Synthesis and antiviral and fungicidal activity evaluation of β-carboline, dihydro-β-carboline, tetrahydro-β-carboline alkaloids, and their derivatives

Article abstract of DOI:10.1021/jf404840x

Six known β-carboline, dihydro-β-carboline, and tetrahydro-β-carboline alkaloids and a series of their derivatives were designed, synthesized, and evaluated for their anti-tobacco mosaic virus (TMV) and fungicidal activities for the first time. All of the alkaloids and some of their derivatives (compounds 3, 4, 14, and 19) exhibited higher anti-TMV activity than the commercial antiviral agent Ribavirin both in vitro and in vivo. Especially, the inactivation, curative, and protection activities of alkaloids Harmalan (62.3, 55.1, and 60.3% at 500 μg/mL) and tetrahydroharmane (64.2, 57.2, and 59.5% at 500 μg/mL) in vivo were much higher than those of Ribavirin (37.4, 36.2, and 38.5% at 500 μg/mL). A new derivative, 14, with optimized physicochemical properties, obviously exhibited higher activities in vivo (50.4, 43.9, and 47.9% at 500 μg/mL) than Ribavirin and other derivatives; therefore, 14 can be used as a new lead structure for the development of anti-TMV drugs. Moreover, most of these compounds exhibited good fungicidal activity against 14 kinds of fungi, especially compounds 4, 7, and 11.

Full text of DOI:10.1021/jf404840x

Article
pubs.acs.org/JAFC
Synthesis and Antiviral and Fungicidal Activity Evaluation of
β‑Carboline, Dihydro-β-carboline, Tetrahydro-β-carboline Alkaloids,
and Their Derivatives
Hongjian Song, Yongxian Liu, Yuxiu Liu, Lizhong Wang, and Qingmin Wang*
State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University,
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, People’s Republic of China
ABSTRACT: Six known β-carboline, dihydro-β-carboline, and tetrahydro-β-carboline alkaloids and a series of their derivatives
were designed, synthesized, and evaluated for their anti-tobacco mosaic virus (TMV) and fungicidal activities for the first time.
All of the alkaloids and some of their derivatives (compounds 3, 4, 14, and 19) exhibited higher anti-TMV activity than the
commercial antiviral agent Ribavirin both in vitro and in vivo. Especially, the inactivation, curative, and protection activities of
alkaloids Harmalan (62.3, 55.1, and 60.3% at 500 μg/mL) and tetrahydroharmane (64.2, 57.2, and 59.5% at 500 μg/mL) in vivo
were much higher than those of Ribavirin (37.4, 36.2, and 38.5% at 500 μg/mL). A new derivative, 14, with optimized
physicochemical properties, obviously exhibited higher activities in vivo (50.4, 43.9, and 47.9% at 500 μg/mL) than Ribavirin and
other derivatives; therefore, 14 can be used as a new lead structure for the development of anti-TMV drugs. Moreover, most of
these compounds exhibited good fungicidal activity against 14 kinds of fungi, especially compounds 4, 7, and 11.
KEYWORDS: β-carboline, dihydro-β-carboline, tetrahydro-β-carboline, alkaloids, anti-TMV, fungicidal activity,
structure−activity relationship
reversible inhibitor of MAO-A.7 The current research on the
INTRODUCTION
■
activity of β-carboline alkaloids and their analogues has mainly
Tobacco mosaic virus (TMV) may cause heavy losses to modern
focused on antitumor,8,9 inhibition of monoamine oxidase,10
agriculture (Figure 1), but its control remains a challenge.1
and antiparasitic activities,11,12 but few studies have focused on
antiviral13−15 and fungicidal activities. In this study, six known
β-carboline, dihydro-β-carboline, and tetrahydro-β-carboline
alkaloids were synthesized, and their anti-TMV and fungicidal
activities were systematically evaluated for the first time. To
study the role of the substituents played in the biological
activities, β-carbolines containing different substituents were
synthesized, and their anti-TMV and fungicidal activities were
also systematically evaluated (Figure 3).
Figure 1. Leaves after infection.
MATERIALS AND METHODS
■
1
Many plant viral inhibitors (Ribavirin, Ningnanmycin, BTH, etc.)
(Figure 2) are used to prevent TMV disease; however, these
chemical pesticides cannot completely cure the infected plant
tissues or fully protect plants from TMV infection under field
conditions. Plant pathogens also lead to great harm to crops;
they can either infect the whole crops or infect crop organs.
Therefore, novel and more practical antiviral and fungicidal
reagents still need to be developed.
Natural products can be used as ideal lead structures to
develop agrochemicals. β-Carboline and its saturated analogue
are common structural motifs in natural products and
pharmaceuticals.2 Harmine and tetrahydroharmine, which are
β-carboline and tetrahydro-β-carboline alkaloids, respectively,
are two representative harmala alkaloids. Harmine was originally
isolated from P. harmala L.3 and found to exhibit a cytotoxic
effect on HL60 and K562 leukemic cell lines.4 Tetrahydrohar-
mine is a fluorescent indole alkaloid present in the tropical liana
species Banisteriopsis caapi,5 and it weakly inhibits serotonin
reuptake.6 Harmaline is a central nervous system stimulant and a
Instruments. H NMR spectra were obtained at 400 MHz using
a Bruker AV400 spectrometer in CDCl3 or DMSO-d6 solution with
tetramethylsilane as the internal standard. HRMS data were obtained
on an FTICR-MS instrument (Ionspec 7.0 T). The melting points
were determined on an X-4 binocular microscope melting point
apparatus (Beijing Tech Instruments Co., Beijing, China) and are
uncorrected.
General Synthesis. All anhydrous solvents were dried and purified
by using standard techniques. The synthetic routes are given in
Schemes 1−5.
Synthesis of Harmalan (1-Methyl-4,9-dihydro-3H-pyrido-
[3,4-b]indole).16 To a solution of tryptamine (0.50 g, 3.13 mmol)
and triethylamine (2 mL) in dichloromethane was added dropwise
acetyl chloride (0.27 g, 3.44 mmol) in dichloromethane (5 mL) at
room temperature, and the mixture was stirred at room temperature
Received: November 6, 2013
Revised: January 3, 2014
Accepted: January 15, 2014
Published: January 24, 2014
© 2014 American Chemical Society
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Figure 2. Virus inhibitors.
Figure 3. Design of target compounds.
Scheme 1. Synthesis of Harmalan, Tetrahydroharmane, and Harmane
for 5 h. Then the mixture was washed with a saturated aqueous
solution of NaHCO3, dried over anhydrous Na2SO4, and filtered, and
the solvent was removed under reduced pressure. The crude was
dissolved in toluene (20 mL) and chloroform (20 mL), phosphorus
oxychloride (3 mL) was added dropwise to the above solution, and
then the mixture was heated at reflux for 7 h. The mixture was adjusted
to pH 9 with a saturated aqueous solution of Na2CO3 after it was
cooled to room temperature. The resulting solution was extracted with
dichloromethane (3 × 20 mL), the combined organic phases were
washed with brine (50 mL), dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo and purified by flash chromatography on
silica gel using dichloromethane and methanol (v/v = 10:1) as eluent
to give harmalan as a brown solid (0.35 g, 60%): mp = 180−183 °C
175 °C (lit.19 mp = 177−178 °C); 1H NMR (400 MHz, DMSO-d6) δ
10.68 (s, 1H, NH), 7.35 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.27 (d, 3JHH
=
8.0 Hz, 1H, Ar−H), 6.98−7.02 (m, 1H, Ar−H), 6.91−6.95 (m, 1H,
Ar−H), 3.99−4.04 (m, 1H, CHNH), 3.33 (s, 1H, CHNH), 3.14−3.19
(m, 1H, CH2NH), 2.81−2.87 (m, 1H, CH2NH), 2.51−2.62 (m, 2H,
3
CH2CH2), 1.36 (d, JHH = 6.8 Hz, 3H, CH3CH); HRMS (ESI) calcd
for C12H15N2 (M + H)+ 187.1230, found 187.1231.
