Discovery of potent N-(isoxazol-5-yl)amides as HSP90 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.09.065

HSP90 is ubiquitously overexpressed in a broad spectrum of human cancers and has been recognized as an attractive target for cancer treatment. Here, we described the fragment screening, synthesis and structure-activity relationship studies of small molecule inhibitors with 4,5-diarylisoxazole scaffold targeting HSP90. Among them, the compound N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-morpholinopiperidin-1-yl)methyl)phenyl)isoxazol-5-yl)cyclopropanecarboxamide (108) showed high affinity for binding to HSP90 (FP binding assay, IC₅₀ Combining double low line 0.030 μM) and inhibited the proliferation of various human cancer cell lines with averaging GI₅₀ about 88 nM. Compound 108 exhibited its functional inhibition of HSP90 by depleting key signaling pathways and concomitantly elevating of HSP70 and HSP27 in U-87MG cells. Further in vivo studies showed that compound 108 strongly suppressed the tumor growth of human glioblastoma xenograft model U-87MG with T/C Combining double low line 18.35% at 50 mg/kg q3w/2.5w. Moreover, compound 108 also exhibited good pharmacokinetic properties. Together, our study implicates that compound 108 is a promising candidate of HSP90 inhibitor and is currently advanced to preclinical study.

Full text of DOI:10.1016/j.ejmech.2014.09.065

Accepted Manuscript
Discovery of potent N-(isoxazol-5-yl)amides as HSP90 inhibitors
Danqi Chen, Aijun Shen, Jian Li, Feng Shi, Wuyan Chen, Jing Ren, Hongchun Liu,
Yechun Xu, Xin Wang, Xinying Yang, Yiming Sun, Min Yang, Jianhua He, Yueqin
Wang, Liping Zhang, Min Huang, Meiyu Geng, Bing Xiong, Jingkang Shen
PII:
S0223-5234(14)00884-8
10.1016/j.ejmech.2014.09.065
EJMECH 7372
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 August 2014
Revised Date: 19 September 2014
Accepted Date: 19 September 2014
Please cite this article as: D. Chen, A. Shen, J. Li, F. Shi, W. Chen, J. Ren, H. Liu, Y. Xu, X. Wang, X.
Yang, Y. Sun, M. Yang, J. He, Y. Wang, L. Zhang, M. Huang, M. Geng, B. Xiong, J. Shen, Discovery of
potent N-(isoxazol-5-yl)amides as HSP90 inhibitors, European Journal of Medicinal Chemistry (2014),
doi: 10.1016/j.ejmech.2014.09.065.
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ACCEPTED MANUSCRIPT
By fragment screening and x-ray crystallography techniques a scaffold of 4,5-diarylisoxazole was
selected and developed to a novel series of HSP90 inhibitors with excellent in vivo activities.

ACCEPTED MANUSCRIPT
Discovery of Potent N-(isoxazol-5-yl)amides as HSP90 Inhibitors
1#
2#
3#
1
4
1
2
Danqi Chen , Aijun Shen , Jian Li , Feng Shi , Wuyan Chen , Jing Ren , Hongchun Liu ,
4
*
1
2
2
3
3
2
Yechun Xu , Xin Wang , Xinying Yang , Yiming Sun , Min Yang , Jianhua He , Yueqin Wang ,
5
2
2*
1*
1*
Liping Zhang , Min Huang , Meiyu Geng , Bing Xiong , Jingkang Shen
1
Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
China
2
Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai
2
01203, China
3
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, 239 Zhangheng Road,
Shanghai 201204, China
4
Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
5
Department of Pharmacology and Glycobiology, School of Medicine and Pharmacy, Ocean
University of China, 5 Yushan Road, Qingdao 266003, People’s Republic of China
#
these authors contributed equally
*
Corresponding authors Tel:86-21-50806600-5412 (B.X) E-mail: bxiong@simm.ac.cn; (Y.X)
ycxu@simm.ac.cn; (M.G) mygeng@simm.ac.cn; (J.S)jkshen@simm.ac.cn
Abstract
HSP90 is ubiquitously overexpressed in a broad spectrum of human cancers and has been
recognized as an attractive target for cancer treatment. Here, we described the fragment screening,
synthesis and structure-activity relationship studies of small molecule inhibitors with
4
,5-diarylisoxazole
scaffold
targeting
HSP90.
Among
them,
the
compound
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-morpholinopiperidin-1-yl)methyl)phenyl)isoxaz
ol-5-yl)cyclopropanecarboxamide (108) showed high affinity for binding to HSP90 (FP binding
assay, IC50 =0.030ꢀM) and inhibited the proliferation of various human cancer cell lines with
1

ACCEPTED MANUSCRIPT
averaging GI50 about 88 nM. Compound 108 exhibited its functional inhibition of HSP90 by
depleting key signaling pathways and concomitantly elevating of HSP70 and HSP27 in U-87MG
cells. Further in vivo studies showed that compound 108 strongly suppressed the tumor growth of
human glioblastoma xenograft model U-87MG with T/C =18.35% at 50mg/kg q3w/2.5w.
Moreover, compound 108 also exhibited good pharmacokinetic properties. Together, our study
implicates that compound 108 is a promising candidate of HSP90 inhibitor and is currently
advanced to preclinical study.
Keywords: HSP90; fragment-based drug discovery; crystallography;
Abbreviations list: CDI, 1,1'-Carbonyldiimidazole; DCM, Dichloromethane; DHP,
3
,4-Dihydro-2H-pyran; DMAP, 4-Dimethylaminopyridine; DMF, N,N-Dimethylformamide;
LHMDS, Lithium Hexamethyldisilazide
1
. Introduction
Heat shock proteins (HSPs) comprise a group of evolutionarily conserved proteins divided into
five main families on the basis of their molecular weight: HSP110, -90, -70, -60, and the small
HSPs. HSPs are required for essential housekeeping functions such as protein folding, assembly,
and transportation. HSPs can also redirect damaged and misfolded proteins towards proteasomal
degradation and promote cell survival by maintaining the structural and functional integrity of
several client proteins that regulate cell proliferation, survival and apoptosis. Among the HSPs,
HSP90 is relatively unique because it plays important roles in maintaining the conformation,
stability and function of an extensive list of signaling proteins involved in pathways of cell
proliferation, cell cycle progression, angiogenesis, invasion and metastasis. Importantly, its client
proteins include many key oncogenic proteins, such as Her2, AKT, CDK4, VEGF, MET and ALK,
which makes it one of the most attractive antitumor targets [1-6]. It has been well believed that the
HSP90 inhibitors can simultaneously affect several abnormal signaling pathways in tumor cells
and may also overcome the notorious cancer resistance issue [7-11].
The first identified HSP90 inhibitor Geldanamycin[12], belonging to the macrocyclic polyketide
class, was discovered from the bacterial species Streptomyces hygroscopicus. Its derivatives
1
7-AAG and 17-DMAG (Figure 1) were semi-synthesized to improve activity and reduce toxicity
2

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[13-14]. PU3, the first totally synthesized inhibitor of HSP90, was developed with the aid of the
structure-based drug design technique [15-16]. Pharmacophore models extracted from PU3 and its
derivatives were applied to develop CNF-2024/BIIB021 which entered clinical trials in 2005
[17-18]. Other unique HSP90 inhibitors such as NVP-AUY922 and SNX-5422 were developed by
Vernalis and Serenex respectively, and both entered clinical trials in 2007 [19-20]. AT13387, a
novel HSP90 inhibitor developed by Astex via fragment-based drug discovery (FBDD) approach
is now in phase II clinic trials [21-24]. STA-9090, developed by Synta, has entered phase III clinic
trials [25]. However, side effects were commonly observed in HSP90 inhibitors, which limited the
clinical studies of these inhibitors. Thus novel structure explorations or modifications are still
necessary[26-30].
Figure 1. Reported HSP90 inhibitors
Here we described our work on identification of novel HSP90 inhibitors. Briefly, a focused
fragment library was established and screened by direct co-crystallization and X-ray
crystallography methods. The structure of N-(isoxazol-5-yl)amides was identified and optimized
within structure-based drug design methods to obtain a novel series of HSP90 inhibitors with good
pharmacokinetic properties and excellent in vitro/vivo antitumor activities.
2
. Results and Discussion
2
.1 Fragment Screening
As demonstrated in previous works from Astex and our group [31], computational docking
followed by X-ray crystallography verification is very efficient to identify weak binding fragments
in FBDD. In current study, an in-house fragment library containing some commercial available
reagents and low molecular weight compounds (about 600 compounds) were docked into the
ATP-binding site of N–terminal HSP90. Ten fragments were selected based on the binding
interaction patterns. Together with other ten synthesized fragment compounds designed on the
basis of known HSP90 inhibitors, totally 20 fragments were subjected to the co-crystallization
experiments, and then diffracted with X-ray crystallography techniques to detect their binding.
The crystals were diffracted at Shanghai Synchrontron Radiation Facility (SSRF). Among them,
3

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fragment 3 was identified to bind at the ATP site of N-terminus of HSP90. The detailed binding
mode was illustrated in Figure 2.
Figure 2. Binding mode of fragments with HSP90.
Fragment 3 contained a resorcinol group, one of the usually used pharmacophores in HSP90
inhibitors, and an isoxazole group. With detailed analysis of the binding mode of fragment 3, we
found that the N and O of the isoxazole ring and the hydroxyl group on 2’-position of resorcinol
interacted with Asp93, Gly97 and Thr184 via the crystal water molecules. The hydroxyl group on
4
’-position of resorcinol interacted with Leu48 and Ser52 via another conserved water molecule.
The O atom on the isoxazole ring and the amino group at 5-position also formed hydrogen bonds
with Gly97 directly. All these interactions formed a network and exhibited a typical binding mode
of resorcinol inhibitors of HSP90.
We found that the structure of 3 had a large portion in common with NVP-AUY922 as they both
had a resorcinol moiety linking to an isoxazole ring. These two molecules overlapped well at the
binding site of HSP90, even the three crystal water molecules (Figure 2B). But as highlighted in
Figure 2C and 2D, fragment 3 reversed the O-N position in the isoxazole ring, and had an
additional amino group at position 5. The similarity with NVP-AUY922 made fragment 3 a
promising hit with predictable good activities while the reversed isoxazole ring might lead some
differences from NVP-AUY922. Thus fragment 3 was considered as a good starting point.
2
.2 Chemistry
4
-Chlororesorcinol and acetic acid took Friedel-Crafts acylation under the catalysis of boron
trifluoride to give compound 5. After benzyl protection of the phenyl hydroxyl groups, compound
was transformed via bromoform reaction to acid 7 [19] which was methylated by CH I to yield 8.
6
3
Under the reaction of LHMDS compound 8 was condensed with anisyl cyanide to give 9, and
followed by cyclization with hydroxylamine hydrochloride to generate 10 [32]. Compound 10
reacted with acryl chlorides and subsequently deprotected by boron trichloride to yield the target
compounds 23-35 (Scheme 1).
4

