB (human carcinoma of the nasopharynx) and MCF7
Scheme 2. syntheses of dehydrophoebine (2a) and dehydrocrebanine (2b). reagent and conditions: (a)
10% Pd-c, acetonitrile, reflux.
Scheme 3. syntheses of oxophoebine (3a′) and oxocrebanine (3b′). reagent and conditions: (a) fremy's
salt, methanol, rt.

JouRNal oF aSIaN NaTuRal PRoduCTS ReSeaRCH
5
Scheme 4. synthesis of uthongine (4). reagent and conditions: (a) ch3i, acetonitrile, reflux.
Table 1. cytotoxic activities of the aporphine alkaloids (1–4) against Mcf7, KB and nci-h187 cancer cell
lines.
Cytotoxicity (IC50, μM)
Compounds
MCF7
KB
NCI-H187
Phoebine (1a)
>50
>50
27.30
>50
>50
>50
>50
29.40
22.62
>50
>50
>50
8.63
>50
4.77
4.30
1.36
>50
>50
>50
>50
>50
>50
13.04
>50
26.29
7.55
0.20
norphoebine (1a′)
dehydrophoebine (2a)
oxophoebine (3a′)
crebanine (1b)
norcrebanine (1b′)
dehydrocrebanine (2b)
oxocrebanine (3b′)
uthongine (4)
10.34
>50
3.05
ellipticinea
ntb
doxorubicina
15.23
aPositive control.
bnot tested.
(human breast cancer) cell lines using MTT assays reported by Skehan et al. [24] and Plumb
et al. [25]. e results of their cytotoxic activities are expressed in IC50 (median inhibitory
concentration) and are summarized in Table 1. In particular, uthongine (4) exhibited the
most potent activity against MCF7 cell line with an IC50 value of 3.05 μM which is signif-
icantly more potent than doxorubicin (IC50 = 15.23 μM) which was used as the positive
control. Moreover, uthongine also showed cytotoxic activity against KB cell line with an IC50
value (4.77 μM) comparable with that of ellipticine (4.30 μM) and exhibited weak cytotoxic-
ity against NCI-H187 cell line with an IC50 value of 23.29 μM. Dehydrocrebanine exhibited
moderate cytotoxic activities against all three cell lines with IC50 values of 8.36 (KB), 10.34
(MCF7) and 13.04 (NCI-H187) μM while dehydrophoebine showed weak activity against
KB (IC50 = 22.62 μM) and MCF7 (IC50 = 27.30 μM) cell lines. Norphoebine also showed
weak activity against a KB cell line with an IC50 value of 29.40 μM. Unfortunately, the rest
of the alkaloids (phoebine, oxophoebine, crebanine, norcrebanine and oxocrebanine) did
not display significant cytotoxic effects (IC50 > 50 μM).
In conclusion, we have achieved the first total syntheses of ( )-norphoebine, dehydro-
phoebine, oxophoebine, dehydrocrebanine, oxocrebanine and uthongine. e successful
cyclization of the isoquinolines (12) was achieved using the palladium-catalyzed biaryl
coupling under microwave radiation as a key step. All synthetic alkaloids were evaluated
for in vitro cytotoxicity against three human cancer cell lines. Our investigation revealed
that the most potent compound in the series is uthongine (4), which showed the highest

6
K.-o. RayaNIl eT al.
cytotoxicity against MCF7 breast cancer cell line. It is interesting to note that the quaternary
oxoaporphine salt (uthongine) appears to enhance a greater degree of anticancer activity
than its core structure (oxocrebanine). In addition, the presence of an aromatic moiety on
ring C (dehydrocrebanine and dehydrophoebine) is possibly crucial in the enhancement
of the cytotoxic activity of these molecules.
3. Experimental
3.1. General experimental procedures
Reagents were obtained from commercial sources (Sigma-Aldrich Corp., St. Louis, MO,
USA; Merck KGaA, Darmstadt, Germany; Acros Organics, Geel, Belgium) and were used
without further purification. All solvents were reagent grade and were dried by distillation
from the appropriate drying reagents immediately prior to use. Tetrahydrofuran was distilled
from sodium and benzophenone under an argon atmosphere. Triethylamine and dichlo-
romethane were distilled from calcium hydride under argon. Starting materials (compounds
5, 7 and 8) were synthesized according to literature procedures [16–18]. Analytical thin-
layer chromatography (TLC) was performed on Merck precoated silica gel 60 F254 plates
(Merck KGaA, Darmstadt, Germany). Compounds were visualized under UV light and/or
by heating the plate afer spraying with a 1% solution of vanillin in 0.1 M sulfuric acid in
methanol. Column chromatography (CC) was carried out on silica gel 60 (70–230 mesh or
230–400 mesh, Merck KGaA, Darmstadt, Germany). Melting points were measured on a
Stuart Scientific SMP 2 melting point apparatus (Bibby Scientific US, Burlington, NJ, USA)
and are uncorrected. Optical rotations were measured on a JASCO P1010 digital polarim-
eter (JASCO, Tokyo, Japan). Ultraviolet (UV) spectra were recorded on a Hewlett Packard
8453 UV–vis spectrometer (Agilent Technologies Inc., Waldbronn, Germany). Infrared (IR)
spectra were obtained on a Perkin Elmer GX FT-IR spectrophotometer (Perkin Elmer Inc.,
Waltham, MA, USA). 1D and 2D nuclear magnetic resonance (NMR) experiments were
recorded on a Bruker AVANCE 300 MHz spectrometer (Bruker AG, Fällanden, Germany)
operating at 300 MHz for proton and 75 MHz for carbon. Mass spectra were recorded on
a Bruker microTOF LC mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany)
and the lock mass calibration was applied for the determination of accurate masses.
