Synthesis and antifungal activity of novel indole-replaced streptochlorin analogues

Article abstract of DOI:10.1016/j.ejmech.2016.12.001

Based on examples of the successful applications in drug discovery of bioisosterism, a series of streptochlorin analogues in which indole has been replaced by other heterocycles has been designed and synthesized, as a continuation of our studies aimed at the discovery of novel streptochlorin analogues with improved antifungal activity. Biological testing showed that most of the indole-replaced streptochlorin analogues were inactive, though compound 6f had a broad spectrum of antifungal activity with significant activity against Alternaria solani. The SAR study demonstrated that indole ring is an essential moiety for the antifungal activity of streptochlorin analogues, promoting the idea of indole ring as a framework that might be exploited in the future.

Full text of DOI:10.1016/j.ejmech.2016.12.001

Accepted Manuscript
Synthesis and antifungal activity of novel indole-replaced streptochlorin analogues
Ming-Zhi Zhang, Chen-Yang Jia, Yu-Cheng Gu, Nick Mulholland, Sarah Turner, David
Beattie, Wei-Hua Zhang, Guang-Fu Yang, John Clough
PII:
S0223-5234(16)31007-8
10.1016/j.ejmech.2016.12.001
EJMECH 9092
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 September 2016
Revised Date: 29 November 2016
Accepted Date: 1 December 2016
Please cite this article as: M.-Z. Zhang, C.-Y. Jia, Y.-C. Gu, N. Mulholland, S. Turner, D. Beattie, W.-H.
Zhang, G.-F. Yang, J. Clough, Synthesis and antifungal activity of novel indole-replaced streptochlorin
analogues, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.001.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis and antifungal activity of novel indole-replaced
streptochlorin analogues
Ming-Zhi Zhang,1,2* Chen-Yang Jia,1,2 Yu-Cheng Gu,3 Nick Mulholland,3 Sarah Turner,3 David
Beattie,3 Wei-Hua Zhang,1,* Guang-Fu Yang2 and John Clough3
1Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural
University, Nanjing 210095, P.R.China
2Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, P.R.China
3Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United
Kingdom
A series of streptochlorin analogues in which indole replaced by other heterocycles has been
synthesized and evaluated for their antifungal activity, the SAR demonstrated indole is essential
for the activity.

ACCEPTED MANUSCRIPT
Synthesis and antifungal activity of novel indole-replaced
streptochlorin analogues
Ming-Zhi Zhang,1,2* Chen-Yang Jia,1,2 Yu-Cheng Gu,3 Nick Mulholland,3 Sarah Turner,3 David
Beattie,3 Wei-Hua Zhang,1,* Guang-Fu Yang2 and John Clough3
1Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing Agricultural
University, Nanjing 210095, P.R.China
2Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, P.R.China
3Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United
Kingdom
───────────
Corresponding Authors:
Dr. Ming-Zhi Zhang, E-mail: mzzhang@njau.edu.cn
Prof. Wei-Hua Zhang, E-mail: njzhangwh@126.com
Nanjing Agricultural University, Nanjing 210095, P.R. China
Abstract:
Based on examples of the successful applications in drug discovery of bioisosterism, a series of
streptochlorin analogues in which indole has been replaced by other heterocycles has been
designed and synthesized, as a continuation of our studies aimed at the discovery of novel
streptochlorin analogues with improved antifungal activity. Biological testing showed that most of
the indole-replaced streptochlorin analogues were inactive, though compound 6f had a broad
spectrum of antifungal activity with significant activity against Alternaria solani. The SAR study
demonstrated that indole ring is an essential moiety for the antifungal activity of streptochlorin
analogues, promoting the idea of indole ring as a framework that might be exploited in the future.
Keywords: Streptochlorin analogues; active heterocycles; synthesis; antifungal activity; SAR.
1. Introduction
Streptochlorin is an indole alkaloid isolated from marine Streptomyces sp[1, 2], belonging to
the family of naturally occurring 5-(3-indolyl)oxazoles, it’s reported to possess a broad spectrum
of biological activity, such as anticancer, antiangiogenic, and antiallergic effects[3-5].

ACCEPTED MANUSCRIPT
Streptochlorin also exhibits selective cytotoxicity against several cancer cell lines, potently
inhibits TNF-α-induced NF-κB activation and it is a potent suppressor of angiogenesis in vitro.
Members of this family, including streptochlorin, pimprinine and other natural products, have also
been claimed to demonstrate a variety of biological activities, including antiviral[6], antibiotic[7],
anticonvulsant[8], and pesticidal activity[9]. Biological activity screening conducted at Syngenta
showed that streptochlorin and pimprinine demonstrated good antifungal activity against Pythium
dissimile, Botrytis cinerea, Zymoseptoria tritici, Pyricularia oryzae, Fusarium culmorum and
Rhizoctonia solani[10], but these two compounds lack potency, rarely extending to the further
study.
In order to find new lead candidates from natural products for further structural optimization,
we have described various structural modifications to streptochlorin in our previous study,
including different substituents at the indole ring and the nitrogen of the indole ring, replacing the
Cl on the oxazole ring by Br or H[10, 11], and replacing the oxazole ring by oxadiazole[12]. In a
continuation of our studies aimed at the discovery of novel streptochlorin analogues though
step-by-step optimization, we now describe this study which has focused on replacement of the
indole ring of streptochlorin with other active heterocycles (Figure 1).
Figure 1. Design strategies of target molecules
As good bioisosteres of indole, other heterocycles such as benzofuran, benzothiophene,
piperonal, naphthalene and quinoline exist widely in commercial drugs and agricultural
chemicals[13-15], displaying a broad spectrum of biological activities (Figure 2). For example,
Vilazodone, containing both an indole and benzofuran ring, is approved for the treatment of major
depressive disorder and is a selective serotonin reuptake inhibitor (SSRI)[16]. Dronedarone,
approved by the FDA in 2009, is a benzofuran-containing antiarrhythmic drug developed by
Sanofi-Aventis[17]. Raloxifene, a benzothiophene-containing drug developed by Eli Lilly and