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Scheme 1
As shown in Scheme 2, starting with diverse 5-substituted methyl 2,4-bis(benzyloxy)benzoate,
compound 56-60 were synthesized according to the methods described in Scheme 1.
Scheme 2
The hydroxyl groups of 1-(2,4-dihydroxyphenyl)ethanone were protected by benzyl groups to give
compound 62 which was submitted to Wittig reaction followed by hydrogenation to generate the
isopropyl substituent. Compound 64 took Vilsmeier reaction and then re-protected by benzyl
groups to yield 66. Coupled with hydroxylamine hydrochloride and then chlorinated by N-
chlorosuccinimide compound 66 turned to the intermediate 68 [33]. Another intermediate 72 was
obtained by hydrolyzation, reduction and dihydropyran protection of commercial available
4
-cyanomethylbenzoic acid methyl ester. Compound 68 and 72 were coupled to generate
isoxazole 73 which was amidated and deprotected to give 75. The hydroxyl group was activated
by methanesulfonyl chloride, substituted with different amines and then debenzylation to yield the
target compounds 93-108.
Scheme 3
2
.3 Structure-Activity Relationships and Structural Studies
Fragment 3 was too small to show strong inhibition. Referring to previous SAR studies on
NVP-AUY922 [19], a methoxyphenyl group was introduced to 4-position on the isoxazole ring of
fragment 3. Considering synthetic difficulties, the isopropyl group was replaced by a chlorine
atom at this initial optimization stage and the dissociative amino group of 5-position was
derivatized to amide to obtain compound 24. This compound showed an IC50 of 0.070 ꢀM in
HSP90 fluorescence polarization (FP) assay (Table 1). Further cellular assay showed that it
effectively inhibited the growth of A549 cancer cell line with GI about 7.198 ꢀM.
5
0
The crystal structure of the complex of compound 24 with HSP90 was solved and illustrated in
Figure 3. The resorcinol moiety and the isoxazole ring of compound 24 had the same binding
5

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mode with fragment 3. The incorporated p-methoxyphenyl group also fitted well within the
binding site of the protein. These results made compound 24 a promising lead for further SAR
study.
Figure 3. Crystal structure of Compound 24 bound to N-terminal HSP90 (PDB ID: 4LWE ).
The initial optimization was focused on 5-position of the isoxazole ring. Amides with diverse
chains and rings were synthesized. As shown in Table 1, when R was small chains such like Me-,
1
Et-, Pr- , Bu-, or cycloaliphatic rings from cyclopropyl to cyclohexyl, most of the compounds
retained the binding affinities (<0.1 ꢀM) on molecular level; when a longer chain was selected for
R1 such as 34, the inhibition activity decreased considerably. However, these compounds showed
large differences in cellular assay. The activities ranged from less than 1.0 ꢀM to over 40 ꢀM.
Among these compounds, 30 with a cyclopropane carbonyl group was the most potent one with
GI 0.761 ꢀM on A549 cell. Compound 35 with a carbamide structure showed no advantage over
50
the amides in the enzymatic FP assay and was no longer concerned.
Table 1. Binding (FP Assay) and Cell Growth Inhibition (SRB Assay) Data of 23-35
From the co-crystal structure of compound 24 with HSP90, it was found that the subsite
accommodating chlorine at the 5’-position of the resorcinol had more space for optimization.
During the following modifications focused on R , methyl and isopropyl group were selected for
2
R considering the availability of raw materials. As shown in Table 2, most of the compounds
1
retained the binding affinities. The compounds with R substituted by H (57) or a relatively big
2
group (tert-butyl, 59) showed decreased inhibition activities, especially in the cellular assay.
Among these compounds, 60 was found to have most potent cellular activity. Together with the
enzymatic binding activity of compound 30, it indicated that the structure with c-Pr at R and i-Pr
1
at R could benefit the cellular permeability. Therefore, these groups were fixed in later
2
optimization.
Table 2. Binding (FP Assay) and Cell Growth Inhibition (SRB Assay) Data of 56-60
6

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To check the binding of 60 with HSP90, the co-crystal structure was solved at 1.7Å resolution.
The resorcinol moiety and the isoxazole ring bind to the same subsite in HSP90, and the
methoxyphenyl group points toward the solvent range (Figure 4A). Therefore, a number of
hydrophilic pieces were used to modify the methoxyphenyl group of compound 60 intending to
increase the solubility as well as to tune the properties of drug-likeness. As shown in Table 3, the
incorporation of hydrophilic groups did not affect molecular activities. Most of them exhibited FP
IC50 better than 0.05 ꢀM, which confirmed that R was a suitable part for pharmacokinetic
3
optimization. Most of the compounds also showed excellent inhibition activities on cellular level,
among which compounds 93, 102, and 108 exhibited the GI50 less than 0.1 ꢀM. We also solved
the co-crystal structure of 93 with HSP90 to verify the binding mode. As illustrated in Figure 4B,
the morpholine ring stretches to the solvent as expected. Based on the cellular activities,
compounds 93, 102 and 108 were selected for further investigation.
Figure 4. A. Complex of compound 60 with HSP90 (PDB ID: 4LWI); B. Complex of compound
93 with HSP90 (PDB ID: 4LWH).
Table 3. Binding (FP Assay) and Cell Growth Inhibition (SRB Assay) Data of 93-108
2
.4 Cancer Cell Lines Panel Test
A panel of 15 human cancer cell lines from diverse tissue origins including lung, liver, colon,
breast, ovary, prostate, kidney, cervix and skin were selected to evaluate the antitumor efficacy of
compounds 93, 102 and 108 using SRB assay. All these three compounds displayed potent
cytotoxicity with average GI50 at 0.057, 0.058 and 0.088 ꢀM respectively, indicating their
broad-spectrum antitumor activities (Table 4).
Table 4. Cell Growth Inhibition of Compounds 93, 102 and 108 in Various Human Cancer Cell
2
.5 hERG Safety Assay.
Blockade of hERG K+ channels is one of the most important molecular mechanisms of cardiac
7

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arrhythmia, which is a common cause of the failures of many antitumor drugs. Therefore,
inhibitory liability on hERG (causing QT prolongation) has become a major safety concern during
the development of therapeutic drugs. Compounds 93, 102 and 108 were subjected to hERG safety
assay. As shown in Table 5, compound 102 revealed higher probability of hERG toxicity than
compounds 93 and 108, therefore was filtered out for further study.
Table 5. HERG safety assay of compounds 93, 102 and 108.
2
.6 Effects on Client Proteins In vitro
Human glioblastoma cell line U-87 MG which had been reported to be highly sensitive to
NVP-AUY922 was selected to evaluate the ability of compounds 93 and 108 of inhibiting Hsp90
in cancer cells. U-87 MG cells were exposed to increased concentrations of compounds 93 and
1
08 for 24 h, after that the expression and/or phosphorylation of various client proteins and heat
shock proteins were determined by western blotting. As shown in Figure 5, molecular
co-chaperone HSP70 and HSP27 were induced effectively, indicating that these compounds
inhibited HSP90 directly. Accordingly, the expression level of c-Met and its downstream signaling,
namely the phosphorylation of Akt and Erk, were reduced at concentration of 50 nmol/L and
barely detectable at 100 nmol/L. All these results were re-obtained by NVP-AUY922 which was
taken as a positive control. Collectively, these data supported directly that the effects of
compounds 93 and 108 on cell proliferation were a consequence of functional inhibition of
HSP90.
Figure 5. Western Blotting analysis of the expression of co-chaperone and client proteins of
HSP90 after incubation with compounds 93 and 108 in U-87 MG cells.
2
.7 In vitro Metabolic Stability. Compounds 93 and 108 were tested on rat and human liver
microsome (RLM and HLM, respectively) tissues to evaluate their in vitro metabolic stability
Table 6). When the value of RLM and HLM is below 100 ꢀL/min/mg protein, the compound is
(
considered to be metabolically stable. As shown in Table 6, compounds 93 and 108 both showed
relatively good stability, especially compound 108. Both compounds had direct inhibition (>50%)
8

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on CYP2D6 at 10 ꢀM concentration. Compound 93 also had obvious inhibition (>50%)) on
-4
CYP2C9. Neither of them showed high inhibition (>200 10 /min) on CYPs in time-dependent
inhibition (TDI) tests. In these tests the results of compounds 93 and 108 were equivalent or
slightly better than NVP-AUY922.
Table 6. Metabolic stability and inhibition of rat and human liver microsomes by compounds 93,
and 108.
2
.8 Pharmacokinetic (PK) Profiles. Pharmacokinetic parameters of compounds 93 and 108 in
mice after i.v. and p.o. dosing were summarized in Table 7. Comparing the parameters of 93 and
1
08, it was found that compound 108 had longer half-life (6.46 hours) and higher AUC value over
3 with iv injection. Therefore compound 108 was advanced to in vivo antitumor study.
9
Table 7. Pharmacokinetic profiles of compounds 93 and 108.
2
.9 In vivo Antitumor Study
According to former results, finally, compound 108 was selected to evaluate its in vivo anti-tumor
activity using mice bearing U-87 MG xenograft tumors whereas NVP-AUY922 was chosen as a
reference compound. Compound 108 in form of hydrochloride salt was intravenously injected at
doses of 25 and 50 mg/kg 3 times per week for 17 consecutive days. As shown in Figure 6,
compound 108 showed statistically significant tumor growth inhibition. The T/C values of
compound 108 were 48.28% at 25 mg/kg and 18.35% at 50 mg/kg, which was obviously better
than NVP-AUY922 (T/C values was 34.06% at 50 mg/kg). Compound 108 was also well tolerated,
exhibiting no serious treatment-related toxicity at these doses and minimal weight loss over the
course of this study.
Figure 6. In vivo anti-tumor effect of compound 108 in U-87 MG xenograft model.
3
. Conclusions
Obtained from fragment screening and with the aids of X-ray crystallography techniques,
9

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fragment 3 was developed to a novel series of HSP90 inhibitors. Compound 108, one in this series,
exhibited strong inhibition of HSP90 in both molecular and cellular level. It also showed good
results in pharmacokinetic and safety experiments, which were not less than the reference
compound NVP-AUY922. Furthermore the compound significantly inhibits the tumor growth in
xenograft cancer model U-87 MG, almost twice strong than NVP-AUY922. These indicated that
although compound 108 had a part in common with NVP-AUY922 the reversed isoxazole ring
and different side chains did make contribution to the activities. The overall properties warrant
compound 108 a promising candidate of Hsp90 inhibitor and this compound is now under further
evaluation.
4
. Experiment and Methods
4
.1 HSP90 expression and purification
The sequence-verified cDNA( NCBI reference sequence: NM_005348.3) encoding the human
full-length Hsp90α protein was inserted into the pFastBac-HTb plasmid( Invitrogen, USA).
Recombinant bacmid was generated by transposition in E.Coli, and transfected into SF9 insert
cells using Cellfectin II reagent (Cat no. 10362100, Invitrogen, USA). Large-scale protein
production was performed in SF9 insert cell cultures infected with high-titer Hsp90α recombinant
o
baculovirus. Cells were harvested by centrifugation after 96h post infection and stored at -80 C
until use. The cell pellet was resuspended in lysis buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 10%
glycerol and 20 mM imidazole). And protein was eluted with the same buffer containing 250 mM
imidazole and concentrated to 1mg/ml. Protein preparations were divided to small aliquots, flash
o
frozen in liquid nitrogen, and stored at -70 C.
4
.2 FP Enzymatic assay
Based on a test set of Hsp90α inhibitors competing with fluorescent geldanamycin( GM-BODIPY)
for binding to Hsp90α, the assays were performed on a multi-mode microplate reader (Synergy4,
Bio-Tek, Company, USA). All experiments were conducted in 384-well black flat-bottomed
polystyrene plates (Corning no. CLS3575) in a total volume of 40uL for each well. All inhibitors
and GM-BODIPY were prepared in DMSO and diluted with the assay buffer (20mM Hepes,
pH7.3, 50mM KCl, 5mM MgCl , 20mM Na MoO , 0.01% NP40, 0.1mg/ml BSA and 2mM DTT).
2
2
4
For each assay, background wells (buffer only), negative controls (GM-BODIPY only) and
1
0

