Total Synthesis of 4,5-Didehydroguadiscine: A Potent Melanogenesis Inhibitor from the Brazilian Medicinal Herb, Hornschuchia obliqua

Article abstract of DOI:10.1021/np500995z

The first total synthesis of the 7,7-dimethylaporphinoid, 4,5-didehydroguadiscine (6), originally isolated from the stems and roots of Hornschuchia oblique (Annonaceae), was achieved by the condensation of homopiperonylamine (7) with an α,α-dimethylphenylacetic acid derivative (8) and subsequent Pschorr reaction of the resulting benzylisoquinoline intermediate (22). The reported ¹³C NMR data were partially revised on the basis of the analysis of HMBC spectra measured under different conditions. The melanogenesis inhibitory activity (IC₅₀ = 4.7 μM) of 6 was 40 times stronger than that of arbutin (174 μM), which was used as reference standard. Furthermore, 6 was the most potent natural melanogenesis inhibitor within this class of compounds. (Chemical Equation Presented).

Full text of DOI:10.1021/np500995z

Article
pubs.acs.org/jnp
Total Synthesis of 4,5-Didehydroguadiscine: A Potent Melanogenesis
Inhibitor from the Brazilian Medicinal Herb, Hornschuchia obliqua
Genzoh Tanabe,† Youta Sugano,† Miki Shirato,† Naoki Sonoda,† Nozomi Tsutsui,† Toshio Morikawa,‡
Kiyofumi Ninomiya,‡ Masayuki Yoshikawa,‡ and Osamu Muraoka*,†
‡
†Faculty of Pharmacy and Pharmaceutical Research and Technology Institute, Kinki University, Higashi-osaka, Osaka 577-8502,
Japan
S
* Supporting Information
ABSTRACT: The first total synthesis of the 7,7-dimethylaporphinoid, 4,5-
didehydroguadiscine (6), originally isolated from the stems and roots of Hornschuchia
oblique (Annonaceae), was achieved by the condensation of homopiperonylamine (7)
with an α,α-dimethylphenylacetic acid derivative (8) and subsequent Pschorr reaction
of the resulting benzylisoquinoline intermediate (22). The reported 13C NMR data
were partially revised on the basis of the analysis of HMBC spectra measured under
different conditions. The melanogenesis inhibitory activity (IC50 = 4.7 μM) of 6 was
40 times stronger than that of arbutin (174 μM), which was used as reference
standard. Furthermore, 6 was the most potent natural melanogenesis inhibitor within
this class of compounds.
he aporphinoids are a diverse family of isoquinoline
alkaloids including more than 300 members.1 They share
to the literature method,5 homopiperonylamine (7) was first
derived from piperonal (9) in good yield through the nitroaldol
reaction with nitromethane followed by LiAlH4 reduction of
the corresponding nitroalkene (10). The propionic acid (8)
was synthesized in the following manner. Commercially
available 3-methyl-4-nitroanisole (11) was condensed with
diethyl oxalate in the presence of tBuOK. Subsequent oxidative
degradation of the corresponding pyruvate by alkaline H2O2
afforded (5-methoxy-2-nitrophenyl)acetic acid6 (12) in 66%
yield. Treatment of the acid with methanol under Fischer
esterification conditions gave methyl (5-methoxy-2-
nitrophenyl)acetate6a,7 (13), which was subjected to a one-
pot α,α-dimethylation in dry THF according to the protocol
reported by Prasad and co-workers.8 Treatment of 13 with 2.2
T
a characteristic tetracyclic motif with different levels of
oxidation on both aromatic rings (Figure 1). A range of
important biological activities has been documented, including
serotonergic, antiplatelet, anticancer, antimalarial, and vaso-
relaxing activities.2 These properties have prompted intensive
structure−activity relationship (SAR) studies over the past
three decades.3 Several aporphinoids have been isolated from
the flower buds and leaves of the sacred lotus (Nelumbo
nucifera, Nymphaeaceae), and some inhibit melanogenesis.3a Of
these aporphinoids, four (1, 2, 3, and 4) significantly inhibited
melanogenesis (IC50 = 13.3−19.3 μM) in theophylline-
stimulated murine B16 melanoma 4A5 cells, whereas a 4,5-
didehydro type aporphinoid, lysicamine (5), was nearly inactive
at the same concentration.3a During the course of our
continued SAR studies on these aporphinoids and their
derivatives, we achieved the first total synthesis of 4,5-
didehydroguadiscine (6), a didehydroaporphinoid constituent
originally isolated from the stems and roots of Hornschuchia
obliqua (Annonaceae).4 Intensive NMR spectroscopic studies
on 6 including HMBC measurements under different
conditions permitted the unambiguous assignment of the 13C
NMR signals and revision of the reported 13C NMR data. The
melanogenesis inhibitory activity of 6 was found ca. 40 times
greater than that of arbutin, which was used as reference
standard.
t
equiv of MeI and BuOK in the presence of 18-Crown-6
resulted in the formation of a ca. 1.4:1 mixture of the target
methyl 2-(5-methoxy-2-nitrophenyl)-2-methylpropionate8 (14)
and the α-monomethylated compound methyl 2-(5-methoxy-2-
nitrophenyl)propionate8,9 (15). The yield of α,α-dimethylation
was improved by treating compound 13 with an excess of
tBuOK (4 equiv) and MeI (5 equiv) to form 14 in 97% yield.
