Identifying novel targets in renal cell carcinoma: Design and synthesis of affinity chromatography reagents

Article abstract of DOI:10.1016/j.bmc.2013.12.028

Two novel scaffolds, 4-pyridylanilinothiazoles (PAT) and 3-pyridylphenylsulfonyl benzamides (PPB), previously identified as selective cytotoxins for von Hippel-Lindau-deficient Renal Carcinoma cells, were used as templates to prepare affinity chromatograp

Full text of DOI:10.1016/j.bmc.2013.12.028

Bioorganic & Medicinal Chemistry 22 (2014) 711–720
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identifying novel targets in renal cell carcinoma: Design
and synthesis of affinity chromatography reagents
Muriel Bonnet a, Jack U. Flanagan a,b, Denise A. Chan c, , Amato J. Giaccia c, Michael P. Hay a,b,
⇑
a Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand
c 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:
Two novel scaffolds, 4-pyridylanilinothiazoles (PAT) and 3-pyridylphenylsulfonyl benzamides (PPB), pre-
viously identified as selective cytotoxins for von Hippel–Lindau-deficient Renal Carcinoma cells, were
used as templates to prepare affinity chromatography reagents to aid the identification of the molecular
targets of these two classes. Structure–activity data and computational models were used to predict pos-
sible points of attachment for linker chains. In the PAT class, Click coupling of long chain azides with
2- and 3-pyridylanilinothiazoleacetylenes gave triazole-linked pyridylanilinothiazoles which did not
retain the VHL-dependent selectivity of parent analogues. For the PPB class, Sonagashira coupling of
4-iodo-(3-pyridylphenylsulfonyl)benzamide with a propargyl hexaethylene glycol carbamate gave an
acetylene which was reduced to the corresponding alkyl 3-pyridylphenylsulfonylbenzamide. This
reagent retained the VHL-dependent selectivity of the parent analogues and was successfully utilized
as an affinity reagent.
Received 11 October 2013
Revised 29 November 2013
Accepted 8 December 2013
Available online 20 December 2013
Keywords:
Click chemistry
Sonogashira cross coupling
Renal cell carcinoma
Von Hippel–Lindau factor
GLUT-1
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
advanced RCC. Common to a majority of RCCs is the loss of function
of the von Hippel–Lindau (VHL) tumour suppressor gene.7 The VHL
Identification of tumour-selective agents is a top priority in
anticancer drug development. One approach is to leverage a genet-
ic abnormality common to a particular tumour type and to identify
agents that are selectively cytotoxic to tumour cells with this ge-
netic abnormality. We recently used such a synthetic lethality ap-
proach1–3 to discover two novel chemotypes that were selectively
cytotoxic to Renal Cell Carcinoma (RCC) cells lacking the von
Hippel Lindau factor (VHL) both in vitro and in vivo.4,5
RCCs are refractory to standard chemo- and radiotherapy and
advanced RCC has an extremely poor prognosis.6 Although new
‘targeted’ anti-angiogenic agents such as sunitinib and sorafinib
have been approved for use against the highly vascularised ad-
vanced RCC, these agents provide limited efficacy and patients
eventually relapse and succumb to their disease. Thus, there is still
a cogent need for drugs with increased efficacy in the treatment of
protein regulates a variety of proteins,8 including the activity of the
Hypoxia Inducible Factor (HIF) family of transcription factors, by
targeting them for degradation. Loss of this control increases HIF
activity and increases transcription of a wide range of genes.9 This
genetic response mimics the impact of tumour hypoxia and pro-
motes reprogramming of tumor metabolism, progression, invasion,
and metastasis, resulting in an aggressive phenotype, poor progno-
sis and resistance to therapeutic agents,10,11 and so the VHL-defi-
cient RCC cell line also provides a model of tumour cells under
hypoxic stress.
The two chemotypes identified in our synthetic lethal screen,
4-pyridylanilinothiazoles (PAT) (1) and 3-pyridylphenylsulfonyl
benzamides (PPB) (2) (Fig. 1) displayed selective cytotoxicity for
VHL-deficient RCC4 cells compared to RCC/VHL VHL-proficient
cells (Table 1) but displayed different phenotypic behaviour. In
the first case, PAT cytotoxicity was independent of HIF-1 status.
PAT compounds induced autophagy, as measured by LC3 immuno-
staining and Western blot analysis, and this led to cell death. Func-
tional analysis of the activity of the PAT class using a yeast deletion
pool implicated proteins involved in Golgi body processing as
important in the induction of autophagy, but failed to unequivo-
cally identify the target protein(s) of the PATs.4
Abbreviations: BOC, tert-butyloxycarbonyl; DCM, dichloromethane; DMF,
dimethylformamide; HTS, high throughput screening; PAT, pyridylanilino thiazole;
PPB, pyridylphenylsulfonyl benzamides; PEG, polyethylene glycol; RCC, renal cell
carcinoma; SAR, structure–activity relationship; TBTA, tris-(benzyltriazolyl)amine;
TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMS, trimethylsilyl.