3.2. Preparation
3.2.1. 2-(2-Bromo-4,5-methylenedioxyphenyl)-N-(2,3,4-trimethoxyphenethyl)
acetamide (9a)
A solution of 5 (14.70 g, 69.58 mmol) and 6 (17.30 g, 66.78 mmol) in xylene (150 ml) was
refluxed for 2 h with water removal using a Dean-Stark trap. e xylene was then removed
under reduced pressure to give a residue which was dissolved in chloroform (200 ml). e
chloroform layer was washed with 5% hydrochloric acid (3×200 ml), water (300 ml), 10%
sodium carbonate (3×200 ml) and then dried over anhydrous sodium sulfate. Removal of
the solvent under reduced pressure gave a residue which was recrystallized from ethanol
to give 9a as white crystals (14.90 g, 49%), mp 136–137 °C. IR (film) υmax cm−1: 3318, 2940,
1650, 1602, 1495, 1481, 1233, 1100, 1036, 924, 799, 674. 1H-NMR (CDCl3) δ: 7.02 (1H, s,
ArH), 6.78 (1H, s, ArH), 6.75 (1H, d, J = 8.4 Hz, ArH), 6.57 (1H, d, J = 8.4 Hz, ArH), 5.99

JouRNal oF aSIaN NaTuRal PRoduCTS ReSeaRCH
7
(2H, s, OCH2O), 5.82 (1H, br s, NH), 3.85 and 3.83 (9H, 2s, 3xOCH3), 3.57 (2H, s, CH2),
3.43 (2H, apparent q, J = 6.6 Hz, CH2), 2.72 (2H, t, J = 6.6 Hz, CH2). 13C-NMR (CDCl3) δ:
169.7, 152.6, 151.7, 147.7, 147.6, 142.2, 127.7, 124.7, 124.5, 115.3, 112.8, 111.0, 107.4, 101.9,
60.9, 60.7, 55.9, 43.7, 40.8, 29.5. HRESIMS: m/z 452.0716 [M + H]+ (calcd for C20H23BrNO6,
452.0709).
3.2.2. 2-(6-Bromo-2,3-dimethoxyphenyl)-N-(3,4-methylenedioxyphenethyl)
acetamide (9b)
A solution of 8 (13.15 g, 44.80 mmol) in ethanol-free chloroform (150 ml) was added to
a mixture of 7 (7.40 g, 44.80 mmol) in ethanol-free chloroform (35 ml) and 10% sodium
hydrogen carbonate (150 ml). e mixture was stirred at room temperature for 2 h. e
chloroform layer was washed with 10% sodium hydrogen carbonate (3×200 ml), water
(3×200 ml), 10% hydrochloric acid (3×200 ml) and water (3×200 ml) and then dried
over anhydrous sodium sulfate. Removal of the solvent under reduced pressure gave a
residue which was recrystallized from ethanol to give 9b as a pale yellow solid (13.84 g,
87%), mp 164–166 °C. IR (film) υmax cm−1: 3282, 2881, 1636, 1546, 1472, 1246, 1007, 924,
799, 595. 1H-NMR (CDCl3) δ: 7.28 (1H, d, J = 8.7 Hz, ArH), 6.76 (1H, d, J = 8.7 Hz, ArH),
6.65 (1H, d, J = 7.8 Hz, ArH), 6.58 (1H, d, J = 1.5 Hz, ArH), 6.51 (1H, dd, J = 7.8, 1.5 Hz,
ArH), 5.91 (2H, s, OCH2O), 5.51 (1H, br s, NH), 3.86 (3H, s, OCH3), 3.80 (3H, s, OCH3),
3.75 (2H, s, CH2), 3.41 (2H, apparent q, J = 6.6 Hz, CH2), 2.66 (2H, t, J = 6.6 Hz, CH2). 13
C-NMR (CDCl3) δ: 169.4, 152.3, 148.3, 147.7, 146.0, 132.6, 129.4, 128.0, 121.7, 115.8, 112.8,
109.1, 108.3, 100.8, 60.9, 55.9, 40.9, 38.2, 35.3. HRESIMS: m/z 422.0624 [M + H]+ (calcd
for C19H21BrNO5, 422.0603).
3.2.3. 1-(2-Bromo-4,5-methylenedioxybenzyl)-5,6,7-trimethoxy-3,4-
dihydroisoquinoline (10a)
A solution of 9a (22.60 g, 49.97 mmol) and phosphorus oxychloride (67.80 g, 442.18 mmol)
in benzene (770 ml) was refluxed for 3 h. e excess reagent and solvent were removed
under reduced pressure and the residue was dissolved in chloroform (770 ml). e mixture
was basified by addition of solid sodium carbonate followed by dropwise addition of water.
e chloroform layer was washed with water (300 ml) and then dried over anhydrous
sodium sulfate. Removal of the solvent under reduced pressure gave a yellow solid which was
recrystallized from ethanol to give 10a as yellow crystals (15.70 g, 72%), mp 135–137 °C. IR
(film) υmax cm−1: 2942, 1675, 1613, 1478, 1238, 1119, 1037, 949, 789, 605. 1H-NMR (CDCl3)
δ: 7.02 (H, s, ArH), 6.83 (1H, s, ArH), 6.79 (1H, s, ArH), 5.93 (2H, s, OCH2O), 4.20 (2H, s,
ArCH2), 3.91 (3H, s, OCH3), 3.84 (3H, s, OCH3), 3.81 (3H, s, OCH3), 3.64 (2H, t, J = 7.2 Hz,
CH2), 2.73 (2H, t, J = 7.2 Hz, CH2). 13C-NMR (CDCl3) δ: 164.0, 151.9, 151.3, 150.0, 147.6,
145.0, 133.0, 124.4, 121.7, 114.1, 113.3, 111.0, 107.0, 102.6, 61.0, 60.6, 60.4, 56.2, 47.9, 18.5.
HRESIMS: m/z 434.0598 [M + H]+ (calcd for C20H21BrNO5, 434.0603).
