SAR studies of 4-pyridyl heterocyclic anilines that selectively induce autophagic cell death in von Hippel-Lindau-deficient renal cell carcinoma cells

Article abstract of DOI:10.1016/j.bmc.2011.04.042

We recently identified a class of pyridyl aniline thiazoles (PAT) that displayed selective cytotoxicity for von Hippel-Lindau (VHL) deficient renal cell carcinoma (RCC) cells in vitro and in vivo. Structure-activity relationship (SAR) studies were used to develop a comparative molecular field analysis (CoMFA) model that related VHL-selective potency to the three-dimensional arrangement of chemical features of the chemotype. We now report the further molecular alignment-guided exploration of the chemotype to discover potent and selective PAT analogues. The contribution of the central thiazole ring was explored using a series of five- and six-membered ring heterocyclic replacements to vary the electronic and steric interactions in the central unit. We also explored a positive steric CoMFA contour adjacent to the pyridyl ring using Pd-catalysed cross-coupling Suzuki-Miyaura, Sonogashira and nucleophilic displacement reactions to prepare of a series of aryl-, alkynyl-, alkoxy- and alkylamino-substituted pyridines, respectively. In vitro potency and selectivity were determined using paired RCC cell lines: the VHL-null cell line RCC4 and the VHL-positive cell line RCC4-VHL. Active analogues selectively induced autophagy in RCC4 cells. We have used the new SAR data to further develop the CoMFA model, and compared this to a 2D-QSAR method. Our progress towards realising the therapeutic potential of this chemotype as a targeted cytotoxic therapy for the treatment of RCC by exploiting the absence of the VHL tumour suppressor gene is reported.

Full text of DOI:10.1016/j.bmc.2011.04.042

Bioorganic & Medicinal Chemistry 19 (2011) 3347–3356
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
SAR studies of 4-pyridyl heterocyclic anilines that selectively induce
autophagic cell death in von Hippel-Lindau-deficient renal cell carcinoma cells
Muriel Bonnet a, Jack U. Flanagan a, Denise A. Chan b, , Edwin W. Lai b, Phuong Nguyen b, Amato J. Giaccia b,
Michael P. Hay a,
⇑
a Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand
b Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We recently identified a class of pyridyl aniline thiazoles (PAT) that displayed selective cytotoxicity for
von Hippel-Lindau (VHL) deficient renal cell carcinoma (RCC) cells in vitro and in vivo. Structure–activity
relationship (SAR) studies were used to develop a comparative molecular field analysis (CoMFA) model
that related VHL-selective potency to the three-dimensional arrangement of chemical features of the
chemotype. We now report the further molecular alignment-guided exploration of the chemotype to dis-
cover potent and selective PAT analogues. The contribution of the central thiazole ring was explored
using a series of five- and six-membered ring heterocyclic replacements to vary the electronic and steric
interactions in the central unit. We also explored a positive steric CoMFA contour adjacent to the pyridyl
ring using Pd-catalysed cross-coupling Suzuki–Miyaura, Sonogashira and nucleophilic displacement
reactions to prepare of a series of aryl-, alkynyl-, alkoxy- and alkylamino-substituted pyridines, respec-
tively. In vitro potency and selectivity were determined using paired RCC cell lines: the VHL-null cell line
RCC4 and the VHL-positive cell line RCC4-VHL. Active analogues selectively induced autophagy in RCC4
cells. We have used the new SAR data to further develop the CoMFA model, and compared this to a
2D-QSAR method. Our progress towards realising the therapeutic potential of this chemotype as a tar-
geted cytotoxic therapy for the treatment of RCC by exploiting the absence of the VHL tumour suppressor
gene is reported.
Received 28 February 2011
Revised 15 April 2011
Accepted 21 April 2011
Available online 24 April 2011
Keywords:
Drug design
Structure–activity relationships
Renal cell carcinoma
Autophagy
von Hippel-Lindau
Suzuki–Miyaura
Sonogashira
QSAR
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
that specifically regulates the protein stability of the hypoxia-induc-
ible factor (HIF) family of transcription factors.5 Loss of VHL results
Advanced clear cell renal cell carcinoma (RCC) is a disease
typified by an aggressive phenotype and poor treatment out-
comes.1,2 Mutation or inactivation of the von Hippel-Lindau (VHL)
tumour suppressor gene occurs in the majority of RCCs,3,4 and this
is associated with tumour growth and invasiveness, resulting in a
poor prognosis for patients. This gene encodes an E3 ubiquitin ligase
in constitutive expression of HIF leading to increased transcription
of genes involved in metabolism, angiogenesis, invasion, metastasis,
and proliferation leading to an aggressive phenotype.6–10 VHL is
also known to be involved in a variety of HIF-independent
processes.11–14
We recently used a synthetic lethality approach15,16 to identify
the pyridyl anilino thiazole (PAT) chemotype 1 by high-throughput
screening of commercial libraries (Fig. 1A). The initial lead 2 was
validated as a selective cytotoxin to VHL-deficient cells using XTT
growth inhibition and clonogenic survival assays.17 As well as
being selectively cytotoxic to VHL-deficient cells in vitro, com-
pound 2 reduced VHL-deficient RCC growth in a xenograft tumour
model in vivo. We demonstrated that the PATs cytotoxicity occurs
in a HIF-independent manner through autophagy, however, the
molecular target of the PAT class remains unknown. Induction of
autophagy to selectively kill tumour cells represents a novel ap-
proach to anti-cancer therapy.18,19
Abbreviations: BINAP, 2,20-bis(diphenylphosphino)-1,10-binaphthyl; CoMFA,
comparative molecular field analysis; DCM, dichloromethane; DIAD, diisopropyl
azodicarboxylate; DMF–DMA, dimethylformamide–dimethylacetal; dppf,
1,10-bis(diphenylphosphino)ferrocene; EDCI, 1-ethyl-3-(3-dimethylaminopro-
pyl) carbodiimide; HQ-SAR, hologram quantitative structure–activity relationship;
HIF, hypoxia-inducible factor; PAT, pyridyl anilinothiazoles; VHL, von Hippel-
Lindau; RCC, renal cell carcinoma; SAR, structure–activity relationship; XTT, 2,3-
bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide.
⇑
Corresponding author. Address: Auckland Cancer Society Research Centre, The
University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. Tel.: +64 9
923 6598; fax: +64 9 373 7502.
E-mail address: m.hay@auckland.ac.nz (M.P. Hay).
Present address: Department of Radiation Oncology, University of California at
San Francisco, San Francisco, CA 94143, USA.
An initial exploration of structure–activity relationship (SAR)
studies was carried out with the synthesis of a range of analogues
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.042

3348
M. Bonnet et al. / Bioorg. Med. Chem. 19 (2011) 3347–3356
We now report further SAR studies in order to identify more
N
N
potent and selective PAT analogues with increased potency and
selectivity for VHL-deficient RCC. The in vitro activity of PATs as
VHL-selective cytotoxins was evaluated using paired RCC cell lines;
VHL-deficient RCC4 cell line and the matched line, RCC4-VHL, with
the VHL function restored. Two features identified in the original
CoMFA contour maps are explored in this study (Fig. 1B). The first
feature is the orientation of the thiazole B-ring and the roles of the
heteroatom substituents, while the second feature is the positive
steric contour (green volume) adjacent to the pyridine ring 3-posi-
tion which has a nearby disfavoured electrostatic red region. We
first investigated the influence of thiazole orientation predicted
by the alignment model on the biological activity by replacing
the thiazole central ring with various heterocycles (3–13). We then
explored the steric region adjacent to the pyridine ring with a
range of PAT derivatives bearing various functionalities at the 2-
and 3-positions (compounds 14–54) of the pyridine ring. We also
report on the impact of additional SAR data on the CoMFA model
and compare its predictive ability to that of a 2D-QSAR method,
Hologram QSAR (HQSAR).21
N
S
N
S
NH
NH
Me
A. PAT chemotype 1
2
B. Molecular alignment model
Figure 1. PAT chemotype and biologically relevant alignment.
