Total synthesis and biological evaluation of nannocystin analogues modified at the polyketide phenyl moiety

Article abstract of DOI:10.1016/j.tetlet.2018.07.028

Total synthesis of a focused set of 10 nannocystin analogues 3a–3j modified at the polyketide phenyl moiety was reported. These compounds were evaluated against three cancer cell lines. Compared with the naturally occurring congener 3a, the other synthetic variants either preserved or lose antiproliferative activity at varying degrees. Moreover, the potent analogues also displayed comparable levels of cytotoxicity toward two normal cell lines.

Full text of DOI:10.1016/j.tetlet.2018.07.028

Accepted Manuscript
Total Synthesis and Biological Evaluation of Nannocystin Analogues Modified
at the Polyketide Phenyl Moiety
Yunfeng Tian, Yahui Ding, Xiaolong Xu, Yanju Bai, Yang Tang, Xin Hao,
Weicheng Zhang, Yue Chen
PII:
S0040-4039(18)30888-8
https://doi.org/10.1016/j.tetlet.2018.07.028
TETL 50137
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 May 2018
8 July 2018
9 July 2018
Please cite this article as: Tian, Y., Ding, Y., Xu, X., Bai, Y., Tang, Y., Hao, X., Zhang, W., Chen, Y., Total Synthesis
and Biological Evaluation of Nannocystin Analogues Modified at the Polyketide Phenyl Moiety, Tetrahedron
Letters (2018), doi: https://doi.org/10.1016/j.tetlet.2018.07.028
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Total Synthesis and Biological Evaluation of Nannocystin
Analogues Modified at the Polyketide Phenyl Moiety
Yunfeng Tian, Yahui Ding, Xiaolong Xu, Yanju Bai, Yang Tang, Xin Hao, Weicheng Zhang,* and
Yue Chen*
* The State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin
Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People's
Republic of China
E-mail: zhangweicheng@nankai.edu.cn, yuechen@nankai.edu.cn
Abstract
Total synthesis of a focused set of 10 nannocystin analogues 3a-3j modified at the polyketide
phenyl moiety was reported. These compounds were evaluated against three cancer cell lines.
Compared with the naturally occurring congener 3a, the other synthetic variants either preserved
or lose antiproliferative activity at varying degrees. Moreover, the potent analogues also displayed
comparable levels of cytotoxicity toward two normal cell lines.
Highlights



10 nannocystin analogues varying at the polyketide phenyl moiety were synthesized
These compounds were tested against both cancer cell lines and normal cell lines
All potent compounds inhibited the growth of both cancerous and normal cell lines
Keywords
nannocystin; structure-activity relationship; anticancer; eukaryotic elongation factor 1A
Nannocystin A (1) was initially isolated from the myxobacterial secondary metabolites1 and
later on reported in two side-by-side papers by Brönstrup et al.2 and Hoepfner et al.3 in 2015 (Fig.
1.). Its structure features a mixed polyketide-tripeptide scaffold bearing nine chiral centers, two
1

continuous E-alkenes, and a unique α,β-epoxy amide. Biological studies2,3 found strong and
differential cytotoxicity of 1 across a broad range of cancer cell lines, with IC50 values ranging
from sub-micromolar (0.5 μM) to low nanomolar (5 nM) levels. Through genetic and proteomic
experiments, Hoepfner et al. deduced eukaryotic translation elongation factor 1A (eEF1A) as the
most possible target.3 This protein is primarily in charge of recruiting amino acyl-tRNA (aa-tRNA)
in the presence of GTP to the ribosomal A site during the elongation step of protein synthesis.4
Correct codon-anticodon recognition between the incoming aa-tRNA and the mRNA template
activates eEF1A to catalyze the hydrolysis of GTP. The resulting GDP and eEF1A dissociate from
the aa-tRNA-loaded ribosome, with subsequent regeneration of a new eEF1A/GTP complex for
the binding and delivery of the next proper aa-tRNA. Didemnin B, a known inhibitor of eEF1A,
was found by Hoepfner et al. to effectively displace its target protein eEF1A off the
nannocystin-based affinity matrix with similar level of competition as 1, which pointed to a
common binding region on the target surface shared by the two different molecules.3 Recently,
this opinion was corroborated by Gago et al. through molecular dynamics calculations, who
proposed detailed molecular modeling structures to rationalize the high-affinity binding of four
structurally distinct compounds, namely didemnin B, ternatin, ansatrienin B, and 1 to eEF1A at the
presumably overlapping binding site.5 Nevertheless, so far there is no systematic study to clarify
in detail whether nannocystins such as 1 target the intracellular eEF1A alone, the GTP-bound
eEF1A as in the case of didemnin B,6 or the ternary complex of eEF1A/GTP/aa-tRNA as found in
the ternatin derivatives.7 Elaborate knowledge on this issue will facilitate direct characterization of
the on-target inhibitory effects of nannocystins.
Endowed with a novel structure and potentially intriguing mode of action,8 1 immediately
captured the attention of synthetic community. Indeed, soon after the two leading publications,2,3
several research teams have successfully achieved total synthesis of 1 or its 2,3-desoxy congener
nannocystin Ax (2) via different routes.9-15 These works have not only established a diversity of
synthetic accesses to the natural nannocystins alongside the fermentation approach, but also
enabled the indispensable tool of site-selective structure permutation in order to modulate
ligand/protein interactions and, if necessary, optimize the various critical drug-like
physicochemical properties. For instance, a “motif-oriented” strategy was adopted by Fürstner et
al. to prepare a collection of 2 and other 10 non-natural nannocystins and probe the SAR of the
2