3.2.4. 1-(6-Bromo-2,3-dimethoxybenzyl)-6,7-methylenedioxy-3,4-
dihydroisoquinoline (10b)
By the same procedure used to prepare 10a, compound 10b was obtained in 96% yield as
a yellow solid, mp 84–86 °C. IR (film) υmax cm−1: 2940, 1637, 1601, 1473, 1268, 1007, 935,
1
737. H-NMR (CDCl3) δ: 7.27 (1H, d, J = 8.7 Hz, ArH), 7.12 (1H, s, ArH), 6.73 (1H, d,
J = 8.7 Hz, ArH), 6.68 (1H, s, ArH), 5.98 (2H, s, OCH2O), 4.23 (2H, s, ArCH2), 3.83 (3H,

8
K.-o. RayaNIl eT al.
s, OCH3), 3.79 (3H, s, OCH3), 3.58 (2H, t, J = 7.2 Hz, CH2), 2.62 (2H, t, J = 7.2 Hz, CH2).
13C-NMR (CDCl3) δ: 164.0, 152.1, 149.0, 148.4, 146.4, 133.3, 132.3, 127.5, 123.3, 116.2,
112.0, 107.9, 105.6, 101.3, 60.8, 55.7, 46.7, 36.8, 26.3. HRESIMS: m/z 404.0456 [M + H]+
(calcd for C19H19BrNO4, 404.0497).
3.2.5. 1-(2-Bromo-4,5-methylenedioxybenzyl)-5,6,7-trimethoxy-1,2,3,4-
tetrahydroisoquinoline (11a)
Sodium borohydride (2.00 g, 52.86 mmol) was added portionwise to a stirred solution of 10a
(16.20 g, 37.30 mmol) in ethanol (560 ml) and the mixture was refluxed for 1 h. Removal
of the ethanol under reduced pressure gave a residue which was shaken with chloroform
(500 ml) and water (500 ml). e chloroform layer was washed with brine and then dried
over anhydrous sodium sulfate. Removal of the solvent gave a yellow solid which was recrys-
tallized from ethanol to give 11a as a yellow solid (7.40 g, 46%), mp 130–131 °C. IR (film)
υ
max cm−1: 3319, 2939, 1603, 1476, 1238, 1117, 1037, 930, 837. 1H-NMR (CDCl3) δ: 7.05 (1H,
s, ArH), 6.79 (1H, s, ArH), 6.62 (1H, s, ArH), 5.98 (2H, s, OCH2O), 4.19 (1H, dd, J = 9.9,
2.3 Hz, CH), 3.88 and 3.84 (9H, 2s, 3xOCH3), 3.30–3.20 (2H, m, CH2), 3.01–2.68 (4H, m,
2xCH2). 13C-NMR (CDCl3) δ: 151.4, 151.1, 147.3, 147.2, 140.5, 134.1, 131.7, 121.6, 114.8,
112.8, 111.3, 105.6, 101.7, 60.8, 60.4, 56.0, 55.4, 42.7, 39.6, 23.8. HRESIMS: m/z 436.0748
[M + H]+ (calcd for C20H23BrNO5, 436.0759).
3.2.6. 1-(6-Bromo-2,3-dimethoxybenzyl)-6,7-methylenedioxy-1,2,3,4-
tetrahydroisoquinoline (11b)
By the same procedure used to prepare 11a, compound 11b was obtained in 94% yield as a
yellow solid, mp 103–105 °C. IR (film) υmax cm−1: 3332, 2938, 1648, 1471, 1247, 1079, 1039,
1010, 934, 800, 737, 612. 1H-NMR (CDCl3) δ: 7.29 (1H, d, J = 8.7 Hz, ArH), 6.86 (1H, s,
ArH), 6.73 (1H, d, J = 8.7 Hz, ArH), 6.56 (1H, s, ArH), 5.89 (2H, s, OCH2O), 4.25 (1H, dd,
J = 10.5, 2.4 Hz, CH), 3.85 (3H, s, OCH3), 3.83 (3H, s, OCH3), 3.66–2.62 (6H, m, 3xCH2).
13C-NMR (CDCl3) δ: 152.2, 148.6, 145.9, 145.7, 133.2, 131.9, 128.3, 128.0, 115.8, 112.0,
108.7, 106.8, 100.6, 60.7, 55.8, 55.5, 39.3, 37.1, 29.8. HRESIMS: m/z 406.0642 [M + H]+
(calcd for C19H21BrNO4, 406.0654).
3.2.7. 1-(2-Bromo-4,5-methylenedioxybenzyl)-2-carbomethoxy-5,6,7-trimethoxy-
1,2,3,4-tetrahydroisoquinoline (12a)
Methyl chloroformate (18.50 g, 195.77 mmol) was added dropwise to a stirred solution of
11a (13.30 g, 30.48 mmol) and triethylamine (26.30 g, 259.91 mmol) in chloroform (370 ml)
at 0–10 °C. Stirring was continued at room temperature for 3 h. e chloroform layer was
washed with 10% hydrochloric acid (3×200 ml), water (200 ml) and brine and then dried
over anhydrous sodium sulfate. Removal of the solvent under reduced pressure gave the
crude product which was recrystallized from ethanol to give 12a as pale yellow crystals
(9.50 g, 63%), mp 131–132 °C. IR (film) υmax cm−1: 2940, 1698, 1604, 1477, 1408, 1245, 1230,
1
1112, 1037, 929, 838, 653. H-NMR (CDCl3) δ: 7.02 and 6.98 (total 1H, 2s, ArH of both
conformers), 6.65 and 6.56 (total 1H, 2s, ArH of both conformers), 6.46 and 6.29 (total 1H,
2s, ArH of both conformers), 5.94 (2H, s, OCH2O), 5.34–5.31 (1H, m, CH), 4.32–4.24 and
3.97–3.90 (total 1H, m, H-5α), 3.86 and 3.85 (6H, 2s, 2xOCH3), 3.80 and 3.72 (total 3H,
2s, OCH3 of both conformers), 3.65 and 3.41 (total 3H, 2s, CO2CH3 of both conformers),
3.48–2.67 (5H, m, H-5β, 2xCH2). 13C-NMR (CDCl3) δ: 155.8, 151.8, 151.1, 147.1, 147.0,

JouRNal oF aSIaN NaTuRal PRoduCTS ReSeaRCH
9
140.9, 131.8, 130.7, 120.7, 115.2, 112.4, 111.1, 105.9, 101.7, 60.9, 60.5, 56.0, 54.1, 52.3, 42.5,
37.2, 22.3. HRESIMS: m/z 494.0785 [M + H]+ (calcd for C22H25BrNO7, 494.0814).