ACCEPTED MANUSCRIPT
marketed as Evista, is used in the prevention of osteoporosis in postmenopausal women as an
oral selective estrogen receptor modulator (SERM)[18]. Oxolinic acid, a piperonyl antibiotic
developed in Japan, shows high bioavailability and is widely used in aquaculture for the treatment
of a variety of systemic bacterial infections of fish[19]. Tadalafil, listed in the Top 100
pharmaceutical drugs by US retail sales under the name Cialis in 2012 and 2013 [20], contains
both indole and piperonyl ring in its structure and is a PDE5 inhibitor for the treatment of men's
erectile dysfunction (ED). Berberine, an isoquinoline-containing natural alkaloid revered and
with a long history in traditional Chinese medicine, was used orally for various parasitic and
fungal infections[21]. Quinine occurs naturally in the bark of the cinchona tree and was the first
effective western treatment for malaria caused by Plasmodium falciparum[22].
Figure 2.Structures of drugs and agrochemicals containing active heterocycles
Replacement of an indole ring with other active heterocycles was proved to be an effective
protocol for structural optimization. There are several examples (Figure 3), such as
indole-containing compound 1, which has been evaluated for anticancer activity against a panel of
60 human cancer cell lines, it showed potent anti-leukemic activity against leukemia MV4-11 cell
lines with an LD50 value of 44 nM. The synthesized compound 2 in which indole is replaced by
benzothiophene showed improved activity with an LD50 value of 18 nM[23]. Fluvastin 3, the first
synthetic indole-containing statin launched by Sandoz pharmaceutical company, opened up the
opportunity of discovering more potent synthetic statins including Pitavastatin 4, the
quinolone-containing blood cholesterol-lowering drug[24].

ACCEPTED MANUSCRIPT
Figure 3. Structural optimization–replacement of indole by alternative heterocycles
2. Materials and methods
2.1. Chemistry
All chemicals were purchased from commercial sources (e.g., Crystal Chemicals, Alfa Aesar
Co.) and used without further purification unless otherwise stated. All solvents and liquid reagents
were dried by standard methods in advance and distilled before use. Melting points were taken on
1
a Büchi B-545 melting point apparatus and are uncorrected. H NMR spectra were recorded on a
VARIAN Mercury-Plus 600 spectrometer in CDCl3 or DMSO-d6with TMS as the internal
reference. 13C NMR spectra were recorded in CDCl3 or DMSO-d6 on a Varian Mercury 400/600
(100/150 MHz) spectrometer and chemical shifts (δ) were given in ppm relative to the center line
of a triplet at 77.0 ppm of CDCl3or 39.5 ppm of DMSO-d6. Mass spectra were determined using a
Trace MS 2000 organic mass spectrometry (EI-MS) or a ThermoFisher Mass platform DSQ II by
electrospray ionization (ESI-MS), and the signals were given in m/z. Reaction yields were not
optimized.
2-Chloro-6-fluorobenzaldehyde
(5a),
isonicotinaldehyde
(5b),
(5e),
benzo[b]thiophene-3-carbaldehyde
(5d), benzo[d][1,3]dioxole-5-carbaldehyde
1-naphthaldehyde (5f), 2-naphthaldehyde (5g) and quinoline-2-carbaldehyde (5h) were purchased
from commercial sources. Benzofuran-3-carbaldehyde (5c) were synthesized by using the reported
method[25],
2-methoxy-6-methylquinoline-3-carbaldehyde (5j), 2-chloro-7-methylquinoline-3-carbaldehyde
(5k), 2-methoxy-7-methylquinoline-3-carbaldehyde (5l),
and
2-chloro-6-methylquinoline-3-carbaldehyde
(5i),