Degassed solvent was essential for an optimal reaction. The α-
oxygenated products methyl 2-hydroxy-2-(5-methoxy-2-
nitrophenyl)propionate (16) and/or 2-methoxy-2-(5-me-
thoxy-2-nitrophenyl)propionate (17) were formed as by-
products unless the mixture of the reactants was degassed
prior to tBuOK addition. Ester 14 was saponified with KOH in
a mixture of H2O and MeOH to form 8 in 96% yield (Scheme
2).
RESULTS AND DISCUSSION
■
On the basis of the retrosynthetic analysis of 4,5-didehy-
droguadiscine (6) (Scheme 1), the starting materials
homopiperonylamine (7) and 2-methyl-2-(5-methoxy-2-
nitrophenyl)propionic acid (8) were synthesized. According
Received: December 11, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np500995z
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Article
Figure 1. Structures of aporphinoids.
Scheme 1. Retrosynthesis of 4,5-Didehydroguadiscine (6)
a
Scheme 2. Syntheses of Building Blocks of 4,5-Didehydroguadiscine (6)
a
Reagents and conditions: (i) CH3NO2, NH4OAc, HOAc, reflux, 2 h; (ii) LiAlH4, THF, reflux, 4 h (71% from 9); (iii) (CO2Et)2, tBuOK, Et2O, rt,
t
30 min, then 10 N aq NaOH, H2O2, 0 °C → rt, 1 h (66%); (iv) MeOH, cat. concd H2SO4, reflux, 3 h (96%); (v) CH3I, 18-Crown-6, BuOK,
degassed THF, −78 °C → rt (97%); (vi) KOH, MeOH-H2O (1/1, v/v), reflux, 7 h (96%).
The coupling of compounds 7 and 8 via the acid chloride, 2-
methyl-2-(5-methoxy-2-nitrophenyl)propionyl chloride (18),
proceeded in poor yield (16%) of the amide, N-homopiper-
onyl-2-methyl-2-(5-methoxy-2-nitrophenyl)propionamide
(19). Therefore, an effective peptide synthetic method
developed by Carpino10 was applied. Thus, treatment of a
mixture of 7 and 8 with 1-hydroxy-7-azabenzotriazole (HOAt),
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochlor-
ide (EDC·HCl) and 1,8-bis(N,N-dimethylamino)naphthalene
(PS: proton sponge) at 70 °C for 3 h in DMF afforded 19 in
73% yield. The intramolecular cyclization reaction of 19 with
POCl3 in toluene and subsequent dehydrogenation of the
dihydroisoquinoline derivative, 3,4-dihydro-1-[1,1-dimethyl-1-
(5-methoxy-2-nitrophenyl)methyl]-6,7-methylenedioxyisoqui-
noline (20), by MnO2 afforded 1-[1-methyl-1-(5-methoxy-2-
nitrophenyl)ethyl]-6,7-methylenedioxyisoquinoline (21) from
19 in 88% yield. Both attempted catalytic hydrogenation of the
nitro group in 21 over Pd−C and treatment of 21 with LiAlH4
formed 1-[1-methyl-1-(5-methoxy-2-aminophenyl)ethyl]-6,7-
methylenedioxyisoquinoline (22) in poor yields and were
accompanied by poorly separable impurities. Thus, target amine
22 was obtained by zinc-mediated reduction in HOAc in 56%
yield. The Pschorr cyclization of the diazonium salt (23)
derived from the diazotization of 22 in the presence of MeOH
B
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as a cosolvent11 gave a mixture of 4,5-didehydroguadiscine (6)
(30%) and 1-[1-methyl-1-(5-methoxyphenyl)ethyl]-6,7-meth-
ylenedioxyisoquinoline (24) (29%). The formation of 24 could
be attributed to one-electron reduction of the diazonium
function of 23 with CuCl followed by hydrogen abstraction of
the resulting aryl radical from methanol.12 Thus, the diazonium
salt 23 was treated with CuCl under methanol-free conditions
to exclusively obtain 4,5-didehydroguadiscine (6) in 58% yield
(Scheme 3).
coupling constant. Similarly, the signal at δC 118.4 assigned to
C-11b has to be revised to δC 112.4, which correlates with H-11
(δH 8.51) in the HMBC spectrum measured at 8 Hz, whereas
no correlation was observed between these signals in the
spectrum measured at 35 Hz. Thus, the δC 151.1 and 112.4
resonances were unambiguously assigned to C-1 and C-11b,
respectively.
Finally, the inhibitory effect of 4,5-didehydroguadiscine (6)
on melanogenesis was tested13 and compared to the related
aporphinoids and arbutin. 4,5-Didehydroguadiscine (6, IC50
=
a
4.7 μM) exhibited inhibitory activity superior to any related
natural aporphinoids (1−4, IC50 = 13.3−19.3 μM3a) (Table 3).
Compound 6 is approximately 40 times as potent as arbutin
(IC50 = 174 μM), which was used as reference standard. It is
also noteworthy that compound 6 is more potent than
lysicamine (5),3a a representative of the 4,5-didehydroaporphi-
noid family. No significant cytotoxicity was observed at the
effective concentrations (95.7 9.3% viability at 3 μM, 86.7
8.1% at 10 μM). Thus, 6 is the most potent natural
melanogenesis inhibitor within this class of compounds.
Further SAR studies addressing the melanogenesis inhibitory
activity and origin of the cytotoxicity of aporphinoids and
related compounds are in progress.