Synthesis of Harmane (1-Methyl-9H-pyrido[3,4-b]indole).20
To a stirred solution of maleic acid (0.53 g, 4.57 mmol) in water at
room temperature was added 1-methyl-2,3,4,9-tetrahydro-1H-pyrido-
[3,4-b]indole (0.85 g, 4.57 mmol), and the mixture was stirred until
the solid dissolved in water (120 mL); then Pd/C (0.85 g) was added,
and the mixture was heated at reflux for 8 h. Then the mixture was
cooled and filtered, and the filtered solid was washed carefully with
water. The combined filtrates were adjusted to pH 9 with aqueous
NaOH and then extracted with dichloromethane (3 × 50 mL), and the
combined organic phases were washed with brine (100 mL), dried
over anhydrous Na2SO4, filtered, and concentrated in vacuo to give
harmane as a white solid (0.50 g, 60%): mp = 244−245 °C (lit.21 mp =
235−238 °C); 1H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H, NH), 8.37
(lit.17 mp = 182−183 °C); H NMR (400 MHz, CDCl3) δ 9.47 (s,
1
1H, NH), 7.60 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.48 (d, 3JHH = 8.4 Hz,
1H, Ar−H), 7.31 (t, 3JHH = 8.0 Hz, 1H, Ar−H), 7.16 (t, 3JHH = 8.0 Hz,
3
1H, Ar−H), 3.88 (t, 3JHH = 8.4 Hz, 2H, CH2), 2.95 (t, JHH = 8.8 Hz,
2H, CH2), 2.53 (s, 3H, CH3); HRMS (ESI) calcd for C12H13N2
(M + H)+ 185.1073, found 185.1077.
Synthesis of Tetrahydroharmane (1-Methyl-2,3,4,9-tetrahy-
dro-1H-pyrido[3,4-b]indole).18 To a solution of 40% aqueous
solution of acetaldehyde (8.1 mL, 43.75 mmol) in water (250 mL) was
added concentrated H2SO4 (5 drops), the mixture was stirred at room
temperature for 0.5 h. Then tryptamine (3.50 g, 21.88 mmol) was
added, and the mixture was heated at reflux for 7 h. The mixture was
adjusted to pH 10 with an aqueous solution of NaOH after it was
cooled to room temperature. The resulting solution was extracted with
dichloromethane (3 × 100 mL), and the combined organic phase was
washed with brine (200 mL), dried over anhydrous Na2SO4, filtered,
concentrated in vacuo, and then purified by flash chromatography on
silica gel using dichloromethane and methanol (v/v = 5:1) as eluent to
give tetrahydroharmane as a brown solid (2.53 g, 62%): mp = 173−
3
3
(d, JHH = 5.2 Hz, 1H, Ar−H), 8.12 (d, JHH = 8.0 Hz, 1H, Ar−H),
7.83 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.52−7.57 (m, 2H, Ar−H), 7.26−
7.32 (m, 1H, Ar−H), 2.84 (s, 3H, CH3); HRMS (ESI) calcd for
C12H11N2 (M + H)+ 183.0917, found 183.0915.
Synthesis of Quaternary Ammonium Salts 1. To a solution of
harmine (0.50 g, 2.36 mmol) in ethyl acetate (120 mL) was added
benzyl bromide (0.48 g, 2.83 mmol), and the mixture was heated at
reflux for 12 h. Then the mixture was cooled and filtered, and the filter
cake was washed with ethyl acetate and dried to give 1 as a white solid
(0.67 g, 74%): mp = 276−277 °C; 1H NMR (400 MHz, DMSO-d6) δ
12.78 (s, 1H, NH), 8.74 (d, 3JHH = 6.4 Hz, 1H, Ar−H), 8.58 (d, 3JHH
=
3
6.8 Hz, 1H, Ar−H), 8.37 (d, JHH = 8.8 Hz, 1H, Ar−H), 7.35−7.44
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3
4
3
(m, 3H, Ar−H), 7.23 (d, JHH = 7.2 Hz, 2H, Ar−H), 7.12 (d, JHH
=
8.05 (d, JHH = 6.4 Hz, 1H, Ar−H), 7.80 (s, 1H, Ar−H), 7.70 (d,
3JHH = 6.4 Hz, 1H, Ar−H), 7.18 (t, 3JHH = 6.4 Hz, 1H, Ar−H), 2.88 (s,
3H, CH3); HRMS (ESI) calcd for C12H10BrN2 (M + H)+ 261.0022,
found 261.0025.
3
4
1.0 Hz, 1H, Ar−H), 7.08 (dd, JHH = 8.8 Hz, JHH = 1.0 Hz, 1H,
Ar−H), 5.98 (s, 2H, CH2), 3.95 (s, 3H, OCH3), 2.98 (s, 3H, CH3).
Synthesis of 2-Benzyl-7-methoxy-1-methyl-2,3,4,9-tetrahy-
dro-1H-pyrido[3,4-b]indole (2). To a solution of 1 (0.67 g,
1.75 mmol) in methanol (150 mL) was added dropwise sodium
borohydride (0.53 g, 14.00 mmol) in methanol (30 mL), and then the
mixture was heated at reflux for 15 h. The solvent was distilled off, and
the residue was diluted with dichloromethane (2 × 50 mL) and water
(50 mL); the combined organic phase was washed with brine (50 mL),
dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and
purified by flash chromatography on silica gel using dichloromethane
and methanol (v/v = 5:1) as eluent to give 2 as a brown oil (0.47 g,
Synthesis of Compounds 6 and 7.25 To a mixture of harmane
(0.40 g, 2.20 mmol) and sodium nitrate (0.93 g, 10.99 mmol) was
added dropwise trifluoroacetic acid (20 mmol) at 0 °C, and then the
mixture was stirred overnight at room temperature. The mixture was
adjusted to pH 10−11 with an aqueous solution of NaHCO3 in an
ice bath and then was extracted by dichloromethane (20 mL × 3).
The combined organic phase was concentrated in vacuo and purified by
flash chromatography on silica gel using dichloromethane and methanol
(v/v = 20:1) as eluent to give 6 (0.24 g) and 7 (0.06 g) .
