Design, synthesis and biological evaluation of 17-arylmethylamine-17- demethoxygeldanamycin derivatives as potent Hsp90 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.07.101

Thirty-three 17-arylmethylamine-substituted derivatives of geldanamycin (GA) were designed, synthesized and evaluated for the anti-proliferation activity on human cancer cell lines, LNCaP and MDA-MB-231. Three derivatives (22, 33 and 34) exhibited potent cytotoxicity with IC₅₀ values range from 0.05 to 0.51 μM against both cell lines. Hepatotoxicity test in mice demonstrated that the levels of both AST and ALT of 34-treated group were lower than that of 17-AAG group. Western blot assay indicated that 34 was more potent than 17-AAG in the down-regulation of Hsp90 client proteins CDK4, Her2, EGFR and Raf. Moreover, 34 showed excellent in vivo antitumor activity in the MDA-MB-231 xenograft nude mice, which is superior to 22 and 33, and 17-AAG, indicating that 34 was a promising antitumor candidate. Additionally, preliminary structure-activity relationship (SAR) and molecular dynamics (MD) simulations of this new series of GA derivatives were also investigated, suggesting a theoretical model of 17-arylmethylamine geldanamycins binding to Hsp90.

Full text of DOI:10.1016/j.ejmech.2014.07.101

European Journal of Medicinal Chemistry 85 (2014) 359e370
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of 17-arylmethylamine-17-
demethoxygeldanamycin derivatives as potent Hsp90 inhibitors
,
Zhenyu Li a, Lejiao Jia a, Jifeng Wang b c, Xingkang Wu a, Huilin Hao a, Hongjiao Xu a,
,
, *
Yunfei Wu a, Guowei Shi b c, Chunhua Lu a, Yuemao Shen a
a Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan,
Shandong 250012, PR China
b Department of Urology, The Fifth People's Hospital of Shanghai, Fudan University, No. 801 Heqing Road, Shanghai 200240, PR China
c Urology Research Center, Fudan University, No. 801 Heqing Road, Shanghai 200240, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty-three 17-arylmethylamine-substituted derivatives of geldanamycin (GA) were designed, synthe-
sized and evaluated for the anti-proliferation activity on human cancer cell lines, LNCaP and MDA-MB-
231. Three derivatives (22, 33 and 34) exhibited potent cytotoxicity with IC50 values range from 0.05 to
Received 23 April 2014
Received in revised form
23 July 2014
Accepted 26 July 2014
Available online 1 August 2014
0.51 mM against both cell lines. Hepatotoxicity test in mice demonstrated that the levels of both AST and
ALT of 34-treated group were lower than that of 17-AAG group. Western blot assay indicated that 34 was
more potent than 17-AAG in the down-regulation of Hsp90 client proteins CDK4, Her2, EGFR and Raf.
Moreover, 34 showed excellent in vivo antitumor activity in the MDA-MB-231 xenograft nude mice,
which is superior to 22 and 33, and 17-AAG, indicating that 34 was a promising antitumor candidate.
Additionally, preliminary structureeactivity relationship (SAR) and molecular dynamics (MD) simula-
tions of this new series of GA derivatives were also investigated, suggesting a theoretical model of 17-
arylmethylamine geldanamycins binding to Hsp90.
Keywords:
Geldanamycin
Hsp90
Antitumor activities
Hepatotoxicity
Molecular dynamics
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
However, it is unmet to develop new generations of GA derivatives
by structure modification.
Geldanamycin (GA), isolated from Streptomyces hygroscopicus
var. geldanus in 1970, was identified as the first natural product
inhibitor of heat shock protein 90 (Hsp90) [1,2]. GA binds to the N-
terminal domain ATP/ATP binding site of Hsp90, destabilizes its
client proteins, and induces massive death of tumor cells by
apoptotic mechanism [3]. Because many of the client proteins are
oncogenic, Hsp90 is an attractive target for anticancer chemo-
therapy [4].
GA was potent against a variety of cancer cell lines [5,6], how-
ever, the use of GA as a chemotherapeutic agent has not proceeded
because it causes liver damage at effective concentrations [7]. So
far, lots of GA derivatives were reported [4,8e14] and some of the
less hepatotoxic derivatives entered into clinical trials, for instance,
17-allylamino-17-demethoxygeldanamycin (tanespimycin, 17-
Because modification of 17-position of GA can not only maintain
the excellent antitumor activity [11,13,14], but also endow com-
pounds with reduced hepatotoxicity, e.g. 17-AAG [17,18]. In this
study, we undertook the design of 17-arylmethylamine-substituted
GA derivatives through application of the structure-based bio-
isosterism approach [19e21] (Fig. 2). The effects of different sub-
stituents (halogens, methoxy group, methyl group, amino group,
hydroxyl group, nitro group and trifluoromethyl group) on the
phenyl group were also investigated. The cytotoxicities of thirty-
three GA derivatives against MDA-MB-231 and LNCap cell lines as
well as their in vivo hepatotoxicity and in vivo antitumor activity
were evaluated.
2. Results and discussion
AAG)
and
17-[2-(dimethylamino)ethyl]amino-17-
demethoxygeldanamycin (alvespimycin, 17-DMAG) (Fig. 1) [15,16].
2.1. Chemistry
The title compounds (2e34) were prepared according to the
similar method as reported in the literature (Scheme 1)
[12e14,22,23]. In total, thirty-three compounds were synthesized
* Corresponding author.
E-mail addresses: yshen@sdu.edu.cn, ahua0966@sdu.edu.cn (Y. Shen).
http://dx.doi.org/10.1016/j.ejmech.2014.07.101
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

360
Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
Fig. 1. Known ansamycin Hsp90 inhibitors.
by substituting GA (1) with a variety of substitutional arylmethyl-
amines at the C-17 position (Table 1). Their structures (2e34) were
characterized by physicochemical, MS, 1H NMR and 13C NMR
spectral data.
potent than that of GA. Interestingly, among the various substituted
17-benzylamine derivatives of GA, varying substitutions at different
positions of the benzyl group remarkably influenced their cyto-
toxicities against LNCaP cell line.
Among electron-withdrawing substituted derivatives, chloro-
substituted derivatives (2e4) exhibited varying inhibitory activ-
ities against LNCaP cell line. The meta-chloro-substituted derivative
3 was less potent than its homologs 2 (2-Cl) and 4 (4-Cl), and
cyano-substituted derivatives (23e24) followed the activity role.
Except for para-bromine-substituted derivative 6 (4-Br), all the
2.2. In vitro anti-proliferative activity
The cytotoxicities of all thirty-three 17-arylmethylamine de-
rivatives of GA were evaluated by MTT assay against the prolifera-
tion of human prostatic cancer cell line LNCaP and human breast
cancer cell line MDA-MB-231 (Tables 1 and 2), and GA and 17-AAG
were used as the positive controls.
para-electron-withdrawing-substituted derivatives (5,
7 and
24e29) showed good anti-proliferation activity against LNCaP.
Moreover, meta-nitro-substituted derivative (30) also produced
excellent anti-proliferative potencies against LNCaP.
On MDA-MB-231 cell line, most of the derivatives showed good
cytotoxicities, which are comparable to that of 17-AAG but less
Fig. 2. Design of novel Hsp90 inhibitors.
Scheme 1. Synthesis route of 17-arylmethylamine-substituted GA derivatives 2e34.

Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
361
Table 1
In vitro cytotoxicity of compounds 2e34 against LNCaP and MDA-MB-231 cells.
Table 2
In vitro cytotoxicity of compounds 31e34 against LNCaP and MDA-MB-231 cells.
Compound
Ar
LNCaP IC50
0.68 0.10
(
m
M)
MDA-MB-231 IC50
0.40 0.06
(mM)
Compound
R
LNCaP IC50
(
m
M)
MDA-MB-231 IC50 (mM)
2
3
4
5
6
7
8
9
2-Cl
3-Cl
4-Cl
4-F
4-Br
4-I
3,4-2F
3,5-2F
2-OMe
3-OMe
4-OMe
2,4-2OMe
3,4-2OMe
3,4,5-3OMe
4-OH, 3-OMe
4-OH
0.41 0.06
8.50 1.17
0.77 0.08
2.28 0.34
>10
1.36 0.15
>10
1.98 0.23
>10
2.67 0.46
7.81 1.03
3.11 0.45
>10
2.45 0.26
0.14 0.02
8.48 1.30
5.70 0.72
5.23 0.54
>10
0.18 0.03
0.05 0.01
1.80 0.20
0.48 0.05
0.85 0.11
0.96 0.08
1.03 0.12
1.34 0.14
0.57 0.06
0.34 0.04
0.16 0.19
0.43 0.15
0.65 0.07
0.38 0.05
0.46 0.02
0.63 0.04
0.24 0.01
0.36 0.05
0.63 0.09
0.35 0.02
0.46 0.07
0.88 0.15
0.18 0.03
0.33 0.08
0.36 0.18
0.64 0.12
0.16 0.01
0.17 0.03
1.67 0.22
0.80 0.13
0.49 0.07
0.56 0.06
0.17 0.02
0.45 0.09
0.25 0.03
0.35 0.05
1.06 0.12
0.13 0.01
0.13 0.02
0.18 0.15
0.69 0.11
0.28 0.25
0.05 0.04
31
32
0.44 0.08
0.41 0.03
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
17-AAG
GA
33
34
0.51 0.05
0.35 0.04
0.08 0.01
0.10 0.01
2-Me
3-Me
4-Me
3-NH2
4-NH2
3-CN
17-demethoxygeldanamycin was potent against both cell lines,
which was comparable to that of 17-AAG (Table 2). Accordingly,
compounds 22, 33 and 34 were selected to undertake a preliminary
study on their hepatotoxicities in mice.
4-CN
2.3. In vivo hepatotoxicity evaluations
4-SO2Me
4-SO2NH2
4-COOMe
4-COOEt
4-OCF3
3-NO2
The alanine aminotransferase (ALT) and aspartate aminotrans-
ferase (AST) are the most commonly used biomarkers for assess-
ment of hepatotoxicity, since the damaged hepatocytes could leak
large amounts of these enzymes into the bloodstream, leading to
the increase of serum ALT and AST [24]. ALT is more specific for
hepatic injury than AST because ALT is present mainly in the cytosol
of the liver and in low concentrations elsewhere, while AST is found
in more types of cell (e.g. heart, intestine, muscle) [25,26]. As
shown in Table 3, the AST and ALT activities of GA treated group
Among electron-donating substituted derivatives, mono-
methoxyl substituted series (10e12) showed a similar level of
inhibitory activities against LNCaP cells. To our surprise, the range
of activity where the amines (21e22) were more potent versus
corresponding monomethoxyl- and monomethyl-substituted de-
rivatives. Moreover, introduction of hydroxyl group to the para-
were 317.2
39.1 U/L and 273.0
25.3 U/L, respectively, which
were both significantly higher than that of vehicle control group
(P < 0.001), indicating the obvious hepatotoxicity of GA. Compared
with GA, the AST and ALT levels of 22-, 33- and 34-treated mice
markedly decreased. To our surprise, the AST and ALT activities of
compound 34-treated group were both lower than 22-, 33-treated
mice, and even reference drug 17-AAG-treated group. These results
suggested that compounds 22, 33 and 34 were promising anti-
tumor agents with low hepatotoxicity, which can be further
developed for the treatment of breast cancer.
position (17, IC50 ¼ 8.48
1.30
mM) significantly decreased the
activity compared to amine 22 (IC50 ¼ 0.05 0.01
mM). Besides,
difluoro-substituted derivatives (8e9) and dimethoxyl-substituted
derivatives (13e16) showed weak activities. It is worth to point out
that para-methoxyl group of 14 (3,4-2OMe) was replaced by hy-
droxyl group yielding 16 (4-OH, 3-OMe), which was at least 70
times more active than 14 against LNCaP cells.
Since we observed that different substitutions at various posi-
tions of N-17-benzyl group had evident influence on the cytotox-
icity of these derivatives against LNCaP cell line, in the second
round modification, we used heterocyclic rings to replace
substituted benzyl groups to obtain compounds 31e34. Five-
membered heterocyclic (31e32) and six-membered heterocyclic
derivatives (33e34) showed moderated and excellent antitumor
activity against both cell lines, respectively (Table 2). Especially,
compound 34, 17-((6-(trifluoromethyl)pyridin-3-yl)methylamino)-
Table 3
The AST and ALT levels of 22, 33, 34, 17-AAG, GA and vehicle treated mice.
Compound
AST (U/L)
ALT (U/L)
22
33
34
17-AAG
215.8 14.4
173.6 11.6
164.1 10.5
237.4 29.4
317.2 39.1
197.2 11.2
74.9 5.7
79.4 5.4
61.0 7.3
66.6 8.1
273.0 25.3
60.7 6.9
GA
Vehicle control

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Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
Table 4
Inhibitory activity of compound 22, 33, 34, 17-AAG and
down-regulation of CDK4, Her2, EGFR and Raf and compensatory
induction of Hsp70. When considered together with its markedly
decreased hepatotoxicity in mice, this suggests that 34 may have a
greater therapeutic window than its parent GA and hence consid-
erable potential for application in the therapy of cancer.