Scheme 3. Synthesis of 4,5-Didehydroguadiscine (6)
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined on an AS ONE ATM-02 micromelting point apparatus
and are uncorrected. IR spectra were measured on an IRAffinity-1
spectrophotometer. NMR spectra were recorded on a JEOL JNM-
ECA 400 (400 MHz 1H, 100 MHz 13C), a JEOL JNM-ECA 500 (500
a
1
1
MHz H, 125 MHz 13C), or a JEOL JNM-ECA 800 (800 MHz H,
200 MHz 13C) instrument. Chemical shifts (δ) and coupling constants
(J) are given in ppm and Hz, respectively. Low-resolution and high-
resolution mass spectra were recorded on a JEOL JMS-700T
spectrometer. Column chromatography was performed over Fuji
Silysia silica gel BW-200. All organic extracts were dried over
anhydrous Na2SO4 prior to evaporation.
Reagents and conditions: (i) SOCl2, benzene, reflux, 2.5 h; (ii) 7,
Et3N, CH2Cl2, rt, 18 h (16% from 8); (iii) EDC·HCl, HOAt, PS,
DMF, 7, 70 °C, 5 h (73%); (iv) POCl3, toluene, reflux 3 h (98%); (v)
MnO2, toluene, reflux 7.5 h (90%); (vi) Zn, NH4Cl, H2O, rt, 2 h
(56%); (vii) NaNO2, 10% H2SO4, H2O, 0 °C, 1 h, then CuCl, 50 °C, 4
h (58%).
1
Synthesis of Homopiperonylamine (7). According to the
literature procedure,5 a mixture of piperonal (9, 3.0 g, 20 mmol),
nitromethane (3.2 mL, 60 mmol), NH4OAc (1.5 g, 30 mmol), and
HOAc (15 mL) was heated under reflux for 2 h. The reaction mixture
was allowed to cool to room temperature, and the precipitate of short
yellow needles (10, 2.7 g, 70%) was collected by filtration and washed
with MeOH. The combined filtrate and washings were neutralized by
addition of NaHCO3, and the resulting mixture was extracted with
EtOAc. The extract was washed with brine and the solvent evaporated
in vacuo to give a pale brown solid (1.1 g), which on trituration with
MeOH gave 10 (200 mg, 5%) as a yellow solid.
The H NMR data of synthetic 6 agreed well with those for
the natural species4 (with deviations ΔδH < 0.04 ppm, Table 1).
However, significant deviations (Δδc = −8.1 − +0.9 ppm) were
observed in the 13C NMR data of the reported and our
synthetic sample (Table 2). Analysis of the HMBC
spectroscopic data led to revision of the reported chemical
shift of C-1 (δc 143.0) to δc 151.1 on the basis of the HMBC
correlation (C−H coupling constant, 8 Hz) of C-1 (δC 151.1)
and H3 (δH 7.01). No correlation was observed between these
two signals in the HMBC spectrum measured at a 35 Hz
1
Table 1. H NMR Spectroscopic Data for 4,5-Didehydroguadiscine (6) in CDCl3 (δ in ppm)
a
b
reported4 (500 MHz)
observed (800 MHz)
HMBC correlations
HMBC correlations
c
position
δH (J in Hz)
ΔδH
3
7.01 s
6.98 s
0.03
0.03
0.01
0.03
0.03
0.01
0.03
0.02
0.04
C-1, C-2, C-11c
C-3, C-5, C-11c
C-3a, C-4, C-6a
C-7, C-9, C10, C-11a
C-8, C11a
C-2, C-11c
4
7.35 d (5.5)
8.44 d (5.5)
7.24 d (2.9)
6.95 dd (8.9, 2.9)
8.52 d (8.8)
1.81 s
7.32 d (5.6)
8.43 d (5.6)
7.21 d (3.2)
6.92 dd (8.8, 3.2)
8.51 d (8.8)
1.78 s
−
5
C-3a, C-4, C-6a
8
C-7, C-9, C10, C-11a
10
C11a
11
C7a, C-9, C-11b
C-6a, C-7, C-7a
C-9
C7a
C(CH3)2
OCH3
OCH2O
C-6a, C-7, C-7a
3.93 s
3.91 s
C-9
6.24 s
6.20 s
C-1, C-2
−
a,b
c
Measured under the long-range JC−H values of 8 Hza and 35 Hzb. Deviations of the chemical shifts between those reported4 and those observed in
the present study.
C
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Table 2. 13C NMR Spectroscopic Data for 4,5-Didehydroguadiscine (6) in CDCl3 (δ in ppm)
reported4 (125 MHz)
observed (200 MHz)
reported4 (125 MHz)
observed (200 MHz)
a
a
position
δC
ΔδC
position
δC
ΔδC
1
143.0
142.6
101.4
135.5
118.8
142.8
162.3
43.0
151.1
142.3
100.9
134.6
118.2
142.4
162.5
42.3
−8.1
0.3
9
161.1
112.3
129.4
121.0
118.4
118.7
33.5
159.7
111.9
128.8
120.4
112.4
118.7
33.2
0.4
0.6
0.6
0.6
6.0
0
2
10
3
0.5
11
3a
4
0.9
11a
0.6
11b
5
0.4
11c
6a
7
−0.2
−0.3
0.5
C(CH3)2
OCH3
OCH2O
0.3
0.3
0.5
55.6
55.3
7a
147.8
147.3
101.6
101.1
8
113.2
112.8
0.4
a
Deviations of the chemical shifts between those reported4 and those observed in the present study.