2. Results
2.1. Chemistry
bearing substitutions on each ring of the PAT chemotype as well as
modifications of the linker units between the domains.20 A quanti-
tative analysis of the SAR was used to determine a possible bioac-
tive conformation for the PAT analogues using comparative
molecular field analysis (CoMFA).
The thiazole ring was replaced by a range of other five-mem-
bered ring heterocycles. The imidazole analogue 3 was synthesized
by condensation22 between bromoketone 5520 and 3-methylphe-
nylguanidinium nitrate 5623 using KOH as base (Scheme 1).
N
N
N
H
N
N
(a)
(c)
Me
NH2
NH
NH
Br
X
X
O
55
Me
Me
Me
3
X = NH
56
X = NH
4 X = O
57 X = O
N
N
N
N
S
(b)
NH2
N
CN
NH
$
58
5
N
H
N
(e)
Me
NHR
N
NH
NH
NH
NH
N
N
N
N
H
Me
59
R = C(=S)NH2
(d)
6
60 R = C(=NH)SCH3
N
N
Me
N
Me
NHR
(e)
N
(f)
N
Me
7
61
R = C(=S)NHCH3
(d)
Me
NCS
62 R = =C(SCH3)NHCH3
(g)
$
H
N
H
N
O
N
Me
NH2
(h)
N
S
63
8
Me
Scheme 1. Reagents and conditions: (a) KOH, NEt3, EtOH, 80 °C; (b) NaOMe, MeOH, then NH4Cl; (c) 3-methylphenyl isothiocyanate, iPr2NEt, DMF, then DIAD; (d) MeI, EtOH;
(e) 4-pyridinecarbohydrazide, pyridine; (f) MeNH2, EtOH; (g) NH2–NH2ꢀH2O, EtOH; (h) isonicotinic acid, EDCI, DCM. $ = commercially available.

M. Bonnet et al. / Bioorg. Med. Chem. 19 (2011) 3347–3356
3349
Similarly, condensation of bromoketone 55 and 3-methylphenylu-
R1
R1
rea 57 afforded oxazole 4. Thiadiazole derivative 5 was synthesized
from 4-pyridinecarbonitrile which was converted to the corre-
sponding amidine 58 by treatment with NaOMe followed by NH4Cl
addition. Subsequent DIAD-induced heterocyclization of amidine
58 with 3-methylphenylisothiocyanate gave thiadiazole 5. Methyl-
ation of thiourea 59,20 gave thiocarbamate 60 which underwent
condensation with 4-pyridinecarbohydrazide to give triazole 6 as
a mixture of regioisomers. Addition of MeNH2 to 3-methylpheny-
lisothiocyanate gave methylated thiourea 61 which was converted
to thiocarbamate 62 by alkylation with MeI, and subsequent con-
densation with 4-pyridinecarbohydrazide afforded triazole 7.
3-Methylphenylisothiocyanate was converted to the correspond-
ing hydrazinecarbothioamide 63 by treatment with hydrazine
hydrate in EtOH and subsequent condensation with isonicotinic
acid in the presence of EDCI gave oxadiazole 8.
A series of six-membered ring heterocyclic or carbocyclic ana-
logues were then prepared to further investigate the geometry of
the central B-ring (Scheme 2). Pyrimidinamine 9 was prepared
by condensation of the enaminoketone 64, obtained by reaction
of 4-acetylpyridine with DMF–DMA, and subsequent reaction with
guanidinium nitrate 56 in the presence of KOH.24 The 2,6-
pyrimidine derivative 10 was synthesized from 2,6-dichloropyrim-
idine by Suzuki cross-coupling reaction with 4-pyridineboronic
acid to afford chloropyrimidine 65, which was reacted with
m-toluidine under Buchwald conditions to give pyrimidine 10.
The pyrazine 11 and pyridine 12 were prepared using similar
synthetic routes. Suzuki reaction of 3-bromoaniline and 4-pyridin-
eboronic acid gave intermediate 68, which underwent Buchwald
reaction with m-bromotoluene to give the carbocyclic analogue 13.
We had previously demonstrated that the introduction of small,
lipophilic moieties at the 2- and 3-position was tolerated, while 3-
aryl (e.g., 3-Ph-4-Ac 27) were selective and 3-acetylenic groups
N
N
(e)
R2
N
NH
O
S
Me
$
R1 = H, R2 = OH
14
R = H
(a, b)
(c)
(d)
69 R1 = H, R2 = N(Me)OMe
70 R1 = H, R2 = CH3
15 R = Ph
71
$
R
R
1 = H, R2 = CH2Br
1 = Ph, R2 = OH
(a, f)
72 R1 = Ph, R2 =CH2Br
Me
Me
N
N
N
(e)
R1
N
S
N
NH
O
16
$
R1 = CH3
Me
(d)
(d)
73 R2 = CH2Br
N
N
N
N
(e)
N
S
R1
NH
O
17
Me
74
R1 = CH3
75 R1 = CH2Br
Scheme 3. Reagents and conditions: (a) (COCl)2, DMF, DCM; (b) Me(OMe)NHꢀHCl,
NEt3, DCM; (c) MeMgBr, THF; (d) Br2, HOAc/HBr; (e) arylthiourea 58, EtOH; (f)
TMSCH2N2, THF; then HBr. $ = commercially available.
(e.g., 3–C„CCH2OMe 29) gave increased potency without lowering
selectivity leading to the positive CoMFA derived contour.20
NMe2
O
N
O
N
H
N
N
Me
Me
(a)
(b)
(d)
N
N
N
N
64
9
$
N
N
N
N
Cl
Cl
Cl
Cl
H
N
(c)
Cl
Cl
N
N
N
N
65
66
67
68
10
$
N
N
H
N
(c)
(e)
N
N
N
Me
N
11
$
$
N
N
N
N
Br
Br
N
Br
H
N
(c)
(c)
(e)
(f)
N
Br
N
Me
Me
12
NH2
H
N
NH2
13
$
Scheme 2. Reagents and conditions: (a) DMF–DMA, reflux; (b) 56, NaOH, iPrOH, reflux; (c) Pd(PPh3)4, 4-pyridineboronic acid, K2CO3, 1,4-dioxane, 80 °C; (d) m-toluidine, p-
TsOH, 1,4-dioxane, reflux; (e) m-toluidine, Pd(OAc)2, BINAP, NaOtBu, toluene, DMF, 90 °C; (f) m-bromotoluene, Pd(OAc)2, BINAP, NaOtBu, toluene, DMF, 90 °C.
$ = commercially available.