ACCEPTED MANUSCRIPT
2-chloro-8-methylquinoline-3-carbaldehyde (5m), 2-methoxy-8-methylquinoline-3-carbaldehyde
(5n) were obtained through the reported methods[26].
Scheme 1: Synthetic routes for the target compounds
2.1.1. Preparation of heterocycle-5-oxazole 6 (Scheme 1)
A stirred mixture of heterocycle-carboxaldehyde 5 (1.0 mmol) and p-toluene-sulfonylmethyl
isocyanide (TosMIC) (1.1 mmol, 0.21 g) in 1:1 DME/ MeOH (15 mL, both anhydrous) was
refluxed with Ambersep® 900(OH) ion exchange resin (2.0 g, exchange capacity 1.18 meq/mL)
for 2 h. The reaction mixture was filtered, the resin was washed by MeOH twice (2×5 mL), and
the combined filtrates were concentrated under reduced pressure to give the crude product, which
was purified by column chromatography on silica gel (eluent: petroleum ether/acetone = 4:1) to
afford the pure compound 6.
2.1.1.1. 5-(2-chloro-6-fluorophenyl)oxazole (6a): a white solid, yield 74%, mp, 93-95℃. 1H NMR
(600 MHz, DMSO-d6): δ 7.07–7.16 (m, 1H), 7.28–7.37 (m, 2H), 7.48 (s, 1H), 8.06 (s, 1H).
ESI-MS: m/z 160.8 (MH+). Anal.Calcd. for C9H5ClFNO: C, 54.71; H, 2.55; N, 7.09; Found: C,
54.66; H, 2.60; N, 7.12.
2.1.1.2. 5-(pyridin-4-yl)oxazole (6b): a white solid, yield 99%, mp, 78-80℃.1H NMR (600 MHz,
CDCl3) δ 7.54 (d, J = 4.8 Hz, 2H), 7.59 (s, 1H), 8.02 (s, 1H), 8.69 (d, J = 4.8 Hz, 2H). Anal.Calcd.
for C8H6N2O: C, 65.75; H, 4.14; N, 19.17; Found: C, 65.82; H, 4.20; N, 19.11.

ACCEPTED MANUSCRIPT
2.1.1.3. 5-(benzofuran-3-yl)oxazole (6c): a white solid, yield 46%, mp, 99-101℃.1H NMR (600
MHz, CDCl3) δ 7.33–7.46 (m, 2H), 7.57 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.99 (d, J =
13.8 Hz, 1H). EI-MS: m/z (%) 185 (M+, 100), 156 (15), 130 (42), 102(28). Anal.Calcd. for
C11H7NO2: C, 71.35; H, 3.81; N, 7.56; Found: C, 71.44; H, 3.78; N, 7.47.
1
2.1.1.4. 5-(benzo[b]thiophen-3-yl)oxazole (6d): a white solid, yield 28%, mp, 109-111℃. H
NMR (600 MHz, CDCl3): δ: 7.34 (s, 1H), 7.36-7.40 (m, 2H), 7.56 (s, 1H), 7.79-7.80 (d, J = 7.8 Hz,
2H), 7. 83-7.84(d, J = 7.8Hz, 1H), 7.93(s, 1H). EI-MS: m/z (%) 201 (M+, 100), 173 (14), 146 (31).
Anal.Calcd. for C11H7NOS: C, 65.65; H, 3.51; N, 6.96; Found: C, 65.56; H, 3.35; N, 6.96.
2.1.1.5. 5-(benzo[d][1,3]dioxol-5-yl)oxazole (6e): a white solid, yield 75%, mp, 70-72℃.1H NMR
(600 MHz, CDCl3) δ 6.02 (s, 2H), 6.87 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 7.18 (d, J = 8.4 Hz, 1H),
7.22 (s, 1H), 7.88 (s, 1H). Anal.Calcd. for C10H7NO3: C, 63.49; H, 3.73; N, 7.40; Found: C, 63.24;
H, 3.77; N, 7.20.
2.1.1.6. 5-(naphthalen-1-yl)oxazole (6f): a white solid, yield 59%, 1H NMR (600 MHz, CDCl3) δ
7.46 (s, 1H) , 7.56 (m, 3H), 7.74 (d, J = 7.2 Hz, 1H), , 7.92 (dd, J = 7.2, 4.2 Hz, 2H), 8.08 (s, 1H),
8.25 (d, J = 8.4 Hz, 1H). Anal.Calcd. for C13H9NO: C, 79.98; H, 4.65; N, 7.17; Found: C, 80.03;
H, 4.58; N, 7.25.
1
2.1.1.7. 5-(naphthalen-2-yl)oxazole (6g): a white crystal, yield 71%, mp, 102-104℃. H NMR
(600 MHz, CDCl3) δ 7.48 (s, 1H), 7.52 (m, 2H), 7.73 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H),
7.89 (d, J = 7.8 Hz, 2H), 7.99 (s, 1H), 8.15 (s, 1H). EI-MS: m/z (%) 195 (M+, 100), 167 (27), 140
(44), 127 (30). Anal.Calcd. for C13H9NO: C, 79.98; H, 4.65; N, 7.17; Found: C, 79.75; H, 4.88; N,
7.07.
1
2.1.1.8. 5-(quinolin-2-yl)oxazole (6h): a white crystal, yield 69%, mp, 60-62℃. H NMR (600
MHz, CDCl3) δ 7.56 (t, J = 7.4 Hz, 1H), 7.72–7.81 (m, 2H), 7.83 (d, J = 7.8 Hz, 1H), 7.87 (s, 1H),
8.07 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H). EI-MS: m/z (%) 196 (M+, 100),
168(78), 140(30), 128 (23). Anal.Calcd. for C12H8N2O: C, 73.46; H, 4.11; N, 14.28; Found: C,
73.30; H, 4.29; N, 14.07.
2.1.1.9. 5-(2-chloro-6-methylquinolin-3-yl)oxazole (6i): a white solid, yield 59%, mp, 161-163℃.
1H NMR (600 MHz, DMSO-d6): δ 2.49(s, 3H), 7.59-7.61 (d, J = 8.4 Hz, 1H), 7.81 (m, 2H), 7.91(s,
1H), 8.60(s, 1H), 8.69(s, 1H). 13C NMR (150 MHz, DMSO-d6): δ 21.2, 120.4, 126.3, 126.9, 127.2,
133.7, 135.8, 137.0, 137.8, 139.2, 144.4, 145.9, 152.8. EI-MS: m/z (%) 240 (M+, 100), 211 (91),
184 (66). Anal.Calcd. for C13H9ClN2O: C, 63.81; H, 3.71; N, 11.45; Found: C, 64.04; H, 3.88; N,