trisubstitued 6,7-alkene and the proximate 5-methyl ether region.15 While the alkene moiety was
tolerant of moderate structural variation (the permissive site), the neighboring methyl ether had to
be retained for high potency (the prohibitive site).
Fig. 1. Structures of naturally occurring nannocystin A (1) and 2,3-desoxy congener nannocystin
Ax (2).
Parallel to Fürstner’s work, we recently synthesized a focused set of 10 nannocystin
analogues including 1, 2, and 3a.16 This study was aimed at understanding the possible binding
role of the (2R,3S)-epoxide in 1. Biological assay, in combination with variable temperature NMR
experiments and computational studies, suggested that the epoxide unit is not likely to interact
directly with the target binding site, but serves to control the macrocycle conformation. In the
docking study,16 two pockets I and II were identified to accommodate the side chain of the
β-OH-L-Val3 residue and the polyketide C-11 phenyl group of 3a respectively (Fig. 2). Pocket I
appears to be hydrophobic in nature: mutation at the β-OH-L-Val3 fragment with an L-valine had a
marginal effect on potency,16 whereas substitution at this position for an L-serine caused a marked
drop in activity.17 Thus it is the two methyl groups rather than the hydroxyl group within the side
chain of the β-OH-L-Val3 segment that accounted for favorable binding to the target. Given the
importance of hydrophobic interactions envisioned in the eEF1A-nannocystin binding
process,3,5,16 it is a natural move for us to investigate the influence of the C-11 phenyl moiety,
which was assumed to interact with the neighboring pocket II according to the docking analysis,16
on the cytotoxicity of nannocystins. Hence we chose the bromo-substituted natural congener 3a as
the reference compound18 and further designed 9 non-natural nannocystins 3b-3j modified at the
3

C-11 phenyl part. Total synthesis of these compounds combined with biological evaluation at the
cellular level would establish a structure-cytotoxicity correlation regarding this subunit, which
could help with elucidating the nannocystin pharmacophore essential for its activity.
Fig. 2. Our previous study on in silico docking of the natural congener 3a into the binding site of
eEF1A identified two pockets I and II, which accommodate the side chain of the β-OH-L-Val3
residue and the polyketide C-11 phenyl group respectively.16
Syntheses of 3a-3j were carried out in a straightforward manner following our previous total
synthesis11 of 1 (Scheme 1). Thus chiral anti aldols 5a-5j were first prepared by converting
optically pure N-propionylsultam 4 to an O-silyl-N,O-ketene acetal intermediate, followed by
TiCl4-mediated stereoselective aldol condensation with different aryl aldehydes.19 Protection of
the alcohols as TBS ethers, cleavage of the sultam auxiliary, and then Wittig olefination afforded
terminal alkenes 8a-8j, which were further deprotected and coupled to a common intermediate
1311 via Mitsunobu reaction leading to Boc-protected intermediates 10a-10j, respectively. After
removal of the Boc group, the resulting products 11a-11j were condensed with another common
intermediate 1416 and finally underwent intramolecular Heck macrocyclization to yield the
designed analogues 3a-3j respectively.
4

Scheme 1. Synthesis of nannocystin analogues 3a-3j modified at the polyketide phenyl region.
Reagents and conditions: (a) TBSOTf, NEt3, CH2Cl2, then ArCHO, TiCl4, CH2Cl2, -78 °C; (b)
TBSOTf, 2,6-lutidine, CH2Cl2, -78 °C; (c) DIBAL-H, CH2Cl2, -78 °C; (d) MePPh3Br, tBuOK,
0 °C; (e) TBAF, THF; (f) 13, PPh3, DEAD, THF; (g) TFA, THF; (h) 14, EDC, HOBT, NaHCO3,
THF; (i) Pd(OAc)2, Cs2CO3, NEt3, DMF.
The cytotoxicity of 3a-3j was examined in three typical cancer cell lines HCT116, HepG2,
and PANC1. To assess the safety profile of these compounds, two normal cell lines FHC and LO2
were also subjected to the test. As shown in Table 1, differential activity was observed in all potent
nannocystin analogues (3a-3e, 3h-3j) across the three cancer cell lines, with the general order of
potency being HCT116 > HepG2 > PANC1. In terms of selective cytotoxicity toward cancerous
5