3350
M. Bonnet et al. / Bioorg. Med. Chem. 19 (2011) 3347–3356
N
R
N
N
(b)
(c)
N
S
N
S
N
S
X
NH
NH
NH
R
18-27 R = 3-Ar
Me
76 X = 3-Br
Me
Me
28 3-N, R = -CH2OH
39-49
77
(a)
R = 2-Ar
X = 2-Br
29 3-N, R = -CH2OMe
79 X = 3-I
30
31
3-N, R = -(CH2)2OH
3-N, R = -(CH2)3OH
(d)
82
3-N, R = H
83 2-N, R = H
N
N
(f)
N
N
N
S
N
(e)
Me
NBoc
NH
R1
S
NH
NH
Me
N
S
BnN
N
N
Me
Me
80 R1 = Br
32 X = O
34 3-N
(c)
X
33
50
2-N
81
X = NMe
R1 = CCCH2OH
N
N
N
N
S
N
N
(g)
(g)
R
X
NH
NH
S
R
S
Me
Me
35
77
51
R = 4-Methylpiperazine
R = 4-Methylpiperazine
X = 2-Br
78 X = 3-F
36 R = -NH(CH2)2NMorpholine
37 R = -O(CH2)2NMorpholine
52 R = -NH(CH2)2NMorpholine
53 R = -O(CH2)2NMorpholine
38
54
R = -O(CH2)3NMe2
R = -O(CH2)3NMe2
Scheme 4. Reagents and conditions: (a) CuI, NaI, H2N(CH2)2NH2, 1,4-dioxane, 110 °C; (b) PdCl2(dppf), Ar-boronic acid or ester, K2CO3, tol/EtOH/H2O/DMF, 90 °C; (c)
PdCl2(PPh3)2, CuI, HC„C–R, NEt3/DMF, 50–70 °C; (d) Boc2O, DMAP, DCM; (e) (i) MsCl, NEt3, DCM, ꢁ20 °C, (ii) morpholine or N-methylpiperazine, K2CO3, DMF, (iii) TFA, DCM;
(f) CuSO4, Na ascorbate, BnN3, DCM/EtOH/H2O, 20 °C; (g) ROH or RNH2, neat, Cs2CO3, 160–180 °C.
Table 1
IC50 values and selectivity ratios for B-ring analogues
N
N
N
X
Z
X
Z
H
N
X
Y
Me
NH
NH
Y
Y
W
Z
Me
Me
7,8
2-6
9-13
No.
X
Y
Z
W
RCC4 IC50
(l
M)
RCC4/VHL IC50
(l
M)
Ratioa
1
2
3
4
5
6
7
8
N
N
N
N
N
N
NMe
O
N
CH
CH
CH
CH
N
N
N
N
CH
CH
N
CH
CH
S
S
NH
O
S
NH
N
N
N
N
7
2.1
>70
40
>10
19
>40
>40
24.8
28.7
>40
>40
6.5
23.1
8.5
7.4
>40
>40
>40
22.2
>40
>40
8.6
28.1
31.8
>40
38.1
ND
ND
>1.6
0.8
ND
ND
1.3
1.2
3.7
>5.4
1.0
9
CH
N
CH
CH
CH
10
11
12
13
CH
N
N
CH
CH
CH
CH
36.9
a
Ratio = IC50 (RCC4/VHL)/IC50 (RCC4).
To further explore this positive contour we initially targeted a
bromoketone 71 which underwent condensation with thiourea
58 to afford 14. Quinoline 15 was synthesized from 2-phenylquin-
oline-4-carboxylic acid by conversion to the acid chloride then
treatment with TMSCH2N2 and the intermediate diazoketone was
reacted with HBr to give the bromoketone 72, which was con-
densed with 58 to give 15. Imidazopyridine 16 was synthesized
by bromination of methylimidazopyridinyl ethanone with Br2/
series of fused heteroaryl rings (14–17, Scheme 3). Quinoline ana-
logue 14 was prepared from quinoline-4-carboxylic acid which
was converted to the corresponding Weinreb amide 69 by reaction
with oxalyl chloride followed by treatment with N,O-dimethyl
hydroxylamine. Subsequent Grignard reaction with MeMgBr
to the ketone 70, then bromination with Br2/HBr gave the

M. Bonnet et al. / Bioorg. Med. Chem. 19 (2011) 3347–3356
3351
Table 2
Table 4
IC50 values and selectivity ratios for fused A-ring analogues
PAT CoMFA model
Model
q2a
SEPb
Compdc
r2d
SEEe
Steric
Electrostatic
R
N
15
2
3
4
5
0.352
0.270
0.345
0.246
0.423
0.313
0.431
0.449
0.428
0.475
0.429
0.463
3
4
3
4
5
6
0.818
0.852
0.809
0.831
0.878
0.893
0.229
0.202
0.231
0.225
0.197
0.182
0.618
0.549
0.607
0.597
0.552
0.510
0.382
0.451
0.393
0.403
0.448
0.490
NH
S
Me
No.
R
RCC4 IC50
12.3
(lM)
RCC4/VHL IC50
20.0
(lM)
Ratioa
1.6
6
N
N
Models 1–6 were constructed as described in the text and the compounds in each
set are listed in the Supplementary data.
14
a
q
2 is the cross-validated r2 derived from Leave-One-Out SAMPLS (LOO SAMPLS)
cross validation analysis as implemented in SYBYL8.2.
b
SEP is the Standard Error of Prediction reported for the LOO SAMPLS analysis.
c
Compd is the number of components used in the model.
15
25.4
>40
>1.6
r2, non-cross-validated correlation coefficient.
d
e
SEE is the Standard Error of Estimate derived from the non-validated analysis.
N
N
Me
positive contour adjacent to the pyridine ring (Scheme 4). These
were obtained by functionalization of the pyridine ring, starting
with halopyridines (76–78)20 that were further elaborated using
Pd-catalysed Suzuki and Sonogashira cross-coupling reactions (in
the case of bromo and iodo compounds) as well as nucleophilic
substitution. In some instances, 3-iodopyridine 79, conveniently
prepared from 3-bromopyridine 76 by Cu-catalysed halogen
exchange in the presence of NaI, was used due to the low reactivity
of the corresponding 3-bromopyridine 76. Halopyridines (76, 77,
79) were reacted with a series of aryl boronic acids or boronate es-
ters in the presence of PdCl2(dppf) to afford the Suzuki products
18–27 and 39–49. Sonogashira reactions of 3-bromopyridine 76
with propargyl alcohol, methyl propargyl ether, 1-butynol and 1-
pentynol, using PdCl2(PPh3)2 and CuI as catalysts gave compounds
28, 29, 30 and 31, respectively. The terminal hydroxyl group was
also replaced with polar amine groups to increase aqueous
16
17
9.5
29.8
15.3
3.1
N
N
>40
<0.4
a
Ratio = IC50 (RCC4/VHL)/IC50 (RCC4).
HBr to give the bromoketone intermediate 73 and subsequent
condensation with thiourea 58. Ketone 74, prepared by 1,3-dipolar
cycloaddition reaction of 3-butyn-2-one and 1-aminopyridinium
iodide,25 was treated with Br2/HBr to give bromoketone 75 which
was condensed with 3-methylphenylthiourea to provide pyrazolo-
pyridine 17.
