Synthesis and antimicrobial characterization of half-calycanthaceous alkaloid derivatives

Article abstract of DOI:10.3390/molecules21091207

A total of 29 novel tetrahydropyrroloindol-based calycanthaceous alkaloid derivatives were synthesized from indole-3-acetonitrile in good yields. The synthesized compounds were evaluated against nine strains of bacteria and a wide range of plant pathogen fungi. Bioassay results revealed that majority of the compounds displayed similar or higher in vitro antimicrobial activities than the positive control. The biological activities also indicated that substituents at R₄ and R₅ significantly affect the activities. Notably, compound c4 was found to be most active among the tested calycanthaceous analogues and might be a novel potential leading compound for further development as an antifungal agent. The results could pave the way for further design and structural modification of calycanthaceous alkaloids as antimicrobial agents.

Full text of DOI:10.3390/molecules21091207

molecules
Article
Synthesis and Antimicrobial Characterization of
Half-Calycanthaceous Alkaloid Derivatives
Shaojun Zheng 1,2,3, Xinping Zhou 2, Shixun Xu 2, Rui Zhu 1, Hongjin Bai 2,* and Jiwen Zhang 2,3,
*
1
School of Environmental and Chemical Engineering, Jiangsu University of Science and Technology,
Zhenjiang 212003, Jiangsu, China; sz281cam@just.edu.cn (S.Z.); zhurui991314@163.com (R.Z.)
2
Key Laboratory of Protection & Utilization of Biological Resources in Tarim Basin of Xinjiang Production
and Construction Corps/College of Life Sciences, Tarim University, Alar 843300, Xinjiang, China;
zhouzhouxinping@163.com (X.Z.); xsx_89@163.com (S.X.)
Key Laboratory of Botanical Pesticide R & D in Shaanxi Province, Yangling 712100, Shaanxi, China
Correspondence: bhj67@163.com (H.B.); nwzjw@nwsuaf.edu.cn (J.Z.);
3
*
Tel.: +86-997-4681-612 (H.B.); +86-29-8709-3987 (J.Z.)
Academic Editor: Kamal Kumar
Received: 3 July 2016; Accepted: 6 September 2016; Published: 9 September 2016
Abstract: A total of 29 novel tetrahydropyrroloindol-based calycanthaceous alkaloid derivatives
were synthesized from indole-3-acetonitrile in good yields. The synthesized compounds were
evaluated against nine strains of bacteria and a wide range of plant pathogen fungi. Bioassay results
revealed that majority of the compounds displayed similar or higher in vitro antimicrobial activities
than the positive control. The biological activities also indicated that substituents at R4 and R5
significantly affect the activities. Notably, compound c4 was found to be most active among the
tested calycanthaceous analogues and might be a novel potential leading compound for further
development as an antifungal agent. The results could pave the way for further design and structural
modification of calycanthaceous alkaloids as antimicrobial agents.
Keywords: calycanthaceous alkaloids; synthesis; biological activity; agrochemicals; SAR
1. Introduction
As a part of conventional agriculture, agrochemicals play an important role in the protection
of vegetable and cereal crops. However, the unrestricted usage of highly toxic agrochemicals over
the past several decades had caused negative effects on environment and poisoned non-targeted
species. Therefore, to reduce the negative impacts of agrochemicals, new compounds with high
efficacy against target species are desired. Natural-product-based libraries provide a rich source for
new agrochemical discovery [
China, North America and Australia [
the roots, leaves, flowers, and fruits of Chimonanthus praecox [
biological activities such as anticonvulsant, antifungal, antiviral, analgesic, antitumor, and inhibition
of melanogenesis properties [10 13]. Due to their broad spectrum of biological properties, a number
of studies towards the synthesis and antimicrobial activity of calycanthaceous alkaloids have been
reported [13 25]. However, the core structure of calycanthaceous alkaloids is underexplored. Therefore,
1–
4
]. Calycanthaceous alkaloids [
], are an important class of alkaloids that can be isolated from
], and have demonstrated widespread
5,6] (Figure 1), mainly distributed in
7,8
9
–
–
we focused on the structural optimization of tetrahydropyrroloindole. It was hoped that a leading
compound could be identified for the discovery of novel antimicrobial agents. Herein, a series of novel
calycanthaceous alkaloids analogs were designed and synthesized using indole-3-acetonitrile as the
starting material.
