Design and synthesis of neolamellarin a derivatives targeting heat shock protein 90

Article abstract of DOI:10.1016/j.ejmech.2017.04.019

In this study, we designed and synthesized a novel family of neolamellarin A derivatives that showed high inhibitory activity toward heat shock protein 90 (Hsp90), a kinase associated with cell proliferation. The 3,4-bis(catechol)pyrrole scaffold and the benzyl group with methoxy modification at N position of pyrrole are essential to the Hsp90 inhibitory activity and cytotoxicity of these compounds. Western blot analysis demonstrated that these compounds induced dramatic depletion of the examined client proteins of Hsp90, and accelerated cancer cell apoptosis. Docking simulations suggested that the binding mode of 9p was similar to that of the VER49009, a potent inhibitor of Hsp90. Further molecular dynamics simulation indicated that the hydrophobic interactions as well as the hydrogen bonds contributed to the high affinity of 9p to Hsp90.

Full text of DOI:10.1016/j.ejmech.2017.04.019

European Journal of Medicinal Chemistry 135 (2017) 24e33
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and synthesis of neolamellarin a derivatives targeting heat
shock protein 90
,
,
,
,
,
, b, *
Long Jiang a b, Ruijuan Yin a b, Xueting Wang a b, Jiajia Dai a b, Jing Li a b, Tao Jiang a
,
, b, **
Rilei Yu a
a Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao
266003, China
b Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266003, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we designed and synthesized a novel family of neolamellarin A derivatives that showed
high inhibitory activity toward heat shock protein 90 (Hsp90), a kinase associated with cell proliferation.
The 3,4-bis(catechol)pyrrole scaffold and the benzyl group with methoxy modification at N position of
pyrrole are essential to the Hsp90 inhibitory activity and cytotoxicity of these compounds. Western blot
analysis demonstrated that these compounds induced dramatic depletion of the examined client pro-
teins of Hsp90, and accelerated cancer cell apoptosis. Docking simulations suggested that the binding
mode of 9p was similar to that of the VER49009, a potent inhibitor of Hsp90. Further molecular dynamics
simulation indicated that the hydrophobic interactions as well as the hydrogen bonds contributed to the
high affinity of 9p to Hsp90.
Received 13 February 2017
Received in revised form
5 April 2017
Accepted 10 April 2017
Available online 15 April 2017
Keywords:
Neolamellarin A derivatives
Hsp90 inhibition
3,4-Bis(catechol)pyrrole scaffold
Docking
© 2017 Elsevier Masson SAS. All rights reserved.
Molecular dynamics simulation
1. Introduction
attention has been attracted by Hsp90 as an important molecular
target for development of novel anticancer agents against several
Heat shock protein 90 (Hsp90) is essential to maintain the
function and stability of cellular proteins representing about 1e2%
of the whole cytosolic proteomic load, and can increase up to 4e6%
under stress conditions [1]. Hsp90 is known to facilitate the sta-
bilization and activation of over 300 client proteins involved in
oncogenic signaling, and in establishing the hallmark traits of
malignancy, including proliferation, evasion of apoptosis, immor-
talization, invasion, angiogenesis, and metastasis [2]. Although it is
highly expressed in normal cells, Hsp90 is used by cancer cells for at
least two purposes: (i) to protect an array of mutated and overex-
pressed oncoproteins (kinases and transcription factors) from
misfolding and degradation and (ii) to relieve cellular stresses
induced by the malignant phenotype [3,4]. Therefore, increasing
solid and hematological malignancies or toward leukemia stem
cells [5].
Currently, more than 10 Hsp90 inhibitors are in different stages
of clinical trials and an impressive growth in scientific and patent
literature confirms the great interest toward this pharmacologic
target from the academy and the pharmaceutical industry [6,7].
However, there are still no FDA approved Hsp90-targeting agents
for human use.
Function of the Hsp90 depends on the hydrolysis of ATP to ADP
in the N-terminal domain of the protein followed by ATP/ADP ex-
change. The inhibitory activity of majority of Hsp90 inhibitors is
originated from a competition with ATP binding to the N-terminus
of the protein [8e10]. Geldanamycin (GA) and its analogues such as
tanespimycin (17-AAG) (Fig. 1-1) and alyespimycin (17-DMAG)
have entered into clinical trial as the first generation of Hsp90 in-
hibitors while the poor solubility and liver toxicity limited their
further clinical development [11]. Benefited from the X-ray crystal
structures of Hsp90 and fragment-based drug discovery, the
second-generation of Hsp90 inhibitors were discovered and were
classified into two major types, the resorcinol and the purine [12].
The class of resorcinol Hsp90 inhibitors were found to be involved
* Corresponding author. Key Laboratory of Marine Drugs, Chinese Ministry of
Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan
Road, Qingdao 266003, China.
** Corresponding author. Key Laboratory of Marine Drugs, Chinese Ministry of
Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan
Road, Qingdao 266003, China.
E-mail addresses: jiangtao@ouc.edu.cn (T. Jiang), ryu@ouc.edu.cn (R. Yu).
http://dx.doi.org/10.1016/j.ejmech.2017.04.019
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

L. Jiang et al. / European Journal of Medicinal Chemistry 135 (2017) 24e33
25
in prominent hydrogen bonding and hydrophobic interactions with
2. Results and discussion
residues belonging to the N-terminal ATP binding cleft of Hsp90
that includes VER49009 (Fig. 1-2) [13], and CCT018159 (Fig. 1-3)
[14]. The purine scaffold including PU-H71 (Fig. 1-4) [15], BIIB021
[16] and CUDC-305 [17] is another important class of Hsp90 in-
hibitors designed according to the structural homology of ATP.
Recently, as a group of marine alkaloids, the lamellarins and
related pyrrole-derived alkaloids have attracted increasing atten-
tion because of their promising biological activities [18e20], such
as antitumor activity, and inhibitory activity on HIV-1 integrase and
MCV topoisomerase. Permethylated lamellarins showed no anti-
tumor activity but potent multidrug resistance reversal (MDR) ac-
tivity by modulating P-gp while the solubility of these compounds
was poor. As a continuation of our previous work in structural
modification of the neolamellarin A [21,22] (Fig. 1-5) and exploring
their biological activity diversity, we designed a novel series of
Hsp90 inhibitors (Fig. 1-6) with a polyphenolic pyrrole scaffold,
linking with the methoxy substituted aromatic fragment, which
was introduced to improve the hydrophobic interactions with the
binding pocket of Hsp90. Although the polyphenolic pyrrole scaf-
fold is well-known in medicinal chemistry as a scaffold for diverse
biomedical applications, the 3,4-diaryl-substituted pyrroles were
not yet investigated as core structures of heterocycles Hsp90 in-
hibitors. This fact is even more surprising since other similar het-
erocycles such as pyrazole [13,14], isoxazole [23], 1,2,3-thiadiazole
[24] have been successfully used in targeting the ATP binding
pocket of Hsp90. Besides, the phenolic groups contribute to the
hydrogen bond network formation with the amino acid residues on
the inhibitor-Hsp90 X-ray structure [25]. The cytotoxicity of these
designed compounds was tested in H1299 human lung cancer cell
lines and the inhibitory activity to the Hsp90 was also evaluated.