1
88%): H NMR (400 MHz, CDCl3) δ 7.68 (s, 1H, NH), 7.50−7.52
1-Methyl-6-nitro-9H-pyrido[3,4-b]indole (6). This compound was
1
(m, 2H, Ar−H), 7.43−7.47 (m, 3H, Ar−H), 7.36−7.40 (m, 1H,
obtained as a pale yellow solid in 72% yield: mp > 300 °C; H NMR
3
4
4
Ar−H), 6.90 (dd, JHH = 8.8 Hz, JHH = 2.4 Hz, 1H, Ar−H), 6.83 (d,
(400 MHz, DMSO-d6) δ 12.36 (s, 1H, NH), 9.30 (d, JHH = 2.0 Hz,
4JHH = 2.4 Hz, 1H, Ar−H), 3.97 (d, JHH = 13.6 Hz, 1H, CH2C6H5),
2
1H, Ar−H), 8.41 (dd, 3JHH = 8.8 Hz, 4JHH = 3.0 Hz, 1H, Ar−H), 8.33
3
2
3
3
3.81 (q, JHH = 6.8 Hz, 1H, CHCH3), 3.77 (d, JHH = 13.6 Hz, 1H,
CH2C6H5), 3.22−3.29 (m, 1H, CH2CH2), 2.85−2.95 (m, 2H,
(d, JHH = 5.6 Hz, 1H, Ar−H), 8.20 (d, JHH = 5.2 Hz, 1H, Ar−H),
7.73 (d, 3JHH = 8.4 Hz, 1H, Ar−H), 2.79 (s, 3H, CH3); HRMS (ESI)
calcd for C12H10N3O2 (M + H)+ 228.0768, found 228.0767.
3
CH2CH2), 2.68−2.74 (m, 1H, CH2CH2), 1.61 (d, JHH = 6.8 Hz,
1H, CHCH3).
1-Methyl-8-nitro-9H-pyrido[3,4-b]indole (7). This compound was
obtained as a yellow solid in 12% yield: mp = 207−210 °C; 1H NMR
Synthesis of 7-Methoxy-1-methyl-2,3,4,4a,9,9a-hexahydro-
1H-pyrido[3,4-b]indole (3).22 To a solution of 2 (0.70 g,
2.29 mmol) in 2,2,2-trifluoroethanol (120 mL) was added Pd/C
(0.70 g), and then the reaction system was evacuated and purged with
hydrogen three times; the mixture was stirred in a hydrogen atmos-
phere overnight at room temperature. The mixture was concentrated
in vacuo and purified by flash chromatography on silica gel using
dichloromethane and methanol (v/v = 10:1) as eluent to give 3 as a
pale yellow solid (0.37 g, 74%): mp = 158−160 °C; 1H NMR
3
(400 MHz, DMSO-d6) δ 11.77 (s, 1H, NH), 8.77 (d, JHH = 7.6 Hz,
3
3
1H, Ar−H), 8.50 (d, JHH = 8.0 Hz, 1H, Ar−H), 8.38 (d, JHH
=
=
5.2 Hz, 1H, Ar−H), 8.11 (d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.48 (t, 3JHH
7.6 Hz, 1H, Ar−H), 2.92 (s, 3H, CH3); HRMS (ESI) calcd for
C12H10N3O2 (M + H)+ 228.0768, found 228.0772.
Synthesis of Harmol (1-Methyl-9H-pyrido[3,4-b]indol-7-ol).
A solution of harmine (0.50 g, 2.36 mmol) in glacial acetic acid (18 mL)
and 40% hydrobromic acid solution (18 mL) was stirred at room
temperature for 1 h, and then the mixture was heated at reflux for 10 h.
After cooling to room temperature, the mixture was adjusted to pH 8
with a saturated aqueous solution of NaHCO3. The yellow slurry was
filtered, and the cake was washed with water to afford harmol as a yellow
solid (0.46 g, 98%): mp > 300 °C (lit.26 mp = 304−307 °C); 1H NMR
(400 MHz, DMSO-d6) δ 11.24 (s, 1H, NH), 9.72 (s, 1H, OH), 8.11
(d, 3JHH = 5.2 Hz, 1H, Ar−H), 7.94 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.75
(d, 3JHH = 5.2 Hz, 1H, Ar−H), 6.90 (d, 4JHH = 1.2 Hz, 1H, Ar−H), 6.69
3
(400 MHz, CDCl3) δ 6.93 (d, JHH = 8.0 Hz, 1H, Ar−H), 6.31 (dd,
3JHH = 8.0 Hz, 4JHH = 2.0 Hz, 1H, Ar−H), 6.26 (d, 4JHH = 2.0 Hz, 1H,
Ar−H), 3.76 (s, 3H, OCH3), 3.49 (s, 1H, NH), 3.39−3.45 (m, 1H,
3
CHCH3), 3.35 (t, JHH = 8.4 Hz, 1H, CH), 2.97−3.02 (m, 1H, CH),
2.69−2.72 (m, 1H, CH2), 2.53−2.60 (m, 1H, CH2), 2.08−2.20 (m,
3
2H, CH2NH), 1.26 (s, 1H, NH), 1.23 (d, JHH = 6.4 Hz, 3H, CH3);
HRMS (ESI) calcd for C13H19N2O (M + H)+ 219.1492, found
219.1494.
3
4
Data for 7-Methoxy-1-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-
b]indole (Tetrahydroharmine). The compound was synthesized using
a similar procedure as for compound 3; the mixture was stirred for 3 h
to give tetrahydroharmine as a white solid (0.56 g, 80%): mp = 195−
(dd, JHH = 8.4 Hz, JHH = 1.6 Hz, 1H, Ar−H), 2.69 (s, 3H, CH3);
HRMS (ESI) calcd for C12H11N2O (M + H)+ 199.0866, found
199.0867.
Synthesis of 1-Methyl-9H-pyrido[3,4-b]indol-7-yl isopropyl-
carbamate (8).27 To a solution of harmol (0.50 g, 2.53 mmol) in
DMF (50 mL) was added isopropyl isocyanate via syringe over
5 min, the mixture was stirred at room temperature for 2 h, then
triethylamine (0.08 g, 0.758 mmol) was added, and the mixture was
stirred overnight at room temperature. Then brine (100 mL) was
added, the aqueous solution was extracted with ethyl acetate (20 mL ×
4), and the combined organic layer was dried over anhydrous Na2SO4,
concentrated in vacuo, and purified by flash chromatography on silica
gel using dichloromethane and methanol (v/v = 20:1) as eluent to
197 °C (lit.23 mp = 198−200 °C); H NMR (400 MHz, CDCl3) δ
1
7.64 (s, 1H, NH), 7.35 (d, 3JHH = 8.8 Hz, 1H, Ar−H), 6.85 (d, 4JHH
=
3
4
2.0 Hz, 1H, Ar−H), 6.77 (dd, JHH = 8.4 Hz, JHH = 2.4 Hz, 1H,
Ar−H), 4.14−4.19 (m, 1H, CHCH3), 3.84 (s, 3H, OCH3), 3.33−3.39
(m, 1H, CH2CH2), 3.01−3.07 (m, 1H, CH2CH2), 2.66−2.78 (m, 2H,
3
CH2CH2), 1.65 (s, 1H, NH), 1.44 (d, JHH = 6.8 Hz, 3H, CH3);
HRMS (ESI) calcd for C13H17N2O (M + H)+ 217.1335, found
217.1337.
Synthesis of Compounds 4 and 5.24 To a solution of harmane
(0.20 g, 1.10 mmol) in glacial acetic acid (10 mL) was added NBS
(0.20 g, 1.10 mmol) at room temperature, and the mixture was stirred
at room temperature for 6 h. Then the mixture was concentrated under
reduced pressure and adjusted to pH 10 with an aqueous solution of
NaHCO3, and the aqueous solution was extracted with dichloro-
methane (20 mL × 4). The combined organic layer was dried over
anhydrous Na2SO4 and then was concentrated in vacuo and purified by
flash chromatography on silica gel using dichloromethane and methanol
(v/v = 40:1→20:1) as eluent to give 4 (0.24 g) and 5 (0.05 g).