3.2.8. 2-Trifluoroacetyl-1-(2-bromo-4,5-methylenedioxybenzyl)-5,6,7-trimethoxy-
1,2,3,4-tetrahydroisoquinoline (12a′)
Trifluoroacetic anhydride (24.00 g, 114.60 mmol) was added dropwise to a stirred mixture
of 11a (5.00 g, 11.46 mmol) and triethylamine (6.96 g, 68.76 mmol) in dichloromethane
(130 ml) at 0–10 °C. Stirring was continued at room temperature for 3 h. Dichloromethane
(100 ml) was added and the organic solution was washed with 10% sodium hydrogen
carbonate (3×100 ml), water (100 ml), 10% hydrochloric acid (3×100 ml) and water
(100 ml). e organic layer was separated and then dried over anhydrous sodium sulfate.
Removal of the solvent gave a red-brown viscous oil which was recrystallized from ethanol
to give 12a′ as a pale yellow solid (2.75 g, 45%), mp 126–127 °C. IR (film) υmax cm−1: 2940,
1690, 1604, 1478, 1351, 1232, 1196, 1142, 1122, 1038, 933, 846, 660. 1H-NMR (CDCl3) δ:
7.00 and 6.99 (total 1H, 2s, ArH of both conformers), 6.64 and 6.54 (total 1H, 2s, ArH of
both conformers), 6.39 and 6.29 (total 1H, 2s, ArH of both conformers), 5.97 and 5.95 (total
2H, 2s, OCH2O of both conformers), 5.73–5.68 (1H, m, CH), 4.08–3.62 (2H, m, CH2), 3.88
(3H, s, OCH3), 3.86 (3H, s, OCH3), 3.77 (3H, s, OCH3), 3.34–2.73 (4H, m, 2xCH2). 13C-
NMR (CDCl3) δ: 155.6, 152.3, 150.9, 147.5, 147.4, 141.2, 130.2, 129.4, 119.4, 118.4, 115.5,
112.7, 110.6, 106.1, 101.8, 60.9, 60.6, 56.0, 54.2, 41.5, 39.9, 23.4. HRESIMS: m/z 532.0576
[M + H]+ (calcd for C22H22BrF3NO6, 532.0582).
3.2.9. 2-Trifluoroacetyl-1-(2-bromo-5,6-dimethoxybenzyl)-6,7-methylenedioxy-
1,2,3,4-tetrahydroiso quinoline (12b′)
By the same procedure used to prepare 12a′, compound 12b′ was obtained in 42% yield as
pale yellow crystals, mp 140–141 °C. IR (film) υmax cm−1: 2943, 1690, 1483, 1472, 1280, 1238,
1196, 1140, 1080, 1039, 1009, 940, 864, 799, 752, 651. 1H-NMR (CDCl3) δ: 7.23 and 7.22
(total 1H, 2d, J = 8.7 Hz, ArH of both conformers), 6.81 (1H, s, ArH), 6.73 and 6.71 (total
1H, 2d, J = 8.7 Hz, ArH of both conformers), 6.58 (1H, s, ArH), 5.94 and 5.93 (total 2H,
2s, OCH2O of both conformers), 5.87 (1H, dd, J = 10.8, 3.6 Hz, CH), 3.90 (3H, s, OCH3),
3.84 (3H, s, OCH3), 4.04–3.83 (2H, m, CH2), 3.42–2.75 (4H, m, 2xCH2). 13C-NMR (CDCl3)
δ: 156.0, 151.8, 149.0, 146.8, 146.5, 130.7, 128.8, 127.4, 126.1, 116.0, 113.1, 108.3, 107.1,
106.7, 101.1, 60.7, 56.0, 53.6, 40.0, 36.5, 29.4. HRESIMS: m/z 502.0479 [M + H]+ (calcd for
C21H20BrF3NO5, 502.0477).
3.2.10. 1-(2-Bromo-5,6-methoxybenzyl)-2-formyl-6,7-methylenedioxy-1,2,3,4-
tetrahydroisoquinoline (12b)
A solution of N,N′-dicyclohexylcarbodiimide (DCC, 8.23 g, 39.89 mmol) in dichlorometh-
ane (88 ml) was added to a stirred solution of 11b (14.57 g, 35.86 mmol) and formic acid
(1.80 g, 39.11 mmol) in dichloromethane (220 ml). e resulting mixture was stirred at room
temperature for 2 h. e mixture was filtered and the filtrate was washed with 5% hydro-
chloric acid (3×250 ml), water (3×250 ml), 5% sodium hydrogen carbonate (3×250 ml)
and water (250 ml). e organic layer was dried over anhydrous sodium sulfate. Removal
of the solvent gave a brown viscous oil which was recrystallized from ethanol to give 12b
as a pale yellow solid (7.00 g, 45%), mp 134–136 °C. IR (film) υmax cm−1: 2941, 2840, 1669,
1
1483, 1471, 1273, 1240, 1217, 1078, 1037, 1008, 931, 801, 643. H-NMR (CDCl3) δ: 7.92

10
K.-o. RayaNIl eT al.
and 7.51 (total 1H, 2s, CHO of both conformers), 7.26 (1H, d, J = 8.7 Hz, ArH), 6.83 (1H,
s, ArH), 6.74 (1H, d, J = 8.7 Hz, ArH), 6.60 (1H, s, ArH), 5.93 (2H, s, OCH2O), 4.80 (1H,
dd, J = 10.8, 2.4 Hz, CH), 4.43–4.37 (1H, m, H-5α), 3.85 (3H, s, OCH3), 3.84 (3H, s, OCH3),
3.44–2.71 (5H, m, H-5β, 2xCH2). 13C-NMR (CDCl3) δ: 160.8, 152.1, 148.7, 146.8, 146.3,
130.9, 129.0, 127.8, 127.4, 115.4, 112.8, 108.7, 106.6, 101.0, 60.6, 56.8, 55.9, 37.4, 34.7, 28.2.