To the best of our knowledge, the antimicrobial activities of the synthetic derivatives are reported
herein for the first time.
Molecules 2016, 21, 1207; doi:10.3390/molecules21091207
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1207
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Figure 1. Structures of calycanthaceous alkaloids.
2. Results and Discussion
2.1. Design and Synthesis of Calycanthaceous Alkaloids Analogues
The synthetic route is outlined in Scheme 1. Intermediates
1–
8
were synthesized according to
the corresponding references and their spectral data were consistent with literature values [26–28].
The analogs of calycanthaceous alkaloids were synthesized using indole-3-acetonitrile as the starting
material via acylation at the N3 position. Twenty nine derivatives were synthesized and characterized
by 1H-NMR, and 13C-NMR spectroscopy and ESI-MS.
Scheme 1. Synthetic route to the title compounds a1–a21 and b1–c4.
2.2. Antimicrobial Acitivity
Antibacterial results are shown in Table 1. MIC and MBC values were determined using the
microdilution method, with gentamicin and streptomycin as positive controls to evaluate the biological
activities of analogues against Gram-positive bacteria (B. cereus, S. aureus, and S. epidermidis).

Molecules 2016, 21, 1207
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Table 1. Antibacterial activity of calycanthaceous alkaloids derivatives against B. cereus, S. aureus,
and S. epidermidis.
MIC µg/mL
MBC µg/mL
Compounds
B. cereus
S. aureus
256
256
−
S. epidermidis
B. cereus
−
S. aureus
−
S. epidermidis
a1
a2
a3
a4
a5
a6
a7
a8
a9
a10
a11
a12
a13
a14
a15
a16
a17
a18
a19
a20
a21
b1
b2
b3
b4
c1
c2
c3
c4
−
62.5
62.5
−
125
125
−
−
−
−
125
−
256
−
−
−
62.5
−
−
125
−
−
−
−
−
256
31.25
62.5
125
125
31.25
15.63
−
−
−
−
−
−
256
256
−
62.5
125
−
62.5
125
256
256
62.5
31.25
−
−
125
256
−
−
−
−
−
−
−
125
−
62.5
−
256
−
125
−
−
−
−
−
256
256
62.5
−
−
−
−
−
−
−
−
−
−
−
−
−
125
−
−
−
−
−
−
−
−
−
−
−
−
−
−
256
256
−
62.5
125
−
−
−
125
256
−
125
−
256
−
−
−
62.5
31.25
7.81
31.25
62.5
125
62.5
62.5
−
125
125
125
256
125
125
125
31.25
62.5
15.63
125
125
125
125
62.5
62.5
125
31.25
15.63
31.25
125
62.5
15.63
62.5
125
256
125
125
−
256
256
256
−
256
256
256
256
125
125
256
62.5
31.25
62.5
256
256
256
62.5
125
31.25
gentamicin
streptomycin
1.96
3.9
Note: The gentamicin and streptomycin were used as the positive controls; “ ” means no inhibition effect. MIC:
−
Minimal Inhibitory Concentration; MBC: Minimum Bactericidal Concentration.
Tables 1 and 2 show which of the synthesized compounds exhibited potent in vitro antimicrobial
activities against B. cereus, S. aureus, and S. epidermidis. Compounds a3
and c1 c4 showed higher degrees of activity against B. cereus than gentamicin, with b3 and a12
being the most effective, with MIC values of 7.81
mL−1 and 15.63 mL−1, respectively.
, a7–a12, a16, a20, b1–b4
–
µg
·
µg
·
Compound a4 showed comparable control efficacy against S. epidermidis to that of gentamicin.
Noticeably, compound c4 revealed potent activity against B. cereus, S. aureus, and S. epidermidis,
with MIC values of 62.5 µg·mL−1, 31.25 µg·mL−1, and 31.25 µg·mL−1, respectively.

Molecules 2016, 21, 1207
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Table 2. Antibacterial activity of calycanthaceous alkaloids derivatives against E. coli, S. typhimurium,
S. flexneri, Escherichia sp., P. aeruginosa, and R. solanacearum.
MIC µg/mL
MBC µg/mL
Compounds
E.c.