6-Bromo-1-methyl-9H-pyrido[3,4-b]indole (4). This compound
1
give 8 as a pale white solid (0.50 g, 70%): mp > 300 °C; H NMR
3
(400 MHz, DMSO-d63) δ 11.58 (s, 1H, NH), 8.20 (d, JHH = 5.2 Hz,
3
1H, Ar−H), 8.16 (d, JHH = 8.8 Hz, 1H, Ar−H), 7.90 (d, JHH = 5.2
Hz, 1H, Ar−H), 7.76 (d, 3JHH = 8.0 Hz, 1H, NHCO), 7.27 (d, 4JHH
=
1.6 Hz, 1H, Ar−H), 6.95 (dd, JHH = 8.4 Hz, 4JHH = 2.0 Hz, 1H, Ar−
3
3
H), 3.65−3.73 (m, 1H, CH), 2.75 (s, 3H, CH3), 1.16 (d, JHH = 6.4
Hz, 6H, (CH3)2CH); HRMS (ESI) calcd for C16H18N3O2 (M + H)+
284.1394, found 284.1399.
Synthesis of 1-Methyl-9H-pyrido[3,4-b]indol-7-yl dimethyl-
carbamate (9). To a solution of harmol (0.40 g, 2.02 mmol) in THF
(150 mL) was added triethylamine (0.31 g, 3.03 mmol), and the
mixture was stirred for 0.5 h. Then dimethylcarbamic chloride was
added dropwise, and the mixture was stirred overnight at room
temperature. Then water (20 mL) and dichloromethane (20 mL) were
added. The separated organic layer was dried over anhydrous Na2SO4,
filtered, concentrated in vacuo, and then purified by flash chromato-
graphy on silica gel using dichloromethane and methanol (v/v = 10:1)
as eluent to give 9 as a pale white solid (0.48 g, 89%): mp = 225−
227 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.63 (s, 1H, NH),
1
was obtained as a white solid in 83% yield: mp = 256−257 °C; H
NMR (400 MHz, CDCl3) δ 8.44 (s, 1H, NH), 8.39 (d, 3JHH = 5.6 Hz,
4
3
1H, Ar−H), 8.24 (d, JHH = 2.0 Hz, 1H, Ar−H), 7.78 (d, JHH = 5.6
3
4
Hz, 1H, Ar−H), 7.63 (dd, JHH = 8.4 Hz, JHH = 2.0 Hz, 1H, Ar−H),
3
7.42 (d, JHH = 8.8 Hz, 1H, Ar−H), 2.83 (s, 3H, CH3); HRMS (ESI)
calcd for C12H10BrN2 (M + H)+ 261.0022, found 261.0026.
8-Bromo-1-methyl-9H-pyrido[3,4-b]indole (5). This compound
was obtained as a yellow solid in 17% yield: mp = 201−204 °C; H
NMR (400 MHz, CDCl3) δ 8.36−8.49 (m, 2H, NH and Ar−H),
1
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3
8.20 (d, JHH = 5.2 Hz, 1H, Ar−H), 8.17 (d, JHH = 8.4 Hz, 1H, Ar−
7.17 (t, 3JHH = 7.2 Hz, 1H, Ar−H), 7.11 (t, 3JHH = 7.2 Hz, 1H, Ar−H),
3
4
H), 7.91 (d, JHH = 5.2 Hz, 1H, Ar−H), 7.30 (d, JHH = 2.0 Hz, 1H,
4.26−4.31 (m, 3H, CH and OCH2), 3.81 (dd, 3JHH = 11.2 Hz, 3JHH
=
3
4
Ar−H), 6.97 (dd, JHH = 8.4 Hz, JHH = 2.0 Hz, 1H, Ar−H), 3.10 (s,
3H, CH3), 2.94 (s, 3H, CH3), 2.75 (s, 3H, CH3); HRMS (ESI) calcd
for C15H16N3O2 (M + H)+ 270.1237, found 270.1240.
4.4 Hz, 1H, CH), 3.13 (dd, 2JHH = 15.2 Hz, 3JHH = 4.0 Hz, 1H, CH2),
3
2.79−2.86 (m, 1H, CH2), 1.52 (d, JHH = 6.4 Hz, 3H, CH3), 1.35 (t,
3JHH = 7.2 Hz, 3H, OCH2CH3); HRMS (ESI) calcd for C15H19N2O2
(M + H)+ 259.1441, found 259.1443.
Compounds 10 and 11 were synthesized using a procedure similar
to that used for compound 9.
Synthesis of Ethyl 1-Methyl-9H-pyrido[3,4-b]indole-3-car-
boxylate (15).29 A mixture of 14 (12.4 g, 47.7 mmol) and sulfur
(3.10 g, 95.4 mmol) in xylene (150 mL) was heated at reflux for 12 h.
Then the mixture was cooled to 0 °C and kept for 1 h, dichloro-
methane (10 mL) was added to the mixture, and pink solid was
precipitated. The mixture was filtered, and the cake was washed with
toluene to afford 15 as a brown solid (8.40 g, 69%): mp = 217−
219 °C; 1H NMR (400 MHz, CDCl3) δ 9.60 (s, 1H, NH), 8.79 (s, 1H,
Data for 1-Methyl-9H-pyrido[3,4-b]indol-7-yl acetate (10). This
compound was obtained as a white solid in 50% yield: mp = 237−
1
240 °C; H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H, NH), 8.23 (d,
3JHH = 2.4 Hz, 1H, Ar−H), 7.78 (d, JHH = 8.4 Hz, 1H, Ar−H), 7.70
3
(d, 3JHH = 2.0 Hz, 1H, Ar−H), 7.16 (s, 1H, Ar−H), 6.93 (d, 3JHH = 8.4
Hz, 1H, Ar−H), 2.76 (s, 3H, CH3), 2.42 (s, 3H, CH3CO); HRMS
(ESI) calcd for C14H13N2O2 (M + H)+ 241.2647, found 241.2650.
Data for Synthesis of 1-Methyl-9H-pyrido[3,4-b]indol-7-yl piv-
alate (11). This compound was obtained as a white solid in 85% yield:
mp = 221−222 °C; 1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H, NH),
3
Ar−H), 8.18(d, JHH = 8.0 Hz, 1H, Ar−H), 7.54−7.60 (m, 2H, Ar−
3
H), 7.32−7.36 (m, 1H, Ar−H), 4.50 (d, JHH = 6.8 Hz, 2H, OCH2),
3
2.79 (s, 3H, CH3), 1.41 (t, JHH = 7.2 Hz, 3H, OCH2CH3); HRMS
3
3
(ESI) calcd for C15H15N2O2 (M + H)+ 255.1128, found 255.1131.