⇑
Corresponding author. 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.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.028

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M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 711–720
N
potential feature for further analogue development and this
N
O
feature was further explored, along with the orientation of the
thiazole B-ring and the roles of the heteroatom substituents, in a
subsequent study.14 We used Suzuki–Miyaura and Sonogashira
Pd-mediated cross coupling reactions and nucleophilic displace-
ment reactions to prepare series of aryl-, alkynyl-, alkoxy- and
alkylamino-substituted pyridines, respectively, to explore the ste-
ric and electronic contours adjacent to the pyridyl ring. Although
we failed to improve the predictivity of the CoMFA model, we
did identify several analogues with increased potency and/or selec-
tivity to 1. The CoMFA model predicts the 2- and 3-positions of the
pyridine ring were the most favorable for functionalization with a
variety of groups at these positions providing potent and selective
compounds. Although, two acetylenic compounds displayed selec-
tivity for VHL-negative cells, a lack of selectivity for higher homo-
logues dissuaded us from using an acetylene linker. Either pyrazole
or triazole groups in the 2-position provided similar or improved
potency and selectivity compared to the parent 1 [IC50 (RCC4) ca.
N
S
tBu
N
H
NH
H
N
S
Me
O
O
1
2
Figure 1. PAT and PPB chemotypes identified from RCC HTS.
Table 1
IC50 values and selectivity ratios in Renal Cell Carcinoma cells
No
RCC4 IC50
(
l
M)
RCC4/VHL IC50
(
l
M)
Ratioa
1b
2
7
2.1
0.16
1.7
>40
>40
2.9
>40
5.8
40
19
>40c
2.6
>250
1.5
ND
ND
1.3
ND
>7
8
>40
>40
3.9
>40
>40
>40
>40
11
16
19
30
32
38
2 l
M, selectivity (RCC4VHL/RCC4), 6 to 30-fold)14 and suggested
7.9
>40
>5
ND
a ‘Click’15 strategy to incorporate a long chain linker suitable for
affinity chromatography.
a
b
c
Ratio = IC50 (RCC4/VHL)/IC50 (RCC4).
Data from Ref. 15.
Solubility prevented determination of IC50 values.
Our SAR studies on the PPB chemotype identified considerable
steric tolerance at the 4-position of the phenyl sulfonamide16
These data suggested molecules with a long chain linker attached
to this position may retain activity and may provide a plausible
strategy for attachment of linkers required for affinity reagents.
The overexpression of the facultative glucose transporter GLUT-1
in VHL-deficient RCC cells, combined with evidence for inhibition
of glucose uptake, led us to consider the possibility that the cyto-
toxicity of the PPBs was mediated through an interaction with
GLUT-1.5 We were able to use a homology model of GLUT-117 to
identify a binding mode consistent with the SAR (Fig. 3) and our
approach to position a long chain linker at the 4-position.
In contrast, PPB cytotoxicity was dependent on HIF-1 status and
resulted in necrotic cell death. The PPBs decreased glycolysis in a
VHL-dependent manner and inhibited the uptake of glucose.5
Further development of these novel chemotypes into viable
anticancer agents is critically dependent on the identification of
the molecular target of action. In this study we report our synthetic
efforts to use structure activity relationships (SAR), in combination
with molecular design, to develop chemical biology tools suitable
for use in an affinity chromatography approach12 for target identi-
fication for both PAT and PPB chemotypes.
3. Results
2. Molecular design
3.1. Chemistry
We expanded on the initial hit compound 1 and explored the
SAR for the PAT chemotype to identify more potent and selective
analogues, but were hampered in this by the lack of an identified
molecular target.13 This handicap led us to use a comparative
molecular field analysis (CoMFA) to determine possible bioactive
conformations to aid our studies. We identified a positive steric
contour (Fig. 2, green volume) adjacent to the pyridine ring as a
3.1.1. PAT synthesis
We elaborated the 2-acetylene PATs using ‘Click’ chemistry to
generate a substituted triazole. We had previously demonstrated
Figure 2. CoMFA model 3D-QSAR conformer model relating PAT conformation to
cytotoxicity. Electrostatic fields are contoured at 80% favored (blue) and 20%
disfavored (red). Steric fields are contoured at 80% favored (green) and 20%
disfavored (yellow).
Figure 3. A representation of PPB S3 (white ball and stick) and fasentin (blue stick)
modelled in the central transport channel of GLUT-1. Possible interactions with
Trp412 and Arg126 are shown.

M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 711–720
713
that Sonogashira cross coupling reaction of bromide 3 with TMS
acetylene in the presence of PdCl2(PPh3)2 and CuI as catalysts gave
an intermediate silylacetylene which was deprotected to give acet-
ylene 4 in 75% yield (Scheme 1). Formation of the benzyltriazole 5
from acetylene 4 and benzyl azide also proceeded in good yield.
Incorporation of a long chain alkyl linker using Click chemistry
was initially investigated for a BOC-protected aminohexylazide.
