Amide-tethered quinoline-resorcinol conjugates as a new class of HSP90 inhibitors suppressing the growth of prostate cancer cells

Article abstract of DOI:10.1016/j.bioorg.2019.103119

The study is focused on the design and synthesis of amide tethered quinoline-resorcinol hybrid constructs as a new class of HSP90 inhibitor. In-vitro studies of the synthetic compounds led to the identification of compound 11, which possesses potent cell growth inhibitory effects against HCT116, Hep3B and PC-3 cell lines, exerted through HSP90 inhibition. Compound 11 triggers degradation of HSP90 client proteins along with concomitant induction of HSP70, demonstrates apoptosis inducing ability and causes G2M phase cell cycle arrest in PC-3 cells. Molecular modeling was used to dock compound 11 into the HSP90 active site and key interactions with the amino acid residues of the HSP90 chaperone protein were determined.

Full text of DOI:10.1016/j.bioorg.2019.103119

Accepted Manuscript
Amide-Tethered Quinoline-Resorcinol Conjugates as a new class of HSP90 In-
hibitors Suppressing the Growth of Prostate Cancer cells
Kunal Nepali, Mei-Hsiang Lin, Min-Wu Chao, Sheng-Jhih Peng, Kai-Cheng
Hsu, Tony Eight Lin, Mei-Chuan Chen, Mei-Jung Lai, Shiow-Lin Pan, Jing-
Ping Liou
PII:
S0045-2068(19)30530-9
https://doi.org/10.1016/j.bioorg.2019.103119
103119
DOI:
Article Number:
Reference:
YBIOO 103119
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 April 2019
9 July 2019
11 July 2019
Please cite this article as: K. Nepali, M-H. Lin, M-W. Chao, S-J. Peng, K-C. Hsu, T. Eight Lin, M-C. Chen, M-J.
Lai, S-L. Pan, J-P. Liou, Amide-Tethered Quinoline-Resorcinol Conjugates as a new class of HSP90 Inhibitors
Suppressing the Growth of Prostate Cancer cells, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103119
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Bioorganic Chemistry
Amide-Tethered Quinoline-Resorcinol Conjugates as a new class of HSP90
Inhibitors Suppressing the Growth of Prostate Cancer cells
Kunal Nepalia,1, Mei-Hsiang Lina,1, Min-Wu Chaob, Sheng-Jhih Penga, Kai-Cheng Hsub,d, Tony
Eight Linb, Mei-Chuan Chenc, Mei-Jung Laid, Shiow-Lin Panb,d, Jing-Ping Liou*,a,c,d,e
* To whom correspondence should be addressed. J. P. Liou: (Phone) 886-2-2736-1661 ext 6130; (e-
mail) jpl@tmu.edu.tw
1 Contributed equally to this work.
a.
School of Pharmacy, College of Pharmacy, Taipei Medical University.
b.
The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and
Technology, Taipei Medical University, Taipei, Taiwan.
c.
Ph.D. Program in Clinical Drug Development of Herbal Medicine
d.
TMU Biomedical Commercialization Center
e.
School of Pharmacy, National Defense Medical Center, Taipei, Taiwan

Abstract: The study is focused on the design and synthesis of amide tethered quinoline-resorcinol
hybrid constructs as a new class of HSP90 inhibitor. In-vitro studies of the synthetic compounds led
to the identification of compound 11, which possesses potent cell growth inhibitory effects against
HCT116, Hep3B and PC-3 cell lines, exerted through HSP90 inhibition. Compound 11 triggers
degradation of HSP90 client proteins along with concomitant induction of HSP70, demonstrates
apoptosis inducing ability and causes G2M phase cell cycle arrest in PC-3 cells. Molecular modeling
was used to dock compound 11 into the HSP90 active site and key interactions with the amino acid
residues of the HSP90 chaperone protein were determined.
Key words: Quinoline, Resorcinol, Chaperone, Cancer, Apoptosis, Heat shock protein

1. Introduction
Heat Shock Protein 90 (HSP90), an ATP dependent molecular chaperone, has been categorized as a
crucial facilitator of oncogene addiction and cancer cell survival [1]. A chaperone protein acts as a
cellular machine responsible for correct folding and maturation of its client proteins and protects the
cellular proteins from degradation by the ubiquitin-proteasome system in conditions of stress [2-7].
HSP90 has been shown to be overexpressed in solid and hematologic tumors which indicates that the
continued activity of HSP90 is required for oncogene-driven tumorigenesis. Thus inhibition of the
chaperone function of HSP90 may lead to combinatorial targeting of multiple oncogenic protein
pathways and thus may overcome the notorious cancer resistance issue [7, 8]. Over the years, Hsp90
inhibition has emerged as a fascinating chemotherapeutic strategy for the treatment of diverse
malignancies [9-11]. The documented activity in this field supports the need to increase the number of
investigations on HSP90 inhibitors and this, in turn may escalate the probability of developing cancer
therapeutics.
Although HSP90 as a therapeutic target for cancer has been extensively validated using geldanamycin
[12, 13, 14], the inclination of the medicinal chemist has shifted towards the design of second
generation small molecule HSP90 inhibitors (Fig. 1). This could be attributed to limitations such as
poor bioavailability and hepatotoxicity that have hampered the clinical growth of ansamycin
antibiotics [15]. Resorcinol-based chemical architectures represent a prominent class of second
generation HSP90 inhibitors [16-18]. Compounds such as AT-13387 (4), STA-9090 (5) and NVP-

AUY922 (6) exemplify HSP90 inhibitors containing a resorcinol moiety [19]. Despite the extensive
efforts of the medicinal chemist towards the development of HSP90 inhibitors exerting anti-tumor
effects in the last decade, clinical growth of these inhibitors was hampered/limited either by efficacy
or adverse events in higher stage trials in addition to pharmaceutical property and commercialization
issues. [19, 20, 21, 22]. As a result, none of the Hsp90 inhibitors are clinically approved yet. The
termination of Phase III clinical trials of STA-9090 attributed to acquired resistance exemplifies a case
where the clinical advancement of a HSP90 inhibitor was halted by moderate efficacy [19, 23]. Overall,
it can be concluded that despite the documented promise in preclinical studies and early phase clinical
trials, HSP 90 inhibitors have not been able to replicate the effectiveness at higher stage clinical
investigations [20, 24-26]. Experts have indicated that the clinical promise of HSP90 inhibition in
cancer is likely to be improved with combination therapies and molecular stratification of patients
[27]. However, from the medicinal chemist’s perspective, the present scenario in HSP90 inhibitory
field makes it quite prudent to expand the pipeline of rationally designed second generation HSP90
inhibitors as new cancer therapeutics. To accomplish this, the design of Hsp90 inhibitors via fusion of
existing antitumor pharmacophores furnishing hybrid scaffolds as new chemotypes is anticipated to
yield conclusive benefits of HSP90 inhibition in cancer. Other than this, the approach of dual
balanced modulation of targets (having a biochemical correlation) may also be able to fully extract the
anticancer potential of the chaperone function inhibition. Our ongoing drug discovery program is