Synthesis of 1-Methyl-9H-pyrido[3,4-b]indole-3-carboxylic
acid (16). To a solution of ester 15 (2.00 g, 7.87 mmol) in alcohol
(60 mL) was added NaOH (0.47 g, 11.81 mmol) in portions, and then
the mixture was heated at reflux for 6 h. Then the mixture was
concentrated under reduced pressure, the residue was dissolved in
water (50 mL), and the aqueous solution was extracted by diethyl
ether (50 mL × 2). Then the solution was adjusted to pH 5−6 with an
aqueous solution of diluted hydrochloric acid (3 M), the slight
yellow slurry was filtered, and the cake was washed with water to
8.30 (d, JHH = 5.2 Hz, 1H, Ar−H), 7.75 (d, JHH = 8.4 Hz, 1H, Ar−
H), 7.54 (d, 3JHH = 4.8 Hz, 1H, Ar−H), 7.09 (s, 1H, Ar−H), 6.86 (d,
3JHH = 8.0 Hz, 1H, Ar−H), 2.73 (s, 3H, CH3), 1.45 (s, 9H, C(CH3)3);
HRMS (ESI) calcd for C17H19N2O2 (M + H)+ 283.1441, found
283.1446.
Synthesis of (S)-1-Methyl-9H-pyrido[3,4-b]indol-7-yl-2-(ben-
zyloxycarbonylamino)-3-methylbutanoate (12). To a solution
of amino acid (0.80 g, 3.03 mmol) in dichloromethane (150 mL)
was added triethylamine (0.41 g, 4.04 mmol), 1-ethyl-3-(3-dimethyla-
minopropyl)carbodiimide (EDCI) (0.76 g, 4.04 mmol), and DMAP
(0.50 g, 4.04 mmol), and then the mixture was stirred overnight at
room temperature. The mixture was washed with a saturated aqueous
solution of NH4Cl (50 mL × 2) and a saturated aqueous solution of
NaHCO3 (50 mL) successively, dried over anhydrous Na2SO4, filtered,
concentrated in vacuo, and then purified by flash chromatography on
silica gel using dichloromethane and methanol (v/v = 20:1) as eluent
1
afford 16 as a yellow solid (1.46 g, 82%): mp > 300 °C; H NMR
(400 MHz, DMSO-d6) δ 12.04 (s, 1H, COOH), 8.77 (s, 1H, Ar−H),
3
3
8.36 (d, JHH = 8.0 Hz, 1H, Ar−H), 7.66 (d, JHH = 8.0 Hz, 1H,
3
3
Ar−H), 7.60 (t, JHH = 7.2 Hz, 1H, Ar−H), 7.30 (d, JHH = 7.2 Hz,
3
1H, Ar−H), 7.31 (d, JHH = 7.2 Hz, 1H, Ar−H), 2.82 (s, 3H, CH3);
HRMS (ESI) calcd for C13H11N2O2 (M + H)+ 227.0815, found
227.0811.
1
to give 12 as a pale white solid (0.80 g, 90%): mp = 69−71 °C; H
NMR (400 MHz, CDCl3) δ 8.54 (s, 1H, NH), 8.34 (d, 3JHH = 5.2 Hz,
Synthesis of (1-Methyl-9H-pyrido[3,4-b]indol-3-yl)methanol
(17). To a solution of ester 15 (2.00 g, 7.40 mmol) in THF (300 mL)
was added LiAlH4 (0.60 g, 15.7 mmol) in portions at 0 °C, and then
the mixture was stirred overnight at room temperature. Then the
reaction was quenched by water, and methanol (100 mL) was added.
After the mixture had been stirred at room temperature for 2 h, the
slurry was filtered, the cake was washed with dichloromethane, the
filtrate was concentrated to afford 17 as a yellow solid (1.58 g, 95%):
3
3
1H, Ar−H), 7.92 (d, JHH = 8.4 Hz, 1H, Ar−H), 7.66 (d, JHH = 4.8
Hz, 1H, Ar−H), 7.35−7.38 (m, 5H, Ar−H), 7.19 (s, 1H, Ar−H), 6.94
3
3
(d, JHH = 8.4 Hz, 1H, Ar−H), 5.39 (d, JHH = 8.4 Hz, 1H, NHCO),
5.17 (s, 2H, CH2), 4.54−4.66 (m, 1H, CHNH), 2.77 (s, 3H, CH3),
3
2.38−2.50 (m, 1H, CH(CH3)2), 1.14 (d, JHH = 6.8 Hz, 3H,
3
CH(CH3)2), 1.09 (d, JHH = 6.8 Hz, 3H, CH(CH3)2); HRMS (ESI)
calcd for C25H26N3O4 (M + H)+ 432.1918, found 432.1920.
1
mp = 195−197 °C; H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H,
Synthesis of (1S,3S)-1-Methyl-2,3,4,9-tetrahydro-1H-pyrido-
[3,4-b]indole-3-carboxylic acid (13).28 To a solution of
L-tryptophan (20.00 g, 98.0 mmol) in water (500 mL) were added con-
centrated H2SO4 (2 mL) and a 40% aqueous solution of acetaldehyde
(20 mL) successively, and the mixture was stirred overnight at room
temperature. Then the mixture was adjusted to pH 6−7 with ammonia
solution (25−28%, w/w), and a white solid precipitated. The white
slurry was filtered, and the cake was washed with water to afford 13 as a
NH), 8.19 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.95 (s, 1H, Ar−H), 7.56 (d,
3JHH = 8.0 Hz, 1H, Ar−H), 7.49−7.53 (m, 1H, Ar−H), 7.18−7.22 (m,
3
3
1H, Ar−H), 5.30 (t, JHH = 6.0 Hz, 1H, OH), 4.67 (d, JHH = 6.0 Hz,
2H, CH2OH), 2.73 (s, 3H, CH3).
Synthesis of 1-Methyl-9H-pyrido[3,4-b]indole-3-carbaldehyde
(18). The mixture of alcohol 17 (1.16 g, 5.47 mmol) and IBX (3.04 g,
10.93 mmol) in DMSO (60 mL) was stirred overnight at room
temperature. After water (200 mL) and dichloromethane (50 mL × 3)
were added, the separated organic layer was dried over anhydrous
Na2SO4, filtered, concentrated in vacuo, and purified by flash
chromatography on silica gel using dichloromethane and methanol
(v/v = 10:1) as eluent to give 18 as a white solid (0.46 g, 40%): mp =
1
white solid (16.7 g, 74%): mp = 278−280 °C; H NMR (400 MHz,
3
DMSO-d6) δ 11.11 (s, 1H, COOH), 7.45 (d, JHH = 7.8 Hz, 1H,
Ar−H), 7.34 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.09 (t, 3JHH = 7.6 Hz, 1H,
Ar−H), 7.00 (t, 3JHH = 7.2 Hz, 1H, Ar−H), 4.52 (q, 3JHH = 6.4 Hz, 1H,
3
3
CH), 3.61 (dd, JHH = 11.6 Hz, JHH = 4.4 Hz, 1H, CH), 3.16 (dd,
1
2JHH = 16.0 Hz, JHH = 4.0 Hz, 1H, CH2), 2.74−2.81 (m, 1H, CH2),
3
194−196 °C; H NMR (400 MHz, DMSO-d6) δ 12.17 (s, 1H, NH),
10.07 (s, 1H, CHO), 8.68 (s, 1H, Ar−H), 8.38 (d, 3JHH = 7.6 Hz, 1H,
1.62 (d, 3JHH = 6.4 Hz, 3H, CH3); (dr = 11.7:1); HRMS (ESI) calcd for
C13H15N2O2 (M + H)+ 231.1128, found 231.1132.