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positive controls (GM-BODIPY in the presence of Hsp90α) and test wells (inhibitors and
GM-BODIPY in the presence of Hsp90α) were included on each assay plate. And the assay plate
o
was incubated at 4 C for 16h. The FP values were measured at room temperature with excitation
wavelength at 485 nm and emission wavelength at 535 nm. All experimental data were analyzed
using the Origin 7.5 software (OriginLab, USA ).
4
.3 HSP90 (residues 9–236) expression, purification and crystallography
.3.1 Protein purification and crystallization
4
A cDNA fragment encoding the N-terminal domain of human Hsp90 (residues 9–236), Hsp90N,
was cloned into a pET28 vector. The recombinant plasmid pET28-Hsp90N was transferred into E.
coli strain BL21 (DE3) for overexpression (Invitrogen, Carlsbad, USA). Bacterial cultures were
grown in LB medium at 37 °C to an OD600 of 0.6 – 0.8, and 0.2 mM
isopropyl-b-D-thiogalactopyranoside was then added for further growth at 30 °C for another 5 h.
The harvested cells were re-suspended in lysis buffer (20 mM Tris/300 mM NaCl/5 mM
b-mercaptoethanol/10% glycerol, pH 7.5) and sonicated. The resulted suspension was centrifuged
and the supernatant was passed through a nickel-bead column (GE Healthcare, Piscataway, USA).
The column was then washed with 20 mM imidazole/ 300 mM NaCl/20 mM Tris/5 mM
b-mercaptoethanol/10% glycerol, pH 7.5 (wash buffer), until no further protein was eluted. The
recombinant Hsp90N was then eluted with 100 mM imidazole/300 mM NaCl/20 mM Tris/5 mM
b-mercaptoethanol/10% glycerol, pH 7.5 (elution buffer). The eluted proteins were concentrated
(Millipore, Billerica, USA) and then injected into a 120 ml Hiload Superdex75 column (GE
Healthcare) from which Hsp90N was eluted with 20 mM Tris/300 mM NaCl/5 mM
b-mercaptoethanol/ 10% glycerol, pH 7.5. The fractions containing Hsp90N were collected and
concentrated to 20 mg/ml for crystallization. The purity of protein was assessed by SDS-PAGE to
be 95%.
Crystals of the Hap90N-RL1 complex (PDB code: 4L8Z) was obtained by the hanging drop
vapor-diffusion method in a 24-well plate. RL1 was added in portions to a final concentration of 2
mM, to a protein solution (20 mg/mL). Then 1 ꢀL of the protein−ligand solution was mixed with 1
ꢀL of reservoir solution consisting of 20%–25% (w/v) PEG 2000 monomethyl ether/200 mM
magnesium chloride/100 mM sodium cacodylate, pH 6.5. The plate was incubated at 4 °C, and
crystals appeared within 3-5 days. The crystals were quickly transferred to a cryosolution
consisting of 25% (w/v) PEG 2000 monomethyl ether/200 mM magnesium chloride/100 mM
sodium cacodylate, pH 6.5, 25% (v/v) glycerol, and then flash frozen in liquid nitrogen. Crystals
of the Hsp90N-S29 complex (PDB code: 4L91) and the Hsp90N-S46 complex (PDB code: 4L94)
were obtained by the hanging-drop vapor-diffusion method in a 24-well plate. Compounds were
1
1

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added incrementally to a final concentration of 2 mM, to a protein solution (20 mg/mL). 1 ꢀL of
the resulting protein−ligand solution was mixed with 2 ꢀL of reservoir solution consisting of
2
0%–25% PEG4000/ 200 mM magnesium chloride/100 mM Tris at pH 8.5. The plate was
incubated at 4 °C, and crystals appeared within 2-7 days. The crystals were quickly transferred to
a cryosolution consisting of 25% PEG4000/ 200 mM magnesium chloride/100 mM Tris at pH 8.5,
2
5% (v/v) glycerol and then flash frozen in liquid nitrogen.
4
.3.2 Data collection, structure determination, and refinement
Crystals were grown and then mounted and flash-frozen in liquid nitrogen for diffraction test and
data collection. All data sets were collected at 100 K on beamline BL17U1 at the Shanghai
Synchrotron Radiation Facility (SSRF, Shanghai, China) and were processed with the HKL2000
software package. The structures were solved by molecular replacement, using the PHENIX. The
search model used for the crystals of apo was the previously reported structure of the Hsp90-ATP
(
PDB code 3T0Z), the structures were refined using PHENIX. With the aid of the program Coot,
compounds, water molecules, and others were fitted into to the initial Fo–Fc map.
.4 Cell Proliferation Assay
4
Cells were seeded in 96-well culture plates. On the next day, cells were exposed to various
concentrations of compounds and further cultured for 72 h. Finally, cell proliferation was
determined using sulforhodamine B26 methods. The inhibition rate was calculated as (1-A515
treated/A515 control) ×100%. The cytotoxicity of compounds was expressed as an IC ,
5
0
determined by the Logit method.
.5 Western Blot Analysis. U-87 MG cells were cultured under regular growth conditions to
4
exponential growth phase. Then the cells were treated with the indicated concentration of
compounds for 24 h at 37 °C and lysed in 1 × SDS sample buffer. Those cell lysates were
subsequently resolved on 10% SDS-PAGE, and transferred to nitrocellulose membranes. The
blots were incubated with specific primary antibodies, then subsequently with anti-rabbit or
anti-mouse IgG horseradish peroxidase. Immunoreactive proteins were detected using an
enhanced chemiluminescence detection reagent.
4
.6 Pharmacokinetic study
Microsomes (Human microsome: Xenotech, Lot No.H0610; Rat microsome: Xenotech, Lot No.
R1000) (0.5 mg/mL) were pre-incubated with 1 ꢀM test compound for 5 min at 37 °C in 0.1 M
1
2

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phosphate buffer (pH 7.4) with 1mM EDTA, and 5 mM MgCl . The reactions were initiated by
2
adding pre-warmed cofactors (1 mM NADPH). After 0, 5, 10, and 30min incubations at 37 °C, the
reactions were stopped by adding an equal volume of cold acetonitrile. The samples were vortexed
for 10 min and then centrifuged at 10,000 g for 10 min. Supernatants were analyzed by
LC/MS/MS for the amount of parent compound remaining, and the corresponding loss of parent
compound also determined by LC/MS/MS.
The five CYP (CYP3A4, CYP2D6, CYP2C9, CYP1A2 and CYP1C19) enzymatic activities were
characterized based on their probe reactions. Incubation mixtures were prepared in a total volume
of 100 ꢀl as follows: 0.2 mg/mL microsome (Human microsome: Xenotech, Lot No.H0610),
NADPH (1 mM), 100 mM phosphate buffer (pH 7.4), probe substrates cocktail (midazolam 10
ꢀM, testosterone 100 ꢀM, dextromethorphan 10 ꢀM, diclofenac 20 ꢀM, phenacetin 100 ꢀM and
mephenytoin 100 ꢀM) and 10 ꢀM tested compound or positive control cocktailꢀketoconazole 10
ꢀMꢁquinidine 10 ꢀMꢁsulfaphenazole 100 ꢀM, α-naphthoflavone 10 ꢀM, tranylcypromine 1000
ꢀM or negative control (PBS). The final concentration of organic reagent in incubation mixtures
o
was less than 1% v/v. There was a 5 min pre-incubation period at 37 C before the reaction was
initiated by adding a NADPH-generating system. Reactions were conducted for 20 min for
CYP3A4, CYP2D6 and CYP1A2. For each probe drug, the percentage of metabolite conversion
was less than 20% of substrate added. The inhibition rate was calculated as: (The formation of the
metabolite of probe substrates with 10 ꢀM tested compound) / (The formation of the metabolite of
probe substrates with PBS) × 100%.
4
.7 In vivo U-87 MG Xenograft model study
Animal experiments were performed according to institutional ethical guidelines of animal care.
Xenograft U-87 MG was tumor cell lines derived. Well-developed tumors were cut into 1-mm3
fragments and transplanted s.c. into the right flank of nude mice using a trocar. When the tumor
3
volume reached 100 to 150 mm , the mice were randomly assigned into control and treatment
groups. Control groups were given vehicle alone, and treatment groups received compounds as
indicated doses via intravenous injection 3 days per week for 17days. The sizes of the tumors were
measured twice per week using microcaliper. The tumor volume (TV) was calculated as: TV =
(
length × width2)/2, and the relative tumor volume (RTV) was calculated as: RTV = V /V , where
t 0
V is the tumor volume on the day measured and V is the tumor volume on the first day of
t
0
1
3