ACCEPTED MANUSCRIPT
11.39.
2.1.1.10. 5-(2-methoxy-6-methylquinolin-3-yl)oxazole (6j): a white solid, yield 29%, mp,
102-104℃. 1H NMR (600 MHz, DMSO-d6): δ 2.51(s, 3H), 4.12 (s, 3H), 7.51-7.52 (d, J = 7.2 Hz,
13
1H), 7.68-7.72 (m, 3H), 8.43 (s, 1H), 8.59(s, 1H). C NMR (150 MHz, DMSO-d6): δ 20.9, 53.7,
112.2, 124.4, 126.2, 126.5, 127.1, 132.2, 133.0, 134.0, 143.1, 145.8, 152.1, 156.6. EI-MS: m/z (%)
240 (M+, 100), 211 (91), 184 (66). Anal.Calcd. for C14H12N2O2: C, 69.99; H, 5.03; N, 11.66;
Found: C, 70.14; H, 5.01; N, 11.60.
2.1.1.11. 5-(2-chloro-7-methylquinolin-3-yl)oxazole (6k): a white solid, yield 34%, mp, 154-156℃.
1H NMR (600 MHz, CDCl3): δ 2.58(s, 3H), 7.44-7.45(d, J = 7.8 Hz, 1H), 7.78-7.81(m, 2H),
7.90(s, 1H), 8.04(s, 1H),8.53 (s, 1H). 13C NMR (150 MHz, DMSO-d6): δ 21.4, 102.7, 119.7, 124.5,
126.6, 128.1、130.1, 136.4, 140.6, 145.2, 146.0, 152.7, 153.2. EI-MS: m/z (%) 244(M+, 100),
181(17), 154 (29). Anal.Calcd. for C13H9ClN2O: C, 63.81; H, 3.71; N, 11.45; Found: C, 63.72; H,
3.94; N, 11.26.
2.1.1.12. 5-(2-methoxy-7-methylquinolin-3-yl)oxazole (6l): a white solid, yield 25%, mp,
1
138-140℃. H NMR (600 MHz, CDCl3): δ 2.54 (s, 3H), 4.22 (s, 3H), 7.26 (s, 1H), 7.68-7.69
(m,3H), 7.97 (s, 1H), 8.38 (s, 1H). 13C NMR (150 MHz, DMSO-d6): δ 21.4, 53.7, 111.5, 122.4,
125.8, 126.2，126.7，127.9,，133.4, 140.4, 145.0，145.9, 151.9，157.1. EI-MS: m/z (%) 240 (M+,
100), 211 (77), 184 (65). Anal.Calcd. for C14H12N2O2: C, 69.99; H, 5.03; N, 11.66; Found: C,
69.90; H, 5.23; N, 11.52.
2.1.1.13. 5-(2-chloro-8-methylquinolin-3-yl)oxazole (6m): a white solid, yield 29%, mp,
1
147-149℃. H NMR (600 MHz, CDCl3): δ 2.65 (s, 3H), 7.59-7.61 (t, J = 6.6 Hz, 1H), 7.72-7.73
(d, J = 6.0 Hz, 1H), 7. 96 (s, 1H), 7.96 (s, 1H),7.99-8.00 (d, J = 7.8 Hz,1H), 8.70 (s, 1H), 8.82 (s,
1H). 13C NMR (150 MHz, DMSO-d6): δ 17.2, 126.3, 126.9, 127.7, 131.5, 135.4, 136.1, 137.0,
139.2, 144.9, 145.4, 152.8, 177.7. EI-MS: m/z (%) 244 (M+, 100), 181 (17), 154 (19). Anal.Calcd.
for C13H9ClN2O: C, 63.81; H, 3.71; N, 11.45; Found: C, 63.85; H, 3.60; N, 11.37.
2.1.1.14. 5-(2-methoxy-8-methylquinolin-3-yl)oxazole (6n): a white solid, yield 17%, mp,
122-124℃. 1H NMR (600 MHz, DMSO-d6): δ 2.62 (s, 3H), 4.14 (s, 3H), 7.34-7.36 (t, J = 7.2 Hz,
1H), 7.53-7.54 (d, J = 6.0 Hz, 1H), 7.70 (s, 1H), 7.79-7.80 (d, J = 7.2 Hz,1H), 8.48 (s, 1H), 8.58 (s,
1H). 13C NMR (150 MHz, DMSO-d6): δ 17.1, 54.0, 111.8, 124.2, 124.3, 126.0, 126.4, 130.2,
133.8, 134.0, 143.4, 145.7, 152.0, 156.1. EI-MS: m/z (%) 240 (M+, 100), 211 (14), 184 (36).
Anal.Calcd. for C14H12N2O2: C, 69.99; H, 5.03; N, 11.66; Found: C, 69.78; H, 5.28; N, 11.47.