Ar−H), 7.68 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.61 (t, 3JHH = 7.6 Hz, 1H,
3
Synthesis of Ethyl (1S,3S)-1-Methyl-2,3,4,9-tetrahydro-1H-
pyrido[3,4-b]indole-3-carboxylate (14). To a solution of acid 13
(16.0 g, 69.0 mmol) in ethanol (500 mL) was added dropwise thionyl
chloride in an ice bath, and then the mixture was heated at reflux for
5 h. The solvent was distilled off, the residue was dissolved in water
(250 mL), and then the mixture was adjusted to pH 9 with an aqueous
solution of NaHCO3. The aqueous was extracted by ethyl acetate
(150 mL × 3), the combined organic phase was washed with brine and
dried over anhydrous Na2SO4, and then the mixture was concentrated
in vacuo to give 14 as a light yellow solid (16.40 g, 92%): mp =
Ar−H), 7.33 (t, JHH = 7.6 Hz, 1H, Ar−H), 2.87 (s, 3H, CH3).
Synthesis of (E)-3-(1-Methyl-9H-pyrido[3,4-b]indol-3-yl)-
acrylic acid (19). To a solution of aldehyde 18 (0.45 g, 2.14 mmol)
and piperidine (3 drops) in pyridine was added malonic acid (0.33 g,
3.21 mmol), and the reaction mixture was heated at 120 °C for 4 h.
Then the mixture was concentrated in vacuo, water was added (50 mL),
the pH was adjusted to 11−12 with an aqueous solution of NaOH, and
then the mixture was extracted with diethyl ether. The aqueous phase
was adjusted to pH 5−6 with dilute hydrochloric acid (3 M), the yellow
slurry was filtered, and the cake was washed with water to afford 19 as a
1
1
136−137 °C; H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H, NH), 7.49
(d, JHH = 8.0 Hz, 1H, Ar−H), 7.33 (d, JHH = 8.0 Hz, 1H, Ar−H),
yellow solid (0.51 g, 94%): mp = 220−223 °C; H NMR (400 MHz,
3
3
DMSO-d6) δ 12.22 (s, 1H, NH), 11.85 (s, 1H, COOH), 8.31 (s, 1H,
1013
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Scheme 2. Synthesis of Tetrahydroharmine and 3
Biological Assay. The anti-TMV and fungicidal activities of the
synthesized compounds were tested using our previously reported
methods.30,31
Scheme 3. Synthesis of Compounds 4−7
RESULTS AND DISCUSSION
■
Synthesis. Harmalan and tetrahydroharmane were obtained
from tryptamine by Bischler−Napieralski and Pictet−Spengler
reaction, respectively. Tetrahydroharmane was oxidized using
the Pd/C−maleic acid system in water to give harmane in 60%
yield (Scheme 1).
In the synthesis of Tetrahydroharmine, harmine was used as
starting material. First, harmine was reacted with benzyl bromide
to give a quaternary ammonium salt 1 in 74% yield, then it
was reduced by sodium borohydride to give tetrahydrocarboline
2 in 88% yield, and finally tetrahydroharmine was obtained
from 2 by debenzylation using palladium−carbon and hydrogen.
The reaction time of debenzylation should be controlled at <3 h.
Otherwise, the product will be over-reduced to give hexahydro-
1H-pyridoindole 3. When the reaction time was extended to
12 h, the product was converted into 3 completely (Scheme 2).
In Scheme 3, the bromination and nitration products were
synthesized from harmane using the reported methods.22−24
Harmol can be obtained from harmnine by a demethylation
reaction (Scheme 4). It was reported that the demethylation
reaction of harmine could be processed by BBr3 in chloroform,
but the reaction yield was low (<30%).32 This was because of
the poor solubility of harmine in conventional demethylation
solvent (dichloromethane, chlorobenzene, etc.); thus, the demethy-
lation reaction was very slow and incomplete. Therefore, in our
trial on the demethylation of harmine, glacial acetic acid and 40%
(m/m) aqueous hydrobromic acid (v/v = 1:1) were used instead of
chloroform, and harmol was obtained in 98% yield, which was used
in the synthesis of O-substituent derivatives 8−12 at the 7-position.
To investigate the influence of the degree of unsaturation of
the C ring and the substituents at 3-position on the anti-TMV
3
3
Ar−H), 8.21 (d, JHH = 7.6 Hz, 1H, Ar−H), 7.72 (d, JHH = 15.2 Hz,
1H, CHCH), 7.62 (d, 3JHH = 7.6 Hz, 1H, Ar−H), 7.56 (t, 3JHH = 7.6 Hz,
1H, Ar−H), 7.28 (t, 3JHH = 7.6 Hz, 1H, Ar−H), 6.78 (d, 3JHH = 15.6 Hz,
1H, CHCH), 2.80 (s, 3H, CH3); HRMS (ESI) calcd for C15H13N2O2
(M + H)+ 253.0972, found 253.0975.
Compounds 20 and 21 were synthesized using a procedure similar
to that used for compound 19.
Data for (E)-3-(1-(Thiophen-2-yl)-9H-pyrido[3,4-b]indol-3-yl)-
acrylic acid (20). This compound was obtained as a yellow solid in
1
85% yield: mp = 248−250 °C; H NMR (400 MHz, DMSO-d6) δ
12.43 (s, 1H, NH), 12.10 (s, 1H, COOH), 8.47 (s, 1H, Ar−H), 8.28
3
3
(d, JHH = 8.0 Hz, 1H, Ar−H), 8.17 (d, JHH = 3.2 Hz, 1H, Ar−H),
3
7.80 (d, JHH = 4.8 Hz, 1H, Ar−H), 7.77−7.80 (m, 2H, Ar−H), 7.62
(t, 3JHH = 7.2 Hz, 1H, Ar−H), 7.33−7.38 (m, 2H, Ar−H and CHCH),
3
3
6.88 (d, JHH = 15.6 Hz, 1H, CHCH), 6.93 (d, JHH = 15.6 Hz, 1H,
CHCH); HRMS (ESI) calcd for C18H13N2O2S (M + H)+ 321.0692,
found 321.0694.
Data for (E)-3-(1-(Pyridin-3-yl)-9H-pyrido[3,4-b]indol-3-yl)acrylic
acid (21). This compound was obtained as a yellow solid in 50% yield:
mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.43 (s, 1H, NH),
4
12.10 (s, 1H, COOH), 9.31 (d, JHH = 1.6 Hz, 1H, Ar−H), 8.81
3
4
(dd, JHH = 4.8 Hz, JHH = 1.2 Hz, 1H, Ar−H), 8.61 (s, 1H, Ar−H),
3
8.56−8.59 (m, 1H, Ar−H), 8.31 (d, JHH = 7.6 Hz, 1H, Ar−H), 7.84
3
3
4
(d, JHH = 15.6 Hz, 1H, CHCH), 7.76 (dd, JHH = 7.6 Hz, JHH
4.8 Hz, 1H, Ar−H), 7.70 (d, 3JHH = 8.0 Hz, 1H, Ar−H), 7.62 (t, 3JHH
7.2 Hz, 1H, Ar−H), 7.35 (t, 3JHH = 7.2 Hz, 1H, Ar−H), 6.93 (d, 3JHH
=
=
=
15.6 Hz, 1H, CHCH); HRMS (ESI) calcd for C19H14N3O2 (M + H)+
316.1081, found 316.1079.
