Design and synthesis of natural product derivatives with selective and improved cytotoxicity based on a sesquiterpene scaffold

Article abstract of DOI:10.1016/j.bmcl.2016.03.022

Brasilamide E (1) is a bisabolane sesquiterpenoid isolated from the solid-substrate fermentation cultures of a plant endophytic fungus Paraconiothyrium brasiliense. The compound specifically inhibited proliferation of the MCF-7 cells, but did not show cyt

Full text of DOI:10.1016/j.bmcl.2016.03.022

Accepted Manuscript
Design and synthesis of natural product derivatives with selective and improved
cytotoxicity based on a sesquiterpene scaffold
Yang Zhang, Zhuowei Zhang, Bo Wang, Ling Liu, Yongsheng Che
PII:
S0960-894X(16)30242-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.022
BMCL 23665
Reference:
Bioorganic & Medicinal Chemistry Letters
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4 March 2016
8 March 2016
Please cite this article as: Zhang, Y., Zhang, Z., Wang, B., Liu, L., Che, Y., Design and synthesis of natural product
derivatives with selective and improved cytotoxicity based on a sesquiterpene scaffold, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.03.022
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Design and synthesis of natural product derivatives with selective
and improved cytotoxicity based on a sesquiterpene scaffold
Yang Zhang†, Zhuowei Zhang†, Bo Wang, Ling Liu, Yongsheng Che*
1

Design and synthesis of natural product derivatives with
selective and improved cytotoxicity based on a sesquiterpene
scaffold
Yang Zhang a,†, Zhuowei Zhang a,†, Bo Wang a, Ling Liu b, Yongsheng Che a,*
a State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute
of Pharmacology & Toxicology, Beijing 100850, P. R. China
b State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, P. R. China
* Corresponding author. Tel./Fax: +86 10 66932679. E-mail address: cheys@im.ac.cn
(Y. Che).
† Contributed equally to this work.
2

Abstract
Brasilamide E (1) is a bisabolane sesquiterpenoid isolated from the solid-substrate
fermentation cultures of a plant endophytic fungus Paraconiothyrium brasiliense.
The compound specifically inhibited proliferation of the MCF-7 cells, but did not
show cytoxicity towards the negative controls HaCaT and NIH3T3 cells (IC50 > 50
µM). To improve its potency while maintain selectivity, a total of 27 derivatives of
1 were designed, synthesized, and evaluated for in vitro cytotoxicity against six tumor
cell lines and the negative control NIH3T3 cells. Among these compounds,
compound 12b showed significantly improved potency against the MCF-7, HeLa, and
HO8910 cells with IC50 values of 0.13–0.25 μM compared to 1 (IC50 8.47–18.00 μM),
and remained nontoxic to the NIH3T3 cells.
Key words: Brasilamide E; Synthesis; Derivatives; Selectivity; Cytotoxicity
3

A common drawback of the existing anticancer drugs is their toxicity to
noncancerous cells, resulting in chemotherapy damage to normal cells and causing
various side effects, such as bone marrow suppression, gastrointestinal toxicity,
anemia, hair loss, and constipation.1 Therefore, effective and safe treatments for
cancer are still desperately needed.2 Despite advances in molecular modeling,
combinatorial chemistry, and other synthetic chemistry fields, natural products
continue to be an important source of drug leads, particularly in the field of anticancer
therapy, with over 50% of clinically used drugs are natural products or their
derivatives.3 Terpenoids are the largest and most diverse group of natural products,
showing various biological activities including potent in vitro and in vivo inhibitory
effects on proliferation of a variety of human tumor cell lines.4–8 As the
sesquiterpene type of natural products, the bisabolane sesquiterpenoids have been
isolated from various sources,9–11 and a number of which have been identified from
fungi as antitumor metabolites.12–17
In our previous study, a series of new bisabolane sesquiterpenoids have been
isolated from the solid-substrate fermentation culture of Paraconiothyrium brasiliense,
a fungus endophytic to Acer truncatum Bunge.18–20 Among those, a metabolite with
the 4-cyclohexylfuran skeleton, named brasilamide E (1, Fig. 1), selectively inhibited
proliferation of MCF-7 (human breast cancer) cells, but did not show detectable
toxicity towards the HaCaT (human keratinocyte) and NIH3T3 (mouse embryo
fibroblast) cells at 50 µM.19 Excellent selectivity between tumor and noncancerous
cells for brasilamide E (1) suggested its potential to be an antitumor lead compound.
In view of this, structural optimization of 1 was initiated to prepare new derivatives
with selectivity and improved potency.
The structure of brasilamide E (1) can be dissected into three fragments, the
4