Two sets of 3- and 2-substituted pyridyl analogues (18–38 and
39–54, respectively) were then synthesized to further explore the
Table 3
IC50 values and selectivity ratios for substituted A-ring analogues
N
2
N
S
R
3
NH
Me
No. 3-R
RCC4 IC50
M)
RCC4/VHL IC50
M)
Ratioa No. 2-R
RCC4 IC50
M)
RCC4/VHL IC50
M)
Ratioa
(
l
(
l
(
l
(l
18
19
20
21
22
23
Ph
4-Pyridyl
Ph-4-CF3
Ph-4-iPr
Ph-4-CONH2
Ph-4-CONpiperazine-N-
methyl
11.6
13.8
12.8
>40
4.1
33.2
34.0
22.6
>40
1.6
>40
2.9
2.5
1.8
ND
1.6
39
40
41
42
43
44
2-Ph
Ph-4-Ac
4-Pyridyl
2-Pyridyl-6-F
5-Pyrimidine
4-(1H-Indole)
19.1
38.8
28.9
>40
26.4
40.0
33.4
38.0
>40
37.0
4.5
2.1
0.9
1.3
ND
1.4
1.5
12.8
>3.1
3.1
24
25
Ph-4-CONH(CH2)3NMe2
Ph-4-SO2CH3
4.0
1.6
3.9
13.4
1.0
8.4
45
46
3-Thiophene
4-(3,5-
8.7
3
>40
36.5
>4.6
12
Dimethyloxazole)
3-pyrazole
26
Ph-4-SO2NHtBu
4.5
3.9
0.24
0.62
1.1
6.3
0.57
2.6
>40
2.6
19.5
22.5
35.1
2.9
27.9
2.8
0.3
2.3
2.2
>40
6.6
27.9
0.88
20.2
5.0
9
11.9
45
2.5
0.1
4.0
0.9
ND
2.5
1.4
2.8
7.4
47
48
49
50
51
52
53
54
0.48
5.2
2.2
2.1
11.5
3.7
7.0
6.0
20.0
8.1
66.1
14.0
31.6
8.0
14
1.6
30
6.7
2.8
2.2
3.0
1.5
27b Ph-4-Ac
4-(1-Methylpyrazole)
4-(1-Benzylpyrazole)
4-(1-Benzyltriazole)
4-Methylpiperazine
NH(CH2)2Nmorpholine
O(CH2)2Nmorpholine
O(CH2)3NMe2
28b C„CCH2OH
29b C„CCH2OMe
30
31
32
33
34
35
36
37
38
C„C(CH2)2OH
C„C(CH2)3OH
C„CCH2Nmorpholine
C„CCH2Npiperazine-4-Me
4-(1-Benzyltriazole)
4-Methylpiperazine
NH(CH2)2Nmorpholine
O(CH2)2Nmorpholine
O(CH2)3NMe2
21.1
8.8
0.32
2.7
a
Ratio = IC50 (RCC4/VHL)/IC50 (RCC4).
Data from Ref. 20.
b

3352
M. Bonnet et al. / Bioorg. Med. Chem. 19 (2011) 3347–3356
plates as described previously.17 The selectivity ratio was calcu-
lated as the intraexperiment ratio IC50 (RCC4/VHL)/IC50 (RCC4)
(Tables 1–4). Several active analogues were also examined for their
ability to induce formation of intracytoplasmic vacuoles to indicate
whether they were killing cells by the induction of autophagy. Cells
were incubated with drug (5
images were taken (Fig. 3).
lM) for 24 h before phase contrast
2.3. Molecular modelling
We previously reported a CoMFA derived 3D-QSAR model that
accounted for 34% (q2 = 0.344) of the variation in the selective
cytotoxicity of the PAT series against a VHL-deficient renal cell line,
and provided a putative bioactive conformation for this compound
series.20 This model (Fig. 1B) was recreated by molecular alignment
of the compound training set previously described against 28
(Table 4, Model 1) and differs marginally from that previously pub-
lished. Here we examine the effect of new PAT analogues on this
model (Table 4).
Incorporating Table 1 compounds of defined IC50 into Model 1
generated Model 2, and had a negative effect on the base model,
reducing the q2 from 0.352 to 0.27. Two conformers for each of
14–16 (Table 2) were assessed against Model 1, these conformers
explored different orientations of the pyridyl A-ring substitutions,
and the orientations with the best q2 values were used to build
Model 3, giving results similar to Model 1. Including 2-pyridyl sub-
stituents in Model 1 had a negative effect on q2, decreasing it from
0.352 (Model 1) to 0.246 (Model 4), 3-pyridyl substituents im-
proved q2 (0.423 vs 0.352). A set of compounds was then selected
across all substitution patterns that, along with those used in
Model 1, represent the complete PAT training set, and give Model
6 (see Supplementary data for composition of the training set).
The q2 of this model was comparable, albeit slightly reduced, to
Model 1 at 0.313, and is only able to account for 31% of the vari-
ance in biological activity.
Figure 2. New biologically relevant alignment.
solubility. Boc protection of 76 using Boc2O in DCM gave bromide
80 which underwent Sonogashira cross-coupling with 1-butynol to
produce intermediate alcohol 81 which was further functionalized
by treatment with MsCl, displacement with morpholine or
N-methylpiperazine, and removal of the Boc group with TFA to give
analogues 32 and 33.
Triazole analogues 34 and 50 were synthesized using ‘Click’
chemistry. Sonogashira cross-coupling reactions between bro-
mides 76 and 77 and TMS acetylene in the presence of PdCl2(PPh3)2
and CuI, followed by removal of the TMS group using K2CO3 gave
acetylenes 82 and 83.20 Reaction of 82 and 83 with benzyl azide
in the presence of copper sulfate and sodium ascorbate gave ben-
zyltriazole analogues 34 and 50, respectively.
In an effort to explore whether a flexible linker could access the
steric contour as effectively as the equivalent soluble acetylenes 32
and 33, a range of alkylamines linked at the 2- and 3-positions via
nitrogen and oxygen linker atoms were prepared. Nucleophilic
substitution of 3-fluoropyridine 78 and 2-bromopyridine 77 with
N-methylpiperazine, 2-morpholinoethanamine, 2-morpholinoeth-
anol, and 3-(dimethylamino)-1-propanol gave the derivatives 35–
38 and 51–54, respectively.
As one of the most important characteristics of a QSAR model is
its ability to predict a target property for molecules not used in its
development, the ability of Model 6 to predict IC50 values was
tested against an external test set of compounds generated from
Tables 1–3 along with additional compounds (from Ref. 20) not
used in the construction of Model 1 (Supplementary data, Table
S1). Many of these compounds either displayed low activity, had
2.2. Biological assays
Cellular growth inhibition was determined by IC50 assays, using
a four-day drug exposure of RCC4 and RCC4/VHL cells in 96-well
Figure 3. Induction of autophagy by PATs in RCC4 cells (RCC4 cells, 20ꢂ, after 5
lM drug exposure for 24 h).

M. Bonnet et al. / Bioorg. Med. Chem. 19 (2011) 3347–3356
3353
Table 5
Analysis of the effect of different fragment distinction sets on HQSAR models
Model
Fragment distinctiona
q2b
SEc
r2d
SEc
Components
HLe
7
8
9
None
A
A B
0.193
0.046
0.333
0.237
0.250
0.361
0.250
0.268
0.212
0.082
0.250
0.281
0.201
0.322
0.028
0.199
0.502
0.529
0.442
0.476
0.484
0.443
0.484
0.467
0.496
0.535
0.472
0.473
0.491
0.456
0.551
0.488
0.408
0.256
0.574
0.572
0.705
0.793
0.773
0.522
0.794
0.788
0.588
0.734
0.659
0.734
0.683
0.472
0.430
0.467
0.353
0.357
0.303
0.252
0.266
0.377
0.254
0.257
0.350
0.288
.0321
0.285
0.314
0.396
6
6
2
3
6
5
6
3
6
6
3
6
4
5
6
3
353
353
353
97
10
11
12
13
14
15
16
17
18
19
20
21
22
A B C
A B C H
A B C D
A B C H D
A B H
A B H D
A B D
A C
A C H
A C H D
A C D
A D
151
199
199
353
151
257
353
257
199
97
257
199
A H D
a
b
c
Fragment distinction; molecular features to be used in distinguishing among fragments, include A, atom, B, bond, C, connections, H, hydrogens, D, donor acceptor.
q2, cross-validated r2.