Finally, computational docking and MD simulation studies were
performed to understand the structure activity relationship of the
polyphenolic neolamellarin A derivatives for inhibition of the
Hsp90.
2.1. Chemistry
In consideration of different substituents at the 3, 4, and N po-
sitions of pyrrole, a convergent and efficient method using a
AgOAc-mediated oxidative coupling reaction in a one-pot synthesis
strategy [26] was adopted. Various phenylacetaldehydes [27] and
benzylamines [28] were prepared respectively for the later use.
These novel compounds were prepared as described and the
synthesis of 9a was taken as an example in Scheme 1, starting from
hydroxybenzaldehyde 1a that was first protected as benzyl ether 2a
with 97% yield. 4-(Benzyloxy)benzaldehyde 2a was converted into
the enol ether 3a with 82% yield as an E/Z mixture through Wittig
reaction with the ylide generated from (methoxymethyl) triphe-
nylphosphonium chloride and t-BuOK. The aldehyde 4a was pro-
vided by the acid-catalyzed hydrolysis of 3a and was used for the
pyrrole synthetic step without further purification due to its
instability. The hydrolysis results showed that TFA, HCl and HClO4
could efficiently catalyze the reaction, and among which TFA was
the most efficient.
Subsequently, 4-methoxybenzaldehyde 5a was submitted to
hydroxylamine hydrochloride and sodium hydroxide in methanol
and water to provide 4-methoxybenzaldehyde oxime 6a with
simple purification as a crude product. Then 6a was reduced with
zinc powder in acetic acid under 60e70 ꢀC to give another key
intermediate benzylamine 7a in 29% yield. The zinc powder should
be added in several portions in case of the excessively rising of
reaction temperature. When the temperature rose to more than
90 ꢀC, some black compounds that were difficult to separate were
generated. In this synthetic route, the newly prepared benzyl-
amines should be submitted to the AgOAc-mediated oxidative
coupling reaction immediately due to their sensibility to the air.
The desired key intermediate, pyrrole 8a, was obtained in 43%
yield as the phenylacetaldehyde 4a and benzylamine 7a were
Fig. 1. Structure of Hsp90 inhibitors. (1e4), show the structures of the known Hsp90 inhibitors; (5) shows the structure of the neolamellarin A; (6) shows the designed compounds
in this study.

26
L. Jiang et al. / European Journal of Medicinal Chemistry 135 (2017) 24e33
Scheme 1. Chemical synthesis of compound 9a.
submitted to the crucial AgOAc-mediated oxidative coupling reac-
tion. Finally, the removal of benzyl-protecting groups, using Pd-C
under hydrogen condition generated the resulting phenolic hy-
droxyl and methoxy substituted target compound 9a in 95% yield
(Scheme 1).
With the aim of exploring the influence of different substituents
at different positions to the biological activity of these compounds,
two different benzyloxy substituted phenylacetaldehydes reacted
with six different methoxy substituted benzylamines to give the
target compounds 9a-g and 9o-r, while other three different
methoxy substituted phenylacetaldehydes reacted with three
different benzyloxy substituted benzylamines to give the target
compounds 9h-n.
percentage of viable cells after 48 h exposure to the compounds 9a-
r, and the testing was repeated three times to determine the IC50
values in the cancer cell line. The results in Table 1 showed that all
the compounds except compound 9k caused micromolar concen-
trations inhibition of the cell viability of the cancer cell. The com-
pounds 9o-r with the 3,4-bis(catechol)pyrrole scaffold were the
most cytotoxic compounds. Catechol substituents at 3 and 4 posi-
tions of pyrrole were critical for the cytotoxicity of these com-
pounds due to the following reasons: 1) the compounds 9a-g with
less phenolic hydroxyl modification had less cytotoxicity, even their
N positions were linked to the same substituents comparing to 9o-
r; 2) benzyl substituted with hydroxyl group(s) at the N position
such as compounds 9h-j, 9l-n showed less cytotoxicity. Indeed, as
shown in Fig. 3, compounds 9o, 9p, 9q, 9e and 9l inhibited the cell
viability of the cancer cell in a concentration-dependent manner.
Besides, the Hsp90 inhibitory activity and IC50 of these compounds
showed evidently correlation (Fig. S1) suggesting that the Hsp90
inhibition is related to the cytotoxicity. However, it was evident that
the potencies of the compounds were moderate in antiproliferative
assay compared to the Hsp90 inhibitory activity. This observation
may be attributed to multidrug resistance-associated P-glycopro-
tein, as permethylated pyrrole-derived alkaloids probably act as a
noncompetitive inhibitor of P-gp-mediated transport [22,29,30],
which perhaps affects the ability of compounds to penetrate cells.
Further study is required to validate this proposed hypothesis. In
summary, the compounds substituted with catechol substituents at
3 and 4 positions of pyrrole were more toxic to the cancer cells, and
the methoxy groups on C ring of these compounds also played
important roles in their cytotoxicity.
2.2. Hsp90 inhibitory activity
We systematically investigated the inhibitory activity of our
synthesized compounds against the human Hsp90 protein. As
shown in Table 1 and Fig. 2, most of the compounds showed
moderate inhibitory activity to recombinant human Hsp90
a pro-
tein in a fluorescence polarization (FP) assay at 0.5 M and com-
m
pound 9p showed the highest inhibitory activity (inhibition
rate ¼ 61.9 2.9%) which was even higher than 17-AAG (inhibition
rate ¼ 52.9 2.1%), one of the most active positive control com-
pounds. The result indicated the competitive binding potency of 9p
to the ATP-binding pocket of Hsp90 was superior to 17-AAG at 0.5
mM. In general, compounds with more phenolic hydroxyl groups
showed better inhibitory activity towards Hps90. Among these
compounds, improved activity was obtained when two catechol
substituents of the 3 and 4 positions of pyrrole were introduced
simultaneously. For instance, derivatives 9o-r showed higher
Hsp90 inhibitory activity than other compounds with merely one
or no catechol substituents. Derivative 9p with two methoxy
groups on C ring afforded a significantly improved binding affinity
2.4. Compounds 9o-q suppressed the Hsp90 client protein
expression
Data in Table 1 showed that the compounds 9o-r had the
highest toxicities towards H1299 cancer cells and 9p showed
comparable inhibitory activity to human Hsp90 to that of 17-AAG.