15 mL and 1 × 5 mL), and the CHCl3 solution was condensed in
vacuo. The residue (2.45 g) was purified by column chromatography
(n-hexane-EtOAc, 30:1 → 10:1 → 3:1) to give compound6a,7 13 (2.31
g, 96%) as a slightly yellowish microcrystalline solid: mp, 64−65 °C;
Table 3. Inhibitory Activity of Aporphinoids 1, 2, 3, 4, 5, 6
and Arbutin against Theophylline-Stimulated Melanogenesis
in B16 Melanoma 4A5 Cells
lit.,6a 66 °C. H NMR (400 MHz, CDCl3) δ: 3.71 (3H, s, CO2CH3),
1
a
a
a
a
a
1
2
3
4
5
6
Arbutin
174
3.90 [3H, s, C6H3(NO2)OCH3], 4.01 (2H, s, H-2), 6.78 (1H, d, J =
2.8, phenyl H-6), 6.91 (1H, dd, J = 9.2, 2.8, phenyl H-4), 8.20 (1H, d, J
= 9.2, phenyl H-3). 13C NMR (100 MHz, CDCl3) δ: 40.4 (C-2), 52.2
(CO2CH3), 55.9 (OCH3), 113.0 (phenyl C-4), 118.6 (phenyl C-6),
128.0 (phenyl C-3), 132.7 (phenyl C-1), 141.5 (phenyl C-2), 163.4
(phenyl C-5), 170.4 (CO2CH3).
IC50 (μM)
15.8
14.5
19.3
13.3
−
4.7
a
See literature (ref 3a).
A solution of 10 (1.6 g, 8.3 mmol) in dry THF (10 mL) was added
dropwise to a suspension of LiAlH4 (900 mg, 24 mmol) in dry THF
(30 mL) at 0 °C. After the mixture was heated under reflux for 4 h, the
excess hydride was decomposed successively with EtOAc and 10%
aqueous NaOH. The resulting mixture was filtered through Celite, and
the residue was washed with Et2O. The combined filtrate and washings
were extracted with Et2O. The extract was washed with brine, and the
solvent was evaporated to give title compound 7 (1.3 g, 95%) as a pale
orange semisolid, which was used in the next step without further
α,α-Dimethylation of Compound 13. A mixture of 13 (1.5 g,
6.67 mmol), freshly distilled MeI (2.1 mL, 34 mmol), 18-Crown-6
(440 mg, 1.67 mmol), and dry THF (90 mL) was degassed by
ultrasonication prior to the addition of tBuOK. Under an Ar
t
atmosphere, the mixture was cooled to −78 °C, and BuOK (2.98 g,
26.7 mmol) was added with stirring. The resulting suspension was
stirred at −78 °C for 1 h and allowed to reach room temperature. After
it was stirred at room temperature for 1 h, the reaction mixture was
poured into cold aqueous NH4Cl (100 mL) and extracted with EtOAc
(3 × 50 mL). The extract was washed with brine and the solvent
evaporated in vacuo to give a pale yellow solid (1.69 g), which on
column chromatography (CHCl3) gave methyl 2-(5-methoxy-2-
nitrophenyl)-2-methylpropionate7,8 (14, 1.64 g, 97%).
1
purification. H NMR (400 MHz, CDCl3) δ: 2.67 (2H, t, J = 6.8,
CH2CH2NH2), 2.91 (2H, t, J = 6.8, CH2CH2NH2), 5.92 (2H, s,
OCH2O), 6.62−6.76 (3H, m, arom.). 13C NMR (100 MHz, CDCl3) δ:
39.6 (CH2CH2NH2), 43.5 (CH2CH2NH2), 100.7 (OCH2O), 108.1/
109.0/121.6 (d, arom.), 133.4/145.8/147.6 (s, arom.).
Synthesis of (5-Methoxy-2-nitrophenyl)acetic Acid (12). To a
In contrast, when the reaction was performed using the reported
molar ratio,8 a mixture of 13 (225 mg, 1.0 mmol), MeI (0.14 mL, 2.2
mmol), 18-Crown-6 (66 mg, 0.25 mmol), degassed dry THF (15 mL),
mixture of 3-methyl-4-nitroanisole (11, 3.0 g, 18.0 mmol), diethyl
t
oxalate (4.9 mL, 36.1 mmol), and dry Et2O (20 mL), BuOK (3.0 g,
26.7 mmol) was added at room temperature. After the mixture was
stirred at room temperature for 30 min, the reaction was quenched by
adding cold H2O (50 mL). To the resulting mixture, 10 N aqueous
NaOH (5 mL) and a 30% aqueous solution of H2O2 (5 mL) were
successively added with ice cooling. After the exothermic reaction
subsided, the mixture was stirred at room temperature for 1 h. The
resulting orange suspension was filtered, and the filtrate was washed
with EtOAc. Na2SO3 was added to the aqueous layer until a negative
test to starch iodide paper was obtained, and the resulting mixture was
acidified with concd HCl to pH 2. The deposited pale yellow solid was
collected by filtration and washed with H2O to give title compound 12
(2.5 g, 66%) as a pale yellow solid, which was recrystallized from
MeOH to give 12 as slightly yellowish prisms: mp, 182−183 °C [lit.6a
t
and BuOK (246 mg, 2.2 mmol) gave a mixture of α,α-dimethylated
ester 14 and the α-monomethylated ester, methyl (5-methoxy-2-
nitrophenyl)propionate9 (15), as a pale yellow oil (248−268 mg, 14/
15 = ca. 1.3−1.5/1).