GA against Hsp90
a.
Compound
IC50 (mM)
22
33
34
17-AAG
GA
0.89 0.17
1.04 0.23
0.43 0.12
0.78 0.08
0.14 0.04
2.6. In vivo antitumor activity evaluations
Due to the excellent in vitro antitumor activity against MDA-MB-
231 cells and low hepatotoxicity in mice, we assessed the activity of
compounds 22, 33 and 34 against the growth of MDA-MB-231 xe-
nografts in nude mice.17-AAG was served as a positive control drug.
When average tumor volume reached about 100 mm3, mice were
randomized into five groups: compounds 22, 33, 34, 17-AAG and
vehicle control groups (6 mice per group). The drugs were injected
via caudal vein every three days at a dose of 10 mg/kg body weight,
whereas the vehicle control group received an equal volume of 5%
glucose injection containing 1% DMSO and 2% lecithin. Tumor
growth inhibition (TGI) and relative increment ratio (T/C) were
calculated at the end of treatment to reveal the antitumor effects in
tumor weight and tumor volume, respectively [27].
2.4. Hsp90 inhibitory activity
To confirm the drug target of the newly synthesized com-
pounds (2e34), the representative compounds 22, 33 and 34 were
selected to perform an Hsp90 binding assay using fluorescence
polarization assay (FPA) with GA and 17-AAG as the reference
drugs. The results revealed that inhibitory activity of compounds
22 and 33 were comparable to 17-AAG (Table 4), while 34 dis-
played inhibitory activity with an IC50 value of 0.43
which is more potent than reference drug 17-AAG
0.12 mM,
the mean tumor weight of control group ꢁ the mean tumor weight of treated group
TGI ¼
the mean tumor weight of control group
(IC50 ¼ 0.78
0.08
m
M). This result indicated that the 17-
Tumor volume (V) was estimated using the equation (V ¼ a ꢀ b2/
2, where a represents the largest tumor diameter and b represents
the perpendicular tumor diameter, respectively). T/C was calculated
according to the following formula:
arylmethylamine-17-demethoxygeldanamycin derivatives can be
classified as Hsp90 inhibitors.
2.5. Inhibition of Hsp90 in cellular systems
T
C
the mean RTV of treated group
the mean RTV of control group
¼
Inhibition of Hsp90 by 34 in cellular systems was performed by
western blot assay. A decrease in Hsp90 client proteins (including
CDK4, Her2, EGFR and Raf) and a compensatory increase in Hsp70
are the two most common biomarkers used for inhibition of Hsp90
in cells [8]. The levels of Hsp70 were all markedly increased when
treated with 34, GA and 17AAG (Fig. 3). Importantly, 34 at the
RTV, namely relative tumor volume ¼ Vt/V0, (Vt: the tumor
volume measured at the end of treatment; V0: the tumor volume
measured at the beginning of treatment).
In the MDA-MB-231 xenograft model, compound 34, with the
higher TGI value (63%) and lower T/C value (31%), demonstrated
superior antitumor activity to its analogues 22 and 33, and the
reference drug 17-AAG (TGI ¼ 57%, T/C ¼ 35%) after treatment for 15
consecutive days (Table 5). The tumor growth curve depicted in
Fig. 4A and the final tumor tissue size visualized in Fig. 4B also
explicitly showed the excellent antitumor potency of 34.
On the basis of the above in vivo experiment results, it was
concluded that relative to 17-AAG, compound 34 exhibited more
potent MDA-MB-231 inhibitory activity and lower in vivo hepato-
toxicity, which could be a promising antitumor candidate for
further study.
concentration of 0.5
mM was more potent than 17-AAG in the
2.7. Molecular modeling
To predict the binding mode of the newly synthesized com-
pounds, a docking study of compound 34 was first performed using
AutoDock Vina [28] and then a molecular dynamics (MD) simula-
tion was performed to refine the docking result by Amber 12. The X-
ray crystal structure of Hsp90 with GA taken from PDB (1YET) was
used as the input structure. Hsp90e34 complex was equilibrated
after 20 ns MD simulation, and the plot of RMSD (in ångstrom) of
the complex was shown in Fig. 5.
Our findings suggested that compound 34 exhibited a similar
binding mode with lead compound GA (Fig. 6) [2]. Compound 34
Fig. 3. Evaluation of 34 as the inhibitor of Hsp90 in the cellular system.

Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
363
Table 5
4
In vivo antitumor activity of 22, 33, 34 and 17-AAG in the MDA-MB-231 xenograft
34 & Hsp90
mode.
3
2
1
0
Compound
TGI (%)
T/C (%)
。
22
33
34
17-AAG
46
50
63
57
55
40
31
35
adopts a compact conformation to bind inside the pocket of ATP/
ADP binding domain of Hsp90. The tip of 34 ansa ring which has the
6-position methoxy group, 7-carbamate group and 8- and 10-
methyl groups, binds near the bottom of the Hsp90 pocket and
makes a high density of vander Waals contacts, whereas the ben-
zoquinone is positioned near the entrance of the pocket and makes
only a few contacts [2]. Importantly, the carbamate amino group
makes a hydrogen bond to one of the Asp83 side chain oxygen
atoms and its carbonyl oxygen atom makes a hydrogen bond with
Thr174. In middle of 34 ansa ring, the 11-hydroxyl group makes a
hydrogen bond contact with Lys48. At the pocket entrance, the 18-
carbonyl oxygen atom of the ansa ring and 17-nitrogen atom are
both make hydrogen bonds to Asp44, and 21-carbonyl oxygen atom
of the ansa ring makes hydrogen bonds to both residues Lys102 and
Gly125. The 1-carbonyl oxygen atom of the ansa ring makes a
hydrogen bond to Phe128. In particular, the phenyl ring of 3,4,5-
trimethoxycinnamoyl group interacts favorably with the Phe124
0
5
10
15
20
Time (ns)
Fig. 5. Plot of RMSD (in ångstrom) for the Hsp90e34 complex during 20 ns MD
simulation.
17-AAG group, indicating that 34 was a promising antitumor
candidate. As an Hsp90 inhibitor, compound 34 also showed
inhibitory concentrations comparable to the reference drug 17-
AAG. In particular, 34 showed excellent in vivo antitumor activity,
which is superior to its analogues 22 and 33, and the reference drug
17-AAG. In addition, docking and MD refinement of the Hsp90e34
complex give us an explanation of theoretical binding model of 17-
arylmethylamine-17-demethoxygeldanamycins. Because of their
excellent in vitro and in vivo potency, these 17-arylmethylamine-
17-demethoxygeldanamycins may have potential and should be
further pursued as next generation Hsp90 inhibitors.
side chain, giving rise to a positive pep stacking interaction.
Taken together, our molecular simulation allowed us to ratio-
nalize the activity profile of the 17-arylmethylaminegeldanamycins
against Hsp90, which provided valuable information for further
design of novel effective Hsp90 inhibitors.
4. Experimental section
4.1. Chemistry
3. Conclusions
All melting points were determined on a micromelting point
apparatus and were uncorrected. 1H NMR and 13C NMR spectra
were recorded on a Bruker Avance (600 MHz) spectrometer.
In our on-going efforts to identify structurally diverse GA de-
rivatives with low hepatotoxicity, we have identified a novel series
of 17-arylmethylamine-17-demethoxygeldanamycins by the
structure-based bioisosterism approach and also explored the SAR
at the entrance of the Hsp90 ATP/ADP binding site. All the GA de-
rivatives were evaluated for their inhibitory activities against
LNCaP and MDA-MB-231 cells. The results showed that three of the
derivatives (22, 33 and 34) exhibited evident in vitro antitumor
Chemical shifts (d) are in parts per million (ppm) downfield from
TMS ( ); multiplicity; observed coupling constant (J) in hertz (Hz);
d
proton count; assignment. Multiplicities are recorded as singlet (s),
doublet (d), doublet of doublet (dd), doublet of triplet (dt), triplet
(t), quarter (q), multiplet (m) and broad singlet (br s) where
appropriate. Mass spectra were obtained on an electrospray ioni-
zation mass spectrometer as the value m/z. Thin-layer chroma-
tography (TLC) was self-made silica gel (GF254) sheets. Flash
chromatography was performed using 200e300 mesh silica gel.
activity with IC50 values range from 0.05 to 0.51 mM against both
cell lines. Hepatotoxicity test in mice demonstrated that the AST
and ALT activity of 34-treated group were both lower than that of
Fig. 4. Growth curve of implanted MDA-MB-231 xenograft in nude mice (A); Picture of dissected MDA-MB-231 tumor tissues (B).

364
Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
34.4, 32.3, 28.5, 23.0, 12.8, 12.7, 12.4; ESI-MS: m/z 692.2 [MþNa]þ
(calcd. 692.3).
4.2.3. 17-(4-Chlorobenzylamino)-17-demethoxygeldanamycin (4)
Purple solid (28.2 mg, 84.1%); mp 210e212 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.14 (s, 1H), 7.37 (d, J ¼ 7.5 Hz, 2H), 7.31 (s, 1H),
7.21 (d, J ¼ 7.7 Hz, 2H), 6.95 (d, J ¼ 11.3 Hz, 1H), 6.58 (t, J ¼ 11.4 Hz,
1H), 6.41 (s,1H), 5.89 (d, J ¼ 9.3 Hz,1H), 5.86 (d, J ¼ 10.5 Hz,1H), 5.19
(s, 1H), 4.83 (br s, 2H), 4.72 (dd, J ¼ 14.7, 5.5 Hz, 1H), 4.60 (dd,
J ¼ 14.6, 4.9 Hz, 1H), 4.31 (d, J ¼ 9.9 Hz, 1H), 4.13 (br s, 1H),
3.59e3.55 (m, 1H), 3.46e3.42 (m, 1H), 3.36 (s, 3H), 3.27 (s, 3H),
2.78e2.69 (m, 1H), 2.65 (d, J ¼ 14.0 Hz, 1H), 2.43e2.34 (m, 1H), 2.03
(s, 3H), 1.80 (s, 3H), 1.78e1.75 (m, 2H), 1.75e1.70 (m, 1H), 1.01 (d,
J ¼ 6.4 Hz, 3H), 0.99 (d, J ¼ 7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d
183.7, 181.2, 168.4, 156.0, 144.5, 141.1, 135.9, 135.1, 134.9, 134.3,
133.7, 132.8, 129.4 (30-C and 70-C), 129.1 (40-C and 60-C), 127.0, 126.6,
109.2, 108.9, 81.7, 81.4, 81.2, 72.6, 57.2, 56.8, 49.3 (10-C), 35.0, 34.5,
32.3, 28.5, 23.0, 12.9, 12.7, 12.4; ESI-MS: m/z 692.2 [MþNa]þ (calcd.
692.3).
Fig. 6. The binding model of Hsp90e34 complex was shown by PyMoL [http://www.
pymol.org/].
4.2.4. 17-(4-Fluorobenzylamino)-17-demethoxygeldanamycin (5)
Purple solid (31.6 mg, 96.7%); mp 108e109 ꢂC; 1H NMR
(600 MHz, CDCl3)
d 9.14 (s, 1H, 1-CONH), 7.30 (s, 1H, 19-H),
7.27e7.24 (m, 2H, 30-H and 70-H), 7.12e7.05 (m, 2H, 40-H and 60-H),
6.95 (d, J ¼ 11.5 Hz,1H, 3-H), 6.58 (t, J ¼ 11.4 Hz,1H, 4-H), 6.39 (s,1H,
17-NH), 5.90e5.84 (m, 2H, 5-H and 9-H), 5.19 (s, 1H, 7-H), 4.91 (br s,
2H, 7-CONH2), 4.73 (dd, J ¼ 14.4, 5.0 Hz, 1H, 10-H), 4.58 (dd, J ¼ 14.4,
4.5 Hz, 1H, 10-H), 4.31 (d, J ¼ 9.9 Hz, 1H, 6-H), 4.20 (br s, 1H, 11-OH),
3.57 (d, J ¼ 8.6 Hz, 1H, 11-H), 3.47e3.41 (m, 1H, 12-H), 3.36 (s, 3H,
12-OMe), 3.27 (s, 3H, 6-OMe), 2.78e2.69 (m, 1H, 10-H), 2.66 (d,
J ¼ 13.7 Hz, 1H, 15-H), 2.42 (dd, J ¼ 14.0, 10.7 Hz, 1H, 15-H), 2.02 (s,
3H, 2-Me), 1.80 (d, J ¼ 0.9 Hz, 3H, 8-Me), 1.78e1.76 (m, 2H, 13-H),
1.75e1.72 (m, 1H, 14-H), 1.01 (d, J ¼ 6.5 Hz, 3H, 10-Me), 0.99 (d,
4.2. General procedure for preparation of compounds 2e34
To geldanamycin (34 mg, 0.06 mmol, 1.0 equiv.) in dichloro-
methane (8 mL) was added substitutional arylmethylamine
(0.12 mmol, 20.0 equiv.). The reaction mixture was stirred for 24 h
at room temperature, and the color changed from yellow to purple.