currently directed towards both these approaches; however this study solely employs the formerly
mentioned strategy focused at the design and synthesis of new HSP90 inhibitors.
O
NH2
N
Cl
N
O
N
N
N
N
N
N
S
I
H2N
N
H2N
N
N
N
S
O
N
O
O
HN
HN
BIIB021 (2)
PU-H71 (1)
CUDC-305 (3)
N
N
H
N
N
O
N
OH
OH
HO
N
O
OH
N
NH
STA-9090 (5)
AT-13387 (4)
F
F
O
F
O
N
O
N
N
NH2
N
N
N
N
N
O
HN
N
HO
NH2
NH2
O
N
O
O
OH
O
N
N
H
TAS-116 (7)
NVP-AUY922 (6)
SNX-5422 (8)
Fig. 1 Chemical structures of HSP90 inhibitors
Identification of key structural motifs and their appropriate fusion has emerged as a rational drug
design strategy in recent years [28]. The resorcinol fragment has been recognized as a crucial structural
unit present in several second generation HSP90 inhibitors (4, 5, 6) (Fig. 1). Specifically, the 4-
isopropyl resorcinol fragment serves as a key binder associated with the ATP binding site of HSP90

proteins by means of an appropriate fit in the hydrophilic and hydrophobic regions of the protein [20,
29-30]. This structural information led us direct our attempts towards the design of resorcinol bearing
adducts as Hsp90 inhibitors. The other functionality selected for inclusion in the hybrid structure
design was quinoline, a privileged heteroaryl scaffold in cancer drug discovery. The application of this
bicyclic heterocycle has been fast spreading in medicinal chemistry owing to its remarkable biological
and synthetic versatility [31-33]. Our recent investigations have comprehensively explored quinoline-
based anticancer scaffolds which exert antiproliferative effects via diverse mechanisms [34-36] and
the results of these studies have been extremely optimistic. This indubitably indicates the feasibility
along with the ease of inclusion of quinolines as a flexible component in diverse antitumor
pharmacophores. Moreover, quinoline-based HSP90 inhibitors have also demonstrated efficacy in
some recently conducted studies [32-34]. Based on these revelations, the design of amide bond tethered
quinoline-resorcinol hybrids was conceived as a logical strategy in the present study.
Recent medicinal chemistry campaigns of our research group have focused primarily on antitumor
constructs bearing bicyclic planar or non-planar/partially hydrogenated (6,6/6,5-fused) heterocycles.
In alignment with our ongoing drug discovery program that involves continuous transposition between
single target and dual target anticancer chemical architectures, quinolone-resorcinol hybrid constructs
as potential Hsp90 inhibitors were designed in the present study (Fig. 2). Employing a fragment linking
approach, the designed hybrids were synthesized and their in-vitro antiproliferative effects were
evaluated. The impact of regio-variations of the fusion, a resorcinol ring on the quinoline core through

an amide linkage, along with the influence of alkyl substitution at the amide NH on the cellular activity
as well as HSP90 inhibition were also investigated.
O
OH
5
8
4
3
NH2
6
7
HO
N
R
2
OH
Amide Linkage
OH
5
8
4
R1
3
2
6
7
N
N
R
OH
O
9-22
9: R = H;
R1 = H (3-Position)
16: R = H;
17: R = H;
18: R = H;
19: R = H;
20: R = H;
21: R = H;
22: R = H;
R1 = CH3 (5-Position)
R1 = H (6-Position)
R1 = CH3 (6-Position)
R1 = C2H5 (6-Position)
R1 = H (8-Position)
10: R = H; R1 = CH3 (3-Position)
11: R = H; R1 = C2H5 (3-Position)
12: R = H; R1 = C3H7 (3-Position)
13: R = CH3; R1 = H (4-Position)
14: R = CH3; R1 = C2H5 (4-Position)
R1 = CH3 (8-Position)
R1 = C2H5 (8-Position)
15: R = H;
R1 = H (5-Position)
Fig. 2 Designed hybrids
2. Results
2.1 Chemistry
The synthetic routes to the target compounds are shown in Schemes 1-3. The quinoline-resorcinol
fused constructs (9, 13, 15, 17 and 20) were synthesized by a synthetic route depicted in Scheme 1.
The quinolines (23-27) bearing the amine functional group at different positions were condensed with
2,4-bis(benzyloxy)-5-isopropylbenzoic acid employing amide coupling mediated by EDC/HOBt . The
intermediates formed (28-32) were debenzylated to yield the final compounds (9, 13, 15, 17 and 20).
A generalized debenzylation procedure using 10% Pd/C in ethanol methanol in a hydrogenation vessel

at 40 - 42 psi could not be applied to all the intermediates (28-32) owing to the tendency of
intermediates 29 and 30 to form tetrahydroquinolines. Thus an alternative strategy was followed using
Pd/C and formic acid to afford the debenzylated adducts (13, 15).
5
8
4
OBn
OH
6
7
3
b
a
H
H
NH2
N
N
2
N
R
N
R
N
R
O
OBn
O
OH
23.
28.
29.
30.
31.
32.
R = H, 3-NH2
R = CH3, 4-NH2
R = H, 5-NH2
R = H, 6-NH2
R = H, 8-NH2
R = H (3-Position)
R = CH3 (4-Position)
R = H (5-Position)
R = H (6-Position)
R = H (8-Position)
9
R = H (3 - Position)
R = CH3 (4- Position)
R = H (5- Position)
R = H (6- Position)
R = H (8- Position)
24.
25.
26.
27.
13
15
17
20
Scheme 1. Reagents and conditions: (a) NMM, EDC, HOBt, DMF, rt; (b) For 9, 17 and 20: Pd/C,
H2, EtOH, rt; for 13,15 : Pd/C, HCOOH, MeOH, reflux.
To further evaluate the effect of N-alkylation of the amide NH on the cellular activity as well as HSP90
inhibition, the methods depicted in Schemes 2 and 3 were employed. A similar trend of intermediates
responding to different reagents and reaction conditions, generating the target compounds as in
Scheme 1 was observed while attempts were made to alkylate the amide NH group. Scheme 2 utilizes
cesium carbonate, sodium hydride, potassium t-butoxide assisted proton abstraction of the
compounds 28, 31 and 32 to generate the alkylated intermediates (33 - 37). The alkylated
intermediates were then subjected to debenzylation with a combination of various reagents and
solvents, furnishing the target compounds 10, 11, 12, 19, and 22.