SE, standard error.
d
e
r2, non-cross-validated correlation coefficient. All models were generated using a fragment atom count range of 4–7 and selected based on best cross-validated r2.
HL, Hologram Length.
Table 6
IC50 values >40 lM, side chain substitutions with more than one
Optimizing fragment size for the HQSAR analysis using fragment distinction (A B C D)
rotatable bond, or unknown activity. Model 6 showed a linear rela-
tionship between experimental and predicted IC50 values for the
training set with a non-validated r2 of 0.89, Standard error of esti-
mation of 0.18 (Table 4 and Supplementary data, Fig. S1), and a
maximum difference between actual and predicted values (resid-
ual) of 0.44 (Supplementary data, Table S2A). While the log activity
range of the Model 6 training set ranged from 4.3 to 6.7, most com-
pounds (82%) were in the smaller range of 4.5–6 and this may con-
tribute to the low q2 value. Comparison of actual and predicted
activities for the test set does not show any correlation (r2 = 0.07)
(Supplementary data, Fig. S1), and many compounds with IC50 val-
Model Fragment
sizea
q2b
SEc
r2d
SEc
Components HLe
23
24
25
26
27
28
29
30
31
32
1–4
2–5
3–6
4–7
5–8
6–9
7–10
8–11
9–12
10–13
0.143 0.505 0.564 0.360
0.162 0.511 0.768 0.269
0.226 0.484 0.661 0.329
0.361 0.443 0.793 0.252
0.276 0.468 0.732 0.285
0.334 0.448 0.721 0.290
0.312 0.463 0.837 0.225
0.375 0.438 0.782 0.259
0.331 0.450 0.718 0.292
0.343 0.449 0.808 0.243
3
6
4
5
4
4
6
5
4
5
353
199
353
199
353
257
257
307
353
257
ues >40 l
M are incorrectly predicted, although an r2 of 0.10 when
a
b
c
Fragment size in atoms.
q2, cross-validated r2.
SE, standard error.
these compounds are excluded also indicates an absence of corre-
lation for the remainder of the test set with defined IC50 values. The
residual values ranged from 0.01 to 1.77 with approximately 55%
of the test set predicted within 0.45 log units, the maximum resid-
ual value observed for the training set, and 60% predicted within
0.5 log units (Supplementary data, Table S2B). This low predictivity
is consistent with the low q2 value. The CoMFA generated contour
maps are illustrated in Fig. 2.
r2, non-cross-validated correlation coefficient. All models were generated using
d
a fragment atom count range of 4–7 and selected based on best cross-validated r2.
e
HL, Hologram Length.
3. Discussion
To determine if the CoMFA model was limited by the alignment
strategy, a 2D-QSAR analysis was also performed using HQSAR.21
As HQSAR models are influenced by fragment size and distinction,
a series of models were generated using a range of parameters
(Table 5). Model 12 was the best fragment distinction combination,
with a q2 of 0.361, and this was slightly improved to 0.375 when
the fragment length was increased (Table 6, Model 26 vs Model
30). Interestingly, HQSAR Model 30 showed a reduced correlation
between actual and predicted values compared to the CoMFA Mod-
el 6, while the Standard Error was slightly higher. The maximum
difference between experimental and predicted activities for the
training set was 0.58 (Supplementary data, Table S3A). External
validation of Model 30 did not show any correlation between pre-
dicted and actual activities (r2 = 0.0009), and many compounds
A putative bioactive three-dimensional conformation of the PAT
series identified by CoMFA was used to guide synthesis of a set of
PAT analogues which were designed to explore the central B-ring
and its role in the alignment of the molecule and to probe the nat-
ure of the steric pocket adjacent to the pyridine A-ring. Previous
exploration of the basic SAR around the aniline C-ring had shown
that small, lipophilic substituents were optimal for activity hence
we chose to use the 3-methylaniline ring.20
The ligand model gave a consistent curved orientation with the
thiazole N placed in the inner surface and the thiazole S on the ex-
posed rim (Fig. 2). Replacement of the thiazole ring with any other
five-membered-ring heterocycle led to either a large drop in po-
tency and selectivity or complete loss of activity (Table 1). Replace-
ment of the thiazole S with another heteroatom (NH and O) led to
inactive analogues (3 and 4). Incorporation of an additional N with-
in the central ring gave much less potent derivatives (e.g., thiadia-
zole 5) while retaining a hint of selectivity, although lack of
aqueous solubility prevent a full determination in the RCC4/VHL
cell line (Table 1). Similarly, triazole analogues displayed low
potency and selectivity. It was not possible to fully evaluate the
with poor activity (IC50 >40 lM) were incorrectly predicted
(Supplementary data, Fig. S2), and the r2 was not improved when
these compounds were excluded. The residual values ranged from
0.06 to 1.44 with approximately 60% of the test set predicted
within 0.58 log units, the maximum residual value observed for
the training set (Supplementary data, Table S3B) and the range in
difference outside this cutoff was 0.60–1.44.

3354
M. Bonnet et al. / Bioorg. Med. Chem. 19 (2011) 3347–3356
influence of the thiazole N on the biological activity as our efforts
towards the synthesis of a thiophene analogue were unsuccessful
with the instability of the desired thiophene product precluding
purification. We compared the effect of the substitution of O for
S by overlaying minimised structures of thiazole 2, oxazole 4 and
thiadiazole 8 and noted that only a small conformational shift
was seen in the case of the oxazole (Supplementary data, Fig. S3).
Evaluation of the six-membered ring analogues showed that
introduction of a phenyl ring totally abolished selectivity (e.g.,
13). Retention of an N-atom in the 2-position of the central ring
was necessary for activity against the VHL-deficient RCC4 line
(e.g., 9, 11, 12), although it was not sufficient for selectivity with
pyrimidine 9 being nonselective. There was no correlation between
the pKa of either the pyridyl N, or the aniline NH, and cytotoxic
potency across the series 1–13.
Taken together, it seems likely that the thiazole N plays a key
role in the binding to the target; isosteric replacement of the thia-
zole S with @CH is tolerated in six-membered rings, but other min-
or modifications of the central ring result in a loss in potency, thus
these analogues were not pursued further.
The putative space around the pyridine ring was explored with
analogues bearing fused rings to provide an extended aromatic
region. We had previously demonstrated20 that a 4-pyridine was
the only regioisomer that retained activity and so the heterocycles
were designed to place an N-atom in a similar spatial location.
These rigid, bulky aromatic systems generally gave low potency
and selectivity (Table 2), suggesting that optimising the steric
interaction might require a more directed approach.
Hence, a set of aryl, alkynyl alkyloxy and alkylamino analogues
were synthesized by elaboration of the pyridine with a variety of
functional groups conveniently introduced by use of Suzuki and
Sonogashira reactions, Click chemistry, as well as direct nucleo-
philic displacement of aryl halides. Functionalization of the pyri-
dine at the 3-position was examined first with simple aryl 18
and pyridyl 19 substituents resulting in decreased potency and
selectivity relative to 2. Addition of a range of lipophilic substitu-
ents at the 4-position of the phenyl group (20–27) gave a range
of activities and suggested the potential for a lipophilic H-bond
acceptor such as sulphone 25 or methyl ketone 27 to provide po-
tency in the low micromolar range and modest selectivity. In con-
trast, more polar H-bond acceptor/donor carboxamides 22 and 24
were less potent and non-selective.