Reaction of tert-butyl (6-azidohexyl)carbamate with acetylene 4
gave triazole 6 in 39% yield which was deprotected to give amine
7 suitable for loading onto beads using N-hydroxysuccinimide cou-
pling chemistry. We also wished to incorporate a longer polyethyl-
ene glycol chain as a linker and used the commercially available
20-azido-3,6,9,12,15,18-hexaoxaeicosan-1-amine in a Click reac-
tion with acetylene 4 to give triazole 8 in 20% yield.
We had shown the aminothiazole NH is necessary for biological
activity and so designed an appropriate negative control
(Scheme 1). Alkylation of bromide 3 with ethyl iodide in DMF gave
9 in 66% yield. Sonogashira cross coupling reaction with TMS acet-
ylene using PdCl2(PPh3)2 and CuI in DMF/NEt3 gave, after deprotec-
tion of the TMS group using potassium carbonate in THF/MeOH,
the acetylene 10. Click reaction with 20-azido-3,6,9,12,15,18-hexa-
oxaeicosan-1-amine gave triazole product 11 in 71% yield.
We also explored the positive steric contour adjacent to the
pyridyl ring via the 3-pyridyl position, although this proved more
challenging due to the low reactivity at this position. The 3-bromo-
pyridyl analogue 12 underwent Sonogashira cross-coupling reac-
tion with TMS acetylene with subsequent deprotection of the
TMS group to give the 3-acetylene 13 in low (10–35%) yields
(Scheme 2). In an alternative strategy, 3-bromopyridine 12 was
converted to the corresponding 3-iodopyridine 14 by a copper-cat-
alyzed halogen exchange reaction. Sonogashira reaction with the
iodide 14 gave acetylene 13 in 52% yield. Since this route adds
an additional step and the overall yield was not significantly high-
er, it was not adopted.
We had previously demonstrated Click reaction of 3-acetylene
13 with benzyl azide gave benzyl triazole 15 in 55% yield.14 In this
instance, Click reaction of acetylene 13 with 20-azido-
3,6,9,12,15,18-hexaoxaeicosan-1-amine using CuSO4ꢀ5H2O and so-
dium ascorbate at room temperature, or at 40 °C, gave no product.
However, addition of tris(benzyltriazolyl)amine (TBTA) ligand18
and increased reaction temperature (60 °C) gave the triazole PAT
derivative 16 in 20% yield.
A negative control was also prepared for the 3-pyridyl isomer
based on the observation that N-alkylation of the aniline abolished
activity against RCC4 cells.13 Alkylation of 3-bromopyridine 12
with ethyl iodide in DMF gave 17 in 60% yield. Sonogashira
cross-coupling reaction with TMS acetylene and subsequent
deprotection of the TMS group gave 3-acetylene 18 in 65% yield.
Finally, Click reaction in the presence of the TBTA ligand at 60 °C
gave triazole product 19 in 41% yield.
3.1.2. PPB synthesis
We were able to readily prepare the propargyl-PEG6-carbamate
2419 efficiently from commercially available hexaethylene glycol
20 (Scheme 3). Glycol 20 was selectively mono-mesylated using
mesyl chloride in the presence of silver oxide. Treatment of the
mesylate 21 with sodium azide gave azide 22 in 99% yield. Reduc-
tion of 22 gave the intermediate amine which was protected as the
carbamate 23 in 80% yield. Reaction of alcohol 23 with propargyl
bromide gave the acetylene 24.
Condensation of the protected benzoic acid 2520 with 3-amino-
pyridine gave the amide 26 which was deprotected and coupled to
4-iodophenylsulfonyl chloride to give the iodide 28 (Scheme 4).
Sonogashira coupling was used to attach long chain linkers to the
PPB core structure. Thus, reaction of 28 with propargyl-PEG6-car-
bamate 24 gave the protected acetylene 29 which was readily
deprotected to give amine 30. We also explored a more flexible lin-
ker with reduction of the acetylene 29 giving carbamate 31 which
was deprotected to give amine 32. The negative control reagent
was prepared using a phenyl replacement for the 3-pyridyl moiety
based on the previously determined SAR which demonstrates
increased potency for 3-pyridyl analogues.16 Thus in a similar
sequence, coupling of benzoic acid 25 with aniline gave the amide
33 which was deprotected and coupled with 4-iodophenylsulfonyl
chloride to give iodide 35. Sonogashira coupling with 24 gave the
acetylene 36 which was reduced and deprotected to give amine 38.