OBn
OBn
OH
R1
N
6
3
R1
N
6
3
H
6
3
N
b
a
N
R
N
R
8
N
R
O
OBn
8
O
OBn
8
O
OH
33
R = H, R1 = CH3 (3 - Position)
R = H, R1 = C2H5 (3- Position)
R = H, R1 = C3H7 (3- Position)
R = H, R1 = C2H5 (6- Position)
R = H, R1 = C2H5 (8- Position)
10
11
12
19
22
R = H, R1 = CH3 (3 - Position)
R = H, R1 = C2H5 (3- Position)
R = H, R1 = C3H7 (3- Position)
R = H, R1 = C2H5 (6- Position)
R = H, R1 = C2H5 (8- Position)
28
31
32
R = H (3 - Position)
R = H (6- Position)
R = H (8- Position)
34
35
36
37
Scheme 2. Reagents and conditions a) For compound 33: CH3I, CS2CO3, ACN, rt; for compound 34:
C2H5I, NaH, DMF, rt; compound 35-37: RI, t-BuOK, THF, rt; (b) for 10: Pd/C, H2, EtOH, rt; for 19:
Pd/C, H2, MeOH + THF, rt; for 11, 12 and 22: Pd/C, HCOOH, MeOH, reflux.
While attempting to accomplish the synthesis of the fused constructs 14, 16, 18 and 21 through Scheme
2 and employing intermediates (29, 30, 31, 32), it was observed that the synthetic methodology could
only produce the target compounds in poor yields. The exact reason for the attenuated reactivity with
some alkyl iodides and simultaneous formation of degradative products in these cases remains unclear,
and a different strategy, shown in Scheme 3 was employed to form the N-alkylated target compounds
(14, 16, 18, 21). The starting materials (13, 15, 17, 20) were treated with tert-butyldimethylsilyl
chloride (TBDMSCl) which led to selective protection of the OH group at the 4′-position. The selective
protection of this OH group could be attributed to the hydrogen bonding of the OH at the 2’-position
with the carbonyl group of the amide bond. The TBDMS protected intermediates (38 - 41) were then
subjected to alkylation using t-BuOK (42 - 45) followed by subsequent deprotection of the tert-
butyldimethylsilyl ethers to furnish the target compounds (14, 16, 18, 21).

OH
OTBDMS
b)
H
a)
H
N
N
N
R
N
R
O
OH
O
OH
13
15
17
20
38 R
39 R
40 R
41 R
R = CH3 (4- Position)
R = H (5- Position)
R = H (6- Position)
R = H (8- Position)
= CH3 (4- Position)
= H (5- Position)
= H (6- Position)
= H (8- Position)
OTBDMS
OH
R1
N
R1
N
c)
N
R
N
R
O
OH
O
OH
14
R = CH3, R1 = C2H5 (4-Position)
R = H, R1 = CH3 (5-Position)
R = H, R1 = CH3 (6-Position)
R = H, R1 = CH3 (8-Position)
42
43
44
45
R = CH3, R1 = C2H5 (4-Position)
R = H, R1 = CH3 (5-Position)
R = H, R1 = CH3 (6-Position)
R = H, R1 = CH3 (8-Position)
16
18
21
Scheme 3. Reagents and conditions (a) TBDMS-Cl, DIPEA, CH2Cl2, rt; (b) alkyl iodide, t-BuOK,
(R1I), THF, rt; (c) TBAF, CH2Cl2, rt
2.2. Biological evaluation
2.2.1 In-vitro cytotoxicity studies
We systemically investigated the synthetic compounds (9-22) for their antiproliferative activity on
three human tumor cell lines, HCT116 colorectal cancer cell lines, Hep3B liver cancer cell lines, and
PC-3 prostate cancer cell lines. BIIB021, 6-chloro-9-((4-methoxy-3,5-dimethyl-pyridin-2-yl)-
methyl)-9H-purin-2-amine (2) and 17-AAG were employed as standards. The results presented in
Table 1 revealed exciting insights regarding the antiproliferative effects of the synthetic compounds.
The compound 11 (N-ethyl-2,4-dihydroxy-5-isopropyl-N-(quinolin-4-yl)benzamide) bearing the
isopropyl resorcinol functionality tethered via an amide bond at position 3 of the quinoline scaffold

displayed substantial cytotoxic effects against HCT116, Hep3B and PC-3 cancer cell lines with GI50
values of 0.17, 0.33 and 0.14 µM. The adduct 11 is endowed with two-fold higher cell growth
inhibitory effects against PC-3 cell lines in comparison to BIIB0201 (2) and is equipotent with 17-
AAG. Conjugate 11 displays a similar inhibitory profile to that of BIIB021 (2) towards the HCT116
cell lines and was more potent than the standard (2) against Hep3B cell lines. A comparative analysis
of the cytotoxic profile of the fused construct (11) with compounds 9, 10 and 12 confirmed the
favorable activity trend encountered with the N-ethylation of the amide NH. Compounds 9 (bearing
unsubstituted amide NH), 10 (NH-CH3, amide) and 12 (NH-C3H7, amide) exhibit relatively diminished
activity profiles against the cell lines tested. A decline in the activity profile was seen on translocating
the resorcinol fragment from position 3 to 4, 6 and 8 (compare 11 with 14, 19 and 22). Keeping the N-
ethyl substitution pattern intact and shifting the site of resorcinol fusion from position 3 to 6 (19)
retained the cytotoxic effects against the PC-3 (GI50 = 0.46 µM), HCT-116 (GI50 = 1.87 µM) and
Hep3B (GI50 = 0.69 µM) cell lines, but the efficacy was less pronounced than that of compound 11.
The 8-quinolinyl substituted compound with an N-ethyl substituent (22) also demonstrated moderate
inhibition of the growth of tumor cell lines. The in-vitro cytotoxicity assay clearly indicates that the
sites of attachment of the quinoline scaffold and the resorcinol fragment are critical for induction of
the antiproliferative effects. Overall, the fusion of the structural motifs at position 3 of the quinoline
moiety and N-ethyl substitution pattern (amide NH) play a key role in inducing the antiproliferative
effects.