HRESIMS: m/z 434.0636 [M + H]+ (calcd for C20H21BrNO5, 434.0603).
3.2.11. Methyl 1,2,3-trimethoxy-9,10-methylenedioxynoraporphine-6-carboxylate
(13a)
To a solution of 12a (100.0 mg, 0.20 mmol) in dimethylacetamide (DMA, 4 ml), were added
palladium(II) acetate (4.5 mg, 0.02 mmol), di-tert-butyl(methyl) phosphonium tetrafluoro
borate (9.9 mg, 0.04 mmol), potassium carbonate (82.9 mg, 0.60 mmol) and pivalic acid
(6.1 mg, 0.06 mmol). e mixture was irradiated in a microwave reactor for 5 min with
the power level at 850 W under an argon atmosphere. Afer cooling to room temperature,
the reaction mixture was filtered over Celite and the solvent was removed under reduced
pressure to give the crude product which was purified by column chromatography with
25% ethyl acetate-hexane to give 13a as a white solid (80.1 mg, 96%), mp 184–185 °C. IR
1
(film) υmax cm−1: 2940, 1700, 1459, 1414, 1338, 1236, 1197, 1150, 1092, 1038, 933, 877.
H-NMR (CDCl3) δ: 7.90 (1H, s, H-11), 6.76 (1H, s, H-8), 5.98 (2H, AB q, J = 0.9 Hz,
OCH2O), 4.71–4.65 (1H, m, H-6a), 4.45–4.41 (1H, m, H-5α), 3.95 (3H, s, OCH3), 3.89
(3H, s, OCH3), 3.76 (3H, s, OCH3), 3.73 (3H, s, CO2CH3), 3.01–2.50 (5H, m, H-5β, 2xCH2).
13C-NMR (CDCl3) δ: 155.9, 149.8, 149.6, 146.6, 146.3, 145.4, 130.6, 128.9, 125.1, 123.7, 123.4,
108.64, 108.6, 100.9, 61.1, 60.9, 60.4, 52.7, 51.9, 38.6, 34.9, 23.8. HRESIMS: m/z 414.1539
[M + H]+ (calcd for C22H24NO7, 414.1553).
3.2.12. 1,2,3-Trimethoxy-9,10-methylenedioxy-6-trifluoroacetylnoraporphine (13a′)
By the same procedure used to prepare 13a, compound 13a′ was obtained in 19% yield as a
white solid, mp 184–185 °C. IR (film) υmax cm−1: 2944, 1689, 1461, 1415, 1340, 1237, 1187,
1144, 1094, 1046, 921, 758. 1H-NMR (CDCl3) δ: 7.91 (1H, s, H-11), 6.75 (1H, s, H-8), 5.98
(2H, AB q, J = 1.2 Hz, OCH2O), 4.98 (1H, dd, J = 13.8 and 4.2 Hz, H-6a), 4.24–4.20 (1H, m,
H-5α), 3.96 (3H, s, OCH3), 3.91 (3H, s, OCH3), 3.75 (3H, s, OCH3), 3.30–2.57 (5H, m, H-5β,
2xCH2). 13C-NMR (CDCl3) δ: 156.1, 150.4, 149.5, 146.8, 146.6, 145.7, 129.6, 127.2, 124.7,
123.5, 122.4, 118.3, 108.8, 108.5, 101.0, 61.1, 61.0, 60.5, 52.4, 41.0, 33.3, 24.2. HRESIMS:
m/z 452.1344 [M + H]+ (calcd for C22H21F3NO6, 452.1321).
3.2.13. 6-Formyl-8,9-dimethoxy-1,2-methylenedioxynoraporphine (13b)
By the same procedure used to prepare 13a, compound 13b was obtained in 94% yield as a
white solid, mp 133–134 °C. IR (film) υmax cm−1: 2959, 2875, 1645, 1492, 1408, 1278, 1239,
1180, 1078, 1034, 933, 810. 1H-NMR (CDCl3) δ: 8.41 and 8.28 (total 1H, 2s, CHO of both
conformers), 7.85 and 7.84 (total 1H, 2d, J = 8.7 Hz, H-11 of both conformers), 6.92 and
6.89 (total 1H, 2d, J = 8.7 Hz, H-10 of both conformers), 6.58 and 6.55 (total 1H, 2s, H-3
of both conformers), 6.09 and 5.97 (total 2H, 2s, OCH2O of both conformers), 4.98 (total
1H, dd, J = 13.8, 4.2 Hz, H-6a of both conformers), 4.56–4.48 (total 1H, m, H-5α of both
conformers), 3.92 and 3.91 (total 3H, 2s, OCH3 of both conformers), 3.85 and 3.82 (total
3H, 2s, OCH3 of both conformers), 3.66–2.40 (total 5H, m, H-5β, 2xCH2 of both con-
formers). 13C-NMR (CDCl3) δ: 162.1, 152.5, 147.0, 146.3, 142.7, 129.8, 128.9, 127.6, 126.5,

JouRNal oF aSIaN NaTuRal PRoduCTS ReSeaRCH
11
124.0, 123.4, 110.5, 106.9, 100.9, 60.7, 55.8, 49.2, 42.1, 31.0, 26.9. HRESIMS: m/z 354.1344
[M + H]+ (calcd for C20H20NO5, 354.1341).
3.2.14. 8,9-Dimethoxy-1,2-methylenedioxy-6-trifluoroacetylnoraporphine (13b′)
By the same procedure used to prepare 12a (but the reaction mixture was recrystallized
from ethanol), compound 13b′ was obtained in 81% yield as a white solid, mp 198–199 °C.