We had previously demonstrated the 3-propargyl analogues 28
and 29 displayed increased potency while maintaining selectiv-
ity,20 thus the higher homologues 30 and 31 were prepared. How-
ever, increasing alkyl chain length was not favourable and resulted
in decreasing potency and selectivity and increasing the terminal
bulk was also disfavoured, with only the morpholine 32 being
comparable to 28 and 29. Incorporation of the benzyltriazole unit
34 abolished activity.
Pyridines bearing flexible solubilising basic amine side chains
generally retained potency but displayed low selectivity (e.g., 35–
38). Curiously, the oxygen-linked amines 37 and 38 were superior
to the nitrogen-linked amines 35 and 36. The data on the 3-series
show that while substituted phenyl groups at the 3-position of the
pyridine gave some active compounds, the most potent analogues
were obtained with the introduction of a small H-bond acceptor
(e.g., CH2OH, CH2OMe) linked via a rigid acetylene.
We finally examined substitutions at the 2-position of the pyr-
idine to see if these could also provide positive steric interactions.
In contrast with the 3-series, use of a variety of six-membered
rings (40–44), gave compounds with low potency and selectivity.
Interestingly, the introduction of a range of five-membered ring
heterocycles (45–50) gave analogues that generally were active
and selective with the only anomaly being the 1-methyl pyrazole
48. The 1-benzylpyrazole derivative 49 had low micromolar
activity and a high selectivity ratio of 30, which was similar to
the benzyltriazole analogue 50, whereas the unsubstituted pyra-
zole analogue 47 showed a four-fold increase of potency while pre-
serving a modest selectivity of 14-fold. Together the data suggests
the presence of a second domain adjacent to the 2-position of the
pyridine ring which can accommodate a five-membered ring. The
exact definition of this domain and the associated H-bond contacts
remain to be fully characterised. Introduction an alkyl chain at the
2-position with solubilising tertiary amines gave compounds
(51–54) with low selectivity.
A series of active compounds (2, 32, 38, 47, and 49) were exam-
ined to confirm that the mechanism of cell death involved the
induction of autophagy. Treatment of RCC4 cells with 5 lM of each
drug for 24 h induced the formation of intracytoplasmic vesicles
confirming VHL-selective induction of autophagy resulting in cell
death (Fig. 3).
CoMFA was used with the data from this and an earlier study to
better understand the effects of substitutions around the PAT core
on activity. The 3D-QSAR model generated could only explain
approximately 30% of the variance in the IC50 data, and this was re-
flected in its lack of predictivity of an external test set of com-
pounds. A similar result was also found when a 2D-QSAR method
(HQSAR) was used, indicating that the alignment strategy used to
generate the CoMFA model was appropriate, and that the SAR data
of the PAT series with respect to autophagy may be quite complex.
Examination of the CoMFA contour maps generated from Model
6 using the complete training set (Fig. 2) shows that, within the
training set, the contouring around the aniline C-ring is similar to
that previously published. The new PAT CoMFA model exhibits a
large region adjacent to the sulfur of the thiazole B-ring of 28 that
suggests a positive electrostatic effect will favour activity. Interest-
ingly, this region has three distinct, yet interconnected regions.
Two of these are found around either side of the B-ring while the
third is more accessible by substitution from the pyridyl ring 3-
position substitution, as substitutions on this ring were all seen
to reduce activity. Around the pyridyl group, increased steric bulk
is positively associated with activity around the 2- and 3-positions,
consistent with the previous model. From the compounds charac-
terised in Table 3, this region is better accessed by substitutions at
the 3-position as suggested by the higher potency across a number
of homologues. Two distinct regions around the pyridyl 2-position
and nitrogen atom indicate contrasting effects of steric bulk on
activity. A region adjacent to the 2-position indicates that in-
creased steric bulk is associated with decreased activity (yellow
contour); although this is contrasted by the 2-pyrazole that has in-
creased bulk at this position yet has the strongest activity recorded
for a 2-position substitution. This region of disfavoured steric bulk
is also accessed by the fused ring compounds (Table 2), for exam-
ple, 15. The pyrazole moiety may contribute to an additional
favourable steric bulk zone adjacent to the N-atom (green contour).
The further development of the CoMFA alignment model has
not produced gains in the ability of the model to explain the QSAR
observed. Despite generating six different models we are unable to
explain more than 31% of the variance observed and did not signif-
icantly improve on this using the alternative HQSAR approach. This
low predictivity may be a consequence of the complexity of the
phenotypic readout which reports the product of many combined
parameters rather than just the interaction of the PAT with the
molecular target. Although we have further explored the SAR
around the pyridyl A-ring and B-ring, the relatively small gains
in potency and selectivity seen with the 2- and 3-substituted pyr-
idyl analogues, coupled with the relatively flat SAR profile ob-
served in our previous study, suggest that the PAT series may
occupy a local minimum and a large random leap may be neces-
sary to improve potency and selectivity substantially. We will
endeavour to use the alignment model generated in this study to

M. Bonnet et al. / Bioorg. Med. Chem. 19 (2011) 3347–3356
3355
conduct a virtual screen in an effort to identify new chemotypes
with VHL-selective activity. In another approach, we plan to ex-
ploit the steric tolerance adjacent to the pyridyl ring to introduce
linker moieties to assist in the preparation of molecular probes
to isolate and identify the molecular target of this PAT chemotype.
phase was washed with H2O (3 ꢂ 50 mL), washed with brine
(50 mL), dried and the solvent was evaporated. The residue was
purified by column chromatography, eluting with an appropriate
blend of EtOAc/pet. ether or aqueous NH3/MeOH/DCM, to give
the Suzuki product.
4.1.6. General method F. Sonogashira reactions with
bromopyridine (compounds 30, 31 and 81)
4. Experimental section
4.1. Chemistry
PdCl2(PPh3)2 (0.03 mmol) was added to a stirred, degassed solu-
tion of bromopyridine (0.58 mmol), alkyne (0.70 mmol) and CuI
(0.03 mmol) in a 1:1 mixture of DMF and NEt3 (4 mL), and the reac-
tion mixture was stirred at 50 °C for 16 h then at 70 °C for 4 h. After
cooling to 20 °C, the mixture was partitioned between EtOAc
(150 mL) and water (50 mL), the organic phase was washed with
water (3 ꢂ 50 mL), washed with brine (50 mL), dried and the sol-
vent was evaporated. The residue was purified by column chroma-
tography, eluting with an appropriate blend of EtOAc/pet. ether, to
give the alkyne.
4.1.1. General method A. Suzuki reaction of 2,6-dichloro
pyrimidine, 2,6-dichloro pyrazine, 2,6-dichloropyridine and 3-
bromoaniline (compounds 65–68)
Boronic acid or ester (10.0 mmol), K2CO3 (25.0 mmol) in H2O
(10 mL) and Pd(PPh3)4 (0.3 mmol) were added sequentially to a
solution of aryl halide (10.0 mmol) in 1,4-dioxane (40 mL). The
reaction mixture was stirred at 90 °C for 2–20 h and was then
cooled to 20 °C and partitioned between EtOAc (200 mL) and H2O
(100 mL). The organic phase was washed with water (3 ꢂ 50 mL),
washed with brine (50 mL), dried and the solvent was evaporated.