Figure 1. Structure of brasilamide E (1), a bisabolane sesquiterpene from the plant
endophytic fungus Paraconiothyrium brasiliense
methylenecyclohexane (ring A), furan (ring B), and E-methylacrylamide moieties (Fig.
1). Modifications were focused on the 4-cyclohexylfuran portion (rings A and B) of
the structure. A total of 27 derivatives (6a–h, 9a–j, and 12a–I; Schemes 1–3) were
designed, synthesized, and evaluated for their in vitro cytotoxicity against a panel of
six human tumor cell lines, MCF-7 (breast cancer), HeLa (cervical carcinoma),
HO8910 (ovarian cancer), A549 (lung cancer), T24 (bladder carcinoma), and
BGC-823 (gastric cancer), with NIH3T3 (mouse embryo fibroblast) cell line as the
negative control.
Since some aromatic bisabolane sesquiterpenes have been reported to modulate
different levels of cellular growth and apoptosis,21–23 the methylenecyclohexane unit
(ring A) in 1 was first replaced by substituted phenyl rings (6a–c; Scheme 1). In
addition, ring A was also replaced by substituted pyridines (6d–f) and cycloalkenes
(6g and h), respectively, to find clues for structure–activity relationship. The key
intermediates 4a–h were synthesized via Suzuki cross-coupling reactions, in which
4-bromofuran-2-carbaldehyde 2a was individually coupled with phenyl, and pyridyl
or alkenyl-boronic acids 3a–h using Pd(Ph3P)4 as catalyst in the presence of Cs2CO3
(Scheme 1).24 Subsequently, intermediates 4a–h reacted with methylmalonic acid in
the presence of catalytic amount of piperidine via Knoevenagel condensation to afford
5

Scheme 1. Synthetic routes for compounds 6a–h. Reagents and conditions: (a)
Pd(Ph3P)4, Cs2CO3, 1,4-dioxane/water (2:1), reflux, 4–6 h; (b) methylmalonic acid,
piperidine cat., pyridine, reflux, 3 h; (c) SOCl2, DMF cat., THF, reflux, 2 h; (d)
NH3∙H2O, THF, 0 °C, 2 h.
the corresponding E-methacrylic acids 5a–h.25 Finally, treatment of 5a–h with
thionyl chloride followed by reaction with freezing ammonia gave the corresponding
products 6a–h (Scheme 1), according to a described procedure with minor
modifications.26 Compounds 6a–h were tested for their cytotoxic effects using the
Cell Counting Kit-8 assays, and the results are summarized in Table 1. In these
compounds, the phenyl derivatives 6a–c were more potent against the MCF-7 cells,
showing IC50 values of 3.34, 1.63, and 2.21 μM, respectively, compared to that of
8.47 μM for 1. In addition, compound 6a was slightly more potent than 1 (IC50 3.28
vs 5.01 μM) towards the HeLa cells, while 6c showed weak cytotoxicity against the
A549 cells, with an IC50 value of 14.91 μM compared to that of greater than 50 μM
for 1. In general, the pyridine derivatives 6d–f were less potent than 1 against
MCF-7, HeLa, and HO8910 cells, showing IC50 values ranging from 8.64 to greater
than 50 µM (Table 1). The cyclohexene derivative 6g showed potency (IC50 6.58
μM) against MCF-7 cells comparable to 1, but was inactive against the HeLa and
6