3.2. Biological assays
The potency and selectivity of the potential affinity reagents
were evaluated in a XTT growth inhibition assay (as IC50: the drug
concentration over a 4 d exposure required to inhibit cell growth
by 50%) in the RCC4 VHL negative and the RCC/VHL positive cell
lines (Table 1) to identify reagents with sufficient selectivity for
target identification studies. Unfortunately, addition of long chain
triazole substituents at the 2-position of the PAT scaffold was
accompanied by loss of selectivity (7) or loss of potency (8 and
11). This was mirrored for analogues with long chain amines at
the 3-position (16 and 19) and these negative results halted our
efforts with this class. In contrast, the PPB reagents 30 and 32
retained potency and selectivity, although somewhat attenuated,
for the RCC4 cell line, whereas the negative control 38 was inactive
as required. Compounds 32 and 38 were conjugated to Affigel-10
resins and used for affinity chromatography on RCC4 cell
homogenates using antibodies against GLUT-1, GLUT-2 and
N
N
N
R
N
S
(a)
R
N
(b)
N
S
N
S
Br
Bn
N
N
N
NH
N
N
N
Me
Me
Me
5
3 R = H
4
R = H
(d)
(b)
9
10
R = Et
R = Et
(b)
N
N
N
S
RHN
R
N
S
O
NH
H2N
N
N
N
6
N
N
Me
Me
6 R = CO2tBu
8
R = H
R = Et
(c)
7
11
R = H
Scheme 1. Reagents and conditions: (a) (i) TMS-acetylene, PdCl2(PPh3)2, CuI, NEt3, DMF, 50 °C; (ii) K2CO3, THF/MeOH, 20 °C; (b) azide, CuSO4ꢀ5H2O, Na ascorbate, DCM/H2O/
EtOH, 20 °C; (c) TFA/DCM, 20 °C; (d) EtI, DMF, 20 °C.

714
M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 711–720
N
N
N
Bn
R1
N
N
N
(a)
(c)
R
N
N
S
R2
N
N
S
NH
N
S
Me
Me
Me
15
12 R1 = Br, R2 = H
13 R = H
(b) (e)
14
17
18
R
R
1 = I, R2 = H
R = Et
1 = H, R2 = Et
(d)
N
O
N
NH3
CF3CO2
N
6
N
R
N
N
S
Me
16
R = H
19 R = Et
Scheme 2. Reagents and conditions: (a) (i) TMS-acetylene, PdCl2(PPh3)2, CuI, NEt3, DMF, 50 °C; (ii) K2CO3, THF/MeOH, 20 °C; (b) CuI, NaI, MeNH(CH2)2NHMe, dioxane, 110 °C;
(c) CuSO4ꢀ5H2O, Na ascorbate, DCM/H2O/DMF, 20 °C, 20 h; (d) azide, CuSO4ꢀ5H2O, Na ascorbate, TBTA, DCM/H2O/DMF, 60 °C, 20 h; (e) EtI, DMF, 20 °C.
O
(e)
O
O
NHCO2tBu
HO
R
5
5
24
20 R = OH
(a)
(b)
21
22
R = OMs
R = N3
(c), (d)
23 R = NHCO2tBu
Scheme 3. Reagents and conditions: (a) MsCl, Ag2O, DCM; (b) NaN3, DMF; (c) H2 (60 psi), Pd/C, EtOH; (d) BOC2O, DCM; (e) MsCl, Et3N, DCM; (f) HCl/MeOH; (g) NaH, propargyl
bromide, nBu4N+Iꢁ, THF.
X
X
O
O
O
(a)
(c)
I
(d)
N
H
N
H
HO
H
N
R
NHCBZ
S
O
O
25
26 X = N, R = NHCBZ
27
28
X = N
(b)
(b)
X = N, R = NH2.HBr
35 X = CH
33 X = CH, R = NHCBZ
34
X = CH, R = NH2.HBr
X
X
O
O
R
O
R
6
N
N
H
O
(f)
H
N
H
N
H
6
S
S
O
O
O
O
31
29
X = N, R = NHCO2tBu
X = N, R = NHCO2tBu
(e)
(g)
(g)
32 X = N, R = NH2
30 X = N, R = NH2
37 X = CH, R = NHCO2tBu
36 X = CH, R = NHCO2tBu
38
X = CH, R = NH2
Scheme 4. Reagents and conditions: (a) (COCl)2, DMF, THF, 50 °C, then 3-aminopyridine or aniline; (b) HBr, HOAc, 20 °C; (c) 4-IPhSO2Cl, pyridine, 20 °C; (d) PdCl2(PPh3)2, 24,
CuI, Et3N, DMF, 50 °C; (e) HCl, MeOH, 20 °C; (f) H2, Pd/C, EtOH, 20 °C; (g) TFA, DCM, 20 °C.
GLUT-3 for identification.5 This demonstrated selective retention
of GLUT-1 protein by reagent 32, but not reagent 38, confirming
GLUT-1 as a molecular target of the PPB chemotype.
screen. The two classes of compound had quite different properties
with the PAT class inducing cell death via autophagy in
a
VHL-dependent manner and the PPB class inducing necrotic cell
death via disruption of glucose metabolism in a HIF-dependent
manner. Our pursuit of chemical biology tools to tease out the
molecular targets was guided by established SAR and interpreta-
tion using molecular modelling. The CoMFA model developed from
the SAR of the PAT class had demonstrated a positive steric contour
adjacent to the pyridyl ring, but attempts to exploit this putative
space gave mixed results. Our efforts to build on analogues bearing
pyrazole or triazole substituents at the 2-position were not suc-
cessful and none of the reagents prepared for the PAT class showed
4. Discussion
One of the risks of using phenotypic screens for drug discovery
is that a molecular target for any hits may not be apparent. Not
only does this hinder the ‘hit-to-lead’ development of the class, it
undermines any potential clinical development of the agents. Here
we have attempted to design chemical biology tools to assist in the
identification of two hits identified from the same phenotypic

M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 711–720
715
any selectivity for VHL-negative cells. That the CoMFA model could
account for only ca 34% of the variation in cytotoxicity data may
contribute to our inability to identify PAT reagents with selectivity
for VHL-deficient cells.