ACCEPTED MANUSCRIPT
treatment. Relative tumor volume was shown on indicated days as the median relative tumor
volume ± SE indicated for groups of mice. T/C values were calculated on the final day of study
for drug-treated compared with vehicle-treated mice as (RTVTreated/RTVControl)×100%.
4
.8 Chemistry
General: Reagents (chemicals) were purchased from Alfa-Aesar (Karlsruhe, Germany), Acros
Geel, Belgium), Aldrich (St. Louis, MO, USA), Adamas-beta (Shanghai, China) and Shanghai
(
Chemical Reagent Company (Shanghai, China) and were used without further purification.
Analytical thin-layer chromatography was performed on HSGF 254 (150–200 mm thickness;
Yantai Huiyou Company, Yantai, Shandong, China). 1H NMR (300 MHz or 400 MHz) spectra
were recorded on a Varian Mercury-300 or 400 High Performance Digital FT-NMR with TMS as
an inter-nal standard. HPLC analysis was performed using a Gilson HPLC system with UV
detection at 214 and 254 nm. LC–MS spectra were obtained on an LCQ Deca XP ion trap mass
spectrometer (Thermo-Finnigan, San Jose, CA, USA). Accurate mass measurements were carried
out on a Q-TOF ultima Globe mass spectrometer (Micromass, Manchester, UK).
1
-(5-Chloro-2,4-dihydroxyphenyl)ethanone (5). 4-Chlororesorcinol (61 g, 0.42 mol) was
suspended in boron trifluoride etherate (290 mL) and added acetic acid (25.1 mL, 0.42 mol)
o
dropwise under argon protection. The mixture was heated to 90 C for 3.5h. Yellow precipitation
formed when the mixture cooled down to room temperature. The mixture was poured into 1 L 10%
(w/v) sodium acetate solution and stirred vigorously for 2.5 hours. The yellow precipitate was
1
filtered, washed with water and dried overnight to yield the title compound 5 (67 g, 85%): H
NMR (300 MHz, DMSO-d ) δ 12.35 (s, 1H), 11.45 (s, 1H), 8.03 (s, 1H), 6.62 (s, 1H), 2.70 (s, 3H);
6
+
MS (EI): 186 [M] .
1
-(2,4-Bis(benzyloxy)-5-chlorophenyl)ethanone (6). Compound 5 (67 g, 0.36 mol) and
potassium carbonate (124 g, 0.9 mol) were suspended in acetonitrile (600 mL) and added benzyl
bromide (100.5 mL, 0.84 mol) dropwise. The mixture was refluxed overnight and cooled to room
temperature. Most of the solvents were removed in vacuo, and the residue was added 1 L water.
White solid formed when the mixture was stirred at room temperature. The solid was filtered,
washed with water and petroleum subsequently, and dried to obtained the title compound as white
1
solid (123.6 g, 94%): H NMR (300 MHz, CDCl ) δ 7.91 (s, 1H), 7.42-7.32 (m, 10H), 6.55 (s, 1H),
3
+
5
.13 (s, 2H), 5.07 (s, 2H), 2.54 (s, 3H); MS (EI): 366[M] .
1
4

ACCEPTED MANUSCRIPT
2
,4-Bis(benzyloxy)-5-chlorobenzoic acid (7). Compound 6 (1.1 g, 3 mmol) was suspended in
sodium hydroxide solution (1.2 g, 30 mmol in 10 mL water) and dioxane (10 mL) and added
bromine (1.44 g, 9 mmol) dropwise. The mixture was stirred at ambient temperature overnight.
Most of the dioxane was removed in vacuo. 2M HCl was added to the residue to make the pH to 2.
Pale-yellow solid formed. The solid was filtered, washed with water, and dried to obtained the title
1
compound (0.9 g, 81%): H NMR (300 MHz, CDCl ) δ 10.50 (br, 1H), 8.20 (s, 1H), 7.45-7.35 (m,
3
+
1
0H), 6.64 (s, 1H), 5.20 (d, 4H); MS (EI): 368[M] .
Methyl 2,4-bis(benzyloxy)-5-chlorobenzoate (8). Compound 7 (1 g, 2.7 mmol) and potassium
carbonate (1 g, 7 mmol) were suspended in acetonitrile (50 mL) and added iodomethane. The
mixture was refluxed for 1h and cooled to room temperature. Most of the solvent was removed in
vacuo. The residue was added 100 mL water and stirred. The solid was filtered, washed with
1
water, and dried to obtained the title compound (0.92g, 88.6%): H NMR (300 MHz, CDCl ) δ
3
+
.
7
3
4
.95 (s, 1H), 7.50-7.30 (m, 10H), 6.57 (s, 1H), 5.12 (d, 4H), 3.88 (s, 3H); MS(ESI): 382.8[M+H]
-(2,4-Bis(benzyloxy)-5-chlorophenyl)-2-(4-methoxyphenyl)-3-oxopropanenitrile (9).
-Methoxybenzyl cyanide (0.2 g, 1.36 mmol) was added dropwise to LHMDS (3.4 mL, 3.4 mmol)
o
o
in 20 mL dry tetrahydrofuran at -78 C under N protection. The mixture was stirred at -78 C for
2
3
0 minutes and added compound 8 (0.5 g, 1.36 mmol) in 10 mL dry tetrahydrofuran dropwise.
o
The mixture was kept stirring at -78 C for 30 minutes and then 2 hours at room temperature. 10
mL 2M HCl was added to stop the reaction. Most of the solvent was removed in vacuo. The
residue was extracted with dichloromethane. The organic layer was washed with brine, dried over
Na SO and evaporated in vacuo. The crude product was purified by flash chromatography on
2
4
1
silica gel to afford the product as yellow oil (0.53g, 78.5%): H NMR (300 MHz, CDCl ) δ 7.74 (s,
3
1
5
3
9
H), 7.63 (d, J=8.8Hz, 2H), 7.45-7.35 (m, 10H), 6.95 (d, J=8.8Hz, 2H), 6.65 (s, 1H) , 6.59 (s, 1H),
-
.15 (s, 2H), 5.09 (s, 2H), 3.84 (s, 3H); MS (ESI): 496.1[M-H] .
-(2,4-Bis(benzyloxy)-5-chlorophenyl)-4-(4-methoxyphenyl)isoxazol-5-amine (10). Compound
(0.15 g, 0.3 mmol) and hydroxylamine hydrochloride (0.042 g, 0.6 mmol) were dissolved in 5
o
mL dry pyridine and stirred at 100 C overnight. The mixture was diluted with dichloromethane,
washed with saturate citric acid/water (1:1, 30 mL×2), water (30mL×1), sat.NaHCO (30mL×2),
3
brine, dried over Na SO and evaporated in vacuo. The crude product was purified by flash
2
4
1
chromatography on silica gel to afford the product as white solid (0.1 gꢁ68.6%): H NMR (300
1
5

ACCEPTED MANUSCRIPT
MHz, CDCl ) δ 7.48 (s, 1H), 7.38-7.30 (m, 5H), 7.24-7.20 (m, 3H), 6.96 (d, J=8.6Hz, 2H),
3
6
3
.89-6.84 (m, 2H), 6.78 (d, J=8.6Hz, 2H), 6.40 (s, 1H), 5.01 (s, 2H), 4.64 (s, 2H), 4.52 (s, 2H),
+
.79 (s, 3H); MS (ESI): 513.1[M+H] .
General procedure for the synthesis of 23-35. Compound 10 (0.2 g, 0.4 mmol) and triethylamine
1 mL) were dissolved in 20 mL dry dichloromethane. Catalytic amount of DMAP was added to
(
the mixture followed by the addition of acyl chlorides (3 eq) dropwise. When stirred for 2 hours at
room temperature, the mixture was diluted with dichloromethane. The organic layer was washed
with saturate citric acid/water (1:1, 30 mL×2), water (30mL×1), sat.NaHCO (30mL×2), brine,
3
dried over Na SO and evaporated in vacuo. The crude product was purified by flash
2
4
chromatography on silica gel to afford the intermediates 11-22. Compound 10-22 (1 eq) was
dissolve in dry dichloromethane followed by addition of 1N BCl in dichloromethane (3 eq) under
3
nitrogen. The mixture was stirred for 2 hours at room temperature and then diluted with ethyl
acetate. The organic layer was washed with sat.NaHCO (30mL×2), brine, dried over Na SO and
3
2
4
evaporated in vacuo. The crude product was purified by flash chromatography on silica gel to
afford the final product 23-35.
4
-(5-Amino-4-(4-methoxyphenyl)isoxazol-3-yl)-6-chlorobenzene-1,3-diol (23). White solid
1
(
70.9%): H NMR (400MHz, CD OD) δ 7.13 (d, J=8.9Hz, 2H), 6.96 (s, 1H), 6.93 (d, J=8.9Hz,
3
+
2
H), 6.44 (s, 1H), 3.79 (s, 3H); MS (ESI): 333.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)acetamide (24). The
1
intermediate 11 was obtained as white solid (88.6%): H NMR (300 MHz, CDCl ) δ 7.50 (s, 1H),
3
7
6
5
.40-7.34 (m, 5H), 7.33 (s, 1H), 7.26-7.23 (m, 3H), 6.97 (d, J=8.6Hz, 2H), 6.85-6.90 (m, 2H),
.78 (d, J=8.6Hz, 2H), 6.43 (s, 1H), 5.04 (s, 2H), 4.63 (s, 2H), 3.81 (s, 3H), 1.60 (s, 3H); MS(ESI):
+
1
55.1[M+H] . The title product was obtained as Amorphous white solid (56.4%): H NMR
(
400MHz, CD OD) δ 7.13 (d, J=8.3Hz, 2H), 7.05 (s, 1H), 6.90 (d, J=8.3Hz, 2H), 6.45 (s, 1H),
3
+
3
.77 (s, 3H) , 2.09 (s, 3H); MS (ESI): 375.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)propionamide (25).
1
Amorphous white solid (two steps totally 64.5%): H NMR (400MHz, CD OD) δ 7.13 (d,
3
J=8.4Hz, 2H), 7.04 (s, 1H), 6.89 (d, J=8.4Hz, 2H), 6.44 (s, 1H), 3.77 (s, 3H), 2.37 (t, J=7.4Hz,
+
2
H), 1.13 (d, J=7.4Hz, 3H); MS (ESI): 389.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)butyramide
(26).
1
6

ACCEPTED MANUSCRIPT
1
Amorphous white solid (two steps totally 58.8%): H NMR (400MHz, CD OD) δ 7.13 (d,
3
J=8.4Hz, 2H), 7.05 (s, 1H), 6.88 (d, J=8.4Hz, 2H), 6.45 (s, 1H), 3.77 (s, 3H), 2.32 (t, J=7.2Hz,
+
2
H), 1.70-1.60 (m, 2H), 0.93 (t, J=7.2Hz, 3H); MS (ESI): 403.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)isobutyramide (27).
1
Amorphous white solid (two steps totally 57.7%): H NMR (400MHz, CD OD) δ 7.12 (d,
3
J=8.4Hz, 2H), 7.05 (s, 1H), 6.88 (d, J=8.4Hz, 2H), 6.45 (s, 1H), 3.77 (s, 3H), 2.66-2.58 (m, 1H),
+
1
.14 (d, J=7.1Hz, 6H); MS (ESI): 403.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)pivalamide
(28).
1
Amorphous white solid (two steps totally 58.4%): H NMR (400MHz, CD OD) δ 7.12 (d,
3
J=8.4Hz, 2H), 7.07 (s, 1H), 6.88 (d, J=8.4Hz, 2H), 6.45 (s, 1H), 3.77 (s, 3H), 1.23 (s, 9H); MS
+
(
ESI): 417.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)-3-methylbutanamid
1
e (29). Amorphous white solid (two steps totally 61.5%): H NMR (400MHz, CD OD) δ 7.14 (d,
3
J=8.4Hz, 2H), 7.05 (s, 1H), 6.89 (d, J=8.4Hz, 2H), 6.45 (s, 1H), 3.77 (s, 3H), 2.22 (d, J=7.3Hz,
+
2
H), 2.12-2.02 (m, 1H), 0.94 (d, J=6.7Hz, 6H); MS(ESI): 417.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)cyclopropanecarbox
1
amide (30). Amorphous white solid (two steps totally 49.1%): H NMR (400MHz, CD OD) δ
3
7
1
.13 (d, J=8.4Hz, 2H), 7.04 (s, 1H), 6.90 (d, J=8.4Hz, 2H), 6.44 (s, 1H), 3.78 (s, 3H), 1.76 (m,
+
H), 0.94-0.85 (m, 4H); MS(ESI): 400.9[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)cyclobutanecarboxa
1
mide (31). Amorphous white solid (two steps totally 34.8%): H NMR (400MHz, CD OD) δ 7.12
3
(d, J=8.4Hz, 2H), 7.05 (s, 1H), 6.89 (d, J=8.4Hz, 2H), 6.45 (s, 1H), 3.78 (s, 3H), 2.32-2.14 (m,
+
5
H), 2.06-1.96 (m, 1H), 1.92-1.82 (m, 1H); MS(ESI): 415.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)cyclopentanecarboxa
1
mide (32). Amorphous white solid (two steps totally 62.6%): H NMR (400MHz, CD OD) δ 7.13
3
(d, J=8.4Hz, 2H), 7.05 (s, 1H), 6.89 (d, J=8.4Hz, 2H), 6.45 (s, 1H), 3.77 (s, 3H), 2.86-2.77 (m,
+
1
H), 1.94-1.83 (m, 2H), 1.80-1.67 (m, 4H), 1.63-1.55 (m, 2H); MS(ESI): 429.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)cyclohexanecarboxa
1
mide (33). Amorphous white solid (two steps totally 70.2%): H NMR (400MHz, CD OD) δ 7.12
3
(d, J=8.4Hz, 2H), 7.05 (s, 1H), 6.89 (d, J=8.4Hz, 2H), 6.45 (s, 1H), 3.78 (s, 3H), 2.41-2.32 (m,
1
7