The residue was purified by column chromatography, eluting with
an appropriate gradient of EtOAc/pet. ether, to give the Suzuki
product.
4.1.7. General method G. Elaboration of propargyl alcohol
(compounds 32 and 33)
MsCl (0.28 mmol) was added to a solution of alcohol 81
(0.24 mmol) and NEt3 (0.36 mmol) in anhydrous DCM (10 mL) at
ꢁ20 °C and the reaction mixture was stirred at ꢁ20 °C for 1 h.
The mixture was diluted with DCM (100 mL), washed with water
(3 ꢂ 50 mL), washed with brine (50 mL), dried and the solvent
was evaporated. Amine (2.4 mmol) was added to a solution of
the crude mesylate in anhydrous THF (5 mL), and the mixture
was heated at 50 °C for 2 h. The mixture was cooled to 20 °C, par-
titioned between EtOAc (100 mL) and water (50 mL), the organic
layer was washed with aqueous NaHCO3 (50 mL), washed with
brine (50 mL), dried and the solvent was evaporated. A mixture
of crude carbamate in DCM (10 mL) and TFA (1 mL) was then stir-
red at 20 °C for 1 h. The solvent was evaporated and the residue
was purified by column chromatography, eluting with appropriate
blend of aqueous NH3/MeOH/DCM, to give the alkyne.
4.1.2. General method B. Buchwald reaction (compounds
11–13)
Pd(OAc)2 (0.37 mmol), BINAP (0.37 mmol) and NaOtBu
(10.30 mmol) were sequentially added to a stirred, degassed solu-
tion of aryl halide (3.68 mmol) and amine (7.36 mmol) in a mixture
of toluene (50 mL) and DMF (5 mL) and the mixture was heated at
90 °C for 4–5 h. The mixture was cooled to 20 °C, diluted with Et2O
(200 mL), washed with water (3 ꢂ 50 mL), washed with brine
(50 mL), dried and the solvent was evaporated. The residue was
purified by column chromatography, eluting with an appropriate
blend of EtOAc/pet. ether, to give the Buchwald product.
4.1.3. General method C. Preparation of pyridyl 2-
bromoethanones (compounds 71, 73 and 75)
4.1.8. General method H. Click reaction (compounds 34 and 50)
CuSO4ꢀ5H2O (0.02 mmol) and sodium ascorbate (0.04 mmol)
were added to a solution of acetylene (0.18 mmol) and benzyl azide
(0.20 mmol) in a 1:1:1 mixture of DCM/EtOH/H2O (3 mL), and the
mixture was stirred at 20 °C for 20 h. The mixture was partitioned
between EtOAc (100 mL) and water (50 mL), extracted with EtOAc
(2 ꢂ 70 mL), the combined organic phase was washed with water
(2 ꢂ 30 mL), washed with brine (30 mL), dried and the solvent was
evaporated. The residue was purified by column chromatography,
eluting with an appropriate blend of MeOH/DCM, to give the
triazole.
Br2 (80 mmol) was added dropwise to a stirred solution of ace-
tylpyridine (80 mmol) in 30% HBr/HOAc (100 mL) at 15 °C. The
mixture was stirred at 40 °C for 1 h and then 75 °C for 1 h. The mix-
ture was cooled to 20 °C, diluted with Et2O (400 mL) and stirred for
30 min. The precipitate was filtered, washed with Et2O (25 mL) and
dried under vacuum to give the bromoethanone as the hydrobro-
mide salt.
4.1.4. General method D. Preparation of thiazoles (compounds
14–17)
A mixture of bromoketone hydrobromide (3 mmol) and phenyl-
thiourea (3 mmol) in EtOH (20 mL) was stirred at reflux tempera-
ture for 1 h. The mixture was cooled to 20 °C, diluted with water
(50 mL), the pH adjusted to ca. 8 with aqueous NH3 and the mix-
ture stirred at 20 °C for 2 h. The precipitate was filtered, washed
with water (5 mL) and dried. The solid was purified by column
chromatography, eluting with an appropriate blend of EtOAc/pet.
ether, to give the thiazole.
4.1.9. General method I. Fluoride and bromide displacement
reactions (compounds 35–38 and 51–54)
A mixture of 3-fluoro or 2-bromopyridine (0.35 mmol) in the
appropriate amine or alcohol (2 mL) was heated in a sealed glass
pressure vessel at 100–180 °C for 16–24 h (in some cases, the reac-
tion was done in the presence of Cs2CO3). The solution was cooled
to 20 °C, partitioned between EtOAc (100 mL) and water (50 mL),
the organic phase was washed with water (2 ꢂ 50 mL), washed
with brine (50 mL), dried and the solvent was evaporated. The res-
idue was purified by column chromatography, eluting with an
appropriate blend of aqueous NH3/MeOH/DCM to give the substi-
tution product.
4.1.5. General method E. Suzuki reactions with bromo- and
iodopyridines (compounds 18–27 and 39–49)
PdCl2(dppf) (0.03 mmol) was added to a stirred, degassed solu-
tion of bromo- or iodopyridine (0.29 mmol), boronic acid or ester
(0.45 mmol) and K2CO3 (3.00 mmol) in a mixture of toluene/
EtOH/H2O/DMF (5:3:1.5:4, 13.5 mL) and the mixture was heated
at 90 °C for 16 h. After cooling to 20 °C, the mixture was partitioned
between EtOAc (100 mL) and water (50 mL), the combined organic
4.2. Cell viability assays
For 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-
5-carboxanilide (XTT) assays, five thousand cells were plated in

3356
M. Bonnet et al. / Bioorg. Med. Chem. 19 (2011) 3347–3356
96-well plates. The next day, vehicle (DMSO) or drug was added by
serial dilution. Four days later the media was aspirated, XTT solu-
tion (0.3 mg/ml of XTT (Sigma), 2.65 mg/ml N-methyl dibenxopyr-
azine methyl sulfate (Sigma) in phenol red-free media) was added,
and the plates were incubated at 37 °C for 1–2 h. Metabolism of
XTT was quantified by measuring the absorbance at 450 nm. IC50
values were calculated using linear interpolation.
the highest q2 value. Using the default fragment size of 4–7 atoms,
the effect of different fragment distinctions on q2 was explored.
The optimal fragment distinction combination was then used to
characterise the effect of fragment size on HQSAR model q2.
Acknowledgements
The authors thank Dr. Maruta Boyd, Dr. Shannon Black, Stefanie
Maurer and Sisira Kumara for technical assistance and acknowl-
edge the Association for International Cancer Research 10-0042
(M.B.), the Maurice Wilkins Centre for Biodiscovery (M.P.H.,
J.U.F.), US NCI-CA-82566 (A.J.G., M.P.H.), NCI-CA-123823 (D.A.C.),
NCI-T32-CA-121940 (E.W.L.) for funding.