Table 1. Antiproliferative activity of compounds 9-22 against human cancer cell lines
Cell viability
(GI50 µM ± SDa)
Compounds
Prostate
PC3
Colorectal
HCT116
Liver
Hep3B
9
> 10
> 10
> 10
10
11
12
> 10
> 10
> 10
0.14 ± 0.01
> 10
0.17 ± 0.01
> 10
0.33 ± 0.03
> 10
> 10
13
> 10
> 10
14
15
16
17
18
19
20
21
22
> 10
ND
ND
> 10
> 10
> 10
1.81±0.48
> 10
> 10
> 10
ND
ND
> 10
> 10
> 10
0.46 ± 0.13
> 10
1.87 ± 0.68
> 10
0.69 ± 0.06
> 10
> 10
> 10
> 10
2.40 ± 0.27
2.45 ± 0.55
0.15 ± 0.02
5.19 ± 0.44
0.52 ± 0.047
BIIB021 (2)
17-AAG
0.28 ± 0.02
0.14 ± 0.04
0.075 ± 0.007
0.19 ± 0.00
aSD: standard deviation, all experiments were independently performed at least three times
2.2.2 HSP90 Inhibition assay
To validate the underlying mechanism of the antiproliferative activity of constructs 9-22, an in-vitro
assay was performed and the HSP90 inhibitory potential was evaluated. The data in Table 2 made it
evident that only the cytotoxic compounds (11, 19, 22) are able to modulate the function of the
chaperone protein. The compounds unable to induce inhibitory effects against the cell lines tested were
also found to be devoid of any activity against HSP90 (IC50 > 10000 nM) with the exception compound
12. Thus the assay results establish a strong dependence of cell growth inhibitory effects of the

conjugates on their capacity to inhibit the chaperone protein. Compound 11, which is endowed with
substantial antiproliferative effects, exhibits significant HSP90 inhibitory activity with an IC50 value
of 149 nM and the results with either of the standards employed were comparable. Compound 22
(quinoline-resorcinol: site of linkage - C8) also inhibited the chaperone activity with IC50 = 172 nM.
In addition to the activity profile of 11, the promising cellular and chaperone function inhibitory profile
of compound 19 is an important finding (Tables 1 and 2). Compound 19 with IC50 = 124 nM was found
to be the most potent HSP90 inhibitor. Correlating the results of antiproliferative activity (Table 1)
with those from the HSP90 inhibitory assay (Table 2) revealed a similar preference of structural
features for HSP90 inhibition as that for cytotoxicity. The N-ethyl substitution with positions 3 or 6 as
the critical point of attachment of the quinoline scaffold and the resorcinol ring were the favored
structural attributes. In light of these assay results, the most potent antiproliferative agent (11) with
substantial HSP90 inhibitory potential was further investigated to ascertain its HSP90-mediated
suppression of prostate cancer cell growth.
Table 2. HSP90 inhibitory activity of compounds 9-22
IC50 ± SD(nMa)
Compounds
HSP90
9
> 10000
> 10000
10
11
149.06 ± 0.63

12
196.06 ± 15.88
> 10000
13
14
> 10000
15
> 10000
16
> 10000
17
> 10000
18
> 10000
19
20
124.22 ± 17.80
> 10000
21
> 10000
22
172.21 ± 3.74
132.75 ± 14.75
127.73 ±17.56
BIIB021 (2)
17-AAG
aSD: standard deviation, all experiments were independently performed at least three
times;
2.2.3 Effect of test compounds on HSP90-regulated client proteins
To further establish whether the most potent fused construct (11) possesses the signatory features
of known HSP90 inhibitors in terms of its capacity to modulate the expression of cellular markers, a
western blot analysis was performed. The ability of compound 11 to trigger the downregulation of
representative HSP90 client protein expression and induction of HSP70 protein levels was determined
in a PC-3 cell line. Western immunoblotting data revealed that 11 induces HSP70 expression and

depletes the protein expression levels of client proteins, such as EGFR, AKT, ERK, FAK and Rb
indicating selective HSP90 inhibition (Fig. 3A). The underlying mechanism of arrest of PC-3 cells by
11 on the G2/M phase was explored and the expressions of cell cycle related proteins were examined.
Induction of p-MPM2 along with cyclin B1 and downregulation of Cdc2 and its phosphorylation of
Tyr15 was observed after a 24 h treatment with compound 11 and BIIB021 (2) (Fig. 3B). These results
suggested that the HSP90 inhibitor (11) induces mitotic arrest in prostate cancer cells. Moreover,
compound 11 triggers cell apoptosis at 48 h leading to PARP and caspase activations as evident from
the levels of cleaved Caspase 3 and PARP (Fig. 3C). The activation of Caspase 3 and PARP is
considered to be a hallmark of apoptosis and the results of the western blot analysis clearly indicate
the apoptosis inducing ability of 11. These observations coupled with the results presented in Table 1
and Table 2 confirm that mediation of the cell growth inhibitory effects of 11 is a consequence of
Hsp90 chaperone inhibition and also confirms the mitotic arrest in prostate cancer cells.

Fig. 3 The effects of 11 on client proteins of HSP90, cell cycle regulated and apoptotic protein levels.
PC3 cells were treated with DMSO (Control; C) or 0.03-3 M of test compounds (11 or BIIB021) for
24 h. Cells were harvested and subjected to western blot analysis for the detection of various HSP90
client proteins (A) and cell cycle related proteins (B). (C) PC3 cells were incubated with DMSO
(Control; C) or 0.03-3 M of test compounds (11 or BIIB021) for 48 h to detect the apoptotic proteins.
Actin was used as the internal control; B: BIIB021. (G256 represents 11)
2.2.4 Effects of test compounds on cell cycle distribution
The effect of 11 on the cell cycle progression was evaluated by flow cytometry to validate the
underlying mechanism of cell growth repression in PC-3 cells. The results of the experiment indicate
that compound 11 and BIIB021 (2) cause cell arrest (G2M phase) at 24 h followed by a concentration-
dependent apoptotic subG1 accumulation at 48 h (Fig. 4A and B). In addition, it was observed that 11
is more potent than BIIB021 (2) as at 0.3 M, it causes the initiation of the increase in G2M and subG1

phase population (Figs. 4C and 4D). These results indicate that the hybrid compound 11 causes dose-
dependent G2M arrest and apoptosis in prostate cancer cells.
Fig. 4 PC3 cells were treated with DMSO or test compounds, 11 (A) and BIIB021(B) as the indicated
concentrations for 24 h and 48 h. Cells were harvested and analyzed by flow cytometry. The percentage
of cell population in G2M (C) and subG1 phase (D). Statistical analysis was performed with the
#
##
Student t test. *P<0.05, **P<0.01, ***P<0.001, P<0.05, P<0.01, ###P<0.001, compared with the
control groups. (MPT0G256 represents 11)