TLC was used to monitor the progress of the reaction. After the
reaction complete, the mixture was diluted with 20 mL of
dichloromethane and washed with 1.5 N HCl (2 ꢀ 20 mL) followed
by brine (3 ꢀ 25 mL). The organic layer was dried over anhydrous
sodium sulfate, filtered, and evaporated to dryness. The residue was
purified by flash column chromatography (silica gel, ethyl acetate/
petroleum ether ¼ 1:1) to afford a purple solid.
J ¼ 7.0 Hz, 3H, 14-Me); 13C NMR (150 MHz, CDCl3)
d 183.8 (18-C),
181.2 (21-C), 168.4 (1-CONH), 162.7 (d, J ¼ 247.7 Hz, 50-C), 156.1 (7-
CONH2), 144.5 (17-C), 141.2 (20-C), 135.9 (5-C), 135.0 (2-C), 133.7 (9-
C), 132.8 (8-C), 132.4 (d, J ¼ 2.7 Hz, 20-C), 129.6 (d, J ¼ 8.2 Hz, 30-C
and 70-C), 127.0 (4-C), 126.6 (3-C), 116.2 (d, J ¼ 21.7 Hz, 40-C and 60-
C), 109.1 (19-C), 108.9 (16-C), 81.7 (7-C), 81.4 (6-C), 81.2 (12-C), 72.6
(11-C), 57.2 (6-OMe), 56.8 (12-OMe), 49.4 (10-C), 35.0 (13-C), 34.5
(15-C), 32.3 (10-C), 28.5 (14-C), 23.0 (14-Me), 13.0 (8-Me), 12.7 (2-
Me), 12.4 (10-Me); ESI-MS: m/z 676.5 [MþNa]þ (calcd. 676.7).
4.2.1. 17-(2-Chlorobenzylamino)-17-demethoxygeldanamycin (2)
Purple solid (27.0 mg, 80.6%); mp 133e135 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.14 (s,1H), 7.43 (d, J ¼ 7.1 Hz,1H), 7.34e7.27 (m,
3H), 7.27 (s, 1H), 6.95 (d, J ¼ 11.4 Hz, 1H), 6.58 (t, J ¼ 11.3 Hz, 1H),
6.48 (s, 1H), 5.90e5.85 (m, 2H), 5.18 (s, 1H), 5.05e4.53 (m, 4H), 4.31
(d, J ¼ 9.8 Hz, 1H), 4.15 (br s, 1H), 3.57e3.56 (m, 1H), 3.44e3.42 (m,
1H), 3.36 (s, 3H), 3.27 (s, 3H), 2.79e2.69 (m, 1H), 2.58 (d, J ¼ 14.1 Hz,
1H), 2.30 (t, J ¼ 12.2 Hz, 1H), 2.03 (s, 3H), 1.80 (s, 3H), 1.77e1.74 (m,
2H), 1.74e1.70 (m, 1H), 1.04 (d, J ¼ 5.6 Hz, 3H), 0.98 (d, J ¼ 6.7 Hz,
4.2.5. 17-(4-Bromobenzylamino)-17-demethoxygeldanamycin (6)
Purple solid (30.1 mg, 84.2%); mp 229e232 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.13 (s, 1H), 7.52 (d, J ¼ 7.5 Hz, 2H), 7.30 (s, 1H),
3H); 13C NMR (150 MHz, CDCl3)
d 183.8, 181.2, 168.4, 156.0, 144.6,
7.15 (d, J ¼ 7.6 Hz, 2H), 6.95 (d, J ¼ 11.3 Hz, 1H), 6.58 (t, J ¼ 11.3 Hz,
1H), 6.41 (s, 1H), 5.88e5.84 (m, 2H), 5.18 (s, 1H), 4.86 (br s, 2H), 4.70
(dd, J ¼ 14.6, 4.9 Hz, 1H), 4.59 (dd, J ¼ 14.7, 4.3 Hz, 1H), 4.31 (d,
J ¼ 9.7 Hz, 1H), 4.04 (br s, 1H), 3.58e3.56 (m, 1H), 3.44e3.42 (m,
1H), 3.36 (s, 3H), 3.27 (s, 3H), 2.80e2.70 (m, 1H), 2.64 (d, J ¼ 14.1 Hz,
1H), 2.42e2.32 (m, 1H), 2.02 (s, 3H), 1.79 (s, 3H), 1.77e1.71 (m, 3H),
141.1,135.9,134.9,134.2 (20-C),133.8,133.3 (30-C),132.8,130.1 (40-C),
129.8, 129.4, 127.5 (70-C), 127.0 (50-C), 126.6 (60-C), 109.3, 109.0, 81.7,
81.4, 81.2, 72.6, 57.2, 56.7, 47.5 (10-C), 34.9, 34.4, 32.3, 28.3, 23.0,
12.8, 12.7, 12.3; ESI-MS: m/z 692.2 [MþNa]þ (calcd. 692.3).
4.2.2. 17-(3-Chlorobenzylamino)-17-demethoxygeldanamycin (3)
Purple solid (27.0 mg, 80.6%); mp 218e220 ꢂC; 1H NMR
(600 MHz, CDCl3) d 9.13 (s, 1H), 7.35e7.30 (m, 3H), 7.27 (s, 1H), 7.15
1.00e0.98 (m, 6H); 13C NMR (150 MHz, CDCl3)
d 183.7, 181.2, 168.3,
156.1, 144.5, 141.1, 135.9 (20-C), 135.7, 134.9, 133.6, 132.8, 132.3 (40-C
and 60-C), 129.3 (30-C and 70-C), 127.0, 126.5, 122.4 (50-C), 109.3,
108.9, 81.6, 81.4, 81.2, 72.6, 57.1, 56.7, 49.3 (10-C), 35.0, 34.4, 32.3,
28.5, 23.0, 12.8, 12.6, 12.4; ESI-MS: m/z 738.3 [MþNa]þ (calcd.
738.6).
(s, 1H), 6.95 (d, J ¼ 11.4 Hz, 1H), 6.58 (t, J ¼ 11.4 Hz, 1H), 6.44 (s, 1H),
5.89e5.84 (m, 2H), 5.18 (s, 1H), 4.89 (br s, 2H), 4.72 (dd, J ¼ 14.8,
5.6 Hz, 1H), 4.61 (dd, J ¼ 14.8, 5.1 Hz, 1H), 4.30 (d, J ¼ 9.9 Hz, 1H),
4.11 (br s, 1H), 3.57e3.55 (m, 1H), 3.44e3.43 (m, 1H), 3.35 (s, 3H),
3.26 (s, 3H), 2.81e2.70 (m, 1H), 2.64 (d, J ¼ 14.0 Hz, 1H), 2.43e2.31
(m, 1H), 2.02 (s, 3H), 1.79 (s, 3H), 1.77e1.75 (m, 2H), 1.74e1.70 (m,
4.2.6. 17-(4-Iodobenzylamino)-17-demethoxygeldanamycin (7)
Purple solid (34.5 mg, 90.6%); mp 140e142 ꢂC; 1H NMR
1H), 1.03e0.96 (m, 6H); 13C NMR (150 MHz, CDCl3)
d
183.7, 181.3,
(600 MHz, CDCl3)
d
9.13 (s, 1H), 7.71 (d, J ¼ 8.3 Hz, 2H), 7.30 (s, 1H),
168.4, 156.1, 144.5, 141.1, 138.7 (20-C), 135.9, 135.1, 134.9 (40-C), 133.7,
132.8, 130.5 (30-C), 128.6 (70-C), 127.8 (50-C), 127.0, 126.6, 125.7 (60-
C), 109.3, 108.9, 81.6, 81.4, 81.2, 72.6, 57.2, 56.8, 49.4 (10-C), 34.9,
7.02 (d, J ¼ 8.2 Hz, 2H), 6.94 (d, J ¼ 11.5 Hz, 1H), 6.58 (t, J ¼ 11.4 Hz,
1H), 6.42 (t, J ¼ 5.5 Hz, 1H), 5.89e5.84 (m, 2H), 5.18 (s, 1H), 4.91 (br
s, 2H), 4.69 (dd, J ¼ 14.8, 5.8 Hz, 1H), 4.57 (dd, J ¼ 14.8, 5.5 Hz, 1H),

Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
365
4.30 (d, J ¼ 9.9 Hz, 1H), 4.13 (br s, 1H), 3.57e3.55 (m, 1H), 3.44e3.42
4.2.10. 17-(3-Methoxybenzylamino)-17-demethoxygeldanamycin
(11)
Purple solid (25.0 mg, 75.1%); mp 120e121 ꢂC; 1H NMR
(600 MHz, CDCl3)
9.16 (s, 1H), 7.33e7.28 (m, 2H, 60-H and 19-H),
(m, 1H), 3.35 (s, 3H), 3.26 (s, 3H), 2.79e2.69 (m, 1H), 2.63 (d,
J ¼ 13.7 Hz, 1H), 2.36 (dd, J ¼ 13.9, 10.6 Hz, 1H), 2.02 (s, 3H), 1.79 (s,
3H), 1.77e1.74 (m, 2H), 1.74e1.69 (m, 1H), 1.00e0.98 (m, 6H); 13C
d
NMR (150 MHz, CDCl3)
d
183.7, 181.2, 168.4, 156.1, 144.5, 141.1, 138.3
6.95 (d, J ¼ 11.4 Hz, 1H), 6.88 (d, J ¼ 8.3 Hz, 1H, 50-H), 6.85 (d,
J ¼ 7.4 Hz, 1H, 70-H), 6.79 (s, 1H, 30-H), 6.59 (t, J ¼ 11.4 Hz, 1H), 6.48
(s, 1H), 5.90 (d, J ¼ 9.3 Hz, 1H), 5.86 (t, J ¼ 10.5 Hz, 1H), 5.19 (s, 1H),
4.82 (br s, 2H), 4.72 (dd, J ¼ 14.5, 4.8 Hz, 1H), 4.59 (dd, J ¼ 14.4,
4.0 Hz, 1H), 4.31 (d, J ¼ 9.9 Hz, 1H), 4.25 (br s, 1H), 3.81 (s, 3H, 40-
OMe), 3.60e3.55 (m, 1H), 3.47e3.42 (m, 1H), 3.36 (s, 3H), 3.27 (s,
3H), 2.80e2.70 (m, 1H), 2.65 (d, J ¼ 14.1 Hz, 1H), 2.52e2.32 (m, 1H),
2.03 (s, 3H), 1.80 (s, 3H), 1.79e1.76 (m, 2H), 1.76e1.71 (m, 1H), 1.02
(d, J ¼ 6.1 Hz, 3H), 0.99 (d, J ¼ 6.7 Hz, 3H); 13C NMR (150 MHz,
(40-C and 60-C), 136.4 (20-C), 135.9, 134.9, 133.7, 132.8, 129.5 (30-C
and 70-C), 127.0, 126.6, 109.3, 108.9, 94.0 (50-C), 81.6, 81.4, 81.2, 72.6,
57.2, 56.8, 49.4 (10-C), 35.0, 34.4, 32.3, 28.5, 23.0,12.9,12.7,12.4; ESI-
MS: m/z 784.4 [MþNa]þ (calcd. 784.6).
4.2.7. 17-(3,4-Difluorobenzylamino)-17-demethoxygeldanamycin
(8)
Purple solid (23.0 mg, 68.5%); mp 128e130 ꢂC; 1H NMR
CDCl3) d
183.8,181.0,168.4,160.2 (40-C),156.0,144.6,141.2,138.1 (20-
(600 MHz, CDCl3)
d
9.11 (s, 1H), 7.31 (s, 1H), 7.18 (dd, J ¼ 18.0, 8.4 Hz,
C), 135.8, 135.0, 133.8, 132.8, 130.3 (60-C), 126.9, 126.6, 119.9 (70-C),
113.6 (50-C),113.5 (30-C),109.0,108.9, 81.7, 81.4, 81.2, 72.6, 57.2, 56.8,
55.3 (40-OMe), 50.0 (10-C), 35.0, 34.5, 32.3, 23.0, 28.5, 12.8, 12.7,
12.3; ESI-MS: m/z 688.3 [MþNa]þ (calcd. 688.8).