IR (film) υmax cm−1: 2900, 1679, 1461, 1272, 1188, 1083, 915, 817, 650. 1H-NMR (CDCl3)
δ: 7.86 (1H, d, J = 8.7 Hz, H-11), 6.90 (1H, d, J = 8.7 Hz, H-10), 6.57 (1H, s, H-3), 6.10 and
5.98 (2H, 2s, OCH2O), 5.05 (1H, dd, J = 13.5, 3.9 Hz, H-6a), 4.25–4.18 (1H, m, H-5α), 3.91
(3H, s, OCH3), 3.86 (3H, s, OCH3), 3.56 (1H, dd, J = 14.4, 4.2 Hz, H-5β), 3.40–2.49 (4H, m,
2xCH2). 13C-NMR (CDCl3) δ: 156.1, 152.6, 147.1, 146.3, 142.8, 129.4, 126.4, 123.8, 123.6,
118.3, 117.4, 114.5, 110.7, 106.7, 101.0, 60.8, 55.8, 52.3, 41.4, 30.4, 26.3. HRESIMS: m/z
422.1239 [M + H]+ (calcd for C21H19F3NO5, 422.1215).
3.2.15. Phoebine (1a)
A mixture of 13a (0.94 g, 2.27 mmol) and lithium aluminum hydride (0.78 g, 20.48 mmol)
in dry tetrahydrofuran (50 ml) was refluxed for 2 h. Water (15 ml) was added dropwise
followed by dilute ammonium hydroxide (5 ml). e pale yellow granular residue was
filtered off and washed with chloroform. e organic phase was separated and then dried
over anhydrous sodium sulfate. Removal of the solvent under reduced pressure gave a pale
brown solid which was chromatograped over silica gel using ethyl acetate as eluent to give
1a as a yellow solid (0.71 g, 84%), mp 91–92 °C. UV (MeOH) λmax/nm (log ε) 220.7 (4.55),
282.2 (4.12), 313.8 (4.14). IR (film) υmax cm−1: 2936, 1485, 1458, 1413, 1373, 1338, 1241,
1096, 1043, 936, 877. 1H-NMR (CDCl3) δ: 7.81 (1H, s, H-11), 6.74 (1H, s, H-8), 5.96 (2H,
AB q, J = 1.2 Hz, OCH2O), 3.94 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.73 (3H, s, OCH3),
2.52 (3H, s, NCH3), 3.10–2.78 and 2.46–2.38 (7H, 2 m, H-6a, 3xCH2). 13C-NMR (CDCl3)
δ: 149.7, 149.4, 146.5, 146.0, 145.2, 130.8, 130.1, 125.4, 122.8, 122.6, 108.4, 108.2, 100.7,
62.8, 60.9, 60.6, 60.4, 53.0, 44.0, 34.8, 23.8. HRESIMS: m/z 370.1633 [M + H]+ (calcd for
C21H24NO5, 370.1654).
3.2.16. Crebanine (1b)
By the same procedure used to prepare 1a, compound 1b was obtained in 97% yield as a
yellow-brown solid, mp 114–115 °C. UV (MeOH) λmax/nm (log ε) 217.6 (4.62), 242.5 (4.21),
279.3 (4.44), 291.0 (4.37), 322.2 (3.77). IR (film) υmax cm−1: 2927, 1601, 1496, 1413, 1282,
1236, 1129, 1073, 1036, 987, 937, 818. 1H-NMR (CDCl3) δ: 7.81 (1H, d, J = 8.7 Hz, H-11),
6.88 (1H, d, J = 8.7 Hz, H-10), 6.52 (1H, s, H-3), 5.99 (2H, AB q, J = 0.9 Hz, OCH2O), 3.90
(3H, s, OCH3), 3.81 (3H, s, OCH3), 3.68 (1H, dd, J = 14.7, 4.2 Hz, H-6a), 3.18–2.25 (6H,
m, 3xCH2), 2.58 (3H, s, NCH3). 13C-NMR (CDCl3) δ: 152.0, 146.5, 145.8, 142.0, 129.7,
126.6, 126.5, 124.6, 123.1, 116.5, 110.2, 106.8, 100.6, 61.9, 60.6, 55.7, 53.6, 43.9, 29.2, 26.9.
HRESIMS: m/z 340.1582 [M + H]+ (calcd for C20H22NO4, 340.1549).
3.2.17. Norphoebine (1a′)
Compound 13a′ (0.14 g, 0.31 mmol) was dissolved in 90% methanol-water (10 ml). Sodium
carbonate (0.067 g, 0.63 mmol) was added and then the mixture was refluxed for 2 h. e
solvent was then removed under reduced pressure and water (20 ml) and 10% sodium
bicarbonate (20 ml) were added to the residue followed by extraction with dichloromethane

12
K.-o. RayaNIl eT al.
(3×20 ml). e organic layer was then dried over anhydrous sodium sulfate. Removal of
the solvent under reduced pressure gave crude norphoebine which was chromatograped
over silica gel using ethyl acetate as eluent to give 1a′ as a purple-brown solid (0.12 g, 99%),
mp 112–113 °C. UV (MeOH) λmax/nm (log ε) 219.8 (4.54), 238.7 (4.27), 282.4 (4.12), 309.9
(4.12). IR (film) υmax cm−1: 3313, 2936, 1734, 1619, 1462, 1411, 1337, 1241, 1199, 1133,
1094, 1039, 934, 878. 1H-NMR (CDCl3) δ: 7.84 (1H, s, H-11), 6.70 (1H, s, H-8), 5.94 (2H,
s, OCH2O), 3.93 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.73 (3H, s, OCH3), 3.76–3.70 (1H,
m, H-6a), 3.40–3.34 (1H, m, H-5α), 2.95–2.57 (5H, m, H-5β, 2xCH2). 13C-NMR (CDCl3) δ:
150.2, 149.5, 146.5, 146.0, 145.4, 131.3, 129.7, 125.5, 122.8, 122.4, 108.5, 108.2, 100.8, 61.0,
60.6, 60.4, 54.0, 42.7, 37.1, 23.7. HRESIMS: m/z 356.1526 [M + H]+ (calcd for C20H22NO5,
356.1498).