Table 1. Cytotoxicity of 1, 6a–h, 9a–j, and 12a–i against six human tumor cell lines and mouse embryo fibroblast cells
IC50 a(μM)
Compound C logP
MCF-7
8.47 ± 0.36
3.34 ± 0.51
1.63 ± 0.13
2.21 ± 0.06
13.16 ± 0.58
10.16 ± 1.78
14.10 ± 3.42
6.58 ± 0.53
15.00 ± 0.72
>50
0.97 ± 0.12
2.68 ± 0.38
>50
1.44 ± 0.07
2.38 ± 0.34
>50
3.30 ± 0.89
6.54 ± 1.60
6.71 ± 0.13
4.92 ± 0.53
0.24 ± 0.05
>50
1.21 ± 0.19
>50
17.11 ± 0.73
>50
HeLa
5.01 ± 1.24
3.28 ± 0.30
12.00 ± 0.35
11.81 ± 0.65
>50
>50
>50
>50
17.60 ± 1.92
4.59 ± 0.75
2.03 ± 0.08
12.65 ± 2.76
>50
20.84 ± 0.74
2.77 ± 0.15
24.96 ± 1.87
5.40 ± 0.35
15.69 ± 3.67
24.80 ± 2.37
19.44 ± 0.58
0.25 ± 0.02
>50
HO8910
18.00 ± 0.24
42.35 ± 2.72
24.22 ± 1.50
>50
8.64 ± 1.68
>50
30.03 ± 4.29
>50
A549
T24
BGC-823
>50
NIH3T3
>50
1
6a
>50
>50
>50
14.91 ± 4.78
6.62 ± 0.72
>50
>50
>50
16.62 ± 1.26
>50
1.65 ± 0.01
>50
>50
>50
>50
>50
13.21 ± 0.98
>50
5.10 ± 0.75
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
2.952
3.451
3.095
1.455
1.954
1.682
3.380
2.821
3.661
3.305
3.422
3.921
3.573
2.330
2.829
2.480
3.776
4.275
3.621
2.875
1.820
3.183
4.320
4.143
3.366
3.403
4.146
–
>50
>50
6b
6c
6d
6e
6f
6g
6h
9a
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
49.29 ± 5.00
>50
1.66 ± 0.34
0.99 ± 0.03
>50
>50
>50
9b
9c
9d
9e
9f
9g
9h
9i
3.32 ± 0.33
>50
0.94 ± 0.16
>50
0.96 ± 0.11
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
3.78 ± 0.21
>50
>50
>50
>50
>50
4.45 ± 0.35
12.60 ± 0.80
26.79 ± 1.33
15.21 ± 0.52
0.13 ± 0.02
>50
2.38 ± 0.17
>50
>50
>50
>50
>50
17.34 ± 0.97
>50
>50
>50
>50
9j
7.65 ± 0.98
>50
>50
>50
12a
12b
12c
12d
12e
12f
12g
12h
12i
Cisplatinb
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
2.90 ± 0.40
>50
>50
>50
>50
3.50 ± 0.42
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
3.05 ± 0.91
>50
46.70 ± 1.35
>50
>50
>50
14.70 ± 0.93
>50
15.97 ± 0.68
>50
>50
1.86 ± 0.18
2.80 ± 0.30
5.79 ± 0.63
a IC50 values were averaged from at least three independent experiments. b Positive control.
7

Scheme 2. Synthetic routes for compounds 9a–j. Reagents and conditions: (a)
Pd(Ph3P)4, Cs2CO3, 1,4-dioxane/water (2:1), reflux, 4–6 h; (b) methylmalonic acid,
piperidine cat., pyridine, reflux, 3 h; (c) SOCl2, DMF cat., THF, reflux, 2 h; (d)
NH3∙H2O, THF, 0 °C, 2 h.
HO8910 cells, while the cyclopentene derivative 6h was less potent against the
MCF-7, HeLa, and HO8910 cells. However, compound 6h was cytotoxic to A549
and NIH3T3 cells, with IC50 values of 16.62 and 49.29 μM, respectively, compared to
those of greater than 50 μM for 1. The above results implied that the more bulky
cyclohexene derivative 6g showed better selectivity than 6h.
Since the phenyl derivatives 6a–c maintained selectivity and showed improved
potency, ring A remained to be phenyl rings in modification of the ring B portion of 1.
It was reported that thiophene, thiazole, and phenyl derivatives showed inhibitory
effects against several human tumor cell lines.27,28 Therefore, compounds 9a–j
(Scheme 2) were designed and synthesized by varying the substitution patterns of the
furan ring (9a, b), and replacing the furan ring with a thiophene (9c–e), thiazole (9f–h)
or an phenyl ring (9i, j). Procedures similar to those used in preparation of 6a–h
8