1H, 70-H), 7.13e7.07 (m, 1H, 60-H), 7.01 (s, 1H, 30-H), 6.94 (d,
J ¼ 11.5 Hz, 1H), 6.58 (t, J ¼ 11.4 Hz, 1H), 6.40 (t, J ¼ 5.6 Hz, 1H),
5.89e5.84 (m, 2H), 5.18 (s, 1H), 4.91 (br s, 2H), 4.70 (dd, J ¼ 14.8,
5.9 Hz, 1H), 4.59 (dd, J ¼ 14.8, 5.6 Hz, 1H), 4.30 (d, J ¼ 9.9 Hz, 1H),
4.06 (br s, 1H), 3.58e3.54 (m, 1H), 3.45e3.42 (m, 1H), 3.35 (s, 3H),
3.26 (s, 3H), 2.76e2.71 (m, 1H), 2.64 (d, J ¼ 13.8 Hz, 1H), 2.33 (dd,
J ¼ 13.9, 10.6 Hz, 1H), 2.02 (s, 3H), 1.79 (s, 3H), 1.77e1.74 (m, 2H),
1.74e1.69 (m, 1H), 1.00e0.97 (m, 6H); 13C NMR (150 MHz, CDCl3)
4.2.11. 17-(4-Methoxybenzylamino)-17-demethoxygeldanamycin
(12)
Purple solid (25.9 mg, 77.8%); mp 209e213 ꢂC; 1H NMR
(600 MHz, CDCl3)
d 9.17 (s, 1H, 1-CONH), 7.29 (s, 1H, 19-H), 7.20 (d,
d
183.7,181.4,168.4,156.1151.3 (dd, J ¼ 62.5,12.6 Hz, 40-C),149.6 (dd,
J ¼ 8.4 Hz, 2H, 30-H and 70-H), 6.96 (d, J ¼ 11.5 Hz, 1H, 3-H), 6.91 (d,
J ¼ 8.4 Hz, 2H, 40-H and 60-H), 6.58 (t, J ¼ 11.4 Hz, 1H, 4-H), 6.37 (s,
1H,17-NH), 5.90 (d, J ¼ 9.4 Hz,1H, 5-H), 5.86 (t, J ¼ 10.5 Hz,1H, 9-H),
5.19 (s, 1H, 7-H), 4.87 (br s, 2H, 7-CONH2), 4.70 (dd, J ¼ 14.2, 5.3 Hz,
1H), 4.53 (dd, J ¼ 14.2, 5.0 Hz, 1H), 4.31 (d, J ¼ 9.9 Hz, 1H, 6-H), 4.22
(br s, 1H, 11-OH), 3.82 (s, 3H, 50-OMe), 3.59e3.57 (m, 1H, 11-H), 3.45
(m, 1H, 12-H), 3.36 (s, 3H, 12-OMe), 3.27 (s, 3H, 6-OMe), 2.78e2.71
(m, 1H, 10-H), 2.68 (d, J ¼ 14.0 Hz, 1H, 15-H), 2.47 (dd, J ¼ 13.7,
10.9 Hz, 1H, 15-H), 2.02 (s, 3H, 2-Me), 1.80 (s, 3H, 8-Me), 1.79e1.77
(m, 2H, 13-H), 1.77e1.73 (m, 1H, 14-H), 1.03 (d, J ¼ 6.2 Hz, 3H, 10-
Me), 1.00 (d, J ¼ 6.9 Hz, 3H, 14-Me); 13C NMR (150 MHz, CDCl3)
J ¼ 62.0, 12.7 Hz, 50-C), 144.4, 141.1, 136.0, 134.9, 133.7 (J ¼ 5.0,
3.7 Hz, 20-C), 133.6, 132.9, 127.0, 126.6, 123.7 (dd, J ¼ 6.4, 3.5 Hz, 70-
C), 118.1 (d, J ¼ 17.5 Hz, 60-C), 116.8 (d, J ¼ 17.6 Hz, 30-C), 109.5, 109.0,
81.6, 81.3, 81.2, 72.6, 57.2, 56.8, 48.9 (10-C), 34.9, 34.4, 32.3, 28.5,
23.0, 12.9, 12.6, 12.4; ESI-MS: m/z 694.4 [MþNa]þ (calcd. 694.7).
4.2.8. 17-(3,5-Difluorobenzylamino)-17-demethoxygeldanamycin
(9)
Purple solid (23.2 mg, 69.1%); mp 130e132 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.11 (s, 1H), 7.32 (s, 1H), 6.94 (d, J ¼ 11.3 Hz, 1H),
6.80e6.77 (m, 3H), 6.58 (t, J ¼ 11.3 Hz, 1H), 6.48 (s, 1H), 5.88e5.84
(m, 2H), 5.18 (s, 1H), 4.91 (br s, 2H), 4.72 (dd, J ¼ 15.3, 5.9 Hz, 1H),
4.65 (dd, J ¼ 15.3, 5.5 Hz, 1H), 4.30 (d, J ¼ 9.8 Hz, 1H), 4.00 (br s, 1H),
3.58e3.54 (m, 1H), 3.44e3.41 (m, 1H), 3.35 (s, 3H), 3.27 (s, 3H),
2.77e2.70 (m, 1H), 2.61 (d, J ¼ 14.0 Hz, 1H), 2.31e2.24 (m, 1H), 2.02
(s, 3H), 1.79 (s, 3H), 1.76e1.73 (m, 2H), 1.73e1.69 (m, 1H), 0.99 (s,
d
183.8 (18-C), 180.9 (21-C), 168.4 (1-C), 159.7 (50-C), 156.1 (7-
CONH2), 144.7 (17-C), 141.3 (20-C), 135.8 (5-C), 135.0 (2-C), 133.8
(9-C), 132.8 (8-C), 129.2 (30-C and 70-C), 128.6 (20-C), 126.9 (C-4),
126.5 (C-3), 114.6 (40-C and 60-C), 108.83 (19-C), 108.80 (16-C), 81.7
(7-C), 81.5 (6-C), 81.2 (12-C), 72.6 (11-C), 57.1 (6-OMe), 56.7 (12-
OMe), 55.4 (50-OMe), 49.8 (10-C), 35.1 (13-C), 34.6 (15-C), 32.3
(10-C), 28.5 (14-C), 23.0 (14-Me), 12.8 (8-Me), 12.6 (2-Me), 12.4 (10-
Me); ESI-MS: m/z 688.3 [MþNa]þ (calcd. 688.8).
3H), 0.98 (s, 3H); 13C NMR (150 MHz, CDCl3)
d 183.7, 181.4, 168.3,
164.2 (d, J ¼ 12.6 Hz), 162.6 (d, J ¼ 12.7 Hz), 156.1, 144.4, 141.0, 140.7
(t, J ¼ 8.7 Hz), 136.0, 134.9, 133.5, 132.9, 127.0, 126.5, 110.3 (d,
J ¼ 5.3 Hz), 110.2 (d, J ¼ 5.2 Hz), 109.6, 109.0, 103.8 (t, J ¼ 25.1 Hz,
1H), 81.6, 81.3, 81.2, 72.6, 57.2, 56.8, 48.9, 34.9, 34.3, 32.3, 28.5, 23.0,
12.9, 12.7, 12.4; ESI-MS: m/z 694.4 [MþNa]þ (calcd. 694.7).
4.2.12. 17-(2,4-Dimethoxybenzylamino)-17-
demethoxygeldanamycin (13)
Purple solid (29.2 mg, 83.9%); mp 237e239 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.19 (s, 1H), 7.09 (d, J ¼ 8.2 Hz, 1H, 70-H), 6.95 (d,
4.2.9. 17-(2-Methoxybenzylamino)-17-demethoxygeldanamycin
(10)
J ¼ 11.4 Hz,1H), 6.58 (t, J ¼ 11.4 Hz,1H), 6.48 (s,1H), 6.46 (d, J ¼ 8.0 Hz,
2H, 40-H and 60-H), 5.92 (d, J ¼ 9.3 Hz, 1H), 5.85 (t, J ¼ 10.5 Hz, 1H),
5.18 (s, 1H), 4.89 (br s, 2H), 4.69 (dd, J ¼ 14.1, 5.2 Hz, 1H), 4.55 (dd,
J ¼ 14.1, 4.6 Hz, 1H), 4.46 (br s, 1H), 4.31 (d, J ¼ 10.0 Hz, 1H), 3.81 (s,
6H, 30-OMe and 50-OMe), 3.59e3.56 (m,1H), 3.47e3.43 (m,1H), 3.37
(s, 3H), 3.26 (s, 3H), 2.79e2.70 (m, 1H), 2.66 (d, J ¼ 14.0 Hz,1H), 2.49
(t, J ¼ 12.2 Hz,1H), 2.02 (s, 3H),1.80 (s, 5H),1.77e1.71 (m,1H),1.04 (d,
J ¼ 6.1 Hz, 3H), 0.99 (d, J ¼ 6.8 Hz, 3H); 13C NMR (150 MHz, CDCl3)
Purple solid (25.7 mg, 77.2%); mp 236e238 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.19 (s, 1H), 7.33 (t, J ¼ 7.7 Hz, 1H, 60-H), 7.27 (s,
1H), 7.19 (d, J ¼ 7.2 Hz, 1H, 70-H), 7.00e6.93 (m, 2H, 50-H and 3-H),
6.92 (d, J ¼ 8.4 Hz, 1H, 40-H), 6.58 (t, J ¼ 11.4 Hz, 2H), 5.91 (d,
J ¼ 9.4 Hz, 1H), 5.86 (t, J ¼ 10.5 Hz, 1H), 4.85 (br s, 2H), 4.75 (d,
J ¼ 14.2 Hz, 1H), 4.66 (d, J ¼ 14.4 Hz, 1H), 4.31 (d, J ¼ 10.0 Hz, 1H),
4.30 (br s, 1H), 3.85 (s, 3H, 30-OMe), 3.59e3.58 (m, 1H), 3.45e3.44
(m, 1H), 3.37 (s, 3H), 3.27 (s, 3H), 2.82e2.70 (m, 1H), 2.64 (d,
J ¼ 14.0 Hz, 1H), 2.53e2.38 (m, 1H), 2.02 (s, 3H), 1.80 (s, 3H),
1.79e1.77 (m, 2H),1.77e1.73 (m,1H),1.04 (d, J ¼ 6.2 Hz, 3H), 0.99 (d,
d
183.9, 180.4, 168.4, 161.2 (50-C), 158.4 (30-C), 156.1, 145.0, 141.4,
135.7,135.0,133.9,132.7,130.4 (70-C),126.8,126.6,117.0 (20-C),108.7,
108.4, 104.3 (60-C), 98.8 (40-C), 81.7, 81.5, 81.3, 72.6, 57.1, 56.7, 55.5
(30-OMe and 50-OMe), 45.3 (10-C), 35.1, 34.6, 32.3, 28.4, 22.9, 12.8,
12.7, 12.3; ESI-MS: m/z 718.4 [MþNa]þ (calcd. 718.8).
J ¼ 6.9 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d 184.0, 180.6, 168.4,
157.2 (30-C), 156.1, 145.1, 141.4, 135.8, 135.0, 133.9, 132.7, 129.8 (70-C),
129.4 (50-C), 126.9, 126.6, 124.7 (20-C), 120.9 (60-C), 110.8 (40-C),
108.8, 108.6, 81.7, 81.5, 81.3, 72.6, 57.1, 56.7, 55.4 (30-OMe), 45.4 (10-
C), 35.1, 34.5, 32.3, 28.4, 22.9, 12.8, 12.6, 12.3; ESI-MS: m/z 688.3
[MþNa]þ (calcd. 688.8).
4.2.13. 17-(3,4-Dimethoxybenzylamino)-17-
demethoxygeldanamycin (14)
Purple solid (24.1 mg, 69.3%); mp 213e214 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.16 (s, 1H), 7.38 (d, J ¼ 7.5 Hz, 1H, 70-H), 7.33 (d,

366
Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
J ¼ 7.1 Hz, 1H, 60-H), 7.29 (s, 1H), 6.95 (d, J ¼ 11.3 Hz, 1H), 6.76 (s, 1H,
30-H), 6.58 (t, J ¼ 11.3 Hz, 1H), 6.39 (s, 1H), 5.90 (d, J ¼ 9.2 Hz, 1H),
5.86 (t, J ¼ 10.5 Hz, 1H), 5.18 (s, 1H), 4.90 (br s, 2H), 4.68 (dd, J ¼ 14.1,
4.7 Hz, 1H), 4.53 (dd, J ¼ 14.1, 4.3 Hz, 1H), 4.31 (d, J ¼ 9.9 Hz, 1H),
4.28 (br s, 1H), 3.89 (d, J ¼ 0.7 Hz, 3H, 40-OMe), 3.88 (d, J ¼ 0.7 Hz,
3H, 50-OMe), 3.60e3.54 (m, 1H), 3.48e3.42 (m, 1H), 3.36 (s, 3H),
3.26 (s, 3H), 2.78e2.71 (m, 1H), 2.68 (d, J ¼ 14.1 Hz, 1H), 2.48 (t,
J ¼ 12.3 Hz, 1H), 2.02 (s, 3H), 1.81e1.77 (m, 5H), 1.76e1.72 (m, 1H),
1.03 (d, J ¼ 6.0 Hz, 3H), 0.99 (d, J ¼ 6.6 Hz, 3H); 13C NMR (150 MHz,
156.4 (7-CONH2 and 50-C), 144.6, 141.4, 135.6, 135.1, 133.8, 132.8,
129.3 (30-C and 70-C), 128.1 (20-C), 126.7 (3-C and 4-C), 116.1 (40-C
and 60-C), 108.9, 108.6, 81.9, 81.4, 81.2, 72.7, 57.1, 56.8, 49.8 (10-C),
35.0, 34.6, 32.3, 28.5, 23.0, 12.8, 12.7, 12.4; ESI-MS: m/z 674.6
[MþNa]þ (calcd. 674.7).