2.2.5 Molecular modeling studies
A molecular docking study was performed to elucidate the interactions between compound 11 and
amino acid residues of the chaperone protein HSP90. Initially, the validation of the docking protocol
was performed by redocking the co-crystallized ligand (Fig 1S. in Supporting Information - SI). This
showed that the redocked ligand has a docking pose similar to that of the co-crystallized ligand and
indicates that the docking protocol used has a favorable prediction acuity. Compound 11 was then
docked into the HSP90 binding site and it was found that it is anchored within the binding site and can
be separated into three distinct segments with respect to its HSP90 docking pose (Fig. 5). At Site 1
(S1), the 2,4‐dihydroxy-5-isopropylbenzoyl moiety is held in place through hydrophobic interactions
with residues Met98, Leu107 and Phe138. In addition, the hydroxyl groups form hydrogen bonds with
Asn51 and Asp93, and the carbonyl forms a hydrogen bond with Thr184. At Site 2 (S2), the quinoline
moiety of compound 11 occupies a hydrophobic pocket with Ala55 and Tyr61. At Site 3 (S3), the two
pharmacophores are connected via an N-ethyl amide bond. This ethanamine moiety has hydrophobic
interactions with Ala55, Ile96, Met98 and Tyr61. In contrast, compounds 9 and 10, with an
unsubstituted amide linkage (-NH-CO-) and an N-methyl amide linkage respectively, do not inhibit
HSP90 as effectively as 11, suggesting that the N-ethyl substitution is a prerequisite for activity.
Together, these interactions shed light on the potency of compound 11 against HSP90.

A
B
Leu107
Phe138
S2
Asn51
Met98
Tyr61
Thr184
S1
Asp93
S3
Ala55
Ile96
Fig. 5 Molecular docking analysis of 11 in HSP90. (A) 11 (blue) is anchored within the HSP90 (gray)
binding site. The three distinct sections of MPTG0G256 are colored as yellow, blue and red, which
are located at sites S1, S2 and S3, respectively. Interacting residues are represented as sticks and
labeled as shown. Hydrogen bonds are denoted by dotted green lines. (B) A 2D representation of 11
docked in HSP90. Hydrogen bonds are denoted by dashed lines. Green lines represent areas of
hydrophobic interactions. Interacting residues are labeled as shown.
2.2.6 In vivo antitumor efficacy in human xenografts
Compound 11 was evaluated for its in vivo efficacy versus tumor xenografts in nude mice bearing the
human PC-3 cancer cell line. PC3-tumor-bearing nude mice were treated with compound 11 (50, 100
and 200 mg/kg/d, by oral gavage qd) and tumor was excised when the tumor size reached 1200 mm3.
It was found that treatment of PC3 xenograft-bearing nude mice with compound 11 resulted in dose
dependent decrease of the tumor progression. Construct 11 led to the suppression of tumor growth by
a tumor growth inhibition factor (% TGI = 50.1%) without loss of their body weight (Fig. 6). Thus the
in vivo animal model experiments demonstrated that compound 11 possess moderate in vivo potential

against prostate cancer.
Fig. 6 In vivo antitumor activity of 11 in PC3 xenograft model. (A) PC3-tumor-bearing nude mice
were treated with vehicle or 11 (50, 100 and 200 mg/kg/d, by oral gavage qd). Tumor was excised
when the tumor size reached 1200 mm3. (B) The body weight of the mice measured daily during the
first week and then twice a week of administration. (MPT0G256 represents 11)
3. Conclusion
A novel class of inhibitors that potently inhibits HSP90 is reported. By fusing key structural motifs
known to induce cell growth inhibitory effects by modulation of the HSP90 chaperone protein, a series
of fused quinoline-resorcinol compounds were designed and synthesized. A correlation of the tumor
growth inhibition exerted through HSP90 inhibition was established from the results of in vitro
cytotoxicity studies, HSP90 inhibitory assays and western blot analysis. The site of linkage of the
resorcinol ring to the quinoline scaffold via an amide linkage and N-alkylation of amide bond was

found to be extremely critical for both the cytotoxicity as well as the modulation of chaperone function.
These attempts led us to speculate about the structure activity relationships with ethylation at the amide
NH and position 3 (the quinoline ring) as the site of attachment being the structural prerequisites for
activity. The main finding of the study is the identification of 11 as an HSP90 inhibitor endowed with
apoptosis inducing ability and substantial in vitro antiproliferative effects against PC-3 cancer cells. In
addition to its induction of HSP90 expression, 11 also downregulates the protein expression levels of
client proteins, such as EGFR, Src, FAK, and RB. The significant inhibition of the HSP90 chaperone
by 11 was rationalized by its molecular docking into active site of HSP90. The outcome of the present
study is consistent with recent reports revealing the promising potential of HSP90 inhibitors against
prostate cancer. These are exciting outcomes in light of the fact that prostate cancer is a leading cause
of cancer-related mortality and the revelations could be useful for designing chemotherapeutic agents
for prostate cancer. Further lead modifications on 11 including synthesis of compounds with diverse
substitution on the quinoline rings along with positioning of other planar bicyclic heteroaryl rings in
the hybrid structure design is under progress.
4. Experimental
4.1 Chemistry
Nuclear magnetic resonance spectra were obtained with Bruker DRX-500 spectrometer operating at
300 MHz, with chemical shift recorded in parts per million (ppm, d) downfield from TMS as an
internal standard. High-resolution mass spectra (HRMS) were measured with a JEOL (JMS-700)