were applied to synthesize these compounds. The 2,5-disubstituted furan derivative
9b showed potent inhibitory effect on proliferation of all six human tumor cell lines
(IC50 0.94–3.32 μM) and NIH3T3 cells (IC50 0.96 μM) (Table 1), indicating that 9b
increase potency, but lacks the desired selectivity when compared to the
2,4-disubstituted furan derivative 6c. The thiophene derivatives 9c–e showed similar
potency to the thiazole derivatives 9f–h, while the phenyl derivatives 9i and 9j were
less potent against the MCF-7, HeLa, and HO8910 cells (Table 1). Different from
compound 1, the thiazole derivative 9h was weakly cytotoxic to A549 cells, with an
IC50 value of 13.21 μM, while the phenyl derivative 9j was cytotoxic to both A549
and T24 cells, showing IC50 values of 5.10 and 7.65 μM, respectively. The
thiophene derivatives 9c and 9e selectively inhibited proliferation of the MCF-7 and
HeLa cells (Table 1).
Subsequently, the effects of substituents on ring A of the thiophene derivatives
were investigated. Similarly to compounds 6a–h and 9a–j, the thiophene derivatives
12a–i bearing various substituents on the phenyl ring (ring A) were prepared
according to procedures described above (Scheme 3). Notably, compounds 12b and
12d, which were substituted with the electron withdrawing 4-cyano and 4-nitro
groups, respectively, showed improved potency compared to 1, when maintained
selectivity between certain tumor cells and the negative control NIH3T3 cells (Table
1). Compound 12d also showed cytotoxicity against the A549 cells with an IC50
value of 3.50 μM. The 2-methyl substituted 12a showed significantly improved
potency against the MCF-7, HeLa, and HO8910 cells with IC50 values of 4.92–19.44
μM, compared to the 4-methyl substituted 9d (IC50 > 50 μM), implying that the
position for substitution on the phenyl ring affected the potency. The 4-chloro
substituted derivative 12f was obviously much less potent than the 4-fluoro
9

Scheme 3. Synthetic routes for compounds 12a–i. Reagents and conditions: (a)
Pd(Ph3P)4, Cs2CO3, 1,4-dioxane/water (2:1), reflux, 4–6 h; (b) methylmalonic acid,
piperidine cat., pyridine, reflux, 3 h; (c) SOCl2, DMF cat., THF, reflux, 2 h; (d)
NH3∙H2O, THF, 0 °C, 2 h.
substituted 9e against the MCF-7 cells (IC50 17.11 vs 1.44 μM). In fact, not all the
derivatives with the electron withdrawing groups improved potency, as found in
compounds 12c and 12e, which have strong electron withdrawing groups
trifluoromethyl and methylsulfonyl, respectively, were actually inactive. The logP
values of the synthesized compounds were calculated by ChemBioDraw Ultra 12.0.2
(Table 1). It was found that the logP values for the active thiophene derivatives (9c,
9e, 12a, 12b, 12d, and 12h) range from 2.875 to 3.621, whereas those for 12c and 12e
were 1.820 and 4.320, respectively. It is likely that only the thiophene derivatives
having both the electron withdrawing groups and suitable logP values showed the
desired cytotoxicity.
In summary, a total of 27 natural product derivatives were designed and
synthesized based on a bisabolane sesquiterpene scaffold. Compounds 9b and 12b
showed significantly improved potency with IC50 values ranging from 0.13 to 3.32
μM, and compound 12b maintained selectivity between three human tumors cell lines
10

(MCF-7, HeLa, and HO8910) and the negative control NIH3T3 cells, suggesting that
it is the most promising derivative as a lead compound for further investigation.
Results from this work suggested that replacement of the methylenecyclohexane ring
with substituted phenyl rings in brasilamide E (1) increased its activity and
maintained selectivity. While the substituents and substitution pattern on ring B
appeared to affect the selectivity. The presence of electron withdrawing groups on
the phenyl ring, plus suitable logP values appear to be essential for most of the
4-phenylthiophene derivatives to maintain their selectivity. These results provide
valuable information for further optimization of 1 as an antitumor lead compound.
Continuing studies to further optimize the activity profile and to elucidate the
mechanism of action are underway and the results will be reported in due course.
Acknowledgments
We gratefully acknowledge financial support from the National Natural Science
Foundation of China (81273395) and the National Program of Drug Research and
Development (2012ZX09301-003).
Supplementary data
Supplementary data (detailed experimental procedures for the synthesis,
cytotoxic activity assay, and the spectral data of the prepared compounds) associated
with this article can be found, in the online version, at
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