Since Hsp90 facilitates the stabilization and activation of over 300
client proteins involved in oncogenic signaling, and in establishing
the hallmark traits of malignancy, we then tested if the observed
cytotoxicity was due to the suppressed Hsp90 client protein
expression in the lung cancer cells H1299 using the compounds 9p,
to recombinant human Hsp90a protein in the FP assay with an IC50
value of 374 nM (Table 2). It was noteworthy that the binding af-
finity of 9p was nearly 2-fold higher than that of 9o and 9o, sug-
gesting the importance of the methoxy groups on its Hsp90
inhibitory activity. Therefore, appropriate number of the methoxy
groups together with moderate CLogP (Table S1) values played an
essential role in Hsp90 inhibition.
9o, and 9q at 7.5 and 15 mM, respectively.
2.3. Cell-viability inhibition
EGFR is the receptor of epithelial growth factor (EGF) and over-
expressed in malignant cells. The EGFR signal pathway plays an
important role in the immortalization, invasion, angiogenesis, and
Human lung cancer cell line (H1299) was used to measure the

L. Jiang et al. / European Journal of Medicinal Chemistry 135 (2017) 24e33
Table 1 (continued )
27
Table 1
Inhibitory activity of Pyrrole-derived Alkaloids against Hsp90
cell line.
a and H1299 cancer
Compound
Structure
Hsp90a
H1299b(
mM)
9k
2.7 1.1%
＞100
Compound
Structure
Hsp90a
H1299b(
0.2 0.01
mM)
17-AAG
52.9 2.1%
9l
14.7 2.2%
19.1 0.1
9a
9b
9c
6.8 3.2%
18.9 2.4%
14.6 1.7%
37.3 0.2
9m
10.9 0.8%
23.4 0.3
26.9 4.8
25.3 0.2
9n
9o
9p
17.9 6.2%
34.1 4.5%
61.9 2.9%
29.2 2.4
8.3 0.6
10.5 1.1
9d
9e
9f
15.6 3.6%
19.9 2.9%
8.6 4.2%
23.1 0.6
29.2 1.4
31.6 0.5
9q
9r
28.4 1.8%
13.2 1.3
9g
9h
11.9 1.1%
25.1 1.0
28.9 5.2%
8.6 4.0
18.5 4.6%
20.5 0.9
a
Binding to Hsp90a was determined by a fluorescence polarization (FP) assay and
the results of the competitive inhibition experiment were expressed as inhibition
mean rates in the concentration of 0.5 M of 17-AAG and compounds 9a-r.
Cytotoxicity on H1299 non-small-cell lung cancer cells. Data were expressed as
IC50 mean values ( SD, n ¼ 3) in micromolar concentrations.
9i
9j
14.8 4.5%
14.8 2.9%
20.8 2.0
m
b
metastasis of malignant cells [31]. AKT is an important downstream
effector of EGFR signaling and the phosphorylation of AKT (p-AKT)
relys on the phosphorylated EGFR (p-EGFR), followed by a survival
signaling cascade. Considering that Hsp90 is responsible for
maintaining the stability of EGFR, inhibition of Hsp90 function will
induce the degradation of these client proteins. As shown in Fig. 4,
the levels of EGFR, AKT, p-EGFR and p-AKT decreased significantly
in a concentration-dependent manner. Collectively, the results
30.6 6.0

28
L. Jiang et al. / European Journal of Medicinal Chemistry 135 (2017) 24e33
Fig. 2. Binding to Hsp90
a
was determined by a fluorescence polarization (FP) assay, the competitive inhibitory activity was expressed as inhibition rates ( SD, n ¼ 3).
Table 2
Compound inhibitory activity against Hsp90
H1299 cells after treating the cells with or without compounds 9p,
a
.
9o, and 9q. As shown in Fig. 4, compounds 9p, 9o, and 9q at 7.5 and
15 M respectively inhibited expression of the cell cycle check point
Compound
Hsp90
a
(FP)a (IC50; nM)
MW
CLogPb
m
protein CDK2 and the level of cleaved PARP increased indicating the
inhibition of the expression of PARP in a concentration-dependent
manner. In summary, the results clearly suggested that compounds
9o-q efficiently inhibited the growth of H1299 cells through sup-
pression of the Hsp90 client proteins.
17-AAG
9ꢀ
9p
309
752
374
852
587
403
433
463
3.30
3.39
3.13
2.77
9q
a
Binding to N-terminal domain of Hsp90
a
was determined by a fluorescence
polarization (FP) assay (5 h).
b
ChemBioDraw Ultra 14.0 was employed for CLogP calculations.
2.5. Molecule docking studies
indicated that compounds 9p, 9o, and 9q disrupted the Hsp90
chaperone function and circumvented the gefitinib-resistance in
H1299 cells through destabilizing of oncogenic proteins such as
EGFR, AKT, p-EGFR and p-AKT. The 9p decreased the expression of
the client proteins more remarkably than that of 9o and 9q.
The proliferation of malignant cells depends on the avoidance of
the apoptosis mechanism. To determine whether the anti-
proliferative effects of compounds 9o, 9p, and 9q on H1299 cells
were related to the induction of apoptosis, we assessed cell cycle
signaling checkpoint proteins, including CDK2 and PARP in
In order to understand the molecular binding mechanism of
these compounds to ATP binding pocket of Hsp90
a, computational
docking was performed using the N-terminal domain of Hsp90
a
(PDB code: 2BSM) [32]. The docking study showed that the binding
mode of 9p was similar to that of the VER49009, with 3 rings well
superimposed with the corresponding rings of the inhibitor (Fig. 5A
and B). The A ring was exposed to the entry of the pocket with
hydrophilic residues and the 3-phenolic hydroxyl group formed
hydrogen bonding interactions with the carboxyl group of Asp 54.