4.2.17. 17-(2-Methylbenzylamino)-17-demethoxygeldanamycin
(18)
Purple solid (29.3 mg, 90.2%); mp 130e132 ꢂC; 1H NMR
CDCl3)
d
183.8, 180.9, 168.4, 156.1, 149.4 (40-C), 149.1 (50-C), 144.6,
(600 MHz, CDCl3) d 9.17 (s, 1H), 7.29 (s, 1H), 7.22e7.17 (m, 1H), 7.23
141.3, 135.9, 135.0, 133.7, 132.8, 129.0 (20-C), 128.8 (70-C), 126.9,
126.6, 120.4 (60-C), 111.5 (30-C), 108.9, 108.8, 81.7, 81.4, 81.2, 72.6,
57.1, 56.8, 56.0 (40-OMe and 50-OMe), 50.2 (10-C), 35.0, 34.6, 32.3,
28.5, 23.0,12.8,12.7,12.4; ESI-MS: m/z 718.5 [MþNa]þ (calcd. 718.8).
(d, J ¼ 7.5 Hz, 1H), 7.22e7.17 (m, 2H), 6.95 (d, J ¼ 11.4 Hz, 1H), 6.58 (t,
J ¼ 11.3 Hz, 1H), 6.27 (s, 1H), 5.91 (d, J ¼ 9.2 Hz, 1H), 5.86 (t,
J ¼ 10.5 Hz, 1H), 5.19 (s, 1H), 4.90 (br s, 2H), 4.74 (dd, J ¼ 14.0, 4.5 Hz,
1H), 4.54 (dd, J ¼ 14.0, 3.6 Hz, 1H), 4.31 (d, J ¼ 9.9 Hz, 1H), 4.30 (br s,
1H), 3.61e3.55 (m, 1H), 3.47e3.43 (m, 1H), 3.37 (s, 3H), 3.27 (s, 3H),
2.78e2.71 (m, 1H), 2.68 (d, J ¼ 14.1 Hz, 1H), 2.47 (t, J ¼ 12.2 Hz, 1H),
2.32 (s, 3H, 30-Me), 2.02 (s, 3H), 1.80 (s, 3H), 1.79e1.77 (m, 2H),
1.77e1.72 (m, 1H), 1.06 (d, J ¼ 5.7 Hz, 3H), 1.00 (d, J ¼ 6.4 Hz, 3H);
4.2.14. 17-(3,4,5-Trimethoxybenzylamino)-17-
demethoxygeldanamycin (15)
Purple solid (34.5 mg, 95.1%); mp 121e123 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.16 (s, 1H), 7.30 (s, 1H), 6.95 (d, J ¼ 11.6 Hz, 1H),
13C NMR (150 MHz, CDCl3)
d 183.7, 181.0, 168.4, 156.1, 144.7, 141.3,
6.58 (t, J ¼ 11.4 Hz, 1H), 6.48 (s, 2H, 30-H and 70-H), 6.40 (s, 1H), 5.90
(d, J ¼ 9.5 Hz, 1H), 5.86 (t, J ¼ 10.5 Hz, 1H), 5.19 (s, 1H), 4.88 (br s,
2H), 4.67 (dd, J ¼ 14.2, 4.5 Hz, 1H), 4.53 (dd, J ¼ 14.2, 4.4 Hz, 1H),
4.31 (d, J ¼ 9.8 Hz,1H), 4.23 (s,1H), 3.86 (s, 6H, 40-OMe and 60-OMe),
3.85 (s, 3H, 50-OMe), 3.60e3.56 (m, 1H), 3.47e3.43 (m, 1H), 3.36 (s,
3H), 3.27 (s, 3H), 2.78e2.71 (m, 1H), 2.69 (d, J ¼ 13.7 Hz, 1H), 2.46
(dd, J ¼ 13.8, 10.8 Hz, 1H), 2.02 (s, 3H), 1.80 (s, 3H), 1.79e1.77 (m,
2H), 1.77e1.73 (m, 1H), 1.03 (d, J ¼ 6.5 Hz, 3H), 0.99 (d, J ¼ 6.9 Hz,
136.3 (20-Me), 135.8, 135.0, 134.4 (30-C), 133.8, 132.8, 131.0 (40-C),
129.0 (50-C), 128.8 (60-C), 126.9, 126.7, 126.6 (70-C), 108.9, 108.8, 81.7,
81.4, 81.2, 72.6, 57.1, 56.7, 48.6 (10-C), 35.0, 34.5, 32.3, 28.4, 23.1, 19.0
(30-Me), 12.8, 12.7, 12.4; ESI-MS: m/z 672.6 [MþNa]þ (calcd. 672.8).
4.2.18. 17-(3-Methylbenzylamino)-17-demethoxygeldanamycin
(19)
Purple solid (31.0 mg, 95.4%); mp 227e229 ꢂC; 1H NMR
3H); 13C NMR (150 MHz, CDCl3)
d
183.8, 181.1, 168.4, 156.1, 153.8 (40-
(600 MHz, CDCl3)
d
9.17 (s, 1H), 7.30 (s, 1H), 7.27 (t, J ¼ 7.4 Hz, 1H, 60-
C and 60-C), 144.7, 141.2, 138.0 (50-C), 135.9, 135.0, 133.7, 132.8, 132.2
(20-C),127.0,126.6,109.0,108.8,104.9 (30-C and 70-C), 81.7, 81.4, 81.2,
72.7, 60.9 (50-OMe), 57.2, 56.8, 56.2 (40-OMe and 60-OMe), 50.7 (10-
C), 35.0, 34.6, 32.3, 28.5, 23.1, 12.8, 12.7, 12.4; ESI-MS: m/z 748.6
[MþNa]þ (calcd. 748.8).
H), 7.16 (d, J ¼ 7.5 Hz, 1H, 70-H), 7.10e7.05 (m, 2H, 30-H and 50-H),
6.95 (d, J ¼ 11.4 Hz, 1H), 6.58 (t, J ¼ 11.4 Hz, 1H), 6.46 (s, 1H), 5.90 (d,
J ¼ 9.3 Hz, 1H), 5.86 (t, J ¼ 10.5 Hz, 1H), 5.19 (s, 1H), 4.89 (br s, 2H),
4.72 (dd, J ¼ 14.3, 5.3 Hz, 1H), 4.56 (dd, J ¼ 14.3, 4.7 Hz, 1H), 4.31 (d,
J ¼ 10.0 Hz, 1H), 4.29 (br s, 1H), 3.60e3.54 (m, 1H), 3.47e3.42 (m,
1H), 3.36 (s, 3H), 3.27 (s, 3H), 2.78e2.71 (m, 1H), 2.67 (d, J ¼ 14.1 Hz,
1H), 2.51e2.44 (m, 1H), 2.36 (s, 3H, 40-Me), 2.02 (s, 3H), 1.80 (s, 3H),
1.79e1.77 (m, 2H), 1.76e1.72 (m, 1H), 1.03 (d, J ¼ 6.1 Hz, 3H), 0.99 (d,
4.2.15. 17-(4-Hydroxy-3-methoxybenzylamino)-17-
demethoxygeldanamycin (16)
Purple solid (32.0 mg, 93.8%); mp 106e108 ꢂC; 1H NMR
J ¼ 6.7 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d 183.8, 181.0, 168.4,
(600 MHz, CDCl3)
d
9.17 (s, 1H), 7.29 (s, 1H), 6.96 (d, J ¼ 11.7 Hz, 1H),
156.1, 144.7, 141.3, 139.0 (40-C), 136.5 (20-C), 135.8, 135.0, 133.8,
132.8, 129.2 (30-C), 129.1 (60-C), 128.5 (50-C), 126.9, 126.6, 124.9 (70-
C), 108.8, 81.7, 81.4, 81.2, 72.6, 57.1, 56.8, 50.2 (10-C), 35.0, 34.5, 32.3,
28.5, 23.0, 21.4 (40-Me), 12.8, 12.7, 12.4; ESI-MS: m/z 672.6 [MþNa]þ
(calcd. 672.8).
6.91 (d, J ¼ 8.0 Hz, 1H), 6.79 (dd, J ¼ 8.1, 1.7 Hz, 1H), 6.74 (d,
J ¼ 1.7 Hz, 1H), 6.58 (t, J ¼ 11.4 Hz, 1H), 6.35 (s, 1H), 5.90 (d,
J ¼ 9.5 Hz, 1H), 5.86 (t, J ¼ 10.5 Hz, 1H), 5.74 (s, 1H), 5.19 (s, 1H), 4.88
(br s, 2H), 4.67 (d, J ¼ 13.1 Hz, 1H), 4.51 (d, J ¼ 14.0 Hz, 1H), 4.31 (d,
J ¼ 9.9 Hz, 1H), 4.23 (br s, 1H), 3.89 (s, 3H), 3.60e3.56 (m, 1H),
3.47e3.43 (m, 1H), 3.37 (s, 3H), 3.27 (s, 3H), 2.78e2.72 (m, 1H), 2.69
(d, J ¼ 13.9 Hz, 1H), 2.48 (dd, J ¼ 13.9, 10.6 Hz, 1H), 2.02 (s, 3H), 1.80
(s, 3H), 1.79e1.77 (m, 2H), 1.77e1.73 (m, 1H), 1.03 (d, J ¼ 6.5 Hz, 3H),
4.2.19. 17-(4-Methylbenzylamino)-17-demethoxygeldanamycin
(20)
Purple solid (24.4 mg, 75.1%); mp 221e222 ꢂC; 1H NMR
1.00 (d, J ¼ 6.9 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d
183.8, 180.9,
(600 MHz, CDCl3)
d
9.16 (s, 1H), 7.29 (s, 1H), 7.19 (d, J ¼ 7.7 Hz, 2H),
168.4,156.1,147.0,145.8,144.7,141.3,135.9,135.0,133.7,132.8,128.4,
126.9, 126.5, 121.2, 114.9, 110.3, 108.9, 108.8, 81.7, 81.5, 81.2, 72.7,
57.1, 56.7, 56.0, 50.3, 35.1, 34.6, 32.3, 28.5, 23.0, 12.8, 12.6, 12.4; ESI-
MS: m/z 704.6 [MþNa]þ (calcd. 704.8).
7.16 (d, J ¼ 7.8 Hz, 2H), 6.95 (d, J ¼ 11.4 Hz, 1H), 6.58 (t, J ¼ 11.4 Hz,
1H), 6.44 (s, 1H), 5.90 (d, J ¼ 9.3 Hz,1H), 5.86 (t, J ¼ 10.5 Hz,1H), 5.19
(s, 1H), 4.90 (br s, 2H), 4.71 (dd, J ¼ 14.3, 5.3 Hz, 1H), 4.56 (dd,
J ¼ 14.3, 4.9 Hz, 1H), 4.31 (d, J ¼ 10.0 Hz, 1H), 4.30 (br s, 1H),
3.59e3.55 (m, 1H), 3.46e3.43 (m, 1H), 3.36 (s, 3H), 3.27 (s, 3H),
2.79e2.70 (m, 1H), 2.66 (d, J ¼ 14.0 Hz, 1H), 2.52e2.42 (m, 1H), 2.36
(s, 3H, 50-Me), 2.02 (s, 3H), 1.80 (s, 3H), 1.79e1.77 (m, 2H), 1.76e1.71
(m, 1H), 1.03 (d, J ¼ 6.3 Hz, 3H), 0.99 (d, J ¼ 6.9 Hz, 3H); 13C NMR
4.2.16. 17-(4-Hydroxybenzylamino)-17-demethoxygeldanamycin
(17)
Purple solid (28.3 mg, 86.8%); mp 114e116 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.09 (s, 1H), 7.27 (s, 1H), 7.16 (d, J ¼ 8.4 Hz, 2H),
(150 MHz, CDCl3) d 183.8, 180.9, 168.4, 156.1, 144.7, 141.3, 138.3,
6.90 (d, J ¼ 11.7 Hz, 1H), 6.87 (d, J ¼ 8.5 Hz, 2H), 6.64 (br s, 1H, 50-
OH), 6.57 (t, J ¼ 11.4 Hz, 1H), 6.39 (t, J ¼ 4.8 Hz, 1H), 5.91 (d,
J ¼ 9.3 Hz, 1H), 5.86 (t, J ¼ 10.5 Hz, 1H), 5.20 (s, 1H), 4.96 (br s, 2H),
4.65 (dd, J ¼ 14.2, 5.1 Hz, 1H), 4.52 (br s, 1H, 11-OH), 4.48 (dd,
J ¼ 14.3, 5.2 Hz, 1H), 4.31 (d, J ¼ 10.0 Hz, 1H), 3.59e3.58 (m, 1H),
3.48e3.47 (m, 1H), 3.37 (s, 3H), 3.27 (s, 3H), 2.79e2.74 (m, 1H), 2.65
(d, J ¼ 13.7 Hz, 1H), 2.53e2.45 (m, 1H), 2.02 (s, 3H), 1.81 (s, 3H),
1.80e1.78 (m, 2H), 1.75e1.69 (m, 1H), 1.02 (d, J ¼ 6.7 Hz, 3H), 1.00 (d,
135.8,135.0,133.8,133.5,132.8,129.9 (30-C and 70-C),127.8 (40-C and
60-C), 126.9, 126.6, 108.8 (16-C and 19-C), 81.7, 81.4, 81.2, 72.6, 57.1,
56.8, 50.0 (10-C), 35.0, 34.5, 32.3, 28.5, 23.0, 21.2 (50-Me), 12.8, 12.7,
12.3; ESI-MS: m/z 672.6 [MþNa]þ (calcd. 672.8).