ACCEPTED MANUSCRIPT
2.1.2. General procedure for the synthesis of target compounds 7-8 (Scheme 1)
To a stirred solution of 6 (1.0 mmol) in THF/CCl4 (1:1, 15 mL) was added NCS or NBS (1.1
mmol) and the resulting mixture was heated at 50 oC for 8 h till full conversion of the substrates
was indicated by TLC analysis, then allowed to cool. The solvent was removed under reduced
pressure and the crude product was purified by flash column chromatography using petroleum
ether/acetone (5:1) as eluent to give the desired compounds 7 and 8, respectively.
1
2.1.2.1. 5-(benzofuran-3-yl)-4-chlorooxazole (7c): a white crystal, yield 35%, mp, 119-121℃. H
NMR (600 MHz, CDCl3) δ 7.38 (m, 2H), 7.47-7.51 (m, 1H), 7.55 (s, 1H), 7.88–7.95 (m, 1H), 8.06
(s, 1H). Anal.Calcd. for C11H6ClNO2: C, 60.16; H, 2.75; N, 6.38; Found: C, 60.21; H, 2.80; N,
6.35.
2.1.2.2. 5-(benzo[b]thiophen-3-yl)-4-chlorooxazole (7d): a white solid, yield 61%，mp, 101-103℃.
1H NMR (600 MHz, CDCl3): δ: 7.43-7.45 (t, J = 7.5 Hz 1H), 7.48-7.50 (t, J = 7.8, 1H), 7.82-7.83
(d, J = 7.8 Hz, 2H), 7. 86-7.87(d, J = 7.8Hz, 1H), 7.97 (s, 1H). EI-MS: m/z (%) 237 (37), 235 (M+,
100), 180 (41), 145 (50). Anal.Calcd. for C11H6ClNOS: C, 56.06; H, 2.57; N, 5.94; Found: C,
56.31; H, 2.30; N, 5.83.
1
2.1.2.3. 5-(benzo[d][1,3]dioxol-5-yl)-4-chlorooxazole (7e): a white crystal, yield 53%, H NMR
(600 MHz, CDCl3) δ 6.03 (s, 2H), 6.91 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H),
7.79 (s, 1H). 13C NMR (600 MHz, DMSO-d6): δ: 102.2, 105.6, 109.5, 120.1, 120.2, 123.0, 143.8,
148.4, 148.5, 150.9. Anal.Calcd. for C10H6ClNO3: C, 53.71; H, 2.70; N, 6.26; Found: C, 53.99; H,
2.88; N, 6.15.
1
2.1.2.4. 5-(benzofuran-3-yl)-4-bromooxazole (8c): a white solid, yield 61%, mp, 122-124℃. H
NMR (600 MHz, CDCl3) δ 7.31–7.40 (m, 2H), 7.50 (d, J = 7.2 Hz, 1H), 7.63 (s, 1H), 7.92 (d, J =
7.2 Hz, 1H), 8.06 (s, 1H). Anal.Calcd. for C11H6ClNO2: C, 50.03; H, 2.29; N, 5.30; Found: C,
49.98; H, 2.32; N, 5.35.
2.1.2.5. 5-(benzo[b]thiophen-3-yl)-4-bromooxazole (8d): a white solid, yield 39%, mp, 98-100℃.
1H NMR (600 MHz, CDCl3): δ: 7.42-7.45 (t, J = 7.2 Hz 1H), 7.48-7.50 (t, J = 7.8 Hz, 1H),
7.82-7.83 (d, J = 7.8 Hz, 1H), 7. 85-7.86(d, J = 8.4 Hz, 1H), 7.94(s, 1H),7.98(s, 1H). 13C NMR
(600 MHz, DMSO-d6): δ: 105.1, 122.9, 123.0, 124.9, 125.2, 126.1, 126.5, 136.5, 137.9, 144.2,
152.3. EI-MS: m/z (%) 281(M+, 100), 280 (100), 172 (26), 145 (71). Anal.Calcd. for C11H6BrNOS:
C, 47.16; H, 2.16; N, 5.00; S, 11.45; Found: C, 47.09; H, 2.22; N, 4.91; S, 11.64.
1
2.1.2.6. 5-(benzo[d][1,3]dioxol-5-yl)-4-bromooxazole (8e): a white crystal, yield 31%, H NMR
(600 MHz, CDCl3) δ 6.02 (s, 2H), 6.87 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 7.22 (s, 1H),

ACCEPTED MANUSCRIPT
7.87 (s, 1H). Anal.Calcd. for C10H6BrNO3: C, 44.81; H, 2.26; N, 5.23; Found: C, 44.87; H, 2.35;
N, 5.32.
1
2.1.2.7. 4-bromo-5-(naphthalen-2-yl)oxazole (8g): a white solid, yield 25%, H NMR (600 MHz,
CDCl3) δ 7.41 (s, 1H), 7.57–7.49 (m, 3H), 7.65 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.88
(d, J = 8.4 Hz, 1H), 8.09 (s, 1H). Anal.Calcd. for C10H6BrNO3: C, 56.96; H, 2.94; N, 5.11; Found:
C, 57.05; H, 2.98; N, 5.21.
2.1.3. X-ray Diffraction Analysis
The crystallographic data have been deposited with the Cambridge Crystallographic Data
Centre (CCDC) as supplementary publication number: 933865 (compound 6g), 930417
(compound 7e), shown in Figure 4. Copies of the data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/, or on application to CCDC, 12 Union Road, Cambridge CB12 1EZ,
UK (Fax +44 1223 336033 or E-mail: deposit@ccdc.cam.ac.uk).
Figure 4. X-ray structures of compounds 6g and 7e
2.2. Biological Assays
The materials and methods used in the biological assays conducted at Syngenta were the
same as those described in our earlier study[12]. Screening consists of seven different high
throughput assays covering a selection of economically important plant pathogenic fungi and was
performed using both leaf pieces and artificial media. The results of the biological testing are
shown in Table 1.
Table 1. Antifungal activity of the target compounds
Species
PHYa
ZYMa
UROa
PYTa
ALTa
20
BOTa
20
NCc
0
GIBa
No.
Rate (ppm)
200
0b
0
100
0
100
0
20
0
20
0
6a
6b
6c
6d
0
0
0
0
NC
0
0
0
0
0
0
55d
0
0
0
0
0
0
0
0