The formation of a small amount of the byproducts methyl 2-
hydroxy-2-(5-methoxy-2-nitrophenyl)propionate (16) and/or methyl
2-methoxy-2-(5-methoxy-2-nitrophenyl)propionate (17) was detected
when the reaction was performed without ultrasonication prior to the
t
addition of BuOK.
α,α-Dimethyl Ester (14). A slightly yellowish microcrystalline solid:
mp, 63−64 °C; lit.,8 61−62 °C. IR (KBr): 2985, 2951, 1721, 1609,
1578, 1508, 1473, 1338, 1319, 1273, 1249, 1153, 1135, 1061, 1037
1
cm−1. H NMR (800 MHz, CDCl3) δ: 1.65 [6H, s, C(CH3)2], 3.65
177 °C; lit.,6b 174−176 °C, lit.6c 175−177 °C]. H NMR (400 MHz,
1
(3H, s, CO2CH3), 3.91 [3H, s, C6H3(NO2)OCH3], 6.85 (1H, dd, J =
8.8, 2.4, phenyl H-4), 7.06 (1H, d, J = 2.4, phenyl H-6), 8.10 (1H, d, J
= 8.8, phenyl H-3). 13C NMR (200 MHz, CDCl3) δ: 27.2 [C(CH3)2],
46.6 [C(CH3)2], 52.0 (CO2CH3), 55.7 [C6H3(NO2)OCH3], 110.6
(phenyl C-4), 114.9 (phenyl C-6), 128.6 (phenyl C-3), 141.5 (phenyl
C-2), 142.4 (phenyl C-1), 163.2 (phenyl C-5), 175.6 (CO2CH3).
FABMS (pos.) m/z: 254 [M + H]+, 276 [M + Na]+. FABHRMS m/z:
276.0875 (calcd for C12H15O5NNa, 276.0848).
α-Monomethyl Ester (15). Colorless prisms (from n-hexane): mp,
58−59 °C; lit.,9 mp, 60−62 °C. IR (KBr): 2951, 2845, 1372, 1612,
1582, 1508, 1477, 1462, 1438, 1339, 1315, 1296, 1242, 1120, 1172,
1076, 1010 cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.59 (3H, d, J = 7.2,
DMSO-d6) δ: 3.86 (3H, s, OCH3), 3.98 (2H, s, H-2), 7.05 (1H, dd, J =
9.0, 2.8, phenyl H-4), 7.09 (1H, d, J = 2.8, phenyl H-6), 8.13 (1H, d, J
= 9.0, phenyl H-3), 12.5 (1H, br s, CO2H). 13C NMR (100 MHz,
DMSO-d6) δ: 39.8 (C-2), 56.2 (OCH3), 113.2 (phenyl C-4), 118.8
(phenyl C-6), 127.6 (phenyl C-3), 133.8 (phenyl C-1), 141.4 (phenyl
C-2), 163.1 (phenyl C-5), 171.3 (CO2H).
Synthesis of Methyl (5-Methoxy-2-nitrophenyl)acetate (13).
A mixture of 12 (2.26 g, 4.7 mmol), concd H2SO4 (0.3 mL), and
MeOH (60 mL) was heated under reflux for 3 h. After cooling, the
reaction mixture was neutralized with NaHCO3. The resulting
suspension was filtered, and the filtrate was concentrated in vacuo to
give a brown semisolid (3.37 g), which was triturated with CHCl3 (2 ×
D
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Journal of Natural Products
Article
CHCH3), 3.68 (3H, s, CO2CH3), 3.89 [3H, s, C6H3(NO2)OCH3],
4.42 (1H, q, J = 7.2, CHCH3), 6.87 (1H, dd, J = 8.8, 3.2, phenyl H-4),
6.91 (1H, d, J = 3.2, phenyl H-6), 8.07 (1H, d, J = 8.8, phenyl H-3).
13C NMR (200 MHz, CDCl3) δ: 17.6 (CHCH3), 41.9 (CHCH3), 52.2
(CO2CH3), 55.8 [C6H3(NO2)OCH3], 112.2 (phenyl C-4), 115.3
(phenyl C-6), 127.9 (phenyl C-3), 138.3 (phenyl C-1), 141.6 (phenyl
C-2), 163.3 (phenyl C-5), 173.6 (CO2CH3). FABMS (pos.) m/z: 240
[M + H]+.
= 2.4, arom.), 7.98 (1H, d, J = 8.8, arom.). 13C NMR (200 MHz,
CDCl3) δ: 27.4 [C(CH3)2], 35.2 (CH2CH2NHCO), 40.9
(CH2CH2NHCO), 47.1 [C(CH3)2], 55.8 (OCH3), 100.8 (OCH2O),
108.2/109.1/110.8/115.6/121.6/128.7 (d, arom.), 132.8/141.9/
142.4/146.0/147.7/163.0 (s, arom), 175.2 (CONH). FABMS (pos.)
m/z: 387 [M + H]+.
Synthesis of 1-[1-Methyl-1-(5-methoxy-2-nitrophenyl)-
ethyl]-6,7-methylenedioxyisoquinoline (21). To a solution of
19 (850 mg, 2.2 mmol) in dry toluene (20 mL), POCl3 (1.0 mL, 11
mmol) was added at room temperature, and the mixture was heated
under reflux for 3 h. After it was cooled, the reaction was quenched
with H2O (50 mL), and the pH of the resulting mixture was adjusted
to ca. 10 with 10% NaOH and extracted with EtOAc (3 × 30 mL).