125
125
−
S.t.
31.25
125
−
S.f.
62.5
125
−
E.s.
62.5
125
−
P.a.
256
125
125
125
R.s.
62.5
−
E.c.
256
256
−
S.t.
62.5
256
−
S.f.
125
256
−
E.s.
125
256
−
P.a.
−
R.s.
a1
a2
a3
a4
a5
a6
a7
a8
a9
a10
a11
a12
a13
a14
a15
a16
a17
a18
a19
a20
a21
b1
b2
b3
b4
c1
c2
c3
c4
125
−
250
250
250
250
125
125
125
250
125
125
250
−
125
7.81
125
256
7.81
31.25
15.63
125
15.63
7.81
−
256
15.63
256
−
−
125
−
125
−
−
−
256
−
256
−
−
−
−
125
−
−
−
−
−
−
62.5
62.5
62.5
125
62.5
62.5
125
−
−
−
−
62.5
62.5
−
125
125
−
−
125
125
−
125
125
−
256
256
−
−
256
256
−
15.63
62.5
31.25
256
31.25
15.63
−
125
−
256
−
−
−
−
−
−
−
−
−
62.5
−
125
125
125
125
125
125
62.5
−
62.5
125
125
125
125
256
62.5
−
125
−
125
−
256
256
256
256
256
256
125
−
125
256
256
256
256
−
256
−
−
−
−
−
−
−
−
62.5
125
256
256
125
62.5
125
256
62.5
−
−
−
125
250
125
125
15.63
31.25
62.5
250
250
250
125
15.63
62.5
31.25
31.25
125
−
−
−
−
−
−
−
−
−
62.5
62.5
7.81
15.63
31.25
125
−
−
256
125
256
−
−
−
−
125
−
−
−
−
−
−
62.5
62.5
−
125
125
125
125
125
125
−
62.5
−
125
125
−
125
125
−
256
256
256
256
256
256
−
125
−
256
256
−
125
−
62.5
62.5
125
62.5
−
125
125
256
125
−
62.5
62.5
62.5
62.5
125
125
125
31.25
125
125
−
125
125
256
−
125
125
125
125
256
256
256
62.5
256
256
−
−
125
62.5
62.5
125
125
62.5
16.5
256
7.81
256
125
125
256
256
125
31.25
−
−
62.5
7.81
31.25
15.63
15.63
62.5
−
125
125
125
125
256
1.96
−
265
256
256
256
−
125
125
62.5
62.5
125
125
−
256
256
125
125
256
256
−
−
−
−
−
31.25
256
62.5
62.5
−
3.9
gentamicin
streptomycin
−
1.96
3.9
125
15.63
3.9
7.81
Note: The gentamicin and streptomycin were used as the positive control. “ ” means no inhibition effect.
−
E.c.: E. coli; S.t.: S. typhimurium; S.f.: S. flexneri; E.S.: Escherichia sp.; P.a.: P. aeruginosa and R.s.: R. solanacearum.
MIC: Minimal Inhibitory Concentration; MBC: Minimum Bactericidal Concentration.
As for inhibitory effects of the synthesized compounds against gram-negative bacteria and
gram-positive bacteria, 23 compounds exhibited high degrees of activity against E. coli, S. typhimurium,
and S. flexneri. 28 compounds showed high degrees of activity against E. coli, P. aeruginosa,
and R. solanacearum. Compounds a1, a8, a11, a19, b1–b3, and c3–c4 exhibited high degrees of
activity against more than two kinds of Gram-negative bacteria than that of gentamicin and
streptomycin. Noticeably, compound c4 showed a broad spectrum and remarkably high activity
against E. coli, S. typhimurium, S. flexneri, Escherichia sp., P. aeruginosa, and R. solanacearum,
with MIC values of 31.25
µ
g
·
mL−1, 62.5
µ
g
·
mL−1, 31.25
µ
g
·
mL−1, 16.5
µ
g
·
mL−1, 62.5
µg ,
mL−1
·
and 1.96 µg·mL−1, respectively.