ACCEPTED MANUSCRIPT
6e
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
49
27
36
36
0
0
0
27
55
55
27
0
0
99
27
55
0
0
27
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
99
6f
6g
0
6h
49
0
0
6i
18
33
0
0
6j
49
0
0
0
0
6k
0
0
0
6l
0
0
0
0
0
6m
0
0
0
0
0
6n
0
0
0
0
0
7c
0
0
0
NC
0
0
7d
0
0
0
0
7e
0
0
55
0
0
0
8c
0
0
0
0
8d
8e
0
0
0
0
0
36
0
49
0
77
55
0
0
27
0
8g
0
Pimprinine
51
27
0
0
Streptochlorin
36
55
99
99
99
aPHY, Phytophthora infestans (on tomato leaf pieces); ZYM, Zymoseptoria tritici (on wheat leaf pieces); URO,
Uromyces viciae-fabae (on bean leaf pieces); PYT, Pythium dissimile; ALT, Alternaria solani; BOT, Botryotinia
fuckeliana; GIB, Gibberella zeae (all in artificial media).
bThe data are the mean of three replicates.
c Data not captured for this replicate (test failure).
d The bold means data equal to or above 55% control.
3. Results and discussion
3.1. Synthetic Chemistry
A series of indole-substituted streptochlorin analogues was efficiently synthesized based on
the methods we had developed in our previous work. The synthetic routes to intermediates 6 and
the streptochlorin analogues 7-8 are depicted in Scheme 1. The substituted
heterocycle-carboxaldehydes 5 were either commercially available, or prepared by reported
methods. In the key step of this sequence, the 5-linked oxazole was installed by Van Leusen’s
[3+2] cyclisation with TosMIC, under the catalytic influence of the ion exchange resin Ambersep®
900(OH), to give the heterocycle-5-oxazoles 6. Some representative compounds of 6a-6n (shown
in Scheme 1) could be converted directly into the chlorinated or brominated streptochlorin
analogues 7 or 8, respectively by treatment with NCS or NBS.
3.2. Antifungal activity and the structure-activity relationships (SAR)

ACCEPTED MANUSCRIPT
The results of the biological testing against seven phytopathogenic fungi are given in Table 1.
For the purposes of an analysis of structure-activity relationships, the antifungal activities of
compounds 6, 7 and 8 were compared to the lead compounds, the natural products streptochlorin
and pimprinine.
In the structure-activity point of view, although the antifungal activity of most of the
indole-replaced streptochlorin analogues has been proven to be rather poor, making it difficult to
extract a clear structure-activity relationship analysis, some broad conclusions can still be drawn.
Firstly, Pythium dissimile and Alternaria solani were noticeably more sensitive to the
indole-replaced streptochlorin analogues than the other tested fungi, and compound 6f was
identified as the most promising candidate, as it possessed significant activity to Alternaria solani
and also a broad antifungal spectrum. Secondly, most of the target compounds were inactive
against the tested fungi, and this is highlighted by compounds 7c and 7d, compared these
structures with streptochlorin, just replaced the N of the indole ring by O or S, but the
corresponding compounds 7c and 7d were totally inactive to all the tested fungi, the other target
compounds also dramatically eliminated the antifungal activity. Thirdly, the effect on the
antifungal activity of halogen atom (Cl, Br) at the 4-position of the oxazole ring was uncertain due
to the absence of biological activity, though the potency of streptochlorin analogues could be
generally improved by introducing a halogen atom in our previous research.
4. Conclusions
In order to find new lead candidates from natural products for further structural optimization,
we have designed and synthesized a series of streptochlorin analogues based on our reported
methods. In this study, the indole ring of streptochlorin was replaced by other heterocycles,
including benzofuran, benzothiophene, piperonal, naphthalene and quinoline. Biological testing
showed that most of the designed compounds had dramatically reduced antifungal activity in
comparison with the natural products, though compound 6f had a broad spectrum of antifungal
activity with significant activity against Alternaria solani. The SAR study demonstrates that
indole moiety has an important effect on the antifungal activity of the streptochlorin analogues,
promoting the idea of the indole ring as a framework that might be exploited in the future. Further
step-by-step structural optimization of streptochlorin analogues is well under way, in order to