The extract was washed with brine and the solvent evaporated in vacuo
to give 3,4-dihydro-1-[1-methyl-1-(5-methoxy-2-nitrophenyl)ethyl]-
6,7-methylenedioxyisoquinoline (20, 794 mg) as a pale brown solid,
which was sufficiently pure for the subsequent reaction.
α-Hydroxy Ester (16). Colorless prisms (from n-hexane-EtOAc):
mp, 85−86 °C. IR (KBr): 3503, 3287, 3201, 2947, 2846, 1720, 1612,
1581, 1508, 1454, 1342, 1323, 1292, 1245, 1200, 1110, 1049, 1033
cm−1. 1H NMR (800 MHz, CDCl3) δ: 1.86 [3H, s, C(OH)CH3], 3.77
(3H, s, CO2CH3), 3.79 (1H, s, OH), 3.92 [3H, s, C6H3(NO2)OCH3],
6.90 (1H, dd, J = 8.8, 3.2, phenyl H-4), 7.24 (1H, d, J = 3.2, phenyl H-
6), 7.99 (1H, d, J = 8.8, phenyl H-3). 13C NMR (200 MHz, CDCl3) δ:
26.7 [C(OH)CH3], 53.0 (CO2CH3), 55.9 [C6H3(NO2)OCH3], 75.0
[C(OCH3)CH3], 112.1 (phenyl C-4), 115.2 (phenyl C-6), 128.1
(phenyl C-3), 139.7 (phenyl C-1), 141.1 (phenyl C-2), 163.1 (phenyl
C-1), 174.3 (CO2CH3). FABMS (pos.) m/z: 256 [M + H]+, 278 [M +
Na]+. FABHRMS m/z: 278.0647 (calcd for C12H15O6NNa, 278.0641).
α-Methoxy Ester (17). Colorless prisms (from Et2O): mp, 77−78
°C. IR (KBr): 2974, 2843, 1612, 1578, 1519, 1458, 1350, 1269, 1195,
To a solution of 20 (300 mg, 0.82 mmol) in toluene (20 mL),
MnO2 (704 mg, 8.1 mmol) was added, and the resulting suspension
was heated under reflux for 7 h. The reaction mixture was filtered by
suction, and the filter cake was washed with EtOAc. The combined
filtrate and washings were condensed in vacuo to give the title
compound 21 (268 mg, 88% from 19) as a pale brown solid, which
was recrystallized from MeOH to give slightly yellowish prisms: mp,
166−167 °C. IR (KBr): 2985, 2912, 1609, 1573, 1519, 1466, 1431,
1
1126, 1103, 1045 cm−1. H NMR (500 MHz, CDCl3) δ: 1.85 [3H, s,
C(OCH3)CH3], 3.28 [3H, s, C(OCH3)CH3], 3.72 (3H, s, CO2CH3),
3.92 [3H, s, C6H3(NO2)OCH3], 6.91 (1H, dd, J = 8.9, 2.6, phenyl H-
4), 7.35 (1H, d, J = 2.6, phenyl H-6), 8.06 (1H, d, J = 8.9, phenyl H-3).
13C NMR (125 MHz, CDCl3) δ: 24.1 [C(OCH3)CH3], 52.2
1
1350, 1242, 1037 cm−1. H NMR (800 MHz, CDCl3) δ: 1.98 [6H, s,
C(CH3)2], 3.90 (3H, s, OCH3), 5.96 (2H, s, OCH2O), 6.64 (1H, s,
arom.), 6.82 (1H, dd, J = 8.8, 2.4, arom.), 7.01 (1H, s, arom.), 7.23
(1H, d, J = 2.4, arom.), 7.37 (1H, d, J = 5.6, arom.), 7.45 (1H, d, J =
8.8, arom.), 8.39 (1H, d, J = 5.6, arom.). 13C NMR (200 MHz, CDCl3)
δ: 30.7 [C(CH3)2], 47.2 [C(CH3)2], 55.7 (OCH3), 101.3 (OCH2O),
102.2/103.6/110.8/114.1/119.8/127.1/140.3 (d, arom.), 122.3/
135.6/144.1/144.6/147.3/149.4/161.7/162.0 (s, arom). FABMS
(pos.) m/z: 367 [M + H]+.
(CO2CH3), 52.7 [C(OCH3)CH3], 55.9 [C6H3(NO2)OCH3], 81.3
[C(OCH3)CH3], 112.7 (phenyl C-4), 113.7 (phenyl C-6), 128.1
(phenyl C-3), 140.0 (phenyl C-1), 141.0 (phenyl C-2), 163.5 (phenyl
C-1), 169.1 (CO2CH3). FABMS (pos.) m/z: 270 [M + H]+, 292 [M +
Na]+. FABHRMS m/z: 292.0793 (calcd for C12H15O6NNa, 292.0797).