Inhibitory effects of calycanthaceous alkaloid analogues against a wide range of plant pathogen
fungi are listed in Table 3. MIC and MFC were examined with amphotericin B and carbendazim as
a positive controlx, to evaluate the activities of the synthesized calycanthaceous alkaloid analogues
against P. capsici, V. dahliae, F. oxysperium sp. vasinfectum, C. orbiculare, P. citrinum, Cytospora juglandis,
A. sflavu, A. solani, C. lunaia, F. oxysporum, and A. niger.

Molecules 2016, 21, 1207
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Table 3. Inhibitory effect of synthesized compounds against a wide variety of plant pathogen fungi.
MIC(µg/mL)
MFC(µg/mL)
Compounds
P.ca. V.d. F.s. C.o.
P.c.
−
C.j.
125
62.5
−
A.sf.
A.s. C.l.
F.o.
−
A.n.
62.5
−
P.ca. V.d. F.s. C.o.
P.c.
−
C.j.
256
125
−
A.sf.
A.s.
−
−
−
−
−
−
−
256
−
−
−
−
−
−
−
−
−
−
−
−
−
−
256
−
−
−
−
−
−
−
−
C.l.
−
F.o.
−
A.n.
125
−
a1
a2
a3
a4
a5
a6
a7
a8
a9
a10
a11
a12
a13
a14
a15
a16
a17
a18
a19
a20
a21
b1
b2
b3
b4
c1
31.25
256
256
−
−
−
−
−
−
−
256 125
256 125
256
256
256
256
−
256
−
62.5
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
256
−
−
−
−
−
−
−
−
−
−
256
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
256
−
−
256
−
−
−
−
−
−
−
−
−
−
256
256
−
−
−
−
−
256
125
−
−
−
−
−
−
−
−
−
−
−
−
−
−
256
−
−
−
−
−
256 62.5
256
256
256
125
256
125
256
256
256
125
125
256
256
125
125
62.5
256
62.5
125
125
62.5
62.5
62.5
125
−
−
125
125
−
256
31.25
−
−
−
−
−
256 62.5 15.63
−
−
−
−
−
−
256
256
256 256
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
125
−
−
−
−
−
256
62.5
125
−
256
−
−
−
125
256
−
−
256 125 31.25
−
−
256
−
−
256
256
−
−
256
−
−
−
−
125 125
62.5
256
125
125
62.5
256
125
62.5
125
256
125
125
125
125
125
256
125
125
62.5
−
−
−
256
−
−
−
−
−
256
−
−
−
−
−
−
−
−
−
−
125
−
−
−
−
−
−
−
−
−
−
−
−
125
256
−
−
−
−
256
−
−
−
256
256
125
−
−
−
125
−
−
−
−
−
−
−
−
−
−
−
−
−
−
256
−
−
−
−
−
−
256
256
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
256
125
256
−
−
−
256
256
−
−
−
−
−
−
−
−
−
−
−
−
−
256
−
−
−
−
−
−
−
−
256
256
125
−
256
256
256
−
−
125 256
256 256
256 256
125 256
−
−
−
−
−
−
−
−
−
−
256
−
−
−
−
−
−
−
256
−
256
256
256
256
256
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
125
256
256
125
125
125
256
62.5
256
256
1.93
−
−
−
−
−
−
−
−
−
−
−
−
−
−
125
62.5
125
125
62.5
31.25
256
−
256 256
125
125
125
−
−
256
256
256
−
−
−
256
256
256
−
−
−
−
125 256 125
125
−
125 125
125 256
256 256
256
−
256
−
−
256
256
−
125
−
−
256
256 256
−
−
−
125
−
−
256
256
125
62.5
−
−
256
125
−
−
−
−
−
−
−
−
−
−
125
125
256 256
125
125
125
62.5
256
−
−
125
256
256
−
256
256
256
125
−
−
c2
c3
c4
A.
−
62.5
125
15.63
7.81
0.97
256 125 31.25 31.25 62.5
−
125
256
31.25
15.63
1.95
256
−
−
−
−
−
−
−
−
256
31.25
125
125
125
0.97
−
−
−
31.25 62.5 256 62.5
−
−
62.5 125
7.81 125
125
−
−
62.5
256
15.63
3.9
62.5 256 125 15.63 256
7.81
−
256 31.25
256 1.95 1.95
−
15.63
−
C.