Meanwhile, the B ring extended to the other hydrophilic entry
Fig. 3. Growth inhibition effect of Compounds 9e, 9l, 9o, 9p, and 9q on H1299 human lung cancer cells. H1299 cells suspended in complete media were seeded in 96-well plates
with 2000 cells/well. After 24 h, media were replaced with 200 mL complete media containing serial concentrations of compounds 9e, 9l, 9o, 9p, and 9q. After another 16 h, growth
inhibition was measured by the fluorescent signal as described in the experimental section. Data mean ( SD, n ¼ 3) indicates the statistical significance in comparison with the
controlled cells (p < 0.01).

L. Jiang et al. / European Journal of Medicinal Chemistry 135 (2017) 24e33
29
one methoxy group altered the molecule conformation resulting in
the loss of hydrogen bonds with the amino acid residues. Mean-
while the binding of 9q with three methoxy groups in C ring
induced an entirely different conformational change as the 3,4,5-
trimethoxybenzyl moiety was turned to the entry.
The phenolic hydroxyl groups also significantly affect the
binding affinity of these compounds. The 9e that possessed the
same N-position substituent (Fig. 5E) of 9p was well superimposed
on VER49009 in a similar manner of the 9p. However, no hydrogen
bond was generated in the binding of 9e to the Hsp90 resulting in
its poor binding potency because of the decreased hydroxyl groups.
Besides, 9l (Fig. 5F) from a series of compounds with phenolic hy-
droxyl groups in C ring showed comparable binding potency loss.
2.6. Molecular dynamics simulation
Fig. 4. Effects of Compounds 9p, 9o, and 9q on EGFR, AKT, p-EGFR and p-AKT, cleaved
PARP and CDK2 in H1299 human lung cancer cells. The cells were seeded in 15-cm
dishes for 24 h, and then cells were treated with Compounds 9p, 9o, 9q at 7.5 and
Molecular dynamic simulations were performed on the complex
of the compound 9p bound with Hsp90. The root-mean-square
deviation (RMSD) (Fig. S3) to the initial conformation tended to
stable after 20 ns and remained at 1.5 Å indicating that the whole
system reached equilibrium. The binding mode of 9p on protein
Hsp90 ATP binding domain was very stable similar to that deter-
mined using docking. The methoxy groups of the C ring were ori-
ented to the ATP binding pocket deeply and the cluster of hydroxyl
groups of A ring and B ring were exposed to the solvent (Fig. 6A).
The phenolic hydroxyl groups of B ring were surrounded by Lys107,
Thr109 and Phe138 within a distance of 4 Å (Fig. 6B) while the A
ring interacted with Leu48, Asn51 and Thr184 (Fig. 6C), respec-
tively. The cluster of hydroxyl groups showed considerable flexi-
bility and generated several hydrogen bonds with Gly108, Asp102,
and Asp54. However, these hydrogen bonds were not stable and
could not coexist simultaneously (Fig. 6D) indicating that the
hydrogen bond interactions were not the main driving force to
sustain the stability of the complex. Nevertheless, the hydrophobic
15
mM, respectively. Control: non-derivative treated cells received an equal volume of
DMSO. After 72 h of incubation, cells were harvested for Western blot analysis.
b-actin
was used as equal loading controls. The experiments were repeated for three times.
coinciding with the p-methoxyphenyl moiety of the VER49009
forming a hydrogen bond between the 4-phenolic hydroxyl and
Asp 102. The C ring (3,4-dimethoxybenzyl) at N position which was
selected to improve the hydrophobic interactions projected into the
relatively hydrophobic pocket and efficiently formed proximal Van
der Waals interactions with Val186, Val150, Met98, and Leu48
(Fig. S2). The methoxy groups in this fragment were excellently
superimposed to the 4-chlororesorcinol ring of VER49009 signifi-
cantly enhancing its binding potency.
Compounds 9o (Figs. 5C) and 9q (Fig. 5D) showed decreased
potency due to the modification of methoxy groups. The 9o showed
favourable superimposition with VER49009 while the C ring with
C
ring was inserted into the pocket deeply surrounded by
Fig. 5. Molecular docking model of VER49009 (magenta), 9p (cyan), 9o (yellow), 9q (blue), 9e (gray), and 9l (orange) bound in the ATP-binding pocket of human Hsp90a (PDB code:
2BSM). The oxygen, nitrogen, and sulfur atoms of compounds are shown in red, blue, and yellow, respectively. The side chains of the binding site are colored according to the atom
types (carbon, green; oxygen, red; nitrogen, blue) and the hydrogen bonds are shown as dashed lines. The docking poses were visualized using PyMOL1.8. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)

30
L. Jiang et al. / European Journal of Medicinal Chemistry 135 (2017) 24e33
Fig. 6. Analysis of the stability of 9p/Hsp90 over 50 ns molecular dynamic simulations. The distances were averaged using a 16 ps window. A), the binding mode of compound 9p (in
orange) in the binding site of Hsp90 ATP (in green) binding pocket. B), shows the distances of 9p–L107 (black), 9p–T109 (red), and 9p–F138 (green). C), shows the distances of 9p–
L48 (black), 9p–N51 (red), and 9p–T184 (green). D), shows the hydrogen bond interactions of 9p–G107 (black), 9p–D102 (red), and 9p–D54(green and blue). E), the distances
between D93 and S52 (black), D93 and T184 (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
hydrophobic residues including Lue48, Phe138, and Val186
(Fig. 6A). Besides, the internal hydrogen bonds formed between
Asp93 and Ser52, and Asp93 and Thr184 (Fig. 6E) might play an
important role in sustaining the binding pocket conformation
further enhancing the binding affinity of compound 9p. In
conclusion, the hydrophobic interactions are the main driving force
for the binding affinity of 9p and the hydrogen bond interactions
are an extra to sustain the stability of 9p in the complex.
4 positions and 3,4-dimethoxybenzyl substituent at N position was
demonstrated to have comparable binding potency to the N-ter-
minus of Hsp90 to that of the 17-AAG. The cell viability tests
showed that the cytotoxicity of compounds 9o-r was much higher
than that of compounds 9a-n in H1299 lung cancer cell lines,
indicating that the catechol substituents at 3 and 4 positions of
pyrrole of compounds 9o-r were critical for their cytotoxicity.