4.2.20. 17-(3-Aminobenzylamino)-17-demethoxygeldanamycin
(21)
Purple solid (29.6 mg, 91.0%); mp 108e110 ꢂC; 1H NMR
J ¼ 7.0 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d
183.6, 180.9, 168.3,
(600 MHz, CDCl3)
d
9.17 (s, 1H), 7.30 (s, 1H), 7.15 (t, J ¼ 7.8 Hz, 1H, 60-

Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
367
H), 6.95 (d, J ¼ 11.5 Hz, 1H), 6.67e6.62 (m, 2H, 30-H and 50-H),
6.61e6.55 (m, 2H, 70-H and 4-H), 6.47 (t, J ¼ 5.2 Hz, 1H), 5.90 (d,
J ¼ 9.4 Hz, 1H), 5.86 (t, J ¼ 10.5 Hz, 1H), 5.18 (s, 1H), 4.91 (br s, 2H),
4.65 (dd, J ¼ 14.4, 5.7 Hz, 1H, 10-H), 4.50 (dd, J ¼ 14.5, 5.4 Hz, 1H, 10-
H), 4.31 (d, J ¼ 10.0 Hz, 1H), 4.30 (br s, 1H), 3.80 (br s, 2H, 40-NH2),
3.61e3.55 (m, 1H), 3.47e3.42 (m, 1H), 3.36 (s, 3H), 3.27 (s, 3H),
2.79e2.70 (m, 1H), 2.65 (d, J ¼ 13.8 Hz, 1H), 2.46 (dd, J ¼ 13.9,
10.9 Hz, 1H), 2.02 (s, 3H), 1.80 (s, 3H), 1.79e1.76 (m, 2H), 1.76e1.72
(m, 1H), 1.02 (d, J ¼ 6.6 Hz, 3H), 0.99 (d, J ¼ 7.0 Hz, 3H); 13C NMR
56.8, 49.2 (10-C), 34.9, 34.3, 32.3, 28.6, 23.0, 12.9, 12.7, 12.5; ESI-MS:
m/z 683.5 [MþNa]þ (calcd. 683.7).
4.2.24. 17-(4-Methylsulfonylbenzylamino)-17-
demethoxygeldanamycin (25)
Purple solid (32.3 mg, 90.1%); mp 105e106 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.09 (s, 1H), 7.94 (d, J ¼ 8.2 Hz, 2H), 7.46 (d,
J ¼ 8.1 Hz, 2H), 7.29 (s, 1H), 6.93 (d, J ¼ 11.4 Hz, 1H), 6.58e6.53 (m,
2H), 5.86e5.53 (m, 2H), 5.16 (s, 1H), 5.07 (br s, 2H), 4.82 (dd,
J ¼ 15.5, 6.1 Hz,1H), 4.77 (dd, J ¼ 15.5, 6.0 Hz,1H), 4.29 (d, J ¼ 9.9 Hz,
1H), 3.99 (br s, 1H), 3.55e3.53 (m, 1H), 3.41e3.40 (m, 1H), 3.33 (s,
3H), 3.25 (s, 3H), 3.06 (s, 3H, 50-SO2Me), 2.76e2.68 (m, 1H), 2.59 (d,
J ¼ 14.1 Hz, 1H), 2.30e2.23 (m, 1H), 2.00 (s, 3H), 1.77 (s, 3H),
1.74e1.69 (m, 3H), 0.98 (d, J ¼ 5.8 Hz, 3H), 0.96 (d, J ¼ 7.0 Hz, 3H);
(150 MHz, CDCl3)
d
183.8, 180.9, 168.4, 156.1, 147.2 (40-C), 144.7,
141.3, 137.8 (20-C), 135.8, 135.0, 133.8, 132.8, 130.2 (60-C), 126.9,
126.6,117.7 (70-C),115.0 (30-C),113.9 (50-C),108.83,108.78, 81.7, 81.4,
81.2, 72.6, 57.2, 56.8, 50.1 (10-C), 35.0, 34.5, 32.3, 28.5, 23.0, 12.8,
12.7, 12.4; ESI-MS: m/z 673.7 [MþNa]þ (calcd. 673.8).
13C NMR (150 MHz, CDCl3)
d 183.8, 181.4, 168.3, 156.4, 144.4, 143.3
(20-C), 141.0, 140.4 (50-C), 136.1, 134.8, 133.5, 132.9, 128.3 (30-C, 40-C,
60-C and 70C), 127.1, 126.5, 109.8, 109.0, 81.6, 81.3, 81.2, 72.6, 57.1,
56.8, 49.0 (10-C), 44.5 (50-SO2Me), 34.9, 34.3, 32.3, 28.5, 23.0, 12.9,
12.6, 12.4; ESI-MS: m/z 736.5 [MþNa]þ (calcd. 736.8).
4.2.21. 17-(4-Aminobenzylamino)-17-demethoxygeldanamycin
(22)
Purple solid (29.0 mg, 89.1%); mp 104e107 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.18 (s, 1H), 7.28 (s, 1H), 7.06 (d, J ¼ 8.3 Hz, 2H),
6.95 (d, J ¼ 11.5 Hz, 1H), 6.68 (d, J ¼ 8.3 Hz, 2H), 6.58 (t, J ¼ 11.4 Hz,
1H), 6.35 (s, 1H), 5.91 (d, J ¼ 9.4 Hz,1H), 5.86 (t, J ¼ 10.5 Hz, 1H), 5.18
(s, 1H), 4.90 (br s, 2H), 4.64 (dd, J ¼ 14.1, 5.4 Hz, 1H), 4.45 (dd,
J ¼ 14.1, 5.1 Hz, 1H), 4.37 (br s, 1H, 11-OH), 4.31 (d, J ¼ 10.4 Hz, 1H),
3.79 (br s, 2H, 50-NH2), 3.59e3.56 (m, 1H), 3.46e3.44 (m, 1H), 3.36
(s, 3H), 3.26 (s, 3H), 2.78e2.71 (m, 1H), 2.68 (d, J ¼ 13.8 Hz,1H), 2.50
(dd, J ¼ 13.8,11.1 Hz,1H), 2.02 (s, 3H),1.80 (s, 3H),1.79e1.77 (m, 2H),
1.76e1.72 (m, 1H), 1.03 (d, J ¼ 6.6 Hz, 3H), 0.99 (d, J ¼ 6.9 Hz, 3H);
4.2.25. 17-(4-Sulfamoylbenzylamino)-17-demethoxygeldanamycin
(26)
Purple solid (28.7 mg, 80.3%); mp 168e170 ꢂC; 1H NMR
(600 MHz, DMSO)
d
9.31 (s, 1H), 7.80 (d, J ¼ 7.7 Hz, 2H), 7.68 (s, 1H),
7.43 (d, J ¼ 7.8 Hz, 2H), 7.02 (s, 1H), 6.96 (s, 1H), 6.76 (s, 1H), 6.60 (s,
1H), 6.35 (s, 1H), 5.53 (s, 1H), 4.95 (s, 1H), 4.77 (s, 2H), 4.38 (s, 1H),
4.12 (s,1H), 3.35 (s, 5H), 3.20 (s, 6H), 2.58 (s,1H), 2.29 (s,1H), 2.10 (d,
J ¼ 7.1 Hz, 1H), 1.93 (s, 3H), 1.89 (s, 1H), 1.63 (s, 3H), 1.47 (s, 1H),
1.44e1.34 (m, 1H), 0.88 (d, J ¼ 5.3 Hz, 3H), 0.80 (d, J ¼ 4.7 Hz, 3H);
ESI-MS: m/z 732.7 [MþNH4]þ (calcd. 732.9).
13C NMR (150 MHz, CDCl3)
d
183.8, 180.8, 168.4, 156.1, 146.7 (50-C),
144.7, 141.3, 135.8, 135.0, 133.8, 132.8, 129.3 (30-C and 70-C), 126.9,
126.6, 126.1 (20-C), 115.5 (40-C and 60-C), 108.8, 108.6, 81.7, 81.5, 81.2,
72.6, 57.1, 56.8, 50.1 (10-C), 35.0, 34.6, 32.3, 28.5, 23.0, 12.8, 12.7,
12.3; ESI-MS: m/z 673.6 [MþNa]þ (calcd. 673.8).
4.2.26. 17-(Methyl 4-aminomethylbenzoate)-17-
demethoxygeldanamycin (27)
Purple solid (20.8 mg, 59.7%); mp 130e132 ꢂC; 1H NMR
4.2.22. 17-(3-Cyanobenzylamino)-17-demethoxygeldanamycin
(23)
(600 MHz, CDCl3)
d
9.12 (s, 1H), 8.10e8.01 (m, 2H, 40-H and 60-H),
Purple solid (21.6 mg, 65.4%); mp 222e224 ꢂC; 1H NMR
7.37e7.32 (m, 2H, 30-H and 70-H), 7.31 (s,1H), 6.94 (d, J ¼ 9.7 Hz,1H),
6.57 (t, J ¼ 10.5 Hz, 1H), 6.52 (s, 1H), 5.92e5.80 (m, 2H), 5.17 (s, 1H),
4.94 (br s, 2H), 4.80 (d, J ¼ 15.1 Hz, 1H, 10-H), 4.71 (d, J ¼ 15.0 Hz, 1H,
10-H), 4.30 (d, J ¼ 6.6 Hz, 1H), 4.10 (br s, 1H), 3.92 (s, 3H, 50-COOMe),
3.59e3.52 (m, 1H), 3.45e3.40 (m, 1H), 3.35 (s, 3H), 3.26 (s, 3H),
2.79e2.68 (m, 1H), 2.61 (d, J ¼ 13.7 Hz, 1H), 2.38e2.30 (m, 1H), 2.02
(s, 3H), 1.79 (s, 3H), 1.76e1.67 (m, 3H), 1.02e0.95 (m, 6H); 13C NMR
(600 MHz, CDCl3)
d
9.11 (s, 1H), 7.65 (d, J ¼ 5.9 Hz, 1H, 50-H), 7.58 (s,
1H, 30-H), 7.54e7.51 (m, 2H, 60-H and 70-H), 7.33 (s, 1H), 6.95 (d,
J ¼ 11.4 Hz, 1H), 6.58 (t, J ¼ 11.3 Hz, 1H), 6.44 (s, 1H), 5.90e5.84 (m,
2H), 5.19 (s, 1H), 4.88 (br s, 2H), 4.78 (d, J ¼ 15.0 Hz, 1H), 4.70 (d,
J ¼ 15.1 Hz, 1H), 4.31 (d, J ¼ 9.8 Hz, 1H), 3.58e3.54 (m, 1H),
3.45e3.41 (m, 1H), 3.36 (s, 3H), 3.27 (s, 3H), 2.77e2.71 (m, 1H), 2.63
(d, J ¼ 14.0 Hz, 1H), 2.33e2.25 (m, 1H), 2.02 (s, 3H), 1.79 (s, 3H),
1.76e1.71 (m, 3H), 0.98 (d, J ¼ 5.6 Hz, 6H); 13C NMR (150 MHz,
(150 MHz, CDCl3)
d
183.8, 181.3, 168.4, 166.5 (50-COOMe), 156.2,
144.6, 141.7, 141.1 (20-C), 135.9, 134.9, 133.6 (40-C), 132.9 (60-C), 130.5
(30-C), 130.2 (70-C), 127.5 (50-C), 127.0, 126.6, 109.4, 108.9, 81.6, 81.3,
81.2, 72.6, 57.1, 56.7, 52.3 (50-COOMe), 49.5 (10-C), 34.9, 34.4, 32.3,
28.5, 23.0, 12.8, 12.6, 12.4; ESI-MS: m/z 716.6 [MþNa]þ (calcd.