1.95
256 125 0.97
0.97
62.5
7.81
3.9
−
125
Notes:
“
−
” means no inhibition effect. Amphotericin B and Carbendazim were used as the positive
vasinfectum, C.o.: C. orbiculare,
control. P.ca.: P. capsici, V.d.: V. dahlia, F.s.: F. oxysperium sp.
P.c.: P. citrinum, C.j.: Cytospora juglandis, A.sf.: A. sflavu, A.s.: A. solani, C.l.: C. lunaia, F.o.: F. oxysporum,
A.n.: A. niger, A.: Amphotericin B, C.: Carbendazim. MIC: minimal inhibitory concentration; MBC: Minimum
Bactericidal Concentration.
It is observed that this series of compounds generally exhibits more effective antimicrobial
activity than the positive control. Twenty three compounds exhibited high degrees of activity against
P. capsici, V. dahliae, F. oxysperium sp. vasinfectum, and C. orbiculare. Nineteen compounds showed high
degrees of activity against P. citrinum, A. sflavu, and A. solani. Nine of the synthesized compounds
manifested high degrees of activity against C. lunaia. 27 compounds illustrated high degrees of
activity against F. oxysporum. Twenty eight compounds manifested high degrees of activity against
A. niger. Compounds a11
amphotericin B and carbendazim, with c4 being the most effective with a MIC value of 62.5
Compounds a3 a4 a7 a8 a18 b2 b3, and c1
than that of carbendazim, with b3 and a12 being the most effective with MIC values of 62.5
,
b2
,
b3, and c4 manifested higher degrees of activity against V. dahliae than
µ
g
·
mL−1
.
,
,
–
,
,
–
–
c2 displayed better degrees of activity against C. lunaia
µg
·
mL−1
.
Compounds a4
,
a7
,
a8
,
a12
,
a15
,
c1
,
c2, and c4 illustrated higher degrees of activity against F. oxysporum
than that of amphotericin B, with a4 being the most effective with MIC value of 15.63 µg·mL−1
.
Compounds a4, a8, c1, and c2 indicated higher degrees of activity against C. lunaia and
F. oxysporum than that of amphotericin B and carbendazim. Compound a7 displayed higher degrees of
activity against Cytospora juglandis, C. lunaia and F. oxysporum than amphotericin B and carbendazim.
Compound a8 illustrated higher degrees of activity against, C. lunaia and F. oxysporum than
amphotericin B and carbendazim. Compound b2 manifested higher degrees of activity against
V. dahliae, C. lunaia and Cytospora juglandis than amphotericin B and carbendazim. Compound b3
illustrated higher degrees of activity against V. dahliae, and C .lunaia than amphotericin B and
carbendazim. Especially, compound c4 displayed higher degrees of activity against C. orbiculare,
and C. lunaia than amphotericin B and carbendazim.
3. Materials and Methods
3.1. Instruments and Chemicals
All reagents and solvents were reagent grade or purified according to standard methods before
use. Analytical thin-layer chromatography (TLC) was performed with silica gel plates using silica gel

Molecules 2016, 21, 1207
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60 GF254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Melting points were measured on
an Electrothermal digital apparatus (Beijing, China) and were uncorrected. The 1H-NMR (500 MHz),
and 13
C NMR (125 MHz) were obtained on an AM-500 FT-NMR spectrometer (Bruker Corporation,
−
Switzerland) with CDCl3 as the solvent and TMS as the internal standard. MS were recorded under ESI
conditions using a LCQ Fleet instrument (Thermo Fisher, Waltham, MA, USA). Optical rotation was
measured by an Autopol II polarimeter (Rudolph, Hackettstown, NJ, USA). Yields were not optimized.
The title compounds were synthesized under a nitrogen atmosphere.
3.2. Synthesis
3.2.1. Synthesis of 2-(2-Oxoindolin-3-yl)acetonitrile (2)
To a stirred solution of indole-3-acetonitrile (3.12 g, 20 mmol) in DMSO (30 mL) was added
◦
hydrogen chloride (37% HCl, VDMSO:VHCl = 1:5) dropwise at 0 C. The resulting mixture was allowed
to warm to room temperature. Then the mixture was stirred for 1 h. The solvents were removed to
obtain the white solid. The solid was crystallized from acetone to provide the desired product
93% yield).