3.2.18. Norcrebanine (1b′)
By the same procedure used to prepare 1a′ (but the mixture was refluxed for 6.5 h and the
product was purified by recrystallization from ethanol), compound 1b′ was obtained in
99% yield as yellow crystals, 101–102 °C. UV (MeOH) λmax/nm (log ε) 218.0 (4.55), 243.8
(4.18), 279.8 (4.39), 289.8 (4.34), 321.4 (3.74). IR (film) υmax cm−1: 3302, 2956, 1602, 1495,
1413, 1272, 1233, 1059, 939, 836, 713. 1H-NMR (CDCl3) δ: 7.78 (1H, d, J = 8.7 Hz, H-11),
6.82 (1H, d, J = 8.7 Hz, H-10), 6.47 (1H, s, H-3), 5.92 (2H, AB q, J = 1.2 Hz, OCH2O), 3.86
(3H, s, OCH3), 3.79 (3H, s, OCH3), 3.82–2.31 (7H, m, H-6a, 3xCH2), 2.17 (1H, br s, NH).
13C-NMR (CDCl3) δ: 152.0, 146.7, 145.7, 141.9, 129.3, 127.0, 126.4, 124.6, 123.2, 116.1,
110.2, 107.2, 100.5, 60.6, 55.7, 53.1, 43.2, 29.4, 29.0. HRESIMS: m/z 326.1378 [M + H]+
(calcd for C19H20NO4, 326.1392).
3.2.19. Dehydrophoebine (2a)
To a solution of 1a (0.11 g, 0.30 mmol) in acetonitrile (10 ml) was added 10% palladium
on carbon (0.088 g, 0.83 mmol). e mixture was heated to reflux for 2 h under an argon
atmosphere. e reaction mixture was filtered and the filtrate was concentrated under
reduced pressure to give the crude product which was recrystallized from ethanol to give
2a as a green solid (0.099 g, 90%), mp 155–156 °C. UV (MeOH) λmax/nm (log ε) 216.4
(4.55), 241.9 (4.76), 256.2 (4.85), 271.1 (4.74), 297.9 (4.29), 335.4 (4.16). IR (film) υmax cm−1:
2936, 1597, 1458, 1402, 1337, 1219, 1141, 1091, 1042, 940, 853, 796. 1H-NMR (CDCl3) δ:
8.90 (1H, s, H-11), 7.03 (1H, s, H-8), 6.60 (1H, s, H-7), 6.01 (2H, s, OCH2O), 4.06 (3H,
s, OCH3), 3.93 (6H, s, OCH3), 3.27–3.21 (4H, m, 2xCH2), 3.02 (3H, s, NCH3). 13C-NMR
(CDCl3) δ: 150.1, 147.3, 146.8, 145.7, 145.1, 142.2, 130.6, 122.3, 121.2, 120.3, 119.2, 105.4,
104.0, 103.5, 100.7, 61.2, 60.8, 60.2, 49.9, 40.5, 24.2. HRESIMS: m/z 368.1469 [M + H]+
(calcd for C21H22NO5, 368.1498).
3.2.20. Dehydrocrebanine (2b)
By the same procedure used to prepare 2a, compound 2b was obtained in 99% yield as a
brown solid, mp 139–140 °C. UV (MeOH) λmax/nm (log ε) 214.1 (4.25), 247.4 (4.19), 271.0
(4.58), 335.2 (3.87). IR (film) υmax cm−1: 2940, 2831, 1604, 1492, 1400, 1303, 1278, 1219, 1105,
1
1059, 1038, 985, 821, 743. H-NMR (CDCl3) δ: 8.63 (1H, d, J = 9.0 Hz, H-11), 7.00 (1H,
d, J = 9.0 Hz, H-10), 6.86 (1H, s, H-3), 6.84 (1H, s, H-7), 6.16 (2H, s, OCH2O), 3.97 (3H,
s, OCH3), 3.96 (3H, s, OCH3), 3.34 (2H, t, J = 6.0 Hz, CH2), 3.18 (2H, d, J = 6.0 Hz, CH2),
3.11 (3H, s, NCH3). 13C-NMR (CDCl3) δ: 150.2, 145.1, 144.1, 141.6, 141.5, 129.5, 127.5,

JouRNal oF aSIaN NaTuRal PRoduCTS ReSeaRCH
13
123.4, 118.6, 118.3, 117.4, 108.5, 106.7, 100.8, 93.8, 60.5, 56.2, 50.5, 40.5, 30.8. HRESIMS:
m/z 338.1358 [M + H]+ (calcd for C20H20NO4, 338.1392).
3.2.21. Oxophoebine (3a′)
A saturated solution of Fremy’s salt in 4% sodium bicarbonate was added dropwise to a
solution of 1a′ (0.11 g, 0.31 mmol) in methanol (10 ml) and the mixture was stirred at
room temperature for 6 h. Dichloromethane (20 ml) was added to the mixture followed
by extraction with water. e organic phase was separated and then dried over anhydrous
sodium sulfate. Removal of the solvent under reduced pressure gave a red viscous oil which
was recrystallized from ethanol to give 3a′ as a red amorphous solid (92.2 mg, 81%), UV
(MeOH) λmax/nm (log ε) 210.9 (4.39), 250.6 (4.22), 278.0 (4.38), 319.9 (3.84). IR (film) υmax
cm−1: 2923, 1644, 1582, 1463, 1402, 1363, 1268, 1116, 1031, 955, 897. 1H-NMR (CDCl3) δ:
8.93 (1H, d, J = 5.4 Hz, H-5), 8.56 (1H, s, H-11), 8.17 (1H, d, J = 5.4 Hz, H-4), 7.93 (1H, s,
H-8), 6.12 (2H, s, OCH2O), 4.19 (3H, s, OCH3), 4.08 (3H, s, OCH3), 4.07 (3H, s, OCH3).