716.8).
CDCl3)
d
183.7, 181.5, 168.4, 156.1, 144.4, 141.0, 138.4 (20-C), 136.0,
134.9,133.5, 132.9, 132.1 (70-C), 131.8 (30-C), 131.1 (50-C), 130.1 (60-C),
127.1, 126.5, 118.2 (40-CN), 113.4 (40-C), 109.7, 109.0, 81.6, 81.3, 81.2,
72.6, 57.2, 56.8, 49.0 (10-C), 34.9, 34.4, 32.3, 28.5, 23.0, 12.9, 12.7,
12.4; ESI-MS: m/z 678.6 [MþNH4]þ (calcd. 678.8).
4.2.27. 17-(Ethyl 4-aminomethylbenzoate)-17-
demethoxygeldanamycin (28)
4.2.23. 17-(4-Cyanobenzylamino)-17-demethoxygeldanamycin
(24)
Purple solid (25.1 mg, 70.6%); mp 242e243 ꢂC; 1H NMR
Purple solid (30.8 mg, 93.2%); mp 119e121 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.12 (s, 1H), 8.06 (d, J ¼ 7.7 Hz, 2H), 7.34 (d,
(600 MHz, CDCl3)
d
9.10 (s, 1H), 7.70 (d, J ¼ 8.2 Hz, 2H), 7.39 (d,
J ¼ 7.6 Hz, 2H), 7.31 (s, 1H), 6.94 (d, J ¼ 11.4 Hz, 1H), 6.58 (t,
J ¼ 11.3 Hz, 1H), 6.52 (s, 1H), 5.93e5.81 (m, 2H), 4.87 (d, J ¼ 58.6 Hz,
2H), 4.80 (d, J ¼ 15.0 Hz, 1H), 4.71 (d, J ¼ 15.2 Hz, 1H), 4.38 (q,
J ¼ 6.9 Hz, 2H), 4.30 (d, J ¼ 9.7 Hz, 1H), 3.56 (d, J ¼ 8.7 Hz, 1H), 3.42
(d, J ¼ 7.9 Hz, 1H), 3.35 (s, 3H), 3.26 (s, 3H), 2.78e2.69 (m, 1H), 2.61
(d, J ¼ 14.2 Hz, 1H), 2.34 (t, J ¼ 12.0 Hz, 1H), 2.02 (s, 3H), 1.79 (s, 3H),
1.74 (s, 3H), 1.39 (t, J ¼ 7.0 Hz, 3H), 1.03e0.95 (m, 6H); 13C NMR
J ¼ 8.1 Hz, 2H), 7.33 (s, 1H), 6.95 (d, J ¼ 11.4 Hz, 1H), 6.58 (t,
J ¼ 11.4 Hz, 1H), 6.49 (t, J ¼ 5.7 Hz, 1H), 5.89e5.84 (m, 2H), 5.19 (s,
1H), 4.90 (br s, 2H), 4.80 (dd, J ¼ 15.6, 6.0 Hz, 1H), 4.74 (dd, J ¼ 15.6,
5.7 Hz, 1H), 4.31 (d, J ¼ 9.9 Hz, 1H), 3.57e3.54 (m, 1H), 3.44e3.41
(m, 1H), 3.35 (s, 3H), 3.27 (s, 3H), 2.78e2.69 (m, 1H), 2.61 (d,
J ¼ 14.0 Hz, 1H), 2.26 (dd, J ¼ 13.7, 10.3 Hz, 1H), 2.02 (s, 3H),
1.74e1.71 (m, 3H), 1.00e0.96 (m, 6H); 13C NMR (150 MHz, CDCl3)
(150 MHz, CDCl3) d
183.8, 181.3, 168.4, 166.1 (50-COOCH2CH3), 156.2,
d
183.7, 181.5, 168.4, 156.1, 144.4, 142.2 (20-C), 141.0, 136.1, 134.9,
144.6, 141.6, 141.1 (20-C), 136.0, 134.9, 133.6, 132.9, 130.4 (40-C and
60-C), 127.4 (30-C and 70-C), 127.0, 126.6, 109.4, 108.9, 81.6, 81.3, 81.2,
72.6, 61.2 (50-COOCH2CH3), 57.2, 56.8, 49.5 (10-C), 34.9, 34.4, 32.3,
133.5, 133.0 (40-C and 60-C), 132.9, 128.1 (30-C and 70-C), 127.1, 126.5,
118.3 (50-CN), 112.3 (50-C), 109.8, 109.0, 81.6, 81.3, 81.2, 72.6, 57.2,

368
Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
28.5, 23.0, 14.3 (50-COOCH2CH3), 12.8, 12.7, 12.4; ESI-MS: m/z 730.7
[MþNa]þ (calcd. 730.8).
156.2, 144.2, 140.9, 136.0, 134.9, 133.5, 132.9, 127.1, 126.5, 110.1,
109.0, 81.6, 81.3, 81.1, 72.7, 60.4, 57.2, 56.8, 40.4, 35.0, 34.6, 32.3,
28.7, 23.1, 12.9, 12.6, 12.5; ESI-MS: m/z 662.5 [MþNa]þ (calcd.
662.7).
4.2.28. 17-(4-Trifluoromethoxybenzylamino)-17-
demethoxygeldanamycin (29)
Purple solid (33.0 mg, 91.3%); mp 210e211 ꢂC; 1H NMR
4.2.32. 17-(4-Chloro-3-pyridinmethanamine)-17-
demethoxygeldanamycin (33)
(600 MHz, CDCl3)
d
9.13 (s, 1H), 7.31 (s, J ¼ 8.6 Hz, 2H), 7.30 (s, 1H),
7.23 (d, J ¼ 8.1 Hz, 2H), 6.94 (d, J ¼ 11.5 Hz, 1H), 6.57 (t, J ¼ 11.4 Hz,
1H), 6.43 (s, 1H), 5.88e5.84 (m, 2H), 5.18 (s, 1H), 5.00 (br s, 2H), 4.75
(dd, J ¼ 14.7, 5.3 Hz, 1H), 4.63 (dd, J ¼ 14.7, 4.9 Hz, 1H), 4.30 (d,
J ¼ 9.9 Hz, 1H), 4.10 (s, 1H), 3.57e3.55 (m, 1H), 3.44e3.42 (m, 1H),
3.35 (s, 3H), 3.26 (s, 3H), 2.77e2.70 (m, 1H), 2.64 (d, J ¼ 14.0 Hz, 1H),
2.42e2.34 (m, 1H), 2.01 (s, 3H), 1.78 (s, 3H), 1.76e1.71 (m, 3H),
Purple solid (25.1 mg, 74.5%); mp 211e213 ꢂC; 1H NMR
(600 MHz, CDCl3) d
9.09 (s, 1H), 8.34 (s, 1H, 30-H), 7.58 (s, 1H, 60-H),
7.37 (s, 1H, 50-H), 7.30 (s, 1H), 6.94 (d, J ¼ 9.3 Hz, 1H), 6.57 (t,
J ¼ 10.6 Hz, 1H), 6.34 (s, 1H), 5.86 (s, 2H), 5.17 (s, 1H), 4.94 (br s, 2H),
4.74 (d, J ¼ 14.9 Hz, 1H), 4.67 (d, J ¼ 14.7 Hz, 1H), 4.30 (d, J ¼ 5.7 Hz,
1H), 3.95 (br s, 1H), 3.57e3.53 (m, 1H), 3.45e3.40 (m, 1H), 3.35 (s,
3H), 3.26 (s, 3H), 2.77e2.71 (m, 1H), 2.65 (d, J ¼ 13.7 Hz, 1H),
2.34e2.28 (m, 1H), 2.01 (s, 3H), 1.78 (s, 3H), 1.77e1.69 (m, 1H),
1.02e0.96 (m, 6H); 13C NMR (150 MHz, CDCl3)
d 183.7, 181.2, 168.4,
156.2, 149.1 (50-C), 144.5, 141.1, 136.0 (20-C), 135.4, 134.9, 133.6,
132.9, 129.2 (30-C and 70-C), 127.0, 126.6, 121.7 (40-C and 60-C), 109.3,
108.9, 81.6, 81.4, 81.2, 72.6, 57.1, 56.7, 49.2 (10-C), 34.9, 34.4, 32.3,
28.5, 23.0, 12.8, 12.6, 12.4; ESI-MS: m/z 742.5 [MþNa]þ (calcd.
742.7).
1.01e0.96 (m, 6H); 13C NMR (150 MHz, CDCl3)
d 183.7, 181.5, 168.3,
156.2, 151.6 (30-C), 149.0 (40-C), 144.3, 140.9, 138.1 (60-C), 136.0,
134.9, 133.5, 133.0, 131.5 (20-C), 127.1, 126.5, 124.8 (50-C), 109.9,
109.0, 81.6, 81.3, 81.1, 72.6, 67.1, 57.2, 56.8, 46.6 (10-C), 34.9, 34.4,
32.3, 28.6, 23.0, 12.9, 12.6, 12.4; ESI-MS: m/z 693.6 [MþNa]þ (calcd.
693.3).
4.2.29. 17-(3-Nitrobenzylamino)-17-demethoxygeldanamycin (30)
Purple solid (25.5 mg, 74.9%); mp 252e253 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.10 (s, 1H), 8.22 (d, J ¼ 7.7 Hz, 1H), 8.16 (s, 1H),
7.63e7.58 (m, 2H), 7.32 (s, 1H), 6.95 (d, J ¼ 11.5 Hz, 1H), 6.58 (t,
J ¼ 11.4 Hz, 1H), 6.48 (t, J ¼ 5.9 Hz, 1H), 5.88e5.85 (m, 2H), 5.18 (s,
1H), 4.86 (br s, 2H), 4.85 (dd, J ¼ 15.2, 6.1 Hz, 1H), 4.79 (dd, J ¼ 15.2,
5.8 Hz, 1H), 4.31 (d, J ¼ 9.9 Hz, 1H), 3.96 (br s, 1H), 3.56e3.55 (m,
1H), 3.43e3.42 (m, 1H), 3.35 (s, 3H), 3.27 (s, 3H), 2.76e2.71 (m, 1H),
2.63 (d, J ¼ 14.1 Hz, 1H), 2.30 (dd, J ¼ 13.7, 10.3 Hz, 1H), 2.02 (s, 3H),
1.79 (s, 3H), 1.76e1.72 (m, 3H), 1.00 (d, J ¼ 6.0 Hz, 3H), 0.98 (d,
4.2.33. 17-((6-(Trifluoromethyl)pyridin-3-yl)methylamino)-17-
demethoxygeldanamy-cin (34)
Purple solid (25.0 mg, 78.2%); mp 133e135 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.08 (s, 1H), 8.68 (s, 1H), 7.80 (d, J ¼ 7.8 Hz, 1H),
7.73 (d, J ¼ 7.9 Hz, 1H), 7.32 (s, 1H), 6.94 (d, J ¼ 11.1 Hz, 1H), 6.57 (t,
J ¼ 11.2 Hz, 1H), 6.42 (s, 1H), 5.92e5.81 (m, 2H), 5.18 (s, 1H),
5.07e4.63 (m, 2H), 4.30 (d, J ¼ 9.2 Hz, 1H), 3.59e3.53 (m, 1H),
3.45e3.39 (m, 1H), 3.35 (s, 3H), 3.27 (s, 3H), 2.78e2.69 (m, 1H), 2.64
(d, J ¼ 14.0 Hz, 1H), 2.27 (t, J ¼ 11.2 Hz, 1H), 2.02 (s, 3H), 1.79 (s, 3H),
1.76e1.71 (m, 3H), 1.03e0.95 (m, 6H); 13C NMR (150 MHz, CDCl3)
J ¼ 7.0 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d 183.7, 181.5, 168.3,
156.1, 148.6 (40-C), 144.4,141.0, 139.0 (20-C), 136.0,134.9,133.5,133.4
(70-C), 132.9, 130.3 (60-C), 127.1, 126.5, 123.4 (30-C), 122.6 (50-C),
109.9, 109.0, 81.6, 81.3, 81.2, 72.6, 57.2, 56.8, 49.0 (10-C), 34.9, 34.4,
32.3, 28.6, 23.1, 12.9, 12.6, 12.4; ESI-MS: m/z 703.5 [MþNa]þ (calcd.
703.7).
d
183.7, 181.6, 168.3, 156.1, 149.2, 148.3, 148.1, 144.3, 140.8, 136.5,
136.1, 136.0, 134.9, 133.4, 133.0, 130.9, 128.8, 127.1, 126.5, 122.2,
120.8, 110.2, 120.4, 109.1, 81.6, 81.2, 81.1, 72.6, 57.2, 56.8, 46.8, 34.9,
34.4, 32.3, 28.6, 23.1, 12.9, 12.6, 12.5; ESI-MS: m/z 727.5 [MþNa]þ
(calcd. 727.7).