Synthesis of 2-Methyl-2-(5-methoxy-2-nitrophenyl)-
propionic Acid (8). A mixture of 14 (1.5 g, 5.92 mmol), KOH
(3.3 g, 58.9 mmol), MeOH (6.0 mL), and H2O (6.0 mL) was heated
under reflux for 7 h. After the mixture was cooled, the reaction mixture
was acidified with 10% HCl (pH ca. 3) and then extracted with EtOAc
(3 × 30 mL). The extract was washed with brine and the solvent
evaporated in vacuo. The residue (1.43 g) was purified by column
chromatography (CHCl3 → CHCl3-MeOH, 50:1) to give compound
8 (1.36 g, 96%) as a pale brown solid. Recrystallization from a mixture
of n-hexane−EtOAc gave 8 as slightly yellowish needles: mp, 190−191
°C. IR (KBr): 3448, 2978, 2939, 2650, 2526, 1717, 1581, 1504, 1342,
Synthesis of 1-[1-Methyl-1-(5-methoxy-2-aminophenyl)-
ethyl]-6,7-methylenedioxyisoquinoline (22). To a solution of
21 (250 mg, 0.74 mmol) in MeOH (20 mL), zinc dust (446 mg, 6.8
mmol) and NH4Cl (547 mg, 10.2 mmol) were successively added, and
the resulting suspension was stirred at room temperature for 2 h. The
reaction mixture was filtered by suction, and the filter cake was washed
with MeOH. The combined filtrate and washings were condensed in
vacuo. The residue (420 mg) was purified by column chromatography
(n-hexane-EtOAc, 20:1 → 10:1 → 5:1) to give the title compound 22
(130 mg, 56%) as a slightly yellowish microcrystalline solid: mp, 155−
157 °C. IR (KBr): 3453, 3333, 3225, 2986, 2913, 1640, 1620, 1578,
1501, 1466, 1427, 1281, 1242, 1041 cm−1. 1H NMR (400 MHz,
CDCl3) δ: 1.84 [6H, s, C(CH3)2], 2.75 (2H, br s, NH2), 3.84 (3H, s,
OCH3), 5.94 (2H, s, CH2O2), 6.40 (1H, d, J = 8.8, arom.), 6.66 (1H,
dd, J = 8.8, 2.8, arom.), 7.00 (1H, s, arom.), 7.22 (1H, s, arom.), 7.24
(1H, d, J = 2.8, arom.), 7.39 (1H, d, J = 5.6, arom.), 8.37 (1H, d, J =
5.6, arom.). 13C NMR (100 MHz, CDCl3) δ: 28.7 [C(CH3)2],
46.4[C(CH3)2], 55.8 (OCH3), 101.3 (OCH2O), 102.7/103.3/111.9/
112.2/118.1/120.0/140.3 (d, arom.), 123.4/134.1/135.5/138.2/
147.7/149.8/153.0/164.5 (s, arom.). FABMS (pos.) m/z: 337 [M +
H]+.
Synthesis of 4,5-Didehydroguadiscine (6). To a solution of 22
(55 mg, 0.15 mmol) in a mixture of H2O (1.7 mL) and 10% H2SO4
(0.6 mL), NaNO2 (21 mg, 0.3 mmol) was added at 0 °C, and the
mixture was stirred at 0 °C for 1 h. The reaction mixture containing
the diazonium salt 23 was heated at 50 °C for another 4 h after the
addition of CuCl (15 mg, 0.15 mmol). The pH of the reaction mixture
was adjusted to ca. 8 by NaHCO3 and extracted with EtOAc. The
extract was washed with brine and the solvent evaporated in vacuo.
The residue (44 mg) was purified by column chromatography (n-
hexane-EtOAc, 50:1 → 30:1) to give 4,5-didehydroguadiscine (6, 27.5
mg, 58%).
1
1319, 1289, 1262, 1195, 1084 cm−1. H NMR (800 MHz, CDCl3) δ:
1.68 [C(CH3)2], 3.91 (3H, s, OCH3), 6.85 (1H, dd, J = 9.6, 3.2 phenyl
H-4), 7.07 (1H, d, J = 3.2, phenyl H-6), 8.15 (1H, d, J = 9.6, phenyl H-
3), 12.5 (1H, br s, CO2H). 13C NMR (200 MHz, CDCl3) δ: 27.0
[C(CH3)2], 46.6 [C(CH3)2], 55.8 (OCH3), 110.9 (phenyl C-4), 115.0
(phenyl C-6), 128.9 (phenyl C-3), 141.2 (phenyl C-2), 142.0 (phenyl
C1), 163.5 (phenyl C-5), 180.3 (CO2H). FABMS (pos.) m/z: 240 [M
+ H]+.
Synthesis of N-Homopiperonyl-2-methyl-2-(5-methoxy-2-
nitrophenyl)propionamide (19). A mixture of amine 8 (1.28 g,
7.8 mmol), acid 9 (1.56 g, 6.5 mmol), 1-hydroxy-7-azabenzotriazole
(HOAt, 980 mg, 7.2 mmol), 1-ethyl-3-(3-dimethylamonopropyl)-
carbodiimide hydrochloride (EDC·HCl, 7.2 mmol), 1,8-bis(N,N-
dimethylamino)naphthalene (PS, 710 mg, 3.3 mmol), and DMF (20
mL) was heated at 70 °C for 5 h. The reaction mixture was poured
into cold H2O (50 mL) and extracted with EtOAc (3 × 50 mL). The
extract was successively washed with 10% HCl, aqueous NaHCO3, and
brine and the solvent evaporated in vacuo. The residue (2.51 g) was
purified by column chromatography (n-hexane-EtOAc, 10:1 → 5:1 →
3:1) to give title compound 19 (1.84 g, 73%) as a pale yellow
microcrystalline solid: mp, 116−117 °C. IR (KBr): 3379, 2932, 2882,
1636, 1520, 1354, 1284, 1250, 1180, 1068, 1041 cm−1. 1H NMR (800
MHz, CDCl3) δ: 1.58 [6H, s, C(CH3)2], 2.71 (2H, t, J = 7.2,
CH2CH2NHCO), 3.43 (2H, t, d, J = 7.2, 6.4, CH2CH2NHCO), 3.90
(3H, s, OCH3), 5.34, (1H, br t, J = 6.4 NH), 5.92 (2H, s, OCH2O),
6.57 (1H, dd, J = 8.0, 1.6, arom.), 6.63 (1H, d, J = 1.6, arom.), 6.68
(1H, d, J = 8.0, arom.), 6.84 (1H, dd, J = 8.8, 2.4, arom.), 7.05 (1H, d, J
When a solution of diazonium salt 23 (prepared by the diazotization
of 22 (70 mg, 0.21 mmol) with NaNO2 (47 mg, 0.41 mmol) in a
mixture of H2O (2.4 mL), MeOH (3.2 mL), and 10% H2SO4 (0.8 mL)
at 0 °C) was treated with CuCl (21 mg, 0.21 mmol), 6 (19.6 mg, 30%)
E
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Journal of Natural Products
Article
was obtained together with 1-[1-methyl-1-(5-methoxyphenyl)ethyl]-
6,7-methylenedioxyisoquinoline (24, 19.7 mg, 29%).