4.3. QSAR modelling
4.3.1. Alignment generation
A minimum energy conformation of 2 was generated using
OMEGA2 (Openeyes Software, USA) with default parameters using
the MMFF94s force field and the rms torsion driving parameter set
at 0.6. All other structures were generated from this conformation
using the SKETCHER (SYBYL8.0, TRIPOS) and minimised using
MAXMIN2 (SYBYL8.0) with the Tripos Force field and Gastieger–
Huckel charges. Minimisation was performed using 1000-steps of
steep decents followed by conjugate gradients until convergence
at 0.05 kcal/(mol Å). A distance dependent dielectric function was
used with a dielectric constant of 80. Molecular alignments were
generated by superimposition of all compounds onto 28 using
ROCS (Openeye Software, USA) with default parameters using both
the shape only and shape in combination with colour force field
method. While the shape only alignment was used for CoMFA,
those compounds poorly aligned as assessed after visual inspec-
tion, were replaced by improved alignments from the shape and
colour force field combination alignment if available. Alternate
proton positions were explored for compound 6, while alternative
conformers based on rotating the B-ring relative to the C-ring were
also explored.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.04.042.
References and notes
1. Motzer, R. J.; Mazumdar, M.; Bacik, J.; Berg, W.; Amsterdam, A.; Ferrara, J. J. Clin.
Oncol. 1999, 17, 2530.
2. Murai, M.; Oya, M. Curr. Opin. Urol. 2004, 14, 229.
3. Gnarra, J. R.; Tory, K.; Weng, Y.; Schmidt, L.; Wei, M. H.; Li, H.; Latif, F.; Liu, S.;
Chen, F.; Duh, F.-M.; Lubensky, I.; Duan, D. R.; Florence, C.; Pozzatti, R.; Walther,
M. M.; Bander, N. H.; Grossman, H. B.; Brauch, H.; Pomer, S.; Brooks, J. D.; Isaacs,
W. B.; Lerman, M. I.; Zbar, B.; Linehan, W. M. Nat. Genet. 1994, 7, 85.
4. Kim, W. Y.; Kaelin, W. G., Jr. J. Clin. Oncol. 2004, 22, 4991.
5. Maxwell, P. H.; Wiesener, M. S.; Chang, G. W.; Clifford, S. C.; Vaux, E. C.;
Cockman, M. E.; Wykoff, C. C.; Pugh, C. W.; Maher, E. R.; Ratcliffe, P. J. Nature
1999, 399, 271.
6. Gnarra, J. R.; Zhou, S.; Merrill, M. J.; Wagner, J. R.; Krumm, A.; Papavassiliou, E.;
Oldfield, E. H.; Klausner, R. D.; Linehan, W. M. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 10589.
7. Iliopoulos, O.; Levy, A. P.; Jiang, C.; Kaelin, W. G., Jr.; Goldberg, M. A. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 10595.
8. Knebelmann, B.; Ananth, S.; Cohen, H. T.; Sukhatme, V. P. Cancer Res. 1998, 58,
226.
9. Staller, P.; Sulitkova, J.; Lisztwan, J.; Moch, H.; Oakeley, E. J.; Krek, W. Nature
2003, 425, 307.
10. Erler, J. T.; Bennewith, K. L.; Nicolau, M.; Dornhofer, N.; Kong, C.; Le, Q. T.; Chi, J.
T.; Jeffrey, S. S.; Giaccia, A. J. Nature 2006, 440, 1222.
11. Ohh, M.; Yauch, R. L.; Lonergan, K. M.; Whaley, J. M.; Stemmer-Rachamimov, A.
O.; Louis, D. N.; Gavin, B. J.; Kley, N.; Kaelin, W. G., Jr.; Iliopoulos, O. Mol. Cell
1998, 1, 959.
12. Hergovich, A.; Lisztwan, J.; Barry, R.; Ballschmieter, P.; Krek, W. Nat. Cell Biol.
2003, 5, 64.
13. Okuda, H.; Saitoh, K.; Hirai, S.; Iwai, K.; Takaki, Y.; Baba, M.; Minato, N.; Ohno,
S.; Shuin, T. J. Biol. Chem. 2001, 276, 43611.
14. Na, X.; Duan, H. O.; Messing, E. M.; Schoen, S. R.; Ryan, C. K.; di Sant’Agnese, P.
A.; Golemis, E. A.; Wu, G. EMBO J. 2003, 22, 4249.
4.3.2. 3D-QSAR analysis
CoMFA was used to determine a relationship between the com-
pounds tested and in vitro cytotoxicity as previously described.20
The SAMPLS Leave-One-Out (LOO) protocol was used to determine
the cross-validated r2 (q2) as a indicator of the predictivity of the
3D-QSAR model along with the optimum number of components,
which was subsequently used in a conventional non-validated
PLS analysis. Only compounds that had defined IC50 data and cyto-
toxicity ratios, and substitutions including up to one rotatable
bond were used. A base CoMFA 3D-QSAR model was created using
a previously described compound set (Model 1)20 and compounds
from each substitution pattern were added into this generating
Models 2–5, and a compound set representing all PAT analogues
was used to generate Model 6. The compound sets used to generate
each model are listed in the Supplementary data.
15. Kaelin, W. G., Jr. Nat. Rev. Cancer 2005, 5, 689.
16. Chan, D. A.; Giaccia, A. J. Cell Cycle 2008, 7, 2987.
17. Turcotte, S.; Sutphin, P. D.; Chan, D. A.; Hay, M. P.; Denny, W. A.; Giaccia, A. J.
Cancer Cell 2008, 14, 90.
18. Turcotte, S.; Giaccia, A. J. Curr. Opin. Cell Biol. 2010, 22, 246.
19. Fleming, A.; Noda, T.; Yoshimori, T.; Rubinstein, D. C. Nat. Chem. Biol. 2011, 7, 9.
20. Hay, M. P.; Turcotte, S.; Flanagan, J. U.; Bonnet, M.; Chan, D. A.; Sutphin, P. D.;
Nguyen, P.; Giaccia, A. J.; Denny, W. A. J. Med. Chem. 2010, 53, 787.
21. Heritage, T. W.; Lowis, D. R. Molecular hologram QSAR. In Rational Drug Design
Novel Methodology and Practical Applications; Parrill, A. L., Reddy, M. R., Eds.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1999;
Vol. 719, pp 212–225.
22. Mahboobi, S.; Sellmer, A.; Eswayah, A.; Elz, S.; Uecker, A.; Böhmer, F.-D. Eur. J.
Med. Chem. 2008, 43, 1444.
23. Tavares, F. X.; Boucheron, S. H.; Griffin, R. J.; Preugschat, F.; Thomson, S. A.;
Wang, T. Y.; Zhou, H.-Q. J. Med. Chem. 2004, 47, 4716.
24. Adams, H.; Batten, S. R.; Davies, G. M.; Duriska, M. B.; Jeffery, J. C.; Jensen, P.; Lu,
J.; Motson, G. R.; Coles, S. J.; Hursthouse, M. B.; Ward, M. D. Dalton Trans. 2005,
1910.
25. Miki, Y.; Nakamura, N.; Hachiken, H.; Takemura, S. J. Heterocycl. Chem. 1989, 26,
1739.
4.3.3. 2D-QSAR analysis
The 2D-Hologram QSAR (HQSAR) method21 as implemented in
SYBYLx1.2 was used to eliminate any dependency of the CoMFA
QSAR model on compound 3D structure, conformation and align-
ment strategy. In this method each compound is broken down into
all possible linear, branched and cyclic structural fragments and
these are counted into arrays of fixed length, a molecular holo-
gram. The bin occupancies associated with the molecular hologram
are descriptor variables that can be related to biological activity by
Partial Least Squares analysis. As HQSAR models can be affected by
hologram length, fragment size and distinction, a series of models
characterising variation in these parameters were generated to
find the optimal QSAR model for the PAT series as determined by