afforded by these agents is increasingly recognised as a novel
therapeutic strategy for the treatment of cancer.28,29
5. Experimental
5.1. Chemistry
On the other hand we had accumulated evidence suggesting
that glucose metabolism was the target process for the PPB class
and this provided a limited number of molecular targets. This
was further clarified through glucose uptake experiments which
demonstrated that the PPB class did inhibit glucose uptake.5 The
biochemical and genetic evidence suggested that GLUT-1 was a po-
tential molecular target and this was confirmed using the PPB
affinity chromatography reagents.5 Subsequently, we modelled a
series of ‘active’ PPB analogues and the known GLUT-1 inhibitor
fasentin21 in the transporter’s central channel to explore potential
interactions between the PPB scaffold and GLUT-1. The GLUT-1
homology model (PDB No. 1SUK) had been generated using the
glycerol 3-phosphate antiporter GlpT structure as an initial
homology template followed by evolutionary homology using glu-
cose-6-phosphate translocase as a template. The resulting struc-
ture accounted for the biochemical and mutagenic evidence, as
well as identifying binding sites for glucose and several inhibi-
tors.21 The active compounds (S1-S6; Supporting Information)
5.1.1. General procedures
Analyses were carried out in the Campbell Microanalytical
Laboratory, University of Otago, Dunedin, NZ. All final products
were analysed by reverse-phase HPLC, (Altima C18 5 lm column,
150 ꢂ 3.2 mm; Alltech Associated, Inc., Deerfield, IL) using an
Agilent HP1100 equipped with a diode-array detector. Mobile
phases were gradients of 80% acetonitrile/20% H2O (v/v) in
45 mM ammonium formate at pH 3.5 and 0.5 mL/min. Final com-
pound purity was determined by monitoring at 330 50 nM and
was >95%. Melting points were determined on an Electrothermal
2300 Melting Point Apparatus. NMR spectra were obtained on a
Bruker Avance 400 spectrometer at 400 MHz for 1H and 100 MHz
for 13C spectra. Spectra were obtained in (CD3)2SO unless other-
wise specified, and were referenced to Me4Si. Chemical shifts and
coupling constants were recorded in units of ppm and Hz, respec-
tively. Assignments were determined using COSY, HSQC, and
HMBC two-dimensional experiments. Low resolution mass spectra
were gathered by direct injection of methanolic solutions into a
Surveyor MSQ mass spectrometer using an atmospheric pressure
chemical ionization (APCI) mode with a corona voltage of 50 V
and a source temperature of 400 °C. High resolution mass spectra
(HRMS) were measured on a Bruker microTOF-QII Hybrid Quadru-
pole Time of Flight (TOF-Q) mass spectrometer interfaced with
either an Electrospray Ionization (ESI) or Atmospheric Pressure
Chemical Ionization (APCI) probe allowing positive or negative ions
detection. Solutions in organic solvents were dried with anhydrous
MgSO4. Solvents were evaporated under reduced pressure on a
rotary evaporator. Thin-layer chromatography was carried out on
aluminium-backed silica gel plates (Merck 60 F254) with visualiza-
tion of components by UV light (254 nm) or exposure to
I2. Column chromatography was carried out on silica gel (Merck
230–400 mesh).
were selected from those displaying <1 lM IC50 values against
the RCC4 cell line and a selectivity for the RCC4 over the RCC4/
VHL cell line of >100. Some predicted binding modes positioned
the 3-pyridyl moiety deep into the central channel towards the
extracellular end, occupying a position similar to that predicted
for fasentin (Fig. 3). In this orientation, the amide carbonyl group
occupies a similar location to amide carbonyl in fasentin, with both
groups potentially able to interact with the side chain of Arg126, a
residue involved in glucose transport.22 In this mode, the ligand’s
central aromatic group is located between the channel aromatic
residues Tyr28, Phe72 and Trp412. Additionally, the sulfonyl group
may also interact with the indole nitrogen of Trp412, another res-
idue associated with glucose transport.23 The various substituents
at the 4-phenyl position were oriented toward the intracellular
entrance of the channel, and the tolerance observed for various
substituents provides the rationale for appending a long chain lin-
ker group at the 4-phenyl position. Other predicted binding modes
indicated the potential for an interaction between the ligands pyr-
idyl N and the side chain amine of Lys38. Such an interaction may
explain loss of potency and selectivity seen for the phenyl deriva-
tive 38. Although the side chain positions were not optimized for
ligand binding in the GLUT1 homology model, the data indicate
the potential for the active compounds to interact with several
key residues that span the solute channel. The proposed binding
mode appears to occupy a similar site predicted for both the re-
cently reported thiazolidine 2,4-diones24 and diphenoxybenzo-
ates25–27 that inhibit glucose uptake through binding to GLUT-1.