13C-NMR (CDCl3) δ: 180.6, 156.2, 153.2, 148.2, 147.9, 147.5, 144.9, 144.0, 131.3, 131.1,
127.7, 121.9, 119.0, 115.7, 107.5, 107.1, 102.1, 61.9, 61.5, 61.1. HRESIMS: m/z 366.0937
[M + H]+ (calcd for C20H16NO6, 366.0977).
3.2.22. Oxocrebanine (3b′)
By the same procedure used to prepare 3a′, compound 3b′ was obtained in 86% yield as
an orange solid, mp 101–102 °C. UV (MeOH) λmax/nm (log ε) 207.4 (4.40), 249.5 (4.38),
272.5 (4.30), 375.2 (3.55). IR (film) υmax cm−1: 2934, 1651, 1567, 1463, 1243, 1037, 968, 833.
1H-NMR (CDCl3) δ: 8.76 (1H, d, J = 5.4 Hz, H-5), 8.22 (1H, d, J = 9.0 Hz, H-11), 7.61 (1H,
d, J = 5.4 Hz, H-4), 7.11 (1H, d, J = 9.0 Hz, H-10), 6.98 (1H, s, H-3), 6.29 (2H, s, OCH2O),
4.00 (3H, s, OCH3), 3.93 (3H, s, OCH3). 13C-NMR (CDCl3) δ: 180.9, 153.4, 152.3, 150.8,
146.9, 144.7, 143.3, 135.9, 126.3, 124.9, 123.9, 121.7, 117.3, 117.2, 107.9, 102.7, 102.0, 61.3,
56.1. HRESIMS: m/z 336.0813 [M + H]+ (calcd for C19H14NO5, 336.0872).
3.2.23. Uthongine (4)
Compound 3b′ (48.3 mg, 0.14 mmol) was dissolved in acetonitrile (5 ml). Iodomethane
(20.5 mg, 0.14 mmol) was added and then the mixture was refluxed for 6 h. e solvent was
removed under reduced pressure. e resulting residue was shaken with water (50 ml) and
dichloromethane (50 ml). e aqueous layer was separated and water was then removed
under vacuum to give 4 as a purple amorphous solid (44.8 mg, 91%), UV (MeOH) λmax/nm
(log ε) 219.1 (4.37), 261.0 (4.23), 291.6 (4.14), 382.1 (3.57). IR (film) υmax cm−1: 2925, 1667,
1594, 1492, 1404, 1271, 1032, 985, 844. 1H-NMR (CD3OD) δ: 9.16 (1H, d, J = 6.0 Hz, H-5),
8.53 (1H, d, J = 6.0 Hz, H-4), 8.44 (1H, d, J = 8.4 Hz, H-11), 7.57 (1H, s, H-3), 7.44 (1H,
d, J = 8.4 Hz, H-10), 6.64 (2H, s, OCH2O), 4.85 (3H, s, NCH3), 4.08 (3H, s, OCH3), 4.04
(3H, s, OCH3). 13C-NMR (CD3OD) δ: 178.4, 157.4, 154.5, 153.2, 150.6, 150.2, 141.1, 139.6,
126.4, 125.3, 124.1, 123.7, 123.1, 118.9, 109.2, 104.8, 102.8, 61.7, 56.4, 48.9. HRESIMS: m/z
350.0999 [M]+ (calcd for C20H16NO5, 350.1023).
3.3. Cytotoxicity bioassays
NCI-H187 (Human small cell lung carcinoma, ATCC CRL-5804) was determined by a
MTT assay [25]. Briefly, the cells were diluted to 105 cells/ml. e test compounds were

14
K.-o. RayaNIl eT al.
diluted in distilled water and added to microtiter plates in a total volume of 100 μl. e plates
were incubated at 37 °C, 5% CO2 for 5 days. Fify microliters of a 2 μg/μl MTT solution
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; iazolyl blue) was added
to each well of the plate. e plates were wrapped with aluminum foil and incubated for 4 h.
Afer the incubation period, the microplates were spun down at 200 x g for 5 min. MTT was
then removed from the wells and the formazan crystals were dissolved in 200 μl of 100%
DMSO and 25 μl of Sorensen’s glycine buffer. e OD was read in a microtiter plate reader
at a wavelength of 570 nm. MCF7 (Human breast adenocarcinoma, ATCC HTB-22) and
KB (Human carcinoma of the nasopharynx, ATCC CCL-17) were determined by a color-
imetric cytotoxicity assay that measured the level of cell growth from the cellular protein
content according to literature [24]. Ellipticin and doxorubicin were used as the positive
controls. DMSO was used as the negative control. Briefly, the cells in a logarithmic growth
phase were harvested and diluted to 105 cells/ml with fresh medium and mixed gently. e
test compounds were diluted in distilled water and placed into microtiter plates in a total
volume of 200 μl. e plates were incubated at 37 °C, 5% CO2 for 72 h. Afer the incuba-
tion period, the cells were fixed by 50% trichloroacetic acid. e plates were incubated at
4 °C for 30 min, washed with tap water and air-dried at room temperature. e plates were
stained with 0.05% sulforhodamine B (SRB) dissolved in 1% acetic acid for 30 min. Afer
the staining period, SRB was removed with 1% acetic acid. e plates were air-dried before
the bound dye was dissolved with 10 mM Tris base for 5 min on a shaker. e OD was read
on a microtiter plate reader at the wavelength of 510 nm.
Acknowledgments
e authors are grateful to the Department of Chemistry, Faculty of Science, Silpakorn University
for a graduate student scholarship to C. Prempree. We would like to thank Jim Speirs for his valuable
suggestions on the preparation of this manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was financially supported by the Faculty of Science, Silpakorn University, Nakorn Pathom,
ailand [grant number SRF-JRG-2557-02].
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