electron impact (EI) mass spectrometer. Flash column chromatography was accomplished with silica
gel (Merck Kieselgel 60, No. 9385, 230e400 mesh ASTM). All reactions were carried out under an
atmosphere of dry N2.
2,4-Bis(benzyloxy)-5-isopropyl-N-(quinolin-3-yl)benzamide (28)
2,4-Bis(benzyloxy)-5-isopropylbenzoic acid (1.28 g, 3.4 mmol), N-methylmorpholine (0.9 mL), 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide) (0.97 g, 5.1 mmol) and hydroxybenzotriazole (0.55 g,
4.1 mmol) were added to a solution of 3-aminoquinoline (23) (0.5 g, 3.4 mmol) in DMF (5 mL),. The
reaction mixture was stirred at rt for 12 h and then diluted with water. Extraction was with EtOAc (3
x 50 mL), and the organic layer was dried over anhydrous MgSO4, filtered, and concentrated. The
residue was purified by silica gel chromatography (EtOAc:n-hexane = 1:3) to afford compound 6 (1.03
g, 61% yield). 1H-NMR (300 MHz, CDCl3) δ 1.29 (d, J = 6.9 Hz, 6H), 3.37 (qui, J = 6.9 Hz, 1H), 5.19
(s, 2H), 5.20 (s, 2H), 7.35-7.64 (m, 13H), 7.74-7.78 (m, 1H), 8.00-8.15 (m, 2H), 8.22 (s, 1H), 8.83 (d,
J =2.4 Hz, 1H), 10.22 (s, 1H).
2,4-Bis(benzyloxy)-5-isopropyl-N-(2-methylquinolin-4-yl)benzamide (29)
The title compound (29) was synthesized in 49% yield in a manner similar to that described for
1
compound 28 using 2-methylquinolin-4-amine (24); H-NMR (300 MHz, MeOD) δ 1.27 (d, J = 6.9
Hz, 6H), 2.68 (s, 3H), 3.34-3.39 (m, 1H), 5.27 (s, 2H), 5.42 (s, 2H), 7.00-7.10 (m, 2H), 7.26-7.62 (m,
12H), 7.86 (d, J = 8.4 Hz, 1H), 8.11 (s, 1H), 8.39 (s, 1H).
2,4-Bis(benzyloxy)-5-isopropyl-N-(quinolin-5-yl)benzamide (30)

The title compound 30 was synthesized in 54% yield in a manner similar to that described for
compound 28 using quinolin-5-amine (25); 1H-NMR (300 MHz, CDCl3) δ 1.29 (d, J = 6.6 Hz, 6H),
3.38 (qui, J = 6.9 Hz, 1H), 5.20 (s, 2H), 5.22 (s, 2H), 6.72 (s, 1H), 7.00-7.07 (m, 1H), 7.34-7.50 (m,
11H), 7.69-7.90 (m, 2H), 8.09 (d, J = 8.4 Hz, 1H), 8.25 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.8 (d, J =
4.2 Hz, 1H).
2,4-Bis(benzyloxy)-5-isopropyl-N-(quinolin-6-yl)benzamide (31)
The title compound 31 was synthesized in 53% yield in a manner similar to that described for
compound 28 using quinolin-6-amine (26); 1H-NMR (300 MHz, CDCl3) δ 1.31 (d, J = 6.9 Hz, 6H),
3.38 (qui, J = 6.9 Hz, 1H), 5.16 (s, 4H), 6.63 (s, 1H), 6.96 (dd, J = 6.6, 9 Hz, 1H), 7.24-7.60 (m, 11H),
7.86 (d, J = 9 Hz, 1H), 8.01 (d, J = 8.1 Hz, 1H), 8.25-8.30 (m, 2H), 8.75 (dd, J = 2.4, 3.9 Hz, 1H),
10.14 (s, 1H).
2,4-Bis(benzyloxy)-5-isopropyl-N-(quinolin-8-yl)benzamide (32)
The title compound (32) was synthesized in 64% yield in a manner similar to that described for
compound 28 using quinolin-8-amine (27); 1H-NMR (300 MHz, CDCl3) δ 1.20 (d, J = 6.6 Hz, 6H),
3.25 (qui, J = 6.9 Hz, 1H), 4.90 (s, 2H), 5.30 (s, 2H), 6.44 (s, 1H), 7.13-8.19 (m, 13H), 8.33 (m, 1H),
8.34 (s, 1H), 8.99 (d, J = 0.9 Hz, 1H), 9.02 (d, J = 0.9 Hz, 1H), 12.25 (s,1H).
2,4-Dihydroxy-5-isopropyl-N-(quinolin-3-yl)benzamide (9)
Catalytic Pd/C was added to the solution of compound 28 (0.5 g, 1 mmol) in EtOH (10 mL), and the
reaction mixture was stirred at rt under H2. After continuous stirring for 6 h, the mixture was filtered

through celite, dried over anhydrous MgSO4, and then concentrated. The residue was purified by silica
gel chromatography (EtOAc:n-hexane = 1 : 1) to afford compound 9 (0.1 g, 31% yield). mp: 232-234
°C. 1H-NMR (300 MHz, DMSO-d6) δ 1.20 (d, J = 6.9 Hz, 6H), 3.13 (qui, J = 6.9 Hz, 1H), 6.43 (s, 1H),
7.55-7.72 (m, 2H), 7.82 (s, 1H), 7.93-8.01 (m, 2H), 8.70 (d, J = 2.4 Hz, 1H), 9.06 (d, J = 2.7 Hz, 1H),
10.22 (s, 1H), 10.58 (s, 1H). HRMS (ESI) for C19H19N2O3 [M+H]+: calcd, 323.1396; found, 323.1395.
LRMS (ESI) [M+H]+ : 323.1.
2,4-Dihydroxy-5-isopropyl-N-(2-methylquinolin-4-yl)benzamide (13)
Catalytic Pd/C, and formic acid (3.2 mL) were added to a solution of compound 29 (0.73 g, 1.41 mmol)
in MeOH (15 mL). The resulting mixture was refluxed for 12 h. After cooling at rt, the reaction mixture
was filtered through celite and the organic layer was dried over anhydrous MgSO4. The organic layer
was then concentrated and the residue was purified by silica gel chromatography (EtOAc:n-hexane =
2 : 1) to afford compound 13 (0.15 g, 31% yield). mp: 216-218 °C. 1H-NMR (300 MHz, MeOD) δ
1.23-1.28 (m, 6H), 2.70 (s, 3H), 3.21 (qui, J = 6.9 Hz, 1H), 6.45 (d, J = 1.5 Hz, 1H), 7.54-7.62 (m,
1H), 7.71-7.79 (m, 1H), 7.94 (d, J = 9 Hz, 2H), 8.06 (d, J = 8.4 Hz, 1H), 8.42-8.47 (m, 1H). HRMS
(ESI) for C20H21N2O3 [M+H]+: calcd, 337.1552; found, 337.1555. LRMS (ESI) [M+H]+: 337.1.
2,4-Dihydroxy-5-isopropyl-N-(quinolin-5-yl)benzamide (15)
Catalytic Pd/C and formic acid (0.9 mL) were added to a solution of compound 30 (0.2 g, 0.40 mmol)
in MeOH (15 mL). The reaction mixture was refluxed for 7 h and then cooled at rt. The mixture was
filtered through celite, dried over anhydrous MgSO4 and then concentrated. The residue was purified