4.2.30. 17-(Thiophen-3-ylmethanamine)-17-
demethoxygeldanamycin (31)
Purple solid (30.0 mg, 93.5%); mp 130e132 ꢂC; 1H NMR
(600 MHz, CDCl3)
d
9.15 (s, 1H), 7.37 (d, J ¼ 2.8 Hz, 1H, 40-H), 7.30 (s,
4.3. In vitro antitumor activity assays
1H), 7.22 (s, 1H, 30-H), 7.02 (s,1H, 50-H), 6.95 (d, J ¼ 11.4 Hz,1H), 6.58
(t, J ¼ 11.2 Hz, 1H), 6.45 (s, 1H), 5.92e5.82 (m, 2H), 5.18 (s, 1H), 4.90
(br s, 2H), 4.76 (d, J ¼ 14.6 Hz, 1H), 4.65 (d, J ¼ 14.6 Hz, 1H), 4.31 (d,
J ¼ 9.9 Hz, 1H), 3.59e3.55 (m, 1H), 3.46e3.42 (m, 1H), 3.36 (s, 3H),
3.26 (s, 3H), 2.78e2.70 (m, 1H), 2.67 (d, J ¼ 14.1 Hz, 1H), 2.43 (t,
J ¼ 12.2 Hz, 1H), 2.02 (s, 3H), 1.79 (s, 3H), 1.78e1.76 (m, 2H),
1.75e1.71 (m, 1H), 1.03e0.97 (m, 6H); 13C NMR (150 MHz, CDCl3)
The cytotoxicity was measured by the MTT assay as described
in the literature [29,30]. Briefly, MDA-MB-231 cells were seeded in
96-well plates at the density of 3 ꢀ 103 cells/well/100
mL. After
24 h of cultivation, the cells were treated in triplicate with various
concentrations of compounds for 72 h in 5% CO2 incubator at
37 ꢂC. Cell viability was measured using MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diph-enyltetrazolium bromide] assay.
d
183.8, 181.0, 168.4, 156.1, 144.5, 141.2, 137.2 (20-C), 135.9, 135.0,
133.7, 132.8, 127.4 (50-C), 126.9, 126.7 (40-C), 126.6, 123.3 (30-C),
109.0, 108.9, 81.7, 81.4, 81.2, 72.6, 57.1, 56.8, 45.3 (10-C), 35.0, 34.5,
32.3, 28.5, 23.0, 12.8, 12.7, 12.4; ESI-MS: m/z 664.5 [MþNa]þ (calcd.
664.8).
In short, 20
The plate was incubated for an additional 4 h, and then the me-
dium was removed. Then, 200 L DMSO was added to each well to
mL of MTT (5 mg/mL) solution were added to each well.
m
dissolve any purple formazan crystals formed. The plates were
vigorously shaken before taking measurement of relative color
intensity. The absorbance of each well was measured by a
microplate reader (Bio-Rad680, USA) at a test wavelength of
570 nm. The cell inhibitory rate was calculated with the following
equation:
4.2.31. 17-((1-Methyl-1H-imidazol-5-yl)methanamine)-17-
demethoxygeldanamycin (32)
Purple solid (25.0 mg, 78.2%); mp 133e135 ꢂC; 1H NMR
(600 MHz, CDCl3) d
9.10 (s, 1H), 7.58 (s, 1H, imidazole-40-H), 7.28 (s,
1H), 7.08 (s, 1H, imidazole-60-H), 6.94 (d, J ¼ 11.1 Hz, 1H), 6.57 (t,
J ¼ 11.3 Hz, 1H), 6.02 (s, 1H), 5.90e5.83 (m, 2H), 5.18 (s, 1H), 4.95 (br
s, 2H), 4.82e4.72 (m, 1H), 4.65e4.53 (m, 1H), 4.30 (d, J ¼ 9.6 Hz,1H),
3.61 (s, 3H, imidazole-30-Me), 3.59e3.55 (m, 1H), 3.47e3.42 (m,
1H), 3.36 (s, 3H), 3.26 (s, 3H), 2.79e2.70 (m, 2H), 2.45 (t, J ¼ 11.9 Hz,
1H), 2.01 (s, 3H), 1.79 (br s, 5H), 1.76e1.70 (m, 1H), 1.04 (s, 3H), 0.99
OD control well ꢁ OD treated well
Inhibition rate ¼
ꢀ 100%
OD control well ꢁ OD blank well
The cytotoxicities of compounds were expressed as IC50 that
was defined as the drug concentration required inhibiting growth
by 50% relative to controls.
(d, J ¼ 6.0 Hz, 3H); 13C NMR (150 MHz, CDCl3)
d 183.7, 181.4, 168.4,

Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
369
4.4. In vivo toxicity assays
leupeptin, 1 mM phenylmethylsulfonyl fluoride). The lysate was
mixed with equal volume of 2ꢀ loading buffer (4% SDS, 10% 2-
mercaptoethanol, 20% glycerol, and 0.2 mg/mL bromophenol blue
in 0.1 M TriseHCl, pH 6.8), and boiled for 10 min immediately. The
boiled lysates were subjected to 8e12% SDS-PAGE, and then
transferred to PVDF membranes (Millipore), which was then
blocked with 5% BSA in phosphate-buffered saline with 0.1%
Tween-20 for 1 h, and incubated overnight with the corresponding
primary antibodies in the blocking solution at 4 ꢂC. Primary anti-
Eighteen male Kunming mice (weighing 18e22 g) were ob-
tained from the Experimental Animal Center of Shandong Univer-
sity (Jinan, China). The experiments were conducted according to
the Guide for the Care and Use of Laboratory Animals and approved
by the Animal Care and Use Committee of Shandong University. All
efforts were made to minimize the number of animals used and
their suffering.
The mice were group-housed in stainless steel cages (9 mice per
cage) in an air-conditioned room with temperature maintained at
bodies were detected using either
ImmunoPure goat anti-rabbit IgG (H
a
Peroxidase-conjugated
L) or Peroxidase-
þ
20
2
ꢂC and 12/12 h light/dark cycle for one week before the
conjugated ImmunoPure Goat Anti-Mouse IgG (H þ L) secondary
experiment. The mice were allowed standard mice chow diet and
drinking water ad libitum throughout the study. The doses for the
compounds were selected on the basis of previously published
study [31]. Totally eighteen mice were randomly divided into six
groups of three mice each and were treated as the following details:
Group I vehicle control mice; Group IIeVI treated with GA, 17-AAG,
22, 33 and 34, respectively (10 mg/kg).
All the Kunming mice were administered intravenously via tail
veins and free accessed to standard mice chow diet and drinking
water. Every day injected one time, continuously for three days.
After that, blood samples were collected for measuring liver en-
zymes. The blood serum samples were obtained from centrifugal-
ization under 8000 rpm for 4 min.
To assess damage to the hepatic parenchyma, levels of serum
ALT and AST were determined using commercial available ALT
Assay Kit and AST Assay Kit (Shanghai Rongsheng Biological Phar-
maceutical Co., Ltd., Shanghai, China) based on a spectrophoto-
metric method, respectively. The blood serum samples were
treated according to the manufacturer's instruction. The ALT and
AST activity were expressed as U/L. All data were expressed as
mean standard deviation (SD).
antibody and enhanced chemiluminescence.
4.7. In vivo antitumor assays
Five-week-old female athymic nude mice (BALB/c-nu) were ob-
tained from Slac Laboratory Animal (Shanghai, China). MDA-MB-
231 cells (5 ꢀ106 cells per animal) were injected into the right flank
of three mice to generate orthotopic xenografts. The tumors were
cut into fragments and the fragments were implanted into the right
flank of nude mice. Two weeks after injection, tumors were palpable
(about 100 mm3) and mice were randomized into treatment and
control groups (6 mice per group). The drugs were injected via
caudal vein every three days at a dose of 10 mg/kg body weight,
whereas the blank control group received an equal volume of 5%
glucose injection containing 1% DMSO and 2% lecithin. 17-AAG was
administrated as a positive control drug. During treatment, subcu-
taneous tumors were measured with a vernier caliper every three
days, and body weight was monitored regularly. Tumor volume was
calculated by the formula (V ¼ ab2/2, where a and b stand for the
longest and shortest diameter, respectively). After treated for 15
days with drugs, the animals were sacrificed and solid tumors were
removed and weighted. The inhibition rate was calculated as
[(averaged tumor weight of the control group ꢁ averaged tumor
weight of drug-treated group)/averaged tumor weight of the control
group] ꢀ 100%. Data were analyzed by Student's two-tailed t test. A P
level <0.05 was considered statistically significant.
4.5. Fluorescence polarization assay (FPA)
FPA was used to evaluate for Hsp90 inhibitors based on
displacement of a fluorescent compound GA-FITC that binds spe-
cifically to the Hsp90 ATP-binding site and was carried out as
described. Briefly, reaction mixes (100
mL) containing 20 mM HEPES
4.8. Molecular modeling
(pH7.3), 50 mM KCl, 2 mM DTT, 5 mM MgCl2, 20 mM Na2MoO4,
0.01% Triton X-100 with 0.1 mg/mL BSA, 50 nM of recombinant
Hsp90
Compound 34 was ultimately converted to the PDBQT format
using AutoDock Tools [http://mgltools.scripps.edu], which is
required for AutoDock Vina [28]. The 3-dimensional (3D) structure
of Hsp90 was downloaded from the Protein Data Bank (PDB ID:
1YET). The molecular docking was conducted using AutoDock Vina,
which uses a unique algorithm that implements a machine learning
approach to its scoring function. Using AutoDock Tools, the PDB
(1YET) structure was converted from a pdb file to a pdbqt file and
the search grid was identified as center_x: 40.695,
center_y: ꢁ46.782 and center_z: 65.693 with dimensions size_x:
16, size_y: 16, and size_z: 16. Then the compound 34 was docked
into the GA binding site of Hsp90. For Vina docking, the default
parameters were used if it was not mentioned. Then a MD study
was performed to revise the docking result.
The Amber 12 [32e34] and AmberTools 13 programs were used
for MD simulations of the selected docked pose. Compound 34 was
first prepared by ACPYPE [35], a tool based on ANTECHAMBER
[36,37] for generating automatic topologies and parameters in
different formats for different molecular mechanics programs,
including calculation of partial charges. Then, the forcefield “leap-
rc.gaff” (generalized amber forcefield) was used to prepare the
ligand, while “leaprc.ff12SB” was used for the receptor. The system
was placed in a rectangular box (with a 10.0 Å boundary) of TIP3P
water using the “SolvateOct” command with the minimum
a 5 nM of GA-FITC
protein (ab80369, abcam®, USA),
(ab141589, abcam®, USA), and varying concentrations of tested
compounds or GA were added in a low binding black 96-well plates
(Corning Costar #3650). After incubation for 5 h, plates were read
on Omega POLARstar Multi-Mode Microplate Reader (BMG Lab-
tech, Germany) with excitation at 485/20 nm and emission at
520 nm. Polarization values (mP) were used to calculate competi-
tive effects, which were represented as percentage of control (%
control)
and
calculated
following
the
formula:
%
control ¼ 100 ꢀ (mPc ꢁ mPf)/(mPb ꢁ mPf), where mPc, mPb, and
mPf are recorded mP values from wells containing tested com-
pounds, control wells containing both GA-FITC and Hsp90, and
wells containing only the fluorescent probe GA-FITC, respectively.
IC50 values were determined as the competitor concentrations at
which 50% of bound GA-FITC was displaced.
4.6. Western blot assay
MDA-MB 231 cells were treated with or without compounds for
different time courses, and then harvested and lysed in ice-cold
lysis buffer (20 mM TriseHCl, pH 7.4, 150 mM NaCl, 1 mM EDTA,
1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
b-
glycerolphosphate, mM sodium orthovanadate, mg/mL
1
1

370
Z. Li et al. / European Journal of Medicinal Chemistry 85 (2014) 359e370
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distance between any solute atoms. Equilibration of the solvated
complex was done by carrying out a short minimization (500 steps
of each steepest descent and conjugate gradient method), 500 ps of
heating, and 50 ps of density equilibration with weak restraints
using the GPU (NVIDIA® Tesla K20c) accelerated PMEMD (Particle
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This work was supported by a grant from the 973 Program
(2010CB833802), NSFC Projects (81373304, 91313303, 81302214),
the Fundamental Research Funds of Shandong University to C.H.
Lu (2014JC027), the Post-graduate Independent Innovation Fund
of Shandong University to Z.Y. Li (YZC12095), Shanghai Nature
Science Foundation of Shanghai Science and Technology Commit-
tee, China (13ZR1432700) and Program for Changjiang Scholars and
Innovative Research Team in University (IRT13028).
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