ACCEPTED MANUSCRIPT
better define their levels of antifungal activity and carry out the following QSAR studies in the
future.
Acknowledgments
We are grateful to the financial support for this work from the National Natural Science
Foundation of China (21602110), Jiangsu Provincial Science Foundation for Youths
(BK20160734) and the Fundamental Research Funds for the Central Universities (KYZ201646,
KJQN201720). We thank the Biology Team at Syngenta for their kind help in screening the
compounds for biological activity.
References
[1] H. Watanabe, S. Amano, J. Yoshida, Y. Takase, S. Miyadoh, T. Sasaki, M. Hatsu, Y. Takeuchi, Y.
Komada, A new antibiotic SF2583A, 4-chloro-5-(3'-indolyl) oxazole, produced by Streptomyces., Meiji
Seika Kenkyu Nenpo, 27 (1988) 55-62.
[2] H.J. Shin, H.S. Jeong, H.S. Lee, S.K. Park, H.M. Kim, H.J. Kwon, Isolation and structure determination
of streptochlorin, an antiproliferative agent from a marine-derived Streptomyces sp. 04DH110, J
Microbiol Biotechnol, 17 (2007) 1403-1406.
[3] L. Li, S. He, L. Ding, Y. Yuan, P. Zhu, S. Epstein, J. Fan, X. Wu, X. Yan, Efficient Preparation of
Streptochlorin from Marine Streptomyces sp. SYYLWHS-1-4 by Combination of Response Surface
Methodology and High-Speed Counter-Current Chromatography, Molecules, 21 (2016) 693-705.
[4] D.W. Shim, H.J. Shin, J.W. Han, W.Y. Shin, X. Sun, E.J. Shim, T.J. Kim, T.B. Kang, K.H. Lee,
Anti-inflammatory effect of Streptochlorin via TRIF-dependent signaling pathways in cellular and
mouse models, Int J Mol Sci, 16 (2015) 6902-6910.
[5] S.H. Lee, H.J. Shin, D.Y. Kim, D.W. Shim, T.J. Kim, S.K. Ye, H.S. Won, S. Koppula, T.B. Kang, K.H. Lee,
Streptochlorin suppresses allergic dermatitis and mast cell activation via regulation of Lyn/Fyn and Syk
signaling pathways in cellular and mouse models, PLoS One, 8 (2013) e74194.
[6] Y. Wei, W. Fang, Z. Wan, K. Wang, Q. Yang, X. Cai, L. Shi, Z. Yang, Antiviral effects against EV71 of
pimprinine and its derivatives isolated from Streptomyces sp., Virol J., 11 (2014) 195-208.
[7] J. Kroiss, M. Kaltenpoth, B. Schneider, M.G. Schwinger, C. Hertweck, R.K. Maddula, E. Strohm, A.
Svatos, Symbiotic Streptomycetes provide antibiotic combination prophylaxis for wasp offspring, Nat
Chem Biol, 6 (2010) 261-263.
[8] S.R. Naik, J. Harindran, A.B. Varde, Pimprinine, an extracellular alkaloid produced by Streptomyces
CDRIL-312: fermentation, isolation and pharmacological activity, J Biotechnol, 88 (2001) 1-10.
[9] A. Ratnakar, K. Marja, M. Pamela, Isolated Bacterial Strain of the Genus Burkholderia and Pesticidal
Metabolites Therefrom-Formulations and Uses. WO2013032693 A2.
[10] M.Z. Zhang, Q. Chen, N. Mulholland, D. Beattie, D. Irwin, Y.C. Gu, G.F. Yang, J. Clough, Synthesis
and fungicidal activity of novel pimprinine analogues, Eur J Med Chem, 53 (2012) 283-291.
[11] M.Z. Zhang, Q. Chen, C.H. Xie, N. Mulholland, S. Turner, D. Irwin, Y.C. Gu, G.F. Yang, J. Clough,
Synthesis and antifungal activity of novel streptochlorin analogues, Eur J Med Chem, 92 (2015)