cells over normal cells, however, each of the potent analogues (3a-3e, 3h-3j) also inhibited the
growth of the noncancerous FHC and LO2 cell lines with comparable activity (IC50 values ranging
from 2.13 nM to 16.95 nM for FHC, from 4.72 nM to 148.8 nM for LO2). This observation is
reminiscent of an earlier study by Brönstrup et al.,2 wherein strong inhibitory activity of 1 was
manifested against not only several cancer cell lines (IC50 values ranging from 1.0 nM to 12 nM)
but also quiescent peripheral blood lymphocytes (IC50 11 nM). These data collectively indicate
nannocystins as strong antiproliferative compounds against both cancerous and normal cells, a
result probably stemming from their underlying mechanism of action3 that globally blocks the
eukaryotic cell protein synthesis at the elongation stage. Hence much effort is in demand in the
future to address the cytotoxic selectivity issue associated with nannocystins before they could be
evolved into therapeutically useful antineoplastic agents.20
Table 1 Inhibitory activities of nannocystin analogues 3a-3j against three cancer cell lines and two
normal cell lines of human origin.a
IC50 (nM)
Compound
HCT116
1.59±0.23
1.00±0.29
1.56±0.39
3.72±2.32
HepG2
PANC1
FHC
LO2
3a
3b
3c
3d
3e
3f
2.98±0.11
2.65±1.41
2.79±0.60
9.61±1.95
7.66±3.97
7.85±2.02
3.18±1.91
2.13±0.77
3.14±1.35
6.21±2.27
4.76±3.46
4.72±2.00
5.17±2.32
15.09±6.53
148.8±0.98
>200
4.72±1.23 30.40±0.11
7.84±3.27 24.53±3.49 21.59±0.01 16.95±6.07
90.89±6.04
>200
>200
>200
>200
>200
>200
3g
3h
3i
>200
>200
2.65±0.61
2.19±0.13
3.85±0.93 18.42±5.01
4.43±0.03 25.66±5.91
2.90±0.51
2.24±0.90
8.03±1.73
9.69±3.66
7.80±4.18
43.21±27.15
3j
5.38±0.47 18.31±6.76 22.35±3.16
a
Histotypes of the tested cell lines: HCT116, colon carcinoma; HepG2, liver hepatocellular
carcinoma; PANC1, pancreatic carcinoma; FHC, normal colorectal mucosa; LO2, normal
hepatocytes.
Also found in the assay is a direct correlation between the structural permutation at the
nannocystin C-11 phenyl moiety and the corresponding cytotoxicity toward each individual cancer
cell line. With regard to the human colon carcinoma HCT116, for instance, a para fluoro (3b) or
6

chloro (3c) substitution at the benzene ring gave equipotent activity (IC50 1.00 nM for 3b, 1.56 nM
for 3c) as compared with that of the control compound 3a (IC50 1.59 nM). Further increase in the
bulkiness of the para substituent (Br for 3d, CF3 for 3e), shuffling the fluoro or chloro group from
para to meta position (3h, 3i), or sprouting at both para and meta positions into a 2-naphthyl
group (3j) at C-11 witnessed a moderate drop of potency (IC50 values ranging from 2.19 nM to
7.84 nM). Finally, when a more hindered 1,1′-biphenyl-4-yl (3f) or a benzyl group was introduced
at this site, dramatic loss of activity occurred. Overall, the observed cytotoxicity and its change
with the nannocystin structure is a consequence of the microscopic interplay between the inhibitor
and the target. Hence our results imply that the target binding site is sensitive to the nannocystin
C-11 substituent and may tolerate small to medium sized substituents at the para or meta position
of the phenyl ring.
In summary, a focused library of 10 nannocystin analogues modified at the polyketide phenyl
moiety was built via total synthesis. These compounds were assessed against three cancer cell
lines. Compared with the parent compound 3a, the other synthetic compounds showed comparable
or deteriorated potency. Moreover, those potent nannocystins also inhibited the growth of two
normal cell lines with comparable levels of activity. Further efforts in our lab are directed toward
elucidating the essential pharmacophore of nannocystins through systematic SAR study.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No.
81573282) and the National Science Fund for Distinguished Young Scholars (Grant No. 81625021)
to Y. Chen, and the National Natural Science Foundation of China (Grant No. 21772101) and the
Natural Science Foundation of Tianjin City (Grant No. 17JCYBJC28400) to W. Zhang.
A. Supplementary Data
Supplementary data related to this article include synthetic details for all intermediates,
products, and their analytical data.
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Highlights



10 nannocystin analogues varying at the polyketide phenyl moiety were synthesized
These compounds were tested against both cancer cell lines and normal cell lines
All potent compounds inhibited the growth of both cancerous and normal cell lines
10

11