Western blot analysis indicated that the exposure of H1299 cancer
cells to compounds 9o-q displayed the characteristic molecular
biomarkers of Hsp90 inhibition such as the downregulation of
oncogenic proteins (EGFR and AKT). Docking simulations suggested
that the binding mode of 9p was well superimposed on the potent
inhibitor VER49009 and further molecular dynamic simulation
proved that hydrophobic interactions between the 3,4-
3. Conclusions
Among different neolamellarin
A derivatives prepared by
AgOAc-mediated oxidative coupling reaction in a one-pot manner
synthesis, the compound 9p carrying catechol substituents at 3 and

L. Jiang et al. / European Journal of Medicinal Chemistry 135 (2017) 24e33
31
dimethoxybenzyl fragment and the hydrophobic residues in the
pocket were the determinants for the binding affinity and the
hydrogen bond interactions additionally contributed to the stability
of the complex. Overall, our results suggest that the catechol sub-
stituents at 3 and 4 positions of pyrrole, methoxybenzyl substituent
at N position of the compounds are critical for their biological
activity.
petroleum ether/ethyl acetate (1: 20) to afford the product 4a-e in
49e75% yield (Scheme S1).
4.1.5. General procedure for synthesis of benzaldehyde oxime
To a stirred solution of hydroxylamine hydrochloride (8 mmol)
in methanol (5 mL) was slowly added sodium hydroxide (8 mmol)
in water (5 mL) and Benzaldehyde 5a-i (4 mmol). After being stir-
red for 1 h at 70 ꢀC, the methanol was evaporated in a vacuum and
the solution was extracted with ethyl acetate (3 ꢁ 10 mL). The
extract was dried with anhydrous sodium sulfate and evaporated in
a vacuum to provide the white crude product 6a-i (Scheme S2).
4. Experimental
4.1. Chemistry
4.1.1. Materials and methods
4.1.6. General procedure for synthesis of benzylamine
Hydroxyl benzaldehyde 1a-c, benzaldehyde 5a-i, benzylamine
7j and other chemicals were purchased from Aladdin Reagent Ltd.
(Shanghai, China). Column chromatography was performed using
silica gel (200e300 mesh) purchased from MeiGao Ltd. (Qingdao,
China). TLC was performed on silica gel plate purchased from Merck
Ltd. (Darmstadt, Germany). Melting points were determined with
an X-4 digital micro melting point tester (Taike Ltd., Beijing, China)
and were uncorrected. 1H NMR, 13C NMR spectra were taken on a
Jeol JNM-ECP 600 spectrometer (Jeol Ltd., Tokyo, Japan) with tet-
ramethylsilane (Me4Si) as the internal standard, and chemical shifts
To a stirred solution of crude product 6a-i in acetic acid (10 mL)
was added zinc powder (20 mmol) in 6 portions under 60e70 ꢀC.
After being stirred for 1 h at 70 ꢀC, the reaction mixture was filtered
immediately and the most solvent was evaporated in a vacuum.
Excess of ammonia was added to the solution to pH 10 and the
solution was extracted with ethyl acetate (3 ꢁ 10 mL). The organic
phase was washed with water and dried with anhydrous sodium
sulfate. After filtration and evaporation under reduced pressure,
purification of the residue by chromatography to provided colorless
7a-i in 29e57% yield (Scheme S2).
were recorded as
d values in ppm. Following abbreviations are
used: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet,
dd ¼ double-doublet. Mass spectra were recorded on a Q-TOF
Global mass spectrometer, and the ionization method for all the
compounds below was electrospray ionization (ESI).
4.1.7. General procedure for synthesis of pyrroles
To a solution of the aldehyde 4a-e (1.0 mmol) in anhydrous THF
(2.5 mL) was added the benzylamine 7a-j (1.0 mmol) under argon
atmosphere. After stirred at room temperature for 0.5 h, AgOAc
(334 mg, 2.0 mmol) and NaOAc (164 mg, 2.0 mmol) were added
successively under argon, then the solution was heated at 60 ꢀC for
8 h. The mixture was cooled down to room temperature, filtered
through a pad of diatomite and the diatomite was washed with
EtOAc. The filtrate was evaporated under reduced pressure and the
resulting residue was purified by flash column chromatography to
give the pure pyrroles. (The 1H and 13C NMR spectra data of
compounds 8a-8r were submitted in the supplementary material).
4.1.2. General procedure for synthesis of benzyl-substituted
benzaldehyde
To a stirred solution of hydroxyl benzaldehyde 1a-c (5 mmol) in
dry DMF (10 mL) were added K2CO3 (10 mmol). After being stirred
for 10 min, benzyl bromide (5 mmol) were added at room tem-
perature. The reaction was slowly heated to 40 ꢀC, after being
stirred for 4 h, the solvent was removed on a rotary evaporator and
the residue was the reaction mixture. Cold water (10 mL) was
poured into the residue, and extracted with DCM (3 ꢁ 10 mL). The
combined organic extracts were washed with brine (10 mL), dried
over anhydrous Na2SO4 and concentrated under reduced pressure.
The residue was purified by flash chromatography on silica gel
(petroleum ether/EtOAc: 10/1) to afford powder of compounds 2a-c
(Scheme S1) respectively.
4.1.8. General procedure for debenzylation
To a stirred solution of 8a-8r (200 mg, 0.36 mmol) in methanol
(10 mL) was added 10% Pd-C (20 mg). After being stirred for 1 h at
70 ꢀC under hydrogen, the reaction mixture was filtered and the
solvent was evaporated in a vacuum to give the product. (The 1H
and 13C NMR spectra data of compounds 9a-9r were submitted in
the supplementary material).
4.1.3. General procedure for synthesis of styrene methyl ether
To a suspension of (methoxymethyl)triphenylphonium chloride
(5.0 mmol) in THF (10 mL) was added t-BuOK (6.0 mmol) in THF
(10 mL) at 0 ꢀC under a nitrogen atmosphere. After 30 min, a so-
lution of benzaldehyde (2a-b, 2d-f) (5.0 mmol) in THF (5 mL) was
added dropwise, and the solution was stirred for 2 h at 0 ꢀC and
then for 4 h at room temperature. The mixture was quenched with
saturated water and extracted with ethyl acetate. The combined
organic extracts were washed with brine, dried over Na2SO4,
filtered, and concentrated to give the crude product. The crude
product was purified by column chromatography on silica gel using
ethyl acetate: petroleumether (1:100) as an eluant to afford the
pure compounds 3a-e in 75e82% yield as white solid (Scheme S1).
4.2. Molecular modeling
Molecular docking was performed using MOE using
AMBER10:EHT forcefield [33]. Compounds 9o, 9p, 9q, 9e and 9l
were drawn in Chem3D Pro saved the format as mol2 and mini-
mized using 10000 steps of steepest minimization in MOE. The X-
ray crystal structures of the N-terminal domain of human Hsp90
(PDB code: 2BSM) was downloaded from the protein data bank
(http://www.rcsb.org). In consideration of the flexibility of the side
chains of the residues at the binding site, the induced fit docking
approach was applied in the docking studies. The produced
conformation of with the best score was selected for the analysis.