In particular, the thiazolidine 2,4-diones are predicted to make
an electrostatic interaction with Trp412 and Arg126 as well as
Compounds 3, 4, 5, 12, 13, 14 and 15 were prepared as previ-
ously described.14,15
5.1.2. tert-Butyl 6-(4-{4-[2-(3-toluidino)-1,3-thiazol-4-yl]-2-
pyridinyl}-1H-1,2,3-triazol-1-yl)hexylcarbamate (6)
CuSO4ꢀ5H2O (16 mg, 0.064 mmol) and sodium ascorbate
(26 mg, 0.13 mmol) were added to a solution of acetylene 4
(185 mg, 0.64 mmol) and tert-butyl 6-azidohexylcarbamate
(160 mg, 0.70 mmol) in a mixture of DCM/H2O/EtOH (1:1:1,
3 mL). The reaction was stirred at 18 °C for 24 h and was then par-
titioned between EtOAc (150 mL) and H2O (50 mL). The organic
phase was washed with H2O (2 ꢂ 10 mL), dried and the solvent
was evaporated. The residue was purified by column chromatogra-
phy, eluting with 2% MeOH/DCM, to give triazole 6 (140 mg, 42%)
as a colourless viscous oil: 1H NMR d 10.31 (s, 1H, NH), 8.62–8.63
(m, 2H, H-50, H-600), 8.54 (dd, J = 1.6, 0.7 Hz, 1H, H-300), 7.80 (dd,
J = 5.1, 1.6 Hz, 1H, H-500), 7.79 (s, 1H, H-5000), 7.57 (br s, 1H, H-20000),
7.51 (br d, J = 8.1 Hz, 1H, H-60000), 7.25 (t, J = 7.8 Hz, 1H, H-50000),
6.83 (d, J = 7.4 Hz, 1H, H-40000), 6.72 (br s, 1H, NHBoc), 4.43 (t,
J = 7.0 Hz, 2H, H-6), 2.89 (q, J = 6.3 Hz, 2H, H-1), 2.35 (s, 3H, CH3),
1.84–1.92 (m, 2H, CH2), 1.36 [s, 11H, C(CH3)3, CH2], 1.27–1.28 (m,
4H, CH2); 13C NMR d 163.6, 155.5, 150.8, 150.1, 147.6, 147.2,
142.0, 140.9, 138.2, 128.9, 123.2, 122.3, 118.9, 117.7, 115.5,
114.2, 107.7, 77.3, 49.5, 29.6, 29.2, 28.2 (3), 25.6, 25.5, 21.3 (1C
form a
p–p
stacking interaction with Tyr2824 Similarly, the diph-
enoxybenzoate WZB114 was modelled binding to Arg126 and
Trp412, as well as Asn34. While these interactions remain to be
formally tested, it is possible that the hydrophobic and polar
groups contributed by these amino acids define a tractable drug
target within the solute channel of GLUT-1, and these may provide
an opportunity for the identification of new GLUT-1 inhibitors.
This study highlights the successful design and synthesis of
chemical biology tools to identify the molecular target of the PPB
class, while also showing that success in these endeavours is not
always guaranteed. Having identified the molecular target of the
PPBs we are now poised to further develop this class towards a
tumour-selective anti-tumour agent. The potential opportunity

716
M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 711–720
not observed); MS m/z 535.5 (MH+, 100%). Anal. Calcd for C28H35N7
H-50), 7.29 (br s, 1H, H-20), 7.26 (br d, J = 7.8 Hz, 1H, H-60), 7.20
(br d, J = 7.5 Hz, 1H, H-40), 4.02 (q, J = 7.1 Hz, 2H, CH2CH3), 2.36 (s,
3H, CH3), 1.22 (t, J = 7.1 Hz, 3H, CH2CH3); MS m/z 374.8/376.8
(MH+, 100%).
1
O2Sꢀ 3H2O: C, 62.31; H, 6.66; N, 18.17. Found: C, 62.39; H, 6.55; N,
17.94.