ACCEPTED MANUSCRIPT
+
1
H), 1.87-1.75 (m, 4H), 1.50-1.38 (m, 2H), 1.37-1.20 (m, 4H); MS(ESI): 443.0[M+H] .
N-(3-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)octanamide
(30).
1
Amorphous white solid (two steps totally 70.0%): H NMR (400MHz, CD OD) δ 7.13 (d,
3
J=8.4Hz, 2H), 7.05 (s, 1H), 6.88 (d, J=8.4Hz, 2H), 6.45 (s, 1H), 3.77 (s, 3H), 2.34 (t, J=7.2Hz,
+
2
H), 1.65-1.56 (m, 2H), 1.35-1.22 (m, 11H); MS(ESI): 459.0[M+H] .
1
-(3-(5-Chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)-3-ethylurea
(35).
1
Amorphous white solid (two steps totally 26.2%): H NMR (400MHz, CD OD) δ 7.16 (d,
3
J=8.9Hz, 2H), 7.02 (s, 1H), 6.91 (d, J=8.9Hz, 2H), 6.45 (s, 1H), 3.78 (s, 3H), 3.16 (t, J=7.6Hz,
+
2
H), 1.07 (d, J=7.6Hz, 3H); MS(ESI): 404.0[M+H] .
Compounds 41-45 were prepared according to the procedure of 9.
3
-(2,4-Bis(benzyloxy)-5-bromophenyl)-2-(4-methoxyphenyl)-3-oxopropanenitrile
(41).
+
Yellow oil (97.2%): MS (EI): 541,543[M] .
3
-(2,4-Bis(benzyloxy)phenyl)-2-(4-methoxyphenyl)-3-oxopropanenitrile (42). Yellow oil
1
(
90.6%): H NMR (300MHz, CDCl ) δ 7.81 (d, J=8.8Hz, 1H), 7.47-7.33 (m, 10H), 7.05 (d,
3
J=8.8Hz, 2H), 6.78 (d, J=8.8Hz, 2H), 6.64-6.57 (m, 2H), 5.80 (s, 1H), 5.19-5.05 (m, 4H), 3.76 (s,
+
3
H); MS(EI): m/z 463[M] .
3
-(2,4-Bis(benzyloxy)-5-ethylphenyl)-2-(4-methoxyphenyl)-3-oxopropanenitrile (43). Yellow
1
oil (95.9%): H NMR (400MHz, CDCl ) δ 7.69 (s, 1H), 7.46-7.33 (m, 10H), 7.09 (d, J=8.7Hz,
3
2
H), 6.79 (d, J=8.7Hz, 2H), 6.48 (s, 1H), 5.85 (s, 1H), 5.18-5.05 (m, 4H), 3.76 (s, 3H), 2.60 (q, J
+
=
7.8 Hz , 2H), 1.17 (t, J =7.8 Hz, 3H); MS(EI): 491[M] .
3
-(2,4-Bis(benzyloxy)-5-isopropylphenyl)-2-(4-methoxyphenyl)-3-oxopropanenitrile
(44).
1
Yellow oil (90.4%): H NMR (400MHz, CDCl ) δ 7.76 (s, 1H), 7.46-7.33 (m, 10H), 7.10 (d,
3
J=8.7Hz, 2H), 6.80 (d, J=8.7Hz, 2H), 6.48 (s, 1H), 5.84 (s, 1H), 5.20-5.04 (m, 4H), 3.76 (s, 3H),
+
3
.24 (m, 1H), 1.20 (t, J = 6.5Hz, 6H); MS(EI): 505[M] .
3
-(2,4-Bis(benzyloxy)-5-(tert-butyl)phenyl)-2-(4-methoxyphenyl)-3-oxopropanenitrile (45).
1
Yellow oil (88%): H NMR (400MHz, CDCl ) δ 7.85 (s, 1H), 7.45-7.34 (m, 10H), 7.11 (d,
3
J=8.7Hz, 2H), 6.80 (d, J=8.7Hz, 2H), 6.49 (s, 1H), 5.83 (s, 1H), 5.20-5.07 (m, 4H), 3.77 (s, 3H),
+
1
.34 (s, 9H); MS(EI): 519[M] .
Compounds 46-50 were prepared according to the procedure of 10.
-(2,4-Bis(benzyloxy)-5-bromophenyl)-4-(4-methoxyphenyl)isoxazol-5-amine (46). Yellow
3
1
8

ACCEPTED MANUSCRIPT
1
solid (54.7%): H NMR (300 MHz, CDCl ) δ 7.67 (s, 1H), 7.40-7.35 (m, 5H), 7.26-7.21 (m, 3H),
3
6
.98 (d, J=8.6Hz, 2H), 6.90-6.86 (m, 2H), 6.79 (d, J=8.6Hz, 2H), 6.40 (s, 1H), 5.02 (s, 2H), 4.66
+
(
s, 2H), 4.54 (s, 2H), 3.80 (s, 3H); MS(ESI): 557.0[M+H] .
3
-(2,4-Bis(benzyloxy)phenyl)-4-(4-methoxyphenyl)isoxazol-5-amine (47). Yellow oil (17.8%):
1
H NMR (300MHz, CDCl ) δ 7.45-7.32 (m, 6H), 7.25-7.19 (m, 3H), 7.00-6.92 (m, 4H), 6.77 (d,
3
J=8.8Hz, 2H), 6.62 (dd, J=1.3, 8.8Hz, 1H), 6.48 (d, J=2.0Hz, 1H), 5.01 (s, 2H), 4.72 (s, 2H), 4.52
+
(
s, 2H), 3.78 (s, 3H); MS(ESI): 479.0[M+H] .
3
-(2,4-Bis(benzyloxy)-5-ethylphenyl)-4-(4-methoxyphenyl)isoxazol-5-amine (48). Yellow oil
1
(
36.9%): H NMR (300MHz, CDCl ) δ 7.41-7.30 (m, 6H), 7.24-7.20 (m, 3H), 6.99 (d, J=8.9Hz,
3
2
2
3
H), 6.96-6.91 (m, 2H), 6.78 (d, J=8.9Hz, 2H), 6.39 (s, 1H), 4.95 (s, 2H), 4.67 (s, 2H), 4.50 (s,
+
H), 3.78 (s, 3H), 2.63 (q, J =7.8 Hz , 2H), 1.18 (t, J =7.8 Hz, 3H); MS(ESI): 507.0[M+H] .
-(2,4-Bis(benzyloxy)-5-isopropylphenyl)-4-(4-methoxyphenyl)isoxazol-5-amine (49). ellow
1
oil (37.1%): H NMR (300MHz, CDCl ) δ 7.40-7.30 (m, 5H), 7.27 (s, 1H), 7.23-7.21 (m, 3H),
3
6
.97 (d, J=8.1Hz, 2H), 6.96-6.92 (m, 2H), 6.78 (d, J=8.1Hz, 2H), 6.38 (s, 1H), 4.94 (s, 2H), 4.68
+
(
s, 2H), 4.49 (s, 2H), 3.78 (s, 3H), 3.29 (m, 1H), 1.18 (d, 6H); MS(ESI): 521.0[M+H] .
3
-(2,4-Bis(benzyloxy)-5-(tert-butyl)phenyl)-4-(4-methoxyphenyl)isoxazol-5-amine
(50).
1
Yellow solid (21.3%): H NMR (300MHz, CDCl ) δ 7.39-7.36 (m, 5H), 7.32 (s, 1H), 7.24-7.21
3
(m, 3H), 6.99 (d, J=8.6Hz, 2H), 6.96-6.92 (m, 2H), 6.79 (d, J=8.6Hz, 2H), 6.40 (s, 1H), 4.96 (s,
+
2
H), 4.68 (s, 2H), 4.49 (s, 2H), 3.79 (s, 3H), 1.33 (d, 9H); MS(ESI): 535.0[M+H] .
Compounds 51-55 were prepared according to the procedure of 11-22.
N-acetyl-N-(3-(2,4-bis(benzyloxy)-5-bromophenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)acetam
ide (51). Yellow solid (90%): 1H NMR (400 MHz, CDCl3) δ 7.71 (s, 1H), 7.41-7.33 (m, 5H),
7
3
.27-7.19 (m, 3H), 7.00-6.86 (m, 4H), 6.76-6.70 (m, 2H), 6.44 (s, 1H), 5.07 (s, 2H), 4.62 (s, 2H),
.77 (s, 3H), 2.15-1.93 (m, 2H), 1.07-0.98 (m, 4H) , 0.87-0.77 (m, 4H); MS(EI): 692,694[M]+.
N-acetyl-N-(3-(2,4-bis(benzyloxy)phenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)acetamide (52).
1
Yellow solid (74%): H NMR (300MHz, CDCl ) δ 7.45-7.32 (m, 6H), 7.25-7.19 (m, 3H),
3
7
5
.00-6.92 (m, 4H), 6.77 (d, J=8.8Hz, 2H), 6.62 (dd, J=1.3, 8.8Hz, 1H), 6.48 (d, J=2.0Hz, 1H),
+
.01 (s, 2H), 4.72 (s, 2H), 3.78 (s, 3H) , 2.25 (s, 6H); MS(EI): 562[M] .
N-acetyl-N-(3-(2,4-bis(benzyloxy)-5-ethylphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)acetamid
1
e (53). Yellow solid (76.3%): H NMR (300MHz, CDCl ) δ 7.41-7.36 (m, 5H), 7.30 (s, 1H),
3
1
9