4.1.4. General procedure for synthesis of phenylacetaldehyde
4.3. Molecular dynamics simulations
To a stirred solution of 3a-e (2 mmol) in CH2Cl2 (200 mL) was
slowly added water (4 mL) and TFA (4 mL) successively at room
temperature. After being stirred for 24 h, the mixture was
quenched with saturated NaHCO3 and extracted with CH2Cl2. The
CH2Cl2 solution was dried over MgSO4, filtered and concentrated.
The residue was purified by chromatography eluted with
Molecular dynamic (MD) simulations were implemented on the
complex of the protein Hsp90 and compound 9p, respectively using
the Amber 16 [34] package as previously described [35]. The force
field applied for the ligand was GAFF and FF14 SB was for the re-
ceptor, respectively. The complex was solvated into an octagon box

32
L. Jiang et al. / European Journal of Medicinal Chemistry 135 (2017) 24e33
of TIP3P water molecules and neutralized using Naþ ahead of the
MD simulations. Subsequently, the unfavorable van der Waals in-
teractions were eliminated via two steps of minimization. The first
step contained 1000 steps of steepest descent minimization and
1000 steps of conjugate gradient minimization on the water mol-
ecules. Second, the whole system was relaxed with 3000 steps of
steepest descent minimization and 3000 steps of conjugate
gradient minimization without the restraint on the solute. During
the energy minimization process the cutoff of the non-bonded in-
teractions was limited to 12 Å. MD was performed after minimi-
zation. First, the solute was restrained and the temperature of the
whole system was gradually increased from 10 to 300 K in 100 ps in
the NVT ensemble. Then the system was equilibrated in the NPT
ensemble where the temperature and pressure were kept at 300 K
and 1 atm respectively. Eventually, in the production process, the
whole system was relaxed and a 50-ns molecular dynamics process
was carried out. For all MD steps, the time step was set to 0.002 ps,
the particle mesh Ewald (PME) method [36] was applied to deal
with long-range electrostatic interactions and the lengths of the
bonds involving hydrogen atoms were fixed with the SHAKE al-
gorithm [37].
4.4.4. Immunoblot analysis
H1299 cells were seeded in 15 cm-petri dishes (8 ꢁ 104 cells/mL,
20 mL/dish). After 24 h, cells were treated with serial concentra-
tions of Compounds 9o, 9p, and 9q. After another 72 h of incubation
at 37 ꢀC, all the cells were collected with cell-scrapers, and incu-
bated on ice for 30 min in cell lysis buffer (Cell Signaling Technol-
ogy, USA). Cell suspensions were centrifugated at 10000 ꢁ g for
10 min at 4 ꢀC, then supernatants were collected. Proteins were
quantified by BCA Protein Assay Kit (Beyotime, Beijing, China). For
immunoblot analysis, equal amount of proteins (25e100
mg,
depending on the proteins of interest) were resolved over 10% or
12% SDS- polyacrylamide gel electrophoresis and transferred to
nitrocellulose membrane. The membranes containing the trans-
ferred protein were blocked in blocking buffer (5% nonfat dry milk,
1% Tween-20 in 20 mM Tris-buffered saline, pH 7.6) for 2 h at room
temperature, and then incubated with appropriate monoclonal
primary antibody in blocking buffer overnight at 4 ꢀC. After incu-
bation with appropriate secondary antibodies, the membranes
were washed three times with Tris buffer with Tween-20, and then
visualized using Western Lightning (PerkinElmer, Waltham, MA,
USA). Antibodies for CDK-2, phospho-EGFR (Tyr1068), phospho-
AKT, cleaved PARP, EGFR, AKT were from Cell Signaling Technol-
ogy (Beverly, MA, USA).
4.4. Biology
Acknowledgment
4.4.1. Fluorescence polarization assay
Fluorescence polarization assays were performed under the
following conditions: each 96-well plate contained 200 nM FITC-
This research was supported by The NSFC (Nos. 81373322 and
81502977), Innovation Project from Qingdao National Laboratory
for Marine Science and Technology (No. 2015ASKJ02) and the NSFC-
Shandong Joint Fund (No. U1406402).
GA, Hsp90
in a final volume of 100
a
(30 nM), and tested inhibitor (initial stock in DMSO)
L. For each assay, background wells (buffer
m
only), tracer controls (free, FITC-GA only), and bound GA controls
(fluorescent GA in the presence of Hsp90) were included on each
assay plate. The fraction of tracer bound to Hsp90 was correlated to
the mP value and plotted against log10 values of competitor con-
centrations. The inhibitory activity was tested in the presence of 0.5
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.04.019.
mM concentration of compounds 9a-r and 17-AAG (5 mL). The
compounds 9o, 9p, 9q and 17-AAG concentrations at which 50% of
bound GA was displaced was obtained by fitting the data. Fluo-
rescence intensity measurements were obtained using a Spec-
traMax M5 (Molecular Devices, Sunnyvale, CA). The excitation and
emission wavelengths were 485 nm and 530 nm, respectively.
References
[1] L.M. Butler, R. Ferraldeschi, H.K. Armstrong, M.M. Centenera, P. Workman,
Maximizing the therapeutic potential of HSP90 inhibitors, Mol. Cancer Res. 13
(2015) 1445e1451.
[2] L. Neckers, P. Workman, Hsp90 molecular chaperone inhibitors: are we there
yet? Clin. Cancer Res. 18 (2012) 64e76.
[3] J. Trepel, M. Mollapour, G. Giaccone, L. Neckers, Targeting the dynamic HSP90
complex in cancer, Nat. Rev. Cancer 10 (2010) 537e549.
[4] P. Workman, F. Burrows, L. Neckers, N. Rosen, Drugging the cancer chaperone
HSP90: combinatorial therapeutic exploitation of oncogene addiction and
tumor stress, Ann. N. Y. Acad. Sci. 1113 (2007) 202e216.
4.4.2. Cell culture
Human lung cancer cell lines H1299 were obtained from the
Type Culture Collection of the Chinese Academy of Sciences
(Shanghai, China). H1299 cells were maintained in RPMI 1640
media (Gibco) supplemented with 5% heat-inactivated FBS (Gibco,
Australia), 100 U/mL of penicillin, and 0.1 mg/mL of streptomycin
(Hyclone) at 37 ꢀC with 5% CO2 and 95% air. Cells were kept sub-
confluent and media were changed every other day. All cells used
were between 3 and 30 passages. DMSO was used as the vehicle to
deliver the compounds, and the final concentration of DMSO in all
experiments was 0.1%.