2 (3.20 g,
3.2.2. Synthesis of 2-(1,3-Dibenzyl-2-oxoindolin-3-yl)acetonitrile (3)
To a solution of NaH (1.8 g, 75 mmol) in THF (20 mL) was added the compound 2 (2.58 g, 15 mmol)
in THF (10 mL) dropwise at 0 ◦C. To the resulting mixture was added benzyl chloride (34.5 mmol,
2.3 eq) in THF (5 mL) dropwise at 0 ◦C. Then the reaction mixture was quenched with ammonium
chloride (1 mL), and extracted three times with ethyl acetate. The organic extracts were combined,
washed with brine, dried over Na2SO4, and concentrated. Purification by flash chromatography on
silica (petroleum ether–ethyl acetate = 4:1) gel afforded compound 3 (3.84 g, 73% yield).
3.2.3. Synthesis of 3a,8-Dibenzyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (4)
To compound 3
(3.52 g, 10 mmol) in THF (20 mL) was added LiAlH4 (60 mmol, 7.5 eq) at 0 ◦C.
The resulting mixture was refluxed for 2 h, then, it was allowed to reach room temperature and stirred
for another 1 h, quenched with H2O (1 mL), and extracted three times with ethyl acetate. The organic
extracts were combined, washed with brine, dried over Na2SO4, and concentrated. Purification by
flash chromatography (petroleum ether–ethyl acetate = 2:1) on silica gel afforded compound
63% yield).
4
(2.17 g,
3.2.4. Synthesis of Compounds a1–a21
◦
To compound
4
in pyridine (10 mL) was added the corresponding desired reagent at 0 C.
The resulting mixture was refluxed for 2 h. The resulting mixture was allowed to warm to room
temperature and then stirred for a 1 h. Then, the reaction mixture was quenched with methanol (1 mL),
and extracted three times with ethyl acetate. The organic extracts were combined, washed with brine,
dried over Na2SO4, and concentrated. Purification by flash chromatography on silica gel afforded the
compounds a1–a21 in yields from 80% to 96%. (Characterization data see Supplementary Materials)
3.2.5. Synthesis of 2-(1-Ethyl-1H-indol-3-yl)acetonitrile (5)
To a solution of NaH (1.8 g, 75 mmol) in THF (20 mL) was added indole-3-acetonitrile (3.12 g,
◦
20 mmol) in DMSO (30 mL) dropwise at 0 C. The resulting mixture was allowed to reach room
temperature for a further 30 min. Then, the resulting mixture was added the compound RBr in
THF (5 mL) dropwise at 0 ◦C. The reaction mixture was quenched with ammonium chloride (1 mL),
and extracted three times with ethyl acetate. The organic extracts were combined, washed with brine,
dried over Na2SO4, and concentrated. Purification by flash chromatography (petroleum ether–ethyl
acetate = 4:1) on silica gel afforded the compound 5 (1.47 g, 47% yield).

Molecules 2016, 21, 1207
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3.2.6. Synthesis of 2-(1-Ethyl-2-oxoindolin-3-yl)acetonitrile (6)
To a stirred solution of
5
(3.12 g, 20 mmol) in DMSO (30 mL) was added hydrogen chloride
◦
(37% HCl, VDMSO:VHCl = 1:5) dropwise at 0 C. The resulting mixture was allowed to warm to
room temperature and then stirred for 1 h. Then, the solvent was removed. Purification by flash
chromatography (petroleum ether–ethyl acetate = 2.5:1) on silica gel afforded the compound
73% yield).
6 (1.17 g,
3.2.7. Synthesis of 2-(1,3-Diethyl-2-oxoindolin-3-yl)acetonitrile (7)
To a solution of NaH (1.8 g, 75 mmol) in THF (20 mL) was added compound 6 (2.58 g, 15 mmol)
in THF (10 mL) dropwise at 0 ◦C. The resulting mixture was added the compound RX in THF (5 mL)
◦
dropwise at 0 C. The reaction mixture was quenched with ammonium chloride (1 mL), and extracted
three times with ethyl acetate. The organic extracts were combined, washed with brine, dried over
Na2SO4, and concentrated. Purification by flash chromatography (petroleum ether–ethyl acetate = 3:1)
on silica gel afforded the compound 7 (1.09 g, 82% yield).