Scheme 4. Synthesis of Harmol and Compounds 8−12
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Scheme 5. Synthesis of Compounds13−16 and 19−21
Table 1. In Vitro Antiviral Activity of Harmine, Harmane, Harmol, Harmalan, Tetrahydroharmane, Tetrahydroharmine, and
Compounds 3, 4, 6−16, and 19−21 against TMV
compd
Ribavirin
concn (μg/mL)
inhibition rate (%)
compd
concn (μg/mL)
inhibition rate (%)
500
100
40
9
500
100
12.6
0
12.9
harmine
500
44.6
10
11
12
13
14
15
16
19
20
21
500
100
30
0
100
20
harmane
500
54.5
500
100
0
0
100
16.4
harmol
500
100
32.6
0
500
100
20.7
0
harmalan
500
58.6
500
100
28.6
0
100
30
tetrahydroharmane
500
63.7
500
46.5
100
28.4
100
21.8
tetrahydroharmine
500
58.2
500
100
33.3
0
100
28.7
3
4
6
7
8
500
100
41.9
12.4
500
100
32.5
0
500
100
44.5
18.2
500
100
25.8
0
500
100
15.7
0
500
100
40.2
0
500
100
30
0
500
100
17.8
0
500
100
31.3
0
activities, compounds 13−16 and 19−21 were synthesized
using the method showed in Scheme 5.
activities of these compounds were obviously higher than that of
the compounds containing a pyridine moiety. For instance, the
activities of harmalan, tetrahydroharmane, and tetrahydrohar-
mine were 58.6, 63.7, and 58.2%, respectively, at 500 μg/mL,
which were much higher than that of Ribavirin (40% at
500 μg/mL), whereas the activity of harmine was 44.6% at
500 μg/mL. The activity of tetrahydro-β-carboline 14 was 46.5%
Antiviral Activities. Most of the tested compounds
displayed good antiviral activity in vitro, of which harmane,
harmalan, tetrahydroharmane, tetrahydroharmine, and 14
exhibited higher inhibition than Ribavirin (Table 1). When the
C ring (Figure 3) was dihydropyridine or tetrahydropyridine, the
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Table 2. In Vivo Antiviral Activity of Harmine, Harmane, Harmol, Harmalan, Tetrahydroharmane, Tetrahydroharmine, and
Compounds 3, 4, 6−16, and 19−21 against TMV
concn
inactivation
effect (%)
curative
protection
effect (%)
concn
inactivation
effect (%)
curative
protection
effect (%)
compd
Ribavirin
(μg/mL)
effect (%)
compd
(μg/mL)
effect (%)
500
100
37.4
13.7
36.2
16.9
38.5
16.2
100
0
0
5.8
9
500
100
12
0
15.6
0
18.7
0
harmine
500
100
40.5
11.4
38.6
15.8
42.4
16.3
10
11
12
13
14
15
16
19
20
21
500
100
20.3
0
24.1
0
27.2
0
harmane
500
50.6
50
57.8
100
17.7
23.7
26.9
500
100
15.3
0
0
0
14.4
0
harmol
500
100
35.1
0
30
0
36.7
9.5
500
100
13.6
0
12.5
0
19.6
0
harmalan
500
62.3
55.1
60.3
100
32.7
34.8
35.6
500
100
30.5
0
31.4
0
27.8
0
tetrahydroharmane
500
64.2
57.2
59.5
100
23.5
20.4
24.6
500
50.4
43.9
47.9
tetrahydroharmine
500
59.8
55.5
54.6
100
25.7
17.5
18.6
100
26.4
27.3
24.2
500
100
26.3
0
28.8
0
23.7
0
3
4
6
7
8
500
100
44.1
17.1
45.2
14.2
37.7
14.9
500
100
27.3
0
29.6
0
21.4
0
500
100
46.3
14.9
47.4
19.3
51.2
20.4
500
100
18.4
0
21.5
0
28.4
0
500
100
21.5
0
18.5
0
26.1
0
500
100
39.6
13.5
36.2
10
35.1
10.2
500
100
36.4
10.5
33.3
0
30.1
5.8
500
100
26.5
0
20.2
0
21.3
0
500
29.5
32.7
40.3
at 500 μg/mL, whereas the activity of the corresponding
β-carboline compound 15 was 33.3% at the same concentration.
When tetrahydroharmine was further reduced to hexahydrohar-
mine (3), the activity (41.9%, 500 μg/mL) was also reduced.
When the skeleton was β-carboline (C ring was pyridine)
(Figure 3), it was detrimental to anti-TMV activity when a
substituent was introduced to the benzene ring (A ring). For
example, the in vitro activity of compound with no substituent
on the benzene ring (harmane) was 54.5% at 500 μg/mL,
whereas the activity was reduced to 44.6 and 32.6% when a
methoxy (harmine) or a hydroxy (harmol) group was introduced
to the 7-position. The derivatives of harmol (8−11) exhibited
the same or lower activities against TMV compared with harmol.
For the compound containing a carbamate moiety, the sub-
stituents on the nitrogen of amide had an obvious effect on the
activity; the activity of a compound containing isopropylamino
(8) (31.3%, 500 μg/mL) was better than that of a compound
containing dimethylamino (9) (12.6%, 500 μg/mL). For the
compounds containing an ester structure, the spatial arrange-
ment of the substituents might have an impact on anti-TMV
activity of the compounds, as the activity of compound
containing acetyl (10) was 30% at 500 μg/mL, whereas when
the substituent was changed to pivaloyl (11), the compound
exhibited no activity against TMV. The activity of compound
containing a bromo at 6-position (4) was equivalent to that of
Ribavirin (40% at 500 μg/mL); when the substituent was
changed to nitro at 6- or 8-position (6 and 7), the activities were
reduced to 15.7 and 30% at 500 μg/mL, respectively.
The substituents on the 3-position of the β-carbolines or
tetrahydro-β-carbolines played an important role in the
activities. The activities of compounds containing ethyl formate
(14) at the 3-position were better than that of compounds
containing carboxylic acid (13 and 16) and acroleic acid
(19−21). Among 19−21, the substituents on the 1-position
also had different effects on the antiviral activity; the 2-thienyl
compound (20) was better than the methyl (19) and 3-pyridyl
(21) compounds.