ACCEPTED MANUSCRIPT
7
5
3
.25-7.22 (m, 3H), 7.00-6.96 (m, 2H), 6.87 (d, J=8.7Hz, 2H), 6.72 (d, J=8.7Hz, 2H), 6.45 (s, 1H),
.01 (s, 2H), 4.66 (s, 2H), 3.76 (s, 3H), 2.66 (q, J =7.8 Hz , 2H), 2.26 (s, 6H), 1.21 (d, J=7.8Hz,
+
H); MS(EI): 590[M] .
N-acetyl-N-(3-(2,4-bis(benzyloxy)-5-(tert-butyl)phenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)ac
+
etamide (54). Yellow solid (80.4%): MS(EI): 618[M] .
Compounds 56-59 were prepared according to the procedure of 23-35.
N-(3-(5-bromo-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)acetamide
(56).
1
Amorphous white solid (36.7%): H NMR (400MHz, CD OD) δ 7.20 (s, 1H), 7.13 (d, J=8.4Hz,
3
+
2
H), 6.90 (d, J=8.4Hz, 2H), 6.44 (s, 1H), 3.78 (s, 3H), 2.08 (s, 3H); MS(ESI): 420.9[M+H] .
N-(3-(2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)acetamide (57). White solid
1
(
77.7%): H NMR (400MHz, CD OD) δ 7.13 (d, J=8.7Hz, 2H), 6.93 (d, J=8.9Hz, 1H), 6.87 (d,
3
J=8.7Hz, 2H), 6.32 (d, J=2.5Hz, 1H), 6.23 (dd, J=2.5, 8.9Hz, 1H), 3.76(s, 3H), 2.08 (s, 3H);
+
MS(ESI): 341.0[M+H] .
N-(3-(5-ethyl-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)acetamide
(58).
1
Amorphous white solid (59.5%): H NMR (400MHz, CD OD) δ 7.16 (d, J=8.4Hz, 2H), 6.91 (d,
3
J=8.4Hz, 2H), 6.78 (s, 1H), 6.33 (s, 1H), 3.79 (s, 3H), 2.36 (q, J=7.3Hz, 2H), 2.08 (s, 3H), 0.94 (t,
+
J=7.3Hz, 3H); MS(ESI): 369.0[M+H] .
N-(3-(5-(tert-butyl)-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)acetamide (59).
1
Amorphous white solid (87.4%): H NMR (400MHz, CD OD) δ 7.17 (d, J=8.4Hz, 2H), 6.95 (d,
3
J=8.4Hz, 2H), 6.90 (s, 1H), 6.33 (s, 1H), 3.80 (s, 3H), 2.07 (s, 3H), 1.09 (s, 9H); MS(ESI):
+
3
97.0[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-methoxyphenyl)isoxazol-5-yl)cyclopropanecarb
oxamide (60). Compound 49 (0.2 g, 0.4 mmol) and triethylamine (1.13 mL, 8 mmol) were
dissolved in 20 mL dry dichloromethane. Catalytic amount of DMAP was added to the mixture
followed by the addition of cyclopropylcarboxyl chloride (0.21 mg, 2 mmol) dropwise. When
stirred for 2 hours at room temperature, the mixture was diluted with dichloromethane. The
organic layer was washed with saturate citric acid/water (1:1, 30 mL×2), water (30mL×1),
sat.NaHCO (30mL×2), brine, dried over Na SO and evaporated in vacuo. The crude product was
3
2
4
purified by flash chromatography on silica gel to afford the intermediates 55. Compound 55 (0.1 g,
.16 mmol) was dissolve in dry dichloromethane (20 mL) followed by addition of 1N BCl in
0
3
2
0

ACCEPTED MANUSCRIPT
dichloromethane (0.5 mL) under nitrogen. The mixture was stirred for 2 hours at room
temperature and then diluted with ethyl acetate. The organic layer was washed with sat.NaHCO3
(
30mL×2), brine, dried over Na SO and evaporated in vacuo. The crude product was purified by
2 4
1
flash chromatography on silica gel to afford the final product 60 (two steps totally 47.6%): H
NMR (400MHz, CD OD) δ 7.16 (d, J=8.2Hz, 2H), 6.94 (d, J=7.8Hz, 2H), 6.81 (s, 1H), 6.33 (s,
3
+
1
H), 3.79 (s, 3H), 3.03 (m, 1H), 1.75 (m, 1H), 0.90 (d, 10H); MS(ESI): 409.1[M+H] .
1
-(2,4-Bis(benzyloxy)phenyl)ethanone (62). Benzyl bromide (483 mL, 4 mol) was added to the
suspension of 2,4-dihydroxyacetophenone (280 g, 1.84 mol) and potassium carbonate anhydrous
560 g, 4 mol) in acetonitrile (1.4 L) dropwise. The mixture was refluxed overnight and cooled to
(
ambient temperature. Most of the solvent was removed in vacuo. The residue was added water (2
L) and stirred at room temperature. White solid was formed. The solid was filtered, washed with
1
water followed by petroleum and dried to yield title compound as white solid (590 g, 96.5%): H
NMR (300 MHz, CDCl ) δ 7.85 (d, J=9.3Hz, 1H), 7.44-7.35 (m, 10H), 6.63-6.60 (m, 2H), 5.11 (s,
3
+
2
H), 5.08 (s, 2H), 2.55 (s, 3H); MS(EI): 332[M] .
(((4-(Prop-1-en-2-yl)-1,3-phenylene)bis(oxy))bis(methylene))dibenzene
(63).
Methyltriphenylphosphonium bromide (826 g, 2.31 mol) was suspended in dry tetrahydrofuran
o
under nitrogen. The suspension was cooled to -5 C and added 2.5 M n-Butyllithium in hexanes
(924 mL, 2.31 mol). After stirred for 0.5 hour the mixture was added compound 62 (590 g, 1.78
o
mol) in dry tetrahydrofuran and stirred for another 0.5 hour at -5 C. The mixture was warmed to
ambient temperature and stirred overnight. Methanol was added to stop the reaction. The solvent
was removed in vacuo. The residue was separated in hexane and water. The organic layer was
concentrated. The crude product was purified by flash chromatography on silica gel to afford the
1
final product as white wax (466g, 79.5%): H NMR (300MHz, CDCl ) δ 7.44-7.29 (m, 10H), 7.14
3
(d, J=8.3Hz, 1H), 6.59 (d, J=2.5Hz, 1H), 6.54 (m, 1H), 5.07 (s, 2H), 5.05 (s,2H), 5.03 (s, 2H),
+
2
.12 (s, 3H); MS(EI): 330[M] .
4
-Isopropylbenzene-1,3-diol (64). Compound 63 (466 g, 1.4 mol) was dissolved in mixed solvent
of dichloromethane/ethanol (1:50, v/v), followed the addition of 10% palladium on carbon (100 g).
o
The mixture was stirred at 60 C under 20 atm. hydrogen pressures for 8 hours. The mixture was
filtered, concentrated and purified by flash chromatography on silica gel to afford the final product
1
as white solid (178 g, 82.9%): H NMR (300 MHz,CDCl ) δ 7.03 (d, J=8.3Hz, 1H), 6.38 (d,
3
2
1

ACCEPTED MANUSCRIPT
J=8.3Hz 1H), 6.29 (d, J=2.5Hz, 1H), 4.74 (brs, 1H), 4.63 (brs, 1H), 3.09 (m,1H), 1.22 (d, 6H);
+
MS(EI): 152[M] .
2
,4-Dihydroxy-5-isopropylbenzaldehyde (65). Phosphorus oxychloride (155 mL, 1.64 mol) was
o
added to 350 mL dry dimethylformamide at -5 C and stirred for 0.5 hour. The mixture was added
compound 64 (100g, 0.65mol) in 200 mL dry dimethylformamide and stirred at room temperature
o
for 1 hour and at 50 C for another 1 hour. After cooled down the mixture was poured into sodium
o
hydroxide (315 g) in 2 L water slowly and stirred at 70 C for 15 minutes. The mixture was cooled
to room temperature and acidified to pH 2-3 by concentrated HCl. The aqueous layer was
extracted with ethyl acetate. The organic layer was washed with brine, dried over Na SO ,
2
4
concentrated and purified by flash chromatography on silica gel to afford the final product as
1
white solid (86.65 g, 73.2%): H NMR(300MHz, CDCl ) δ 11.29 (s, 1H), 9.70 (s, 1H), 7.34 (s,
3
+
1
H), 6.60 (s, 1H), 6.36 (s, 1H), 3.18 (m, 1H), 1.26 (d, 6H); MS(EI): 180[M] .
2
,4-Bis(benzyloxy)-5-isopropylbenzaldehyde (66). Compound 66 was prepared according to the
1
procedure of 62 (82.7%): H NMR(300MHz, CDCl ) δ 10.40 (s, 1H), 7.76 (s, 1H), 7.43-7.33 (s,
3
1
3
0H), 6.52 (s, 1H), 6.36 (s, 1H), 5.13 (s, 2H), 5.11 (s, 2H), 3.29 (m, 1H), 1.23 (d, 6H); MS(EI):
+
60[M] .
(2,4-Bis(benzyloxy)-5-isopropylbenzaldehyde oxime (67). Compound 66 (143.2 g, 0.4 mol),
hydroxylamine hydrochloride (55.5 g, 0.8 mol) and sodium hydroxide (32 g, 0.8 mol) were
o
suspended in the mixture of ethanol 600 mL and water 300 mL. The mixture was stirred at 100 C
for 5 hours and cooled to room temperature. Most of the solvent was removed in vacuo. The
residue was added 2 L water and stirred for 0.5 hour. The precipitant was filtered, washed with
1
water and petroleum and dried to yield the title compound as white solid (140.1 g, 93.9%): H
NMR(300MHz, CDCl ) δ 8.51 (s, 1H), 7.62 (s, 1H), 7.41-7.31 (s, 10H), 6.51 (s, 1H), 5.05 (d, 4H) ,
3
+
3
.30 (m, 1H), 1.22 (d, 6H); MS(EI): 375[M] .
4
-(Cyanomethyl)benzoic acid (70). Methyl 4-(cyanomethyl)benzoate (25 g, 0.14 mol) dissolved
in 300 mL methanol was add 160 mL 1 N NaOH and refluxed for3 hours. Most of the solvent was
removed in vacuo. The residue was added 500 mL water and acidified to pH 2-3 with
concentrated HCl. The generated solid was filtered, washed with water and dried to give the title
1
compound as white solid (21.2 g, 92.2%): H NMR (400 MHz, DMSO-d ) δ 12.89 (brs, 1H), 7.87
6
+
(
d, J=8.2Hz, 2H), 7.37 (d, J=8.2Hz, 2H), 3.45 (s, 2H); MS(EI): 161[M] .
2
2