5.1.3. 6-(4-{4-[2-(3-Methylphenyl)-1,3-thiazol-4-yl]-2-
pyridinyl}-1H-1,2,3-triazol-1-yl)-1-hexanammonium
trifluoroacetate (7)
5.1.6. N-Ethyl-4-(2-ethynyl-4-pyridinyl)-N-(3-methylphenyl)-
1,3-thiazol-2-amine (10)
PdCl2(PPh3)2 (21 mg, 0.03 mmol) was added to a purged solu-
tion of bromopyridine 9 (203 mg, 0.54 mmol), TMS-acetylene
(0.43 mL, 3.0 mmol), and CuI (6 mg, 0.03 mmol) in a mixture of
DMF and NEt3 (1:1; 4 mL), and the reaction mixture was stirred
at 50 °C for 2 h. The reaction mixture was cooled to 18 °C, and par-
titioned between EtOAc (200 mL) and H2O (50 mL). The organic
phase was washed with brine (50 mL), dried and the solvent was
evaporated. The residue was dissolved in a mixture of THF/MeOH
(1:1; 20 mL), K2CO3 (75 mg, 0.54 mmol) was added and the mix-
ture was stirred at 18 °C for 1 h. The solvent was evaporated and
the residue purified by column chromatography, eluting with
20% EtOAc/pet. ether, to give acetylene 10 (145 mg, 84%) as a col-
ourless oil which was used directly: 1H NMR d 8.56 (dd, J = 5.2,
0.7 Hz, 1H, H-600), 7.96 (dd, J = 1.6, 0.7 Hz, 1H, H-300), 7.82 (dd,
J = 5.2, 1.7 Hz, 1H, H-500), 7.59 (s, 1H, H-5), 7.39 (t, J = 7.7 Hz, 1H,
H-50), 7.29 (br s, 1H, H-20), 7.25 (br d, J = 7.7 Hz, 1H, H-60), 7.19
(br d, J = 7.6 Hz, 1H, H-40), 4.32 (s, 1H, C„CH), 4.03 (q, J = 7.1 Hz,
2H, CH2CH3), 2.35 (s, 3H, CH3), 1.22 (t, 3H, CH2CH3); 13C NMR d
169.2, 150.6, 147.0, 144.0, 142.2, 141.9, 139.7, 129.9, 128.2,
127.1, 123.8, 123.3, 119.8, 107.7, 83.3, 80.0, 47.2, 20.8, 12.7; MS
m/z 320.7 (MH+, 100%).
TFA (0.44 mL, 5.8 mmol) was added to a solution of carba-
mate 6 (125 mg, 0.23 mmol) in DCM (15 mL), and the reaction
mixture was stirred at 18 °C for 3 h. The solvent was evaporated
and the residue was dried under high vacuum. The residue was
triturated with iPr2O, filtered and dried to give the trifluoracetate
salt 7 (130 mg, 100%) as an orange solid: mp (iPr2O) 179–181 °C;
1H NMR d 10.33 (s, 1H, NH), 8.68 (s, 1H, H-5000), 8.64 (br d,
J = 5.3 Hz, 1H, H-600), 8.57 (d, J = 0.9 Hz, 1H, H-300), 7.85 (dd,
J = 5.3, 1.7 Hz, 1H, H-500), 7.84 (s, 1H, H-5), 7.67 (br s, 3H, NH3),
7.58 (br s, 1H, H-20), 7.51 (br d, J = 8.1 Hz, 1H, H-60), 7.25
(t, J = 7.8 Hz, 1H, H-50), 6.83 (d, J = 7.5 Hz, 1H, H-40), 4.47
(t, J = 6.9 Hz, 2H, H-10000); 13C NMR
d
163.7, 158.1
(q, J = 35.0 Hz), 150.1, 149.5, 147.4, 146.6, 142.6, 140.9, 138.3,
128.9, 123.5, 122.4, 119.1, 117.7, 115.7, 114.3, 108.4, 49.5,
29.3, 26.7, 25.3, 25.1 (2), 21.3; MS m/z 435.2 (MH+, 100%). Anal.
Calcd for C28H35N7O2Sꢀ2.25CF3CO2H: C, 47.86; H, 4.27; N, 14.21.
Found: C, 47.98; H, 4.37; N, 14.13.
5.1.4. 4-{2-[1-(20-Amino-3,6,9,12,15,18-hexaoxaicos-1-yl)-1H-
1,2,3-triazol-4-yl]-4-pyridinyl}-N-(3-methylphenyl)-1,3-thiazol-
2-amine (8)
CuSO4ꢀ5H2O (5 mg, 0.02 mmol) and sodium ascorbate (8 mg,
0.04 mmol) were added to a suspension of acetylene 4 (50 mg,
0.17 mmol) and 20-azido-3,6,9,12,15,18-hexaoxaeicosan-1-amine
5.1.7. 4-{2-[1-(20-Amino-3,6,9,12,15,18-hexaoxaicos-1-yl)-1H-
1,2,3-triazol-4-yl]-4-pyridinyl}-N-ethyl-N-(3-methylphenyl)-
1,3-thiazol-2-amine (11)
(60 mg, 0.17 mmol) in
a mixture of DCM/H2O/EtOH (1:1:1;
CuSO4ꢀ5H2O (10 mg, 0.04 mmol) and sodium ascorbate (16 mg,
0.08 mmol) were added to a suspension of acetylene 10 (120 mg,
0.37 mmol) and 20-azido-3,6,9,12,15,18-hexaoxaeicosan-1-amine
(72 mg, 0.21 mmol) in a mixture of DCM/H2O/EtOH (1:1:1; 3 mL),
and the reaction mixture was stirred at 18 °C for 20 h. The mixture
was diluted with 20% MeOH/DCM (100 mL), SiO2 was added and
the solvent was evaporated. The residue was purified by column
chromatography, eluting with 0.5% NH4OH (28%)/1.5% MeOH/
EtOAc, to give triazole 11 (90 mg, 71%) as a tan oil: 1H NMR d
8.59 (dd, J = 5.2, 0.7 Hz, 1H, H-600), 8.58 (s, 1H, H-5000), 8.46 (dd,
J = 1.6, 0.7 Hz, 1H, H-300), 7.74 (dd, J = 5.2, 1.7 Hz, 1H, H-500), 4.58
(s, 1H, H-5), 7.40 (t, J = 7.7 Hz, 1H, H-50), 7.31 (br s, 1H, H-20),
7.27 (br d, J = 7.8 Hz, 1H, H-60), 7.20 (br d, J = 7.6 Hz, 1H, H-40),
4.62 (t, J = 5.2 Hz, 2H, H-10000), 4.07 (q, J = 7.0 Hz, 2H, CH2CH3), 3.89
(t, J = 5.2 Hz, 2H, H-20000), 3.56 (m, 2H, OCH2), 3.45–3.51 (m, 18 H,
OCH2), 3.34 (t, J = 5.8 Hz, 2H, H-190000), 2.64 (t, J = 5.8 Hz, 2H,
H-200000), 2.36 (s, 3H, CH3), 1.25 (t, J = 7.0 Hz, 3H, CH2CH3), NH2
not observed; 13C NMR d 169.2, 150.7, 150.0, 147.9, 147.2, 144.1,
142.2, 139.7, 129.8, 128.1, 127.1, 123.75, 123.71, 119.0, 115.4,
107.1, 72.8, 69.73 (3), 69.72 (4), 69.6 (2), 69.5, 68.6, 49.6, 47.1,
41.2, 20.8, 12.8; MS m/z 672.4 (MH+, 100%); HRMS Calcd for
1.5 mL), and the reaction mixture was stirred at 18 °C for 20 h.