3.2.8. Synthesis of 3a,8-Diethyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole (8)
To compound
7
(0.8 g, 2.8 mmol) in THF (20 mL) was added LiAlH4 (21 mmol,7.5 eq) at 0 ◦C.
The resulting mixture was refluxed for 2 h, then allowed to warm to room temperature for a further
1 h. Then, the reaction mixture was quenched with H2O (1 mL), and extracted three times with ethyl
acetate. The organic extracts were combined, washed with brine, dried over Na2SO4, and concentrated.
Purification by flash chromatography (petroleum ether–ethyl acetate = 1.5:1) on silica gel afforded
compound 8 (0.45 g, 47% yield).
3.2.9. Synthesis of Compounds b1–c4
To a stirred solution of compound
8
in pyridine (10 mL) was added the corresponding desired
◦
reagents at 0 C. The resulting mixture was heated to reflux for 2 h. The resulting mixture was
allowed to warm to rt. Then, the mixture was stirred for 1 h. The reaction mixture was quenched with
methanol (1 mL), and extracted three times with ethyl acetate. The organic extracts were combined,
washed with brine, dried over Na2SO4, and concentrated. Purification by flash chromatography on
silica gel afforded the compounds b1–c4 in yields, from 91% to 94%). (Characterization data see
Supplementary Materials)
3.3. Biological Activity
The antimicrobial activity of calycanthaceous alkaloids analogues were measured according to
the previously reported method [29,30].
The tested compounds dissolved in 5% dimethyl sulfoxide (DMSO), to a concentration of
1.02 mg/mL, 100
µL of the solutions were added to the first well and serially diluted from first well by
taking 100 L into second. This two-fold dilution was continued down the plate and 100
µ
µL from the
8th column of the plated discarded. The 9th column of the plate was reserved for negative control wells
(without inocula) and the 10th column, for the positive growth control wells (without antibacterial
agent). The antibacterial concentrations were 256, 128, 64, 32, 16, 8, 4 and 2 µg/mL, respectively.
The antibacterial test plates were incubated aerobically at 37 ◦C for 24 h, the antifungal test plates
were incubated aerobically at 28 ◦C for 48 h. The MICs, MBC and MFC were examined. MBC and
MFC were determined by plating 10 µL from each negative well and from the positive growth control
on LB Agar and Sabouraud Dextrose Agar. MBC and MFC were defined as the lowest concentration
yielding negative subcultures or only on colony. All tests were performed in triplicate and repeated if
the results differed.

Molecules 2016, 21, 1207
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4. Conclusions
Twenty nine novel tetrahydropyrroloindole-based calycanthaceous alkaloid derivatives were
synthesized using indole-3-acetonitrile as the starting material via acylation at the N3 position, and their
antimicrobial activity against nine strains of bacteria and a wide range of plant pathogen fungi were
evaluated. According to the bioassay results, most of the title compounds showed moderate to
excellent activities against the selected bacteria and a wide variety of plant pathogen fungi and
were more effective than the positive controls. The compounds with substituted aliphatic groups
at positions R4 and R5, showed higher antimicrobial activities than with the aromatic substituents.
This suggested that substituents at R4 and R5 significantly affect the activities of title calycanthaceous
alkaloid analogues. Notably, compound c4 displayed the broadest and most effective activities
among the tested calycanthaceous analogues and might be a novel potential leading compound
for further development of antifungal agents. These results will pave the way for further design,
structural modification, and development of calycanthaceous alkaloids as antimicrobial agents.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
9/1207/s1.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (31360079,
21372185 and 21502073), the Natural Science Foundation of Jiangsu Province (Grants No BK 20150465),
and Collaborative Innovation Center of Ship Technology in Jiangsu Colleges and Universities/Marine Equipment
and Technology Institute for Jiangsu University of Science and Technology (HZ2016012).
Author Contributions: S.Z., H.B. and J.Z. designed research; S.Z. and S.X. performed research; X.Z. and R.Z.
performed statistical analysis; S.Z. wrote the paper; H.B. and J.Z. reviewed the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).