[5] N. Ho, A. Li, S. Li, H. Zhang, Heat shock protein 90 and role of its chemical
inhibitors in treatment of hematologic malignancies, Pharmaceuticals
5
(2012) 779e801.
[6] J.M. Patki, S.S. Pawar, HSP90: chaperone-me-not, Pathol. Oncol. Res. 19 (2013)
631e640.
[7] F. Jiang, H.-J. Wang, Y.-H. Jin, Q. Zhang, Z.-H. Wang, J.-M. Jia, F. Liu, L. Wang, Q.-
C. Bao, D.-D. Li, Q.-D. You, X.-L. Xu, Novel tetrahydropyrido[4,3-d]pyrimidines
as potent inhibitors of chaperone heat shock protein 90, J. Med. Chem. 59
(2016) 10498e10519.
€
[8] T. Didenko, A.M.S. Duarte, G.E. Karagoz, S.G.D. Rüdiger, Hsp90 structure and
function studied by NMR spectroscopy, Biochim. Biophys. Acta 1823 (2012)
636e647.
[9] C.K. Vaughan, P.W. Piper, L.H. Pearl, C. Prodromou, A common conforma-
tionally coupled ATPase mechanism for yeast and human cytoplasmic
HSP90s,, FEBS J. 276 (2009) 199e209.
[10] D.B. Solit, G. Chiosis, Development and application of Hsp90 inhibitors, Drug
Discov. Today 13 (2008) 38e43.
[11] G. Mehmet, O. Aykut, T. Lutfi, D. Ali, K. Irfan, T. Yusuf, Design, synthesis, and
evaluation of heat shock protein 90 inhibitors in human breast cancer and its
metastasis, Curr. Pharm. Biotechnol. 17 (2016) 1231e1245.
[12] J.H. Lee, S.C. Shin, S.H. Seo, Y.H. Seo, N. Jeong, C.W. Kim, E.E. Kim, G. Keum,
Synthesis and in vitro antiproliferative activity of C5-benzyl substituted 2-
amino-pyrrolo[2,3-d]pyrimidines as potent Hsp90 inhibitors, Bioorg. Med.
Chem. Lett. 27 (2017) 237e241.
4.4.3. Cell proliferation assay
H1299 (2000 cells/well) were seeded in 96-well plates. After
24 h, cells were treated with serial concentrations of the com-
pounds in 200
mL of serum complete media. After 48 h, cells were
added 20 L of resazurin (2 mg/mL dissolved in water, Sigma) to the
m
media. After 16 h of incubation at 37 ꢀC, the fluorescent signal was
monitored using 544 nm excitation wavelength and 595 nm
emission wavelength by Spectramax M5 plate reader (Molecular
Devices). The relative fluorescence unit (RFU) generated from the
assay was proportional to the number of living cells in each well.
[13] S.A. Eccles, A. Massey, F.I. Raynaud, S.Y. Sharp, G. Box, M. Valenti, L. Patterson,

L. Jiang et al. / European Journal of Medicinal Chemistry 135 (2017) 24e33
33
A. de Haven Brandon, S. Gowan, F. Boxall, W. Aherne, M. Rowlands, A. Hayes,
V. Martins, F. Urban, K. Boxall, C. Prodromou, L. Pearl, K. James, T.P. Matthews,
K.M. Cheung, A. Kalusa, K. Jones, E. McDonald, X. Barril, P.A. Brough,
J.E. Cansfield, B. Dymock, M.J. Drysdale, H. Finch, R. Howes, R.E. Hubbard,
A. Surgenor, P. Webb, M. Wood, L. Wright, P. Workman, NVP-AUY922: a novel
heat shock protein 90 inhibitor active against xenograft tumor growth,
angiogenesis, and metastasis, Cancer Res. 68 (2008) 2850e2860.
D. Matulis, 5-Aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3-thiadiazoles
as inhibitors of Hsp90 chaperone, Bioorg. Med. Chem. Lett. 19 (2009)
1089e1092.
[25] R. Baruchello, D. Simoni, G. Grisolia, G. Barbato, P. Marchetti, R. Rondanin,
S. Mangiola, G. Giannini, T. Brunetti, D. Alloatti, G. Gallo, A. Ciacci, L. Vesci,
M. Castorina, F.M. Milazzo, M.L. Cervoni, M.B. Guglielmi, M. Barbarino,
ꢀ
R. Fodera, C. Pisano, W. Cabri, Novel 3,4-isoxazolediamides as potent in-
[14] T. Nakashima, T. Ishii, H. Tagaya, T. Seike, H. Nakagawa, Y. Kanda, S. Akinaga,
S. Soga, Y. Shiotsu, New molecular and biological mechanism of antitumor
activities of KW-2478, a novel nonansamycin heat shock protein 90 inhibitor,
in multiple myeloma cells, Clin. Cancer Res. 16 (2010) 2792e2802.
[15] E. Caldas-Lopes, L. Cerchietti, J.H. Ahn, C.C. Clement, A.I. Robles, A. Rodina,
K. Moulick, T. Taldone, A. Gozman, Y. Guo, N. Wu, E. de Stanchina, J. White,
S.S. Gross, Y. Ma, L. Varticovski, A. Melnick, G. Chiosis, Hsp90 inhibitor PU-
H71, a multimodal inhibitor of malignancy, induces complete responses in
triple-negative breast cancer models, Proc. Natl. Acad. Sci. USA. 106 (2009)
8368e8373.
[16] K. Lundgren, H. Zhang, J. Brekken, N. Huser, R.E. Powell, N. Timple, D.J. Busch,
L. Neely, J.L. Sensintaffar, Y.C. Yang, A. McKenzie, J. Friedman, R. Scannevin,
A. Kamal, K. Hong, S.R. Kasibhatla, M.F. Boehm, F.J. Burrows, BIIB021, an orally
available, fully synthetic small-molecule inhibitor of the heat shock protein
Hsp90, Mol. Cancer Ther. 8 (2009) 921e929.
hibitors of chaperone heat shock protein 90, J. Med. Chem. 54 (2011)
8592e8604.
[26] Q. Li, A. Fan, Z. Lu, Y. Cui, W. Lin, Y. Jia, One-pot AgOAc-mediated synthesis of
polysubstituted pyrroles from primary amines and aldehydes: application to
the total synthesis of purpurone, Org. Lett. 12 (2010) 4066e4069.