by silica gel chromatography (EtOAc:n-hexane = 1 : 1) to afford compound 15 (0.06 g, 45% yield).
mp: 222-224 oC. 1H-NMR (300 MHz, MeOD) δ 1.26 (d, J = 7.2 Hz, 6H), 3.18-3.24 (m, 1H), 6.40 (s,
1H), 7.56-7.63 (m, 1H), 7.78-7.99 (m, 4H), 8.48 (d, J = 9 Hz, 1H), 8.88 (d, J = 3.9 Hz, 1H). HRMS
(ESI) for C19H19N2O3 [M+H]+: calcd, 323.1396; found, 323.1395. LRMS (ESI) [M+H]+: 323.1.
2,4-Dihydroxy-5-isopropyl-N-(quinolin-6-yl)benzamide (17)
The title compound 17 was synthesized in 63% yield using compound 31 in a manner similar to that
o
1
described for compound 9; mp: 162-164 C; H-NMR (300 MHz, DMSO-d6) δ 1.20 (d, J = 6.9 Hz,
6H), 3.14 (qui, J = 6.9 Hz, 1H), 6.43 (s, 1H), 7.50 (q, J = 4.2 Hz, 1H), 7.82 (s, 1H), 7.92-8.05 (m, 2H),
8.28-8.42 (m, 2H), 8.78-8.84 (m, 1H), 10.18 (s, 1H), 10.46 (s, 1H). HRMS (ESI) for C19H19N2O3
[M+H]+: calcd, 323.1396; found, 323.1396. LRMS (ESI) [M+H]+: 323.1.
2,4-Dihydroxy-5-isopropyl-N-(quinolin-8-yl)benzamide (20)
The title compound 20 was synthesized in 52% yield using compound 32 in a manner similar to that
described for compound 9; mp: 172-174 oC; 1H-NMR (300 MHz, MeOD) δ 1.27 (d, J = 6.9 Hz, 6H),
3.23 (qui, J = 6.9 Hz, 1H), 6.41 (s, 1H), 7.49-7.60 (m, 3H), 7.81 (s, 1H), 8.26 (dd, J = 1.8, 8.4 Hz, 1H),
8.80 (dd, J = 2.1, 6.9 Hz, 1H), 8.85 (dd, J =1.5, 4.2 Hz, 1H). HRMS (ESI) for C19H19N2O3 [M+H]+:
calcd, 323.1396; found, 323.1389. LRMS (ESI) [M+H]+: 323.1.
2,4-Bis(benzyloxy)-5-isopropyl-N-methyl-N-(quinolin-3-yl)benzamide (33)
Cesium carbonate (0.65 g, 1.98 mmol) was added to a solution of compound 28 (1 g, 1.98 mmol) in
MeCN (5 mL). The mixture was stirred for 30 min before adding MeI (0.246 mL, 3.96 mmol). The

reaction mixture was stirred for 12 h and then diluted with water. The mixture was extracted with
EtOAc and the organic layer was dried over anhydrous MgSO4, filtered, and concentrated. The mixture
was purified by silica gel chromatography (EtOAc:n-hexane = 1 : 4) to afford compound 33 (0.08 g,
8% yield). 1H-NMR (300 MHz, CDCl3) δ 1.12 (d, J = 6.9 Hz, 6H), 3.14-3.29 (m, 1H), 3.51 (s, 3H),
4.72 (s, 2H), 4.84 (s, 2H), 7.24-7.40 (m, 11H), 7.48-7.56 (m, 3H), 7.60-7.70 (m, 2H), 8.00 (d, J = 9.0
Hz, 1H), 8.53 (s, 1H).
2,4-Bis(benzyloxy)-N-ethyl-5-isopropyl-N-(quinolin-3-yl)benzamide (34)
Sodium hydride (0.05 g, 2 mmol) was added to a solution of compound 28 (0.5 g, 1 mmol) in DMF (5
mL), and the reaction mixture was stirred for 20 min at 0°C. EtI (0.25 mL, 3.10 mmol) was then added
and stirring was continued for 4h at rt. The reaction mixture was diluted with water and extracted with
EtOAc. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated. The residue
obtained was used in a subsequent reaction without purification.
2,4-Bis(benzyloxy)-5-isopropyl-N-propyl-N-(quinolin-3-yl)benzamide (35)
Potassium t-butoxide (0.15 g, 1.35 mmol) was added to a solution of compound 28 (0.45 g, 0.9 mmol)
in THF (5 mL). The mixture was stirred for 10 min before adding n-PrI (0.437 mL, 4.48 mmol). The
reaction mixture was then stirred at rt for 5h. Water was added to the reaction mixture which was then
extracted with EtOAc. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated.
The mixture was purified by silica gel chromatography (EtOAc:n-hexane = 1 : 3) to afford compound
1
35 (0.44 g, 90% yield). H-NMR (300 MHz, CDCl3) δ 0.86-0.96 (m, 3H), 1.13 (d, J =6.6 Hz, 6H),

1.58-1.68 (m, 2H), 3.20 (t, J = 6.6 Hz, 1H), 3.92 (s, 2H), 4.76 (s, 2H), 4.83 (s, 2H), 6.15 (s, 1H), 7.25-
7.40 (m, 11H), 7.45-7.57 (m, 2H), 7.60-7.69 (m, 2H), 8.02 (d, J = 8.4 Hz, 1H), 8.54 (s, 1H).
2, 4-Bis(benzyloxy)-N-ethyl-5-isopropyl-N-(quinolin-6-yl)benzamide (36)
The title compound 36 was synthesized in 85% yield in a manner similar to that described for
compound 35 using compound 31; 1H-NMR (300 MHz, CDCl3) δ 0.99 (d, J = 6.9 Hz, 6H), 1.13 (m,
5H), 3.06-3.15 (m, 1H), 3.91 (d, J = 6.0 Hz, 2H), 4.73 (d, J = 14.1 Hz, 4H), 7.06-7.28 (m, 13H), 7.75
(t, J = 6.9 Hz, 2H), 8.73 (dd, J =1.5, 3.9 Hz, 1H).
2,4-Bis(benzyloxy)-N-ethyl-5-isopropyl-N-(quinolin-8-yl)benzamide (37)
The title compound 37 was synthesized in 43% yield in a manner similar to that described for
compound 35 using compound 32; 1H-NMR (300 MHz, CDCl3) δ 0.4 (d, J = 6.9 Hz, 3H), 0.87 (d, J =
6.6 Hz, 3H), 1.20 (t, J = 7.2 Hz, 3H), 3.62-3.73 (m, 1H), 4.59-4.66 (m, 1H), 4.77 (s, 2H), 4.90-5.05
(m, 2H), 6.18 (s, 1H), 6.84 (s, 1H), 7.20-7.47 (m, 14H), 7.59 (d, J = 8.1 Hz, 1H), 8.03 (d, J = 7.2 Hz,
1H), 8.98 (d, J = 2.7 Hz, 1H).
2,4-Dihydroxy-5-isopropyl-N-methyl-N-(quinolin-3-yl)benzamide (10)
The title compound 10 was synthesized in 79% yield in a manner similar to that described for
compound 9 using compound 33; mp: 236-238 °C; 1H-NMR (300 MHz, (CD3)2CO) δ 1.27 (d, J = 7.2
Hz, 6H), 3.26 (qui, J = 7.2 Hz, 1H), 3.84 (s, 3H), 6.53 (s, 1H), 7.29-7.37 (m, 1H), 7.55-7.59 (m, 2H),
7.70-7.77 (m, 2H), 8.82 (s, 1H), 9.20 (s, 1H), 10.20 (s, 1H), 11.35 (s, 1H). HRMS (ESI) for C20H21N2O3
[M+H]+: calcd, 337.1552; found, 337.1554. LRMS (ESI) [M+H]+: 337.1.