Further bioassay was conducted to investigate their inactiva-
tion, curative, and protection effects in vivo (Table 2). The
result showed that most of the compounds exhibited good to
excellent antiviral activity and similar structure−activity relation-
ships to the activity in vitro. Harmane, harmalan, tetrahy-
droharmane, tetrahydroharmine, 3, 4, and 14 exhibited higher
activities than Ribavirin; especially the activities of compounds
harmalan (62.3, 55.1, and 60.3% at 500 μg/mL) and
tetrahydroharmane (64.2, 57.2, and 59.5% at 500 μg/mL)
1016
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Table 3. Fungicidal Activity of Harmine, Harmane, Harmol, Harmalan, Tetrahydroharmane, Tetrahydroharmine, and
Compounds 3, 4, 6−16, and 19−21 against 14 Kinds of Fungus
fungicidal activity (%)/50 mg/kg
a
compd
harmine
F.C
C.H
P.P
A.S
F.G
F.M
S.S
9.8
P.C
R.C
B.M
W.A
P.I
R.S
B.C
19.2
19.2
26.9
23.1
19.2
19.2
19.0
65.4
23.8
61.9
34.6
23.1
14.3
52.4
38.5
19.2
28.6
23.8
19.0
23.1
33.3
4.8
69.2
92.3
46.2
76.9
61.5
76.9
28.6
84.6
7.1
11.9
61.9
23.8
2.4
15.8
68.4
26.3
26.3
26.3
21.1
50.0
73.7
33.3
88.9
21.1
15.8
33.3
66.7
36.8
21.1
55.6
61.1
33.3
10.5
22.2
22.2
26.2
23.8
23.8
21.4
14.3
11.9
57.1
76.2
50.0
64.3
23.8
38.1
35.7
78.6
35.7
26.2
21.4
71.4
35.7
14.3
78.6
42.9
100
42.9
35.7
28.6
28.6
14.3
14.3
35.7
64.3
42.9
71.4
14.3
21.4
35.7
50.0
21.4
21.4
42.9
35.7
35.7
28.6
64.3
28.6
<50
100
12.5
15.6
25.0
0.0
44.7
42.1
42.1
55.3
42.1
34.2
56.3
68.4
59.4
71.9
36.8
36.8
62.5
90.6
84.2
42.1
59.4
78.1
53.1
63.2
81.3
53.1
22.7
36.4
31.8
27.3
18.2
27.3
21.1
86.4
31.6
94.7
22.7
27.3
21.1
78.9
36.4
22.7
26.3
36.8
21.1
22.7
73.7
15.8
100
35.7
46.4
46.4
25.0
32.1
25.0
33.3
92.9
23.8
85.7
39.3
35.7
33.3
71.4
50.0
35.7
38.1
47.6
38.1
25.0
66.7
23.8
100
22.7
9.1
36.8
26.3
50.0
19.7
52.6
26.3
39.3
96.1
10.7
71.4
6.6
21.2
12.1
9.1
harmane
33.3
5.9
harmol
22.7
31.8
18.2
22.7
25.0
45.5
25.0
70.0
18.2
9.1
harmalan
19.6
9.8
15.2
12.1
27.3
36.4
45.5
54.5
59.1
0.0
tetrahydro-harmane
26.2
14.3
94.1
61.9
35.3
88.2
2.4
0.0
tetrahydro-harmine
19.6
56.0
41.2
56.0
76.0
9.8
9.4
3
36.4
93.8
36.4
90.9
25.0
9.4
4
6
7
71.4
53.8
53.8
35.7
71.4
61.5
30.8
50.0
35.7
50.0
84.6
21.4
0.0
8
9
26.2
5.9
7.8
46.1
0.0
12.1
36.4
54.5
51.5
21.2
68.2
13.6
63.6
12.1
68.2
4.5
10
76.0
80.0
13.7
7.8
18.2
93.9
50.0
18.8
60.6
51.5
60.6
18.8
66.7
9.1
20.0
50.0
45.5
27.3
30.0
35.0
25.0
22.7
55.0
15.0
11
76.5
78.6
23.8
29.4
52.9
82.4
47.6
58.8
23.5
42.9
52.6
26.3
33.9
39.3
42.9
39.5
17.9
0.0
12
13
14
68.0
68.0
72.0
9.8
15
16
19
20
76.0
76.0
21
carbendazim
chlorothalonil
<50
100
<50
73.3
<50
100
<50
73.3
100
<50
<50
100
100
100
100
86.4
100
100
<50
100
<50
91.3
91.3
a
F.C, Fusarium oxysporium f. sp.cucumeris; C.H, Cercospora arachidicola Hori; P.P, Physalospora piricola; A.S, Alternaria solani; F.G, Fusarium
graminearum; F.M, Fusarium moniliforme; S.S, Sclerotinia sclerotiorum; P.C, Phytophthora capsici; R.C, Rhizoctonia cerealis; B.M, Bipolaris maydis; W.A,
watermelon anthracnose; P.I, Phytophthora infestans; R.S, Rhizoctonia solani; B.C, Botrytis cinerea.
were much higher than that of Ribavirin (37.4, 36.2, and 38.5%
at 500 μg/mL); harmine, harmol, 7, and 20 showed activities
comparable to that of Ribavirin. It was interesting that the
compound containing an ester on the 3-position (14) exhibited
obviously higher activities (50.4, 43.9, and 47.9% at 500 μg/mL)
than compounds having other substituents on the 3-position.
In consideration of the better solubility and stability of com-
pound 14, it can be used as a new lead structure for further
development of anti-TMV reagents.
Fungicidal Activities. These alkaloids and their derivatives
also exhibited good fungicidal activities against 14 kinds of plant
fungi (Table 3). When the C ring was pyridine, the compounds
exhibited higher activities than those with dihydropyridine or
tetrahydropyridine as the C ring. When the skeleton was
β-carboline (C ring was pyridine), the types and positions of
the substituents on the benzene ring (A ring) had a significant
influence on the fungicidal activities. For instance, compound 7
(1-methyl-8-nitro-9H-pyrido[3,4-b]indole) exhibited excellent
fungicidal activity against most of the tested fungi, whereas
compound 6 (1-methyl-6-nitro-9H-pyrido[3,4-b]indole) ex-
hibited only moderate activity; compound 11, containing a
pivaloyloxy group, exhibited higher fungicidal activity than the
compound containing acetoxy (10). Some of these compounds
selectively exhibited fungicidal activities to certain fungi. For
example, the activities of harmane (against Cercospora arach-
idicola Hori) and compound 3 (against Physalospora piricola)
were >90%, which was much higher than that against other fungi.
In summary, 6 known β-carboline, dihydro-β-carboline, or
tetrahydro-β-carboline alkaloids and a series of their derivatives
were designed, synthesized, and first assayed for their anti-TMV
activities both in vitro and in vivo and for their fungicidal
activity against 14 fungi. All of the compounds exhibited good
to excellent anti-TMV activities. Especially, the inactivation,
curative, and protection activities of compounds harmalan
(62.3, 55.1, and 60.3%, respectively, at 500 μg/mL) and
tetrahydroharmane (64.2, 57.2, and 59.5% at 500 μg/mL) in
vivo were much higher than those of Ribavirin (37.4, 36.2, and
38.5% at 500 μg/mL). Compound 14 also exhibited obviously
higher activities in vivo (50.4, 50.0, and 57.8% at 500 μg/mL)
than Ribavirin and other derivatives, and its solubility and
stability were better than those of the natural products, so it can
be used as a lead structure for the development of anti-TMV
drugs. Moreover, some of these compounds, especially com-
pounds 4, 7, and 11, exhibited good fungicidal activity against
14 kinds of fungi. Further studies on structural optimization are
in progress in our laboratory.
AUTHOR INFORMATION
■
Corresponding Author
*(Q.W.) Phone: +86-(0)22-23503952. Fax: +86-(0)22-
23503952. E-mail: wang98h@263.netm wangqm@nankai.edu.cn.
Funding
This work was supported by the National Key Project for Basic
Research (2010CB126106), the National Natural Science
Foundation of China (21132003, 21121002, 21372131), and
the Specialized Research Fund for the Doctoral Program of
Higher Education (20130031110017).
Notes
The authors declare no competing financial interest.
1017
dx.doi.org/10.1021/jf404840x | J. Agric. Food Chem. 2014, 62, 1010−1018

Journal of Agricultural and Food Chemistry
Article
(19) Milen, M.; Hazai, L.; Kolonits, P.; Kalaus, G.; Szabo,
́
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