dissolved in DMSO, and the final concentration in the medium was
0.1%.
Cell viability (%) was calculated using the following formula, where
A and B indicate the optical density of vehicle-treated and test
compound-treated groups, respectively.
4,5-Didehydroguadiscine (6). Colorless needles (from MeOH):
mp, 160−161 °C; lit.,4 brownish amorphous powder, melting point
was not reported. IR (KBr): 2967, 2928, 1609, 1570, 1512, 1501,1450,
1420, 1377, 1354, 1315, 1296, 1250, 1219, 1049 cm−1. FABMS (pos.)
m/z: 320 [M + H]+. FABHRMS m/z: 320.1279 (calcd for
C20H18O3N, 320.1287). 1H and 13C NMR spectra of 6 are summarized
in Table 1 and 2.
Compound (24). Colorless plates: mp, 109−110 °C. IR (KBr):
2974, 2908, 1601, 1578, 1465, 1461, 1257, 1211, 1041 cm−1. 1H NMR
(500 MHz, CDCl3) δ: 1.81 [6H, s, C(CH3)2], 3.72 (3H, s, OCH3),
5.94 (2H, s, OCH2O), 6.72 (1H, ddd, J = 8.0, 2.6, 0.9, arom.), 6.77
(1H, dd-like, J = 2.6, 2.2, arom.), 6.79 (1H, ddd, J = 8.0, 2.2, 0.9,
arom.), 6.92 (1H, s, H-8), 7.00 (1H, s, H-5), 7.19 (1H, dd, J = 8.0, 8.0,
arom.), 7.34 (1H, d, J = 5.5, H-4), 8.38 (1H, d, J = 5.5, H-3). 13C
NMR (125 MHz, CDCl3) δ: 30.9 [C(CH3)2], 47.4 [C(CH3)2], 55.1
(OCH3), 101.2 (OCH2O), 103.2/104.1/110.4/112.3/118.4/119.8/
129.6/140.0 (d, arom.), 123.2/135.7/147.0/149.3/152.2/159.8/164.4
(s, arom.). FABMS (pos.) m/z: 322 [M + H]+. FABHRMS m/z:
322.1430 (calcd for C20H20O3N, 322.1443).
Reagents for Bioassay. Dulbecco’s modified Eagle’s medium
(DMEM, 4.5 g/L glucose) was purchased from Sigma-Aldrich (St.
Louis, MO, U.S.A.); fetal bovine serum (FBS), penicillin, and
streptomycin were purchased from Gibco (Invitrogen, Carlsbad, CA,
U.S.A.); and the other chemicals were purchased from Wako Pure
Chemical Co., Ltd. (Osaka, Japan). The 48-well multiplate and 96-well
microplate (Sumilon) were purchased from Sumitomo Bakelite Co.,
Ltd. (Tokyo, Japan).
Cell viability (%) = B/A × 100
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/np500995z.
AUTHOR INFORMATION
Corresponding Author
*E-mail: muraoka@phar.kindai.ac.jp. Tel: +81 6 6721 2332 (ext
5570).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the “High-Tech Research Center”
Project for Private Universities: matching fund subsidy from
MEXT (Ministry of Education, Culture, Sports. Science and
Technology) (to O.M.). The work was also supported by a
grant-in aid for scientific research by JSPS (the Japan Society
for the Promotion of Science) (to G.T. and T.M.). The authors
thank the American Journal Expert for their help in
proofreading of the manuscript.
Cell Culture. Murine B16 melanoma 4A5 cells (RCB0557)14 were
obtained from Riken Cell Bank (Tsukuba, Japan), and the cells were
grown in DMEM supplemented with 10% FBS, penicillin G (100
units/mL), and streptomycin (100 μg/mL) at 37 °C in 5% CO2/air.
The cells were harvested by incubation in phosphate-buffered saline
(−) [PBS (−)] containing 0.05% (w/v) EDTA and 0.02% trypsin for
approximately 3 min at 37 °C and were used for subsequent bioassays.
Melanogenesis. The effects of compounds on melanogenesis were
examined using B16 melanoma 4A5 cells according to the reported
protocol.13 The melanoma cells (8.0 × 103 cells/200 μL/well) were
seeded into 48-well multiplates. After 24 h of culture, a test compound
and 1 mM theophylline were added and incubated for 72 h. After
incubation, the medium was removed, and 105 μL/well of distilled
H2O was added. The cells were homogenized by sonication and lysed
with 6 M NaOH (20 μL/well).
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