N-Ethyl-2,4-dihydroxy-5-isopropyl-N-(quinolin-3-yl)benzamide (11)
Catalytic Pd/C and formic acid (0.9 mL) were added to a solution of compound 34 (0.21 g, 0.39 mmol)
in MeOH (15 mL). The resulting mixture was refluxed for 12 h and then cooled to rt. The mixture was
filtered through celite, dried over anhydrous MgSO4 and then concentrated. Purification was done by
silica gel chromatography (EtOAc:n-hexane = 1 : 4) to afford compound 11 (0.10 g, 73% yield); mp:
1
207-209 oC; H-NMR (300 MHz, MeOD) δ 0.70 (d, J = 6.9 Hz, 6H), 1.26 (t, J = 7.2 Hz, 3H), 2.80-
2.94 (m, 1H), 4.04-4.14 (m, 2H), 6.11 (s, 1H), 6.67(s, 1H), 7.57-7.65 (m, 1H), 7.70-7.79 (m, 1H), 7.88
(d, J = 8.4 Hz, 1H), 7.96 (d, J = 8.7Hz, 1H), 8.21 (d, J = 2.4 Hz, 1H), 8.55 (d, J = 2.4 Hz, 1H). HRMS
(ESI) for C21H23N2O3 [M+H]+: calcd, 351.1709; found, 351.1706. LRMS (ESI) [M+H]+: 351.1.
2,4-Dihydroxy-5-isopropyl-N-propyl-N-(quinolin-3-yl)benzamide (12)
The title compound 12 was synthesized in 13% yield in a manner similar to that described for
1
compound 13 using compound 35; mp: 197-199 oC; H-NMR (300 MHz, CDCl3) δ 0.42 (d, J = 6.9
Hz, 6H), 0.96 (t, J = 7.2 Hz, 3H), 1.65-1.79 (m, 2H), 2.75 (qui, J = 6.9 Hz, 1H), 3.93-4.01 (m, 2H),
6.29 (s, 1H), 6.37 (s, 1H), 7.53-7.63 (m, 1H), 7.67-7.81 (m, 2H), 7.96 (d, J = 2.4 Hz, 1H), 8.08 (d, J =
8.4 Hz, 1H), 8.58 (d, J = 2.7 Hz, 1H), 10.93 (s, 1H). HRMS (ESI) for C22H23N2O3 [M-H] +: calcd,
363.1709; found, 363.1727. LRMS (ESI) [M]+: 364.9.
N-Ethyl-2,4-dihydroxy-5-isopropyl-N-(quinolin-6-yl)benzamide (19)
Catalytic Pd/C was added to a solution of compound 36 (0.36 g, 0.68 mmol) in MeOH (3 mL) and
THF (10 mL), The reaction mixture was stirred at rt for 5 h under H2 and then filtered through celite.

The organic layer was dried over anhydrous MgSO4, concentrated and the residue obtained was
purified by silica gel chromatography (EtOAc:n-hexane = 1 : 2) to afford compound 19 (0.09 g, 38%
yield); mp: 220-221 oC; 1H-NMR (300 MHz, DMSO-d6) δ 0.57 (d, J = 6.6 Hz, 6H), 1.14 (t, J = 6.9
Hz, 3H), 2.71-2.77 (m, 1H), 3.93 (q, J = 6.9 Hz, 2H), 6.12 (s, 1H), 6.53 (s, 1H), 7.49 (q, J = 4.2 Hz,
1H), 7.58 (dd, J = 2.4, 9 Hz, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.94 (d, J =9 Hz, 1H), 8.23-8.28 (m, 1H),
8.83-8.87 (m, 1H). HRMS (ESI) for C21H23N2O3 [M+H]+: calcd, 351.1709; found, 351.1711. LRMS
(ESI) [M+H]+: 351.1.
N-Ethyl-2,4-dihydroxy-5-isopropyl-N-(quinolin-8-yl)benzamide (22)
The title compound 22 was synthesized in 66% yield in a manner similar to that described for
compound 13 using compound 37; mp: 82-84 °C; 1H-NMR (300 MHz, CDCl3) δ 0.39 (bs, 6H), 1.21
(qui, J = 7.2 Hz, 3H), 2.66 (m, 1H), 4.10 (q, J = 7.2 Hz, 2H), 6.20 (s, 1H), 6.40 (s, 1H), 6.46 (s, 1H),
7.25-7.50 (m, 3H), 7.75 (dd, J = 1.8, 7.8 Hz, 1H), 8.19 (dd, J = 1.8, 8.4 Hz, 1H), 8.96 (dd, J = 1.8, 4.2
Hz, 1H), 11.23 (s, 1H). HRMS (ESI) for C21H23N2O3 [M+H]+: calcd, 351.1709; found, 351.1711.
LRMS (ESI) [M+H]+: 351.1.
4-((tert-Butyldimethylsilyl)oxy)-2-hydroxy-5-isopropyl-N-(2-methylquinolin-4-yl)benzamide (38)
tert-Butyldimethylsilyl chloride (0.13 g, 0.89 mmol) and N,N-diisopropylethylamine (0.12 g, 0.89
mmol) were added to a solution of compound 13 (0.15 g, 0.44 mmol) in dry CH2Cl2 (5 mL),. The
reaction mixture was stirred at rt for 6 h and then water was added. The organic layer was separated,
dried over anhydrous MgSO4, and concentrated. The residue was purified by silica gel chromatography