[27] Q. Li, J. Jiang, A. Fan, Y. Cui, Y. Jia, Total synthesis of lamellarins D, H, and R and
ningalin B, Org. Lett. 13 (2011) 312e315.
[28] N. Fujikawa, T. Ohta, T. Yamaguchi, T. Fukuda, F. Ishibashi, M. Iwao, Total
synthesis of lamellarins D, L, and N, Tetrahedron 62 (2006) 594e604.
[29] J.Y. Lee, K.J. Yoon, Y.S. Lee, Catechol-Substituted l-Chicoric acid analogues as
HIV integrase inhibitors, Bioorg. Med. Chem. Lett. 13 (2003) 4331e4334.
[30] D.L. Boger, D.R. Soenen, C.W. Boyce, M.P. Hedrick, Q. Jin, Total synthesis of
ningalin B utilizing a heterocyclic azadiene diels alder reaction and discovery
of a new class of potent multidrug resistant (MDR) reversal agents, J. Org.
Chem. 65 (2000) 2479e2483.
€
[17] R. Bao, C.J. Lai, D.G. Wang, H. Qu, L. Yin, B. Zifcak, X. Tao, J. Wang, R. Atoyan,
M. Samson, J. Forrester, G.X. Xu, S. DellaRocca, M. Borek, H.X. Zhai, X. Cai,
C. Qian, Targeting heat shock protein 90 with CUDC-305 overcomes erlotinib
[31] F.R. Hirsch, P.A. Janne, W.E. Eberhardt, F. Cappuzzo, N. Thatcher, R. Pirker,
H. Choy, E.S. Kim, L. Paz-Ares, D.R. Gandara, Y.-L. Wu, M.-J. Ahn, T. Mitsudomi,
F.A. Shepherd, T.S. Mok, Epidermal growth factor receptor inhibition in lung
cancer: status 2012, J. Thorac. Oncol. 8 (2013) 373e384.
resistance in non-small cell lung cancer, Mol. Cancer Ther.
8 (2009)
3296e3306.
[32] B.W. Dymock, X. Barril, P.A. Brough, J.E. Cansfield, A. Massey, E. McDonald,
R.E. Hubbard, A. Surgenor, S.D. Roughley, P. Webb, P. Workman, L. Wright,
M.J. Drysdale, Novel, potent small-molecule inhibitors of the molecular
chaperone Hsp90 discovered through structure-based design, J. Med. Chem.
48 (2005) 4212e4215.
[33] V. Santiago, C. Giorgio, M. Stefano, Medicinal chemistry and the molecular
operating environment (MOE): application of QSAR and molecular docking to
drug discovery, Curr. Top. Med. Chem. 8 (2008) 1555e1572.
[34] D.A. Case, R.M. Betz, D.S. Cerutti, T.E. Cheatham III, T.A. Darden, R.E. Duke,
T.J. Giese, H. Gohlke, A.W. Goetz, N. Homeyer, S. Izadi, P. Janowski, J. Kaus,
A. Kovalenko, T.S. Lee, S. LeGrand, P. Li, C. Lin, T. Luchko, R. Luo, B. Madej,
D. Mermelstein, K.M. Merz, G. Monard, H. Nguyen, H.T. Nguyen, I. Omelyan,
A. Onufriev, D.R. Roe, A. Roitberg, C. Sagui, C.L. Simmerling, W.M. Botello-
Smith, J. Swails, R.C. Walker, J. Wang, R.M. Wolf, X. Wu, L. Xiao, P.A. Kollman,
AMBER 2016, University of California, San Francisco, 2016.
[18] H. Fan, J. Peng, M.T. Hamann, J.F. Hu, Lamellarins and related pyrrole-derived
alkaloids from marine organisms, Chem. Rev. 108 (2008) 264e287.
[19] D. Baunbaek, N. Trinkler, Y. Ferandin, O. Lozach, P. Ploypradith, S. Rucirawat,
F. Ishibashi, M. Iwao, L. Meijer, Anticancer alkaloid lamellarins inhibit protein
kinases, Mar. Drugs 6 (2008) 514e527.
[20] K. Hasse, A.C. Willis, M.G. Banwell, Modular total synthesis of lamellarin G
trimethyl ether and lamellarin S, Eur. J. Org. Chem. 1 (2011) 88e99.
[21] R. Yin, L. Jiang, S. Wan, T. Jiang, Efficient synthesis of permethylated de-
rivatives of neolamellarin A, a pyrrolic marine natural product, J. Ocean. Univ.
China 14 (2015) 329e334.
[22] P.Y. Zhang, I.L.K. Wong, C.S.W. Yan, X.Y. Zhang, T. Jiang, L.M.C. Chow, S.B. Wan,
Design and synthesis of permethyl ningalin B analogues: potent multidrug
resistance (MDR) reversal agents of cancer cells, J. Med. Chem. 53 (2010)
5108e5120.
[23] P.A. Brough, W. Aherne, X. Barril, J. Borgognoni, K. Boxall, J.E. Cansfield, K.-
M.J. Cheung, I. Collins, N.G.M. Davies, M.J. Drysdale, B. Dymock, S.A. Eccles,
H. Finch, A. Fink, A. Hayes, R. Howes, R.E. Hubbard, K. James, A.M. Jordan,
A. Lockie, V. Martins, A. Massey, T.P. Matthews, E. McDonald, C.J. Northfield,
L.H. Pearl, C. Prodromou, S. Ray, F.I. Raynaud, S.D. Roughley, S.Y. Sharp,
A. Surgenor, D.L. Walmsley, P. Webb, M. Wood, P. Workman, L. Wright, 4,5-
Diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for
the treatment of cancer, J. Med. Chem. 51 (2008) 196e218.
[35] R. Yu, D.J. Craik, Q. Kaas, Blockade of neuronal a7-nAChR by a-conotoxin ImI
explained by computational scanning and energy calculations, PLoS Comp.
Biol. 7 (2011) e1002011.
[36] T. Darden, D. York, L. Pedersen, Particle mesh Ewald: an N$log(N) method for
Ewald sums in large systems, J. Chem. Phys. 98 (1993) 10089e10092.
[37] J.P. Ryckaert, G. Ciccotti, H.J.C. Berendsen, Numerical integration of the car-
tesian equations of motion of a system with constraints: molecular dynamics
of n-alkanes, J. Comput. Phys. 23 (1977) 327e341.
[24] I. Cikotiene, E. Kazlauskas, J. Matuliene, V. Michailoviene, J. Torresan, J. Jachno,