ACCEPTED MANUSCRIPT
2
-(4-(Hydroxymethyl)phenyl)acetonitrile (71). 1,1'-Carbonyldiimidazole (23.3 g, 0.14 mol) was
added to compound 70 (21.2 g, 0.13 mol) in 600 mL dry tetrahydrofuran portion-wise. The
mixture was stirred at room temperature till no bubble was observed. The mixture was added
sodium borohydride (15 g, 0.39 mol) in 800 mL water dropwise and stirred at room temperature
for 4 hours. The mixture was extracted with ethyl acetate. The organic layer was washed with
brine, dried over Na SO , concentrated and purified by flash chromatography on silica gel to
2
4
1
afford the final product as colorless oil (14.36 g, 74.2%): H NMR(300MHz, CDCl ) δ 7.37 (d,
3
+
J=8.5Hz, 2H), 7.30 (d, J=8.5Hz, 2H), 4.68 (s, 2H), 3.74 (s, 2H); MS(EI): 147[M] .
2
-(4-(((Tetrahydro-2H-pyran-2-yl)oxy)methyl)phenyl)acetonitrile (72). Compound 71 (14.36
g, 98 mmol), 3,4-Dihydro-2H-pyran (10.05 mL, 110 mmol) and p-toluenesulfonic acid
monohydrate (0.345 g, 1.8 mmol) were dissolved in 400 mL dichloromethane and stirred at room
temperature for 1 hour. The mixture was diluted with dichloromethane, washed with saturated
sodium bicarbonate (300 mL×2), brine (300 mL), dried over Na SO , concentrated and purified by
2
4
1
flash chromatography on silica gel to afford the final product as colorless oil ( 20.11 g, 89.1%): H
NMR(300MHz, CDCl ) δ 7.38 (d, J=8.3Hz, 2H), 7.30 (d, J=8.3Hz, 2H), 4.78 (d, J=11.2Hz, 1H),
3
4
1
3
.69 (t, J=3.4Hz, 1H), 4.49 (d, J=11.2Hz, 1H), 3.93-3.87 (m, 1H), 3.74 (s, 2H), 3.58-3.51 (m, 1H),
+
.90-1.80 (m, 1H), 1.78-1.70 (m, 1H), 1.68-1.51 (m, 4H); MS(EI): 231[M] .
-(2,4-Bis(benzyloxy)-5-isopropylphenyl)-4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)phen
yl)isoxazol-5-amine (73). Compound 67 (140 g, 0.37 mol) dissolved in 500 mL dry
dimethylformamide was added N- chlorosuccinimide (60 g, 0.45 mol). After stirred at room
temperature for 5 hours, the mixture was diluted with dichloromethane, washed with brine, dried
over Na SO and concentrated. The crude product of compound 68 was unstable and used
2
4
immediately in the next step without further purification. Compound 72 (180 mg, 0.78 mmol)
dissolved in 300 mL dry tetrahydrofuran was added tert-butyllithium (1.6 M in pentane, 1 mL, 1.6
o
mmmol) at -78 C under nitrogen and stirred for 20 minutes followed by the addition of compound
o
6
8 (320 mg, 0.78 mmol) in 100 mL dry tetrahydrofuran. The mixture was kept stirring at -78 C
for 30 minutes and added 50 mL water to stop the reaction. Most of the tetrahydrofuran was
removed and the residue was extracted with dichloromethane. The organic layer was washed with
brine, dried over Na SO , concentrated and purified by flash chromatography on silica gel to
2
4
1
afford the final product as yellow solid (286 mg, 60.7%). H NMR(300MHz, CDCl ) δ 7.40-7.19
3
2
3

ACCEPTED MANUSCRIPT
(m, 11H), 7.03 (d, J=8.1Hz, 2H), 6.92-6.88 (m, 2H), 6.39 (s, 1H), 4.94 (s, 2H), 4.77 (d, J=11.9Hz,
1
3
6
H), 4.72 (t, J=3.4Hz, 1H), 4.65 (s, 2H), 4.58 (s, 2H), 4.46 (d, J=11.9Hz, 1H), 3.97-3.89 (m, 1H),
.60-3.53 (m, 1H), 3.30 (m, 1H), 1.92-1.81 (m, 1H), 1.79-1.71 (m, 1H), 1.69-1.51 (m, 4H), 1.18 (d,
H).
N-(3-(2,4-bis(benzyloxy)-5-isopropylphenyl)-4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)p
henyl)isoxazol-5-yl)-N-(cyclopropanecarbonyl)cyclopropanecarboxamide (74). Compound 73
(0.2 g, 0.33 mmol) and triethylamine (1.13 mL, 8 mmol) were dissolved in 100 mL dry
dichloromethane. Catalytic amount of DMAP was added to the mixture followed by the addition
of cyclopropylcarboxyl chloride (0.21 mg, 2 mmol) dropwise. When stirred for 2 hours at room
temperature, the mixture was diluted with dichloromethane. The organic layer was washed with
saturate citric acid/water (1:1, 30 mL×2), water (30mL×1), sat.NaHCO (30mL×2), brine, dried
3
over Na SO and evaporated in vacuo. The crude product was purified by flash chromatography
2
4
1
on silica gel to afford title compound as yellow solid (204 mg, 83.3%): H NMR(300MHz, CDCl )
3
δ 7.42-7.32 (m, 6H), 7.25-7.19 (m, 5H), 7.03 (d, J=7.8Hz, 2H), 6.98-6.93 (m, 2H), 6.46 (s, 1H),
5
3
4
.01 (s, 2H), 4.77 (d, J=12.5Hz, 1H), 4.71 (t, J=3.6Hz, 1H), 4.65 (s, 2H), 4.47 (d, J=12.1Hz, 1H),
.96-3.88 (m, 1H), 3.60-3.51 (m, 1H), 3.30 (m, 1H), 1.91-1.52 (m, 8H), 1.21 (d, 6H), 0.98-0.92 (m,
+
H), 0.87-0.82 (m, 4H); MS(ESI): 741.3[M+H] .
N-(3-(2,4-bis(benzyloxy)-5-isopropylphenyl)-4-(4-(hydroxymethyl)phenyl)isoxazol-5-yl)-N-(c
yclopropanecarbonyl)cyclopropanecarboxamide (75). Compound 74 (2g, 2.7mmol) and
p-toluenesulfonic acid monohydrate (0.05 g, 0.27 mmol) was dissolved in methanol 100 mL and
tetrahydrofuran 100 mL and stirred at room temperature for 1 hour. The mixture was diluted with
dichloromethane, washed with saturated sodium bicarbonate (100 mL×2), brine (100 mL), dried
over Na SO , concentrated and purified by flash chromatography on silica gel to afford the final
2
4
1
product as yellow solid (1.325 g, 74.7%). H NMR(300MHz, CDCl ) δ 7.42-7.33 (m, 6H),
3
7
4
6
4
.26-7.18 (m, 5H), 7.03 (d, J=8.1Hz, 2H), 6.99-6.95 (m, 2H), 6.46 (s, 1H), 5.02 (s, 2H), 4.66 (s,
H), 3.30 (m, 1H), 2.10-2.00 (m, 2H), 1.22 (d, 6H), 1.08-1.02 (m, 4H), 0.88-0.81 (m, 4H); MS(EI):
+
56[M] .
-(3-(2,4-Bis(benzyloxy)-5-isopropylphenyl)-5-(cyclopropanecarboxamido)isoxazol-4-yl)benz
yl methanesulfonate (76). Compound 75 (200mg, 0.3mmol) and triethylamine (1.13 mL, 8 mmol)
dissolved in dry dichloromethane was added methanesulfonyl chloride (0.116 mL, 1.5 mmol) and
2
4

ACCEPTED MANUSCRIPT
stirred at room temperature for 2 hours. The mixture was diluted with dichloromethane, washed
with saturate citric acid/water (1:1, 100 mL×2), water (100mL×1), sat.NaHCO (100mL×2), brine,
3
dried over Na SO and concentrated to give the title compound as pale-yellow solid (134mg,
2
4
6
7.7%). The compound was used without purification.
General procedure for the synthesis of 93-108. Compound 76 (1 mmol) and amines (2.5 eq) were
dissolved in 20 mL acetonitrile and stirred overnight at room temperature. The mixture was
concentrated and added 50 mL water. The aqueous layer was extracted with dichloromethane. The
organic layer was washed with brine, dried over Na SO and concentrated to give 77-92 without
2
4
purification. Compound 77-92 (1 eq) was dissolve in dry dichloromethane followed by addition of
N BCl in dichloromethane (3 eq) under nitrogen. The mixture was stirred for 2 hours at room
1
3
temperature and then diluted with ethyl acetate. The organic layer was washed with sat.NaHCO3,
brine, dried over Na SO and evaporated in vacuo. The crude product was purified by flash
2
4
chromatography on silica gel to afford the final product 93-108.
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(morpholinomethyl)phenyl)isoxazol-5-yl)cyclop
1
ropanecarboxamide (93). White solid (44.1%): H NMR (400MHz, CD OD) δ 7.39 (d, J=7.8Hz,
3
2
H), 7.25 (d, J=7.8Hz, 2H), 6.80 (s, 1H), 6.33 (s, 1H), 3.71 (t, J=4.5Hz, 4H), 3.67 (s, 2H), 3.03 (m,
1
3
6
1
H), 2.60 (brs, 4H), 1.74 (m, 1H), 0.89 (m, 10H); C NMR (100MHz, d -DMSO) δ 173.0, 161.5,
1
57.3, 156.7, 154.0, 131.3, 131.2, 128.1, 127.8, 127.6, 125.4, 110.0, 106.3, 102.6, 66.4, 63.0, 58.6,
+
+
5
0.6, 25.6, 22.6, 7.9; MS(ESI): 478.2[M+H] ; HRMS calcd for C H N O : 478.2342 (M+H)
27 32 3 5
Found: 478.2338.
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(piperidin-1-ylmethyl)phenyl)isoxazol-5-yl)cycl
1
opropanecarboxamide (94). White solid (29.4%): H NMR (400MHz, CD OD) δ 7.47 (d,
3
J=8.2Hz, 2H), 7.32 (d, J=8.2Hz, 2H), 6.82 (s, 1H), 6.32 (s, 1H), 4.13 (s, 2H), 3.10-3.01 (m, 5H),
+
1
.83-1.73 (m, 5H), 1.63 (brs, 2H), 0.95 (m, 6H), 0.87 (m, 4H); MS(ESI): 476.2[M+H] .
N-(4-(4-((diethylamino)methyl)phenyl)-3-(2,4-dihydroxy-5-isopropylphenyl)isoxazol-5-yl)cyc
1
lopropanecarboxamide (95). White solid (38.8%): H NMR (400MHz, CD OD) δ 7.49 (d,
3
J=7.9Hz, 2H), 7.34 (d, J=7.9Hz, 2H), 6.84 (s, 1H), 6.33 (s, 1H), 4.24 (s, 2H), 3.15-3.03 (m, 5H),
1
.76 (m, 1H), 1.30 (t, J=7.3Hz, 6H), 0.97 (d, J=6.7Hz, 6H), 0.88 (d, J=6.0Hz, 4H); MS(ESI):
+
4
64.1[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-methylpiperazin-1-yl)methyl)phenyl)isoxaz
2
5