ACCEPTED MANUSCRIPT
776-783.
[12] M.Z. Zhang, N. Mulholland, D. Beattie, D. Irwin, Y.C. Gu, Q. Chen, G.F. Yang, J. Clough, Synthesis
and antifungal activity of 3-(1,3,4-oxadiazol-5-yl)-indoles and 3-(1,3,4-oxadiazol-5-yl)methyl-indoles,
Eur J Med Chem, 63 (2013) 22-32.
[13] M.E. Welsch, S.A. Snyder, B.R. Stockwell, Privileged scaffolds for library design and drug discovery,
Curr Opin Chem Biol, 14 (2010) 347-361.
[14] M. Njoroge, N.M. Njuguna, P. Mutai, D.S. Ongarora, P.W. Smith, K. Chibale, Recent approaches to
chemical discovery and development against malaria and the neglected tropical diseases human
African trypanosomiasis and schistosomiasis, Chem Rev, 114 (2014) 11138-11163.
[15] T. Pacher, C. Seger, D. Engelmeier, S. Vajrodaya, O. Hofer, H. Greger, Antifungal stilbenoids from
Stemona collinsae, J Nat Prod, 65 (2002) 820-827.
[16] C.R. Hopkins, ACS chemical neuroscience molecule spotlight on viibryd (Vilazodone), ACS Chem
Neurosci, 2 (2011) 554.
[17] P.J. Zimetbaum, Dronedarone for atrial fibrillation--an odyssey, N Engl J Med, 360 (2009)
1811-1813.
[18] S. Dadiboyena, Recent advances in the synthesis of raloxifene: a selective estrogen receptor
modulator, Eur J Med Chem, 51 (2012) 17-34.
[19] O.B. Samuelsen, O. Bergh, A. Ervik, A single-dose pharmacokinetic study of oxolinic acid and
vetoquinol, an oxolinic acid ester, in cod, Gadus morhua L., held in sea water at 8 degrees C and in
vitro antibacterial activity of oxolinic acid against Vibrio anguillarum strains isolated from diseased cod,
J Fish Dis, 26 (2003) 339-347.
[20] E.A.I.E. Vitaku, J.T. Njarðarson, Compiled and Produced by the Njardarson Group (The University
of Arizona), http://cbc.arizona.edu/njardarson/group/top-pharmaceuticals-poster, (2013).
[21] J. Yin, H. Xing, J. Ye, Efficacy of berberine in patients with type 2 diabetes mellitus, Metabolism, 57
(2008) 712-717.
[22] S. Heeb, M.P. Fletcher, S.R. Chhabra, S.P. Diggle, P. Williams, M. Camara, Quinolones: from
antibiotics to autoinducers, FEMS Microbiol Rev, 35 (2011) 247-274.
[23] N.R. Penthala, H. Zong, A. Ketkar, N.R. Madadi, V. Janganati, R.L. Eoff, M.L. Guzman, P.A. Crooks,
Synthesis, anticancer activity and molecular docking studies on
a series of heterocyclic
trans-cyanocombretastatin analogues as antitubulin agents, Eur J Med Chem, 92 (2015) 212-220.
[24] P.J. Neuvonen, J.T. Backman, M. Niemi, Pharmacokinetic comparison of the potential
over-the-counter statins simvastatin, lovastatin, fluvastatin and pravastatin, Clin Pharmacokinet, 47
(2008) 463-474.
[25] P.V. Podea, M.I. Toşa, C. Paizs, F.D. Irimie, Chemoenzymatic preparation of enantiopure
L-benzofuranyl- and L-benzo[b]thiophenyl alanines, Tetrahedron: Asymmetry, 19 (2008) 500-511.
[26] D.W. Wang, H.Y. Lin, R.J. Cao, T. Chen, F.X. Wu, G.F. Hao, Q. Chen, W.C. Yang, G.F. Yang, Synthesis
and Herbicidal Activity of Triketone-Quinoline Hybrids as Novel 4-Hydroxyphenylpyruvate Dioxygenase
Inhibitors, J Agric Food Chem, 63 (2015) 5587-5596.

ACCEPTED MANUSCRIPT
Figure 1. Design strategies of target molecules
Figure 2.Structures of drugs and agrochemicals containing active heterocycles
Figure 3. Structural optimization–replacement of indole by alternative heterocycles
Figure 4. X-ray structures of compounds 6g and 7e
Scheme 1: Synthetic routes for the target compounds
Table 1. Antifungal activity of the target compounds

ACCEPTED MANUSCRIPT
Figure 1. Design strategies of target molecules
Figure 2.Structures of drugs and agrochemicals containing active heterocycles
Figure 3. Structural optimization–replacement of indole by alternative heterocycles

ACCEPTED MANUSCRIPT
Figure 4. X-ray structures of compounds 6g and 7e
Scheme 1: Synthetic routes for the target compounds

ACCEPTED MANUSCRIPT
Table 1. Antifungal activity of the target compounds
Species
PHYa
200
0b
0
ZYMa
100
0
UROa
100
0
PYTa
20
0
ALTa
20
0
BOTa
20
NCc
0
GIBa
20
0
No.
Rate (ppm)
6a
6b
6c
0
0
0
NC
0
0
0
0
0
0
0
0
6d
6e
0
0
0
0
55d
0
0
0
27
36
36
0
0
27
55
55
27
0
0
0
0
6f
0
0
99
27
55
0
27
0
0
6g
0
0
0
6h
6i
0
49
0
0
0
0
18
33
0
0
0
6j
0
49
0
0
0
0
0
6k
6l
0
0
0
0
0
0
0
0
0
0
0
0
6m
6n
7c
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
NC
0
0
0
7d
7e
0
0
0
0
0
0
0
0
0
55
0
0
0
0
8c
0
0
0
0
0
0
8d
8e
0
0
0
0
0
0
0
0
36
0
49
0
77
55
0
0
27
0
0
8g
0
0
0
Pimprinine
0
51
27
0
0
0
Streptochlorin
49
36
55
99
99
99
99
aPHY, Phytophthora infestans (on tomato leaf pieces); ZYM, Zymoseptoria tritici (on wheat leaf pieces); URO,
Uromyces viciae-fabae (on bean leaf pieces); PYT, Pythium dissimile; ALT, Alternaria solani; BOT, Botryotinia
fuckeliana; GIB, Gibberella zeae (all in artificial media).
bThe data are the mean of three replicates.
c Data not captured for this replicate (test failure).
d The bold means data equal to or above 55% control.

ACCEPTED MANUSCRIPT
Highlights
A series of streptochlorin analogues in which indole has been replaced by other
heterocycles has been designed and synthesized.
Biological testing showed most of the designed streptochlorin analogues were
inactive.
SAR study demonstrated that indole ring is essential for the antifungal activity of
streptochlorin analogues, promoting the idea of indole as the framework that might be
exploited in the future.