ACCEPTED MANUSCRIPT
1
ol-5-yl)cyclopropanecarboxamide (96). White solid (28.5%): H NMR (400MHz, CD OD) δ
3
7
1
4
.37 (d, J=8.2Hz, 2H), 7.24 (d, J=8.2Hz, 2H), 6.80 (s, 1H), 6.34 (s, 1H), 3.60 (s, 2H), 3.04 (m,
H), 2.92 (brs, 4H), 2.64 (brs, 4H), 2.60 (s, 3H), 1.75 (m, 1H), 0.92-0.85 (m, 10H); MS(ESI):
+
91.3[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(thiomorpholinomethyl)phenyl)isoxazol-5-yl)cy
1
clopropanecarboxamide (97). White solid (36.4%): H NMR (400MHz, CD OD) δ 7.35 (d,
3
J=8.3Hz, 2H), 7.22 (d, J=8.3Hz, 2H), 6.80 (s, 1H), 6.34 (s, 1H), 3.56 (s, 2H), 3.03 (m, 1H),
+
2
.75-2.71 (m, 4H), 2.67-2.63 (m, 4H), 1.74 (m, 1H), 0.91-0.85 (m, 10H); MS(ESI): 494.2[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((diisopropylamino)methyl)phenyl)isoxazol-5-y
1
l)cyclopropanecarboxamide (98). White solid (36.6%): H NMR (400MHz, CD OD) δ 7.47 (d,
3
J=8.1Hz, 2H), 7.30 (d, J=8.1Hz, 2H), 6.84 (s, 1H), 6.32 (s, 1H), 4.20 (brs, 2H), 3.58 (brs, 2H),
3
.06 (m, 1H), 1.75 (m, 1H), 1.36-1.26 (m, 12H), 0.96 (d, 6H), 0.87 (d, 4H); MS(ESI):
+
4
92.2[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((methyl(phenethyl)amino)methyl)phenyl)isoxa
1
zol-5-yl)cyclopropanecarboxamide (99). White solid (24.7%): H NMR (400MHz, CD OD) δ
3
7
2
5
.37 (d, J=8.2Hz, 2H), 7.29-7.24 (m, 4H), 7.21-7.16 (m, 3H), 6.82 (s, 1H), 6.33 (s, 1H), 3.81 (s,
H), 3.02 (m, 1H), 2.93-2.80 (m, 4H), 2.45 (s, 3H), 1.74 (m, 1H), 0.92-0.86 (m, 10H); MS(ESI):
+
26.3[M+H] .
N-(4-(4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)-3-(2,4-dihydroxy-5-isopropylphen
1
yl)isoxazol-5-yl)cyclopropanecarboxamide (100). White solid (28.6%): H NMR (400MHz,
CD OD) δ 7.44 (d, J=7.7Hz, 2H), 7.26 (d, J=7.7Hz, 2H), 7.12-7.09 (s, 3H), 7.01-6.97 (s, 1H),
3
6
.82 (s, 1H), 6.34 (s, 1H), 3.76 (s, 2H), 3.68 (s, 2H), 3.02 (m, 1H), 2.91 (t, J=5.8Hz, 2H), 2.81 (t,
+
J=5.4Hz, 2H), 1.75 (m, 1H), 0.92-0.84 (m, 10H); MS(ESI): 524.3[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((2,6-dimethylmorpholino)methyl)phenyl)isoxa
1
zol-5-yl)cyclopropanecarboxamide (101). White solid (25.7%): H NMR (400MHz, CD OD) δ
3
7
.36 (d, J=8.3Hz, 2H), 7.23 (d, J=8.3Hz, 2H), 6.81 (s, 1H), 6.34 (s, 1H), 3.72-3.64 (m, 2H), 3.54
(s, 2H), 3.03 (m, 1H), 2.77 (d, J=10.7Hz, 2H), 1.82-1.71 (m, 3H), 1.11 (d, 6H), 0.91-0.85 (m,
+
1
0H); MS(ESI): 506.3[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((3,5-dimethylpiperidin-1-yl)methyl)phenyl)iso
1
xazol-5-yl)cyclopropanecarboxamide (102). White solid (33.7%): H NMR (400MHz, CD OD)
3
2
6

ACCEPTED MANUSCRIPT
δ 7.43 (d, J=8.4Hz, 2H), 7.29 (d, J=8.4Hz, 2H), 6.83 (s, 1H), 6.34 (s, 1H), 3.93 (s, 2H), 3.12-3.03
+
(
m, 3H), 2.08-2.01 (m, 2H), 1.86-1.74 (m, 5H), 0.95-0.86 (m, 16H); MS(ESI): 502.3[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((2-methylpiperidin-1-yl)methyl)phenyl)isoxazo
1
l-5-yl)cyclopropanecarboxamide (103). White solid (42.9%): H NMR (400MHz, CD OD) δ
3
7
3
3
.46 (d, J=8.1Hz, 2H), 7.32 (d, J=8.1Hz, 2H), 6.83 (s, 1H), 6.33 (s, 1H), 4.44 (d, J=12.9Hz, 1H),
.87 (d, J=12.9Hz, 1H), 3.13-3.02 (m, 3H), 2.69-2.61 (m, 1H), 1.93-1.86 (m, 1H), 1.82-1.72 (m,
+
H), 1.69-1.57 (m, 2H), 1.54-1.42 (m, 4H), 0.95 (d, 6H), 0.88 (d, 4H); MS(ESI): 490.3[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-(dimethylamino)piperidin-1-yl)methyl)phen
1
yl)isoxazol-5-yl)cyclopropanecarboxamide (104). White solid (34.7%): H NMR (400MHz,
CD OD) δ 7.36 (d, J=8.2Hz, 2H), 7.24 (d, J=8.2Hz, 2H), 6.79 (s, 1H), 6.34 (s, 1H), 3.61 (s, 2H),
3
3
.20-2.99 (m, 4H), 2.82 (s, 6H), 2.18 (t, J=12.4Hz, 2H), 2.06 (d, J=10Hz, 2H), 1.80-1.68 (m, 3H),
+
0
.95-0.83 (m, 10H); MS(ESI): m/z 519.3[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(((2-(dimethylamino)ethyl)(methyl)amino)meth
1
yl)phenyl)isoxazol-5-yl)cyclopropanecarboxamide (105). White solid (34.5%): H NMR
(
400MHz, CD OD) δ 7.43 (d, J=7.6Hz, 2H), 7.26 (d, J=7.6Hz, 2H), 6.84 (s, 1H), 6.33 (s, 1H),
3
3
6
.74 (brs, 2H), 3.13-3.02 (m, 3H), 2.76 (brs, 2H), 2.67 (s, 6H), 2.34 (s, 3H), 1.76 (m, 1H), 0.94 (d,
+
H), 0.91-0.86 (m, 4H); MS(ESI): 491.3[M+H] .
N-(4-(4-((cyclohexyl(methyl)amino)methyl)phenyl)-3-(2,4-dihydroxy-5-isopropylphenyl)isox
1
azol-5-yl)cyclopropanecarboxamide (106). White solid (43.7%): H NMR (400MHz, CD OD) δ
3
7
1
2
.48 (d, J=8.3Hz, 2H), 7.35 (d, J=8.3Hz, 2H), 6.85 (s, 1H), 6.32 (s, 1H), 4.28 (s, 2H), 3.23 (m,
H), 3.08 (m, 1H), 2.68 (s, 3H), 2.11 (d, J=11.3Hz, 2H), 1.95 (d, J=11.3Hz, 2H), 1.80-1.69 (m,
H), 1.64-1.52 (m, 2H), 1.43-1.31 (m, 2H), 1.30-1.21 (m, 1H), 0.99 (d, 6H), 0.90-0.85 (m, 4H);
+
MS(ESI): 504.3[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-(pyrrolidin-1-yl)piperidin-1-yl)methyl)phen
1
yl)isoxazol-5-yl)cyclopropanecarboxamide (107). White solid (27.5%): H NMR (400MHz,
CD OD) δ 7.37 (d, J=8.0Hz, 2H), 7.24 (d, J=8.0Hz, 2H), 6.80 (s, 1H), 6.35 (s, 1H), 3.59 (s, 2H),
3
3
.40-3.29 (brs, 4H), 3.16-2.99 (m, 4H), 2.20-2.02 (m, 8H), 1.80-1.68 (m, 3H), 0.93-0.84 (m, 10H);
+
MS(ESI): 545.4[M+H] .
N-(3-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-((4-morpholinopiperidin-1-yl)methyl)phenyl)is
oxazol-5-yl)cyclopropanecarboxamide (108). White solid (30.3%). The hydrochloride salt was
2
7

ACCEPTED MANUSCRIPT
prepared as follows: 400 mg compound 108 was added 25 mL hydrogen chloride solution 4.0 M
in dioxane. The solution was stirred for 4h at room temperature before the solvent was removed.
1
The residue was dried in vacuo to give the final product (100%). H NMR (400MHz, CD OD) δ
3
7
3
0
1
4
5
.39 (d, J=8.0Hz, 2H), 7.25 (d, J=8.0Hz, 2H), 6.80 (s, 1H), 6.32 (s, 1H), 3.69 (t, J=4.8Hz, 4H),
.56 (s, 2H), 3.02 (m, 3H), 2.58 (m, 4H), 2.22 (m, 1H), 2.08 (m, 2H), 1.75 (m, 1H), 1.53 (m, 2H),
1
3
6
.95-0.84 (m, 10H); C NMR (100MHz, d -DMSO) δ 172.9, 161.4, 157.3, 156.7, 154.0, 131.3,
31.2, 128.2, 128.0, 127.6, 125.4, 110.1, 106.3, 102.7, 72.3, 71.9, 66.4, 63.2, 62.7, 60.2, 59.6, 58.2,
+
9.5, 48.4, 25.6, 22.9, 22.6, 13.7, 7.9; MS(ESI): 561.3[M+H] ; HRMS calcd for C H N O :
3
2
41
4
5
61.3077 (M+H)+ Found: 561.3074.
Acknowledgement
The authors wish to thank Dr. Haiyan Liu for her help in performing the in vitro PK study.
Grateful thanks for financial support from “Interdisciplinary Cooperation Team” Program for
Science and Technology Innovation of the Chinese Academy of Sciences; The National Natural
Science Foundation of China (Grant No. 81072580, 81273368 and 21272246);
the National Science & Technology Major Project "Key New Drug Creation and Manufacturing Pr
ogram" of China (Grant No. 2014ZX09507-002); China Marine Commonweal Research Project
(201005022-5); National Science and Technology Major Project of the Ministry of Science and
Technology of China(No.2012ZX09301001-007 to MG).
References and Notes
[
1] M.E. Feder, G.E. Hofmann, HEAT-SHOCK PROTEINS, MOLECULAR CHAPERONES,
AND THE STRESS RESPONSE: Evolutionary and Ecological Physiology, Annu. Rev. Physiol.
1 (1999) 243-282.
2] D. Picard, Heat-shock protein 90, a chaperone for folding and regulation, Cell. Mol. Life Sci.
9 (2002) 1640-1649.
3] C. Garrido, S. Gurbuxani, L. Ravagnan, G. Kroemer, Heat Shock Proteins: Endogenous
Modulators of Apoptotic Cell Death, Biochem. Biophys. Res. Commun. 286 (2001) 433-442.
4] L. Neckers, P. Workman, Hsp90 Molecular Chaperone Inhibitors: Are We There Yet? Clin.
Cancer Res. 18 (2012) 64-76.
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