The mixture was then diluted with EtOAc (100 mL) and washed
with H2O (3 ꢂ 10 mL), washed with brine (10 mL), dried and the
solvent was evaporated. The residue was purified by column chro-
matography, eluting with 0.5% NH4OH/1.5% MeOH/EtOAc, to give
triazole 8 (20 mg, 18%) as a tan glass: 1H NMR d 10.32 (br s, 1H,
NH), 8.63 (dd, J = 5.2, 0.6 Hz, 1H, H-600), 8.58 (s, 1H, H-5000), 8.55
(dd, J =1.6, 0.7 Hz, 1H, H-300), 7.78–7.81 (m, 2H, H-5, H-500), 7.58
(s, 1H, H-20), 7.50 (br d, J = 8.1 Hz, 1H, H-60), 7.25 (t, J = 7.8 Hz,
1H, H-50), 6.83 (d, J = 7.5 Hz, 1H, H-40), 6.62 (br s, 2H, NH2), 4.63
(t, J = 5.2 Hz, 2H, H-1), 3.90 (t, J = 5.2 Hz, 2H, H-2), 3.56 (m, 2H,
OCH2), 3.49–3.52 (m, 2H, OCH2), 3.44–3.48 (m, 16 H, OCH2), 3.33
(t, J = 5.8 Hz, 2H, H-19), 2.63 (br t, J = 5.4 Hz, 2H, H-20), 2.35 (s,
3H, CH3); 13C NMR d 163.6, 150.7, 150.1, 147.6, 147.2, 142.0,
140.9, 138.2, 128.9, 123.7, 122.3, 118.9, 117.7, 115.5, 114.2,
107.7, 72.8, 69.7 (2), 69.7 (4), 69.6 (2), 69.5 (2), 68.6, 49.6, 41.2,
21.3; MS m/z 642.1 (MH+, 100%); HRMS (ESI) Calcd for C31H44N7O6S
(MH+) m/z 642.3068; found m/z 642.3067.
5.1.5. 4-(2-Bromo-4-pyridinyl)-N-ethyl-N-(3-methylphenyl)-
1,3-thiazol-2-amine (9)
C
33H48N7O6S (MH+) m/z 670.3381; found m/z 670.3394.
K2CO3 (97 mg, 0.7 mmol) and EtI (56 lL, 0.7 mmol) were added
to a stirred solution of bromopyridine 3 (200 mg, 0.58 mmol) in
anhydrous DMF (3 mL) and the reaction mixture was stirred at
18 °C for 20 h. The mixture was partitioned between EtOAc
(100 mL) and H2O (50 mL). The organic phase was washed with
H2O (3 ꢂ 20 mL), brine (20 mL), dried and the solvent was evapo-
rated. The residue was purified by column chromatography, elut-
ing with 20% EtOAc/pet. ether, to give amine 9 (142 mg, 66%) as
a colourless oil which was used directly: 1H NMR d 8.38 (dd,
J = 5.1, 0.4 Hz, 1H, H-60), 8.02 (d, J = 0.8 Hz, 1H, H-300), 7.85 (dd,
J = 5.1, 1.4 Hz, 1H, H-500), 7.64 (s, 1H, H-5), 7.40 (t, J = 7.7 Hz, 1H,
5.1.8. 20-(4-{4-[2-(3-Methylphenyl)-1,3-thiazol-4-yl]-3-
pyridinyl}-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18-hexaoxaicosan-
1-ammonium trifluoroacetate (16)
CuSO4ꢀ5H2O (4 mg, 0.09 mmol), sodium ascorbate (6 mg,
0.03 mmol) and TBTA (16 mg, 0.03 mmol) were added to a suspen-
sion of acetylene 13 (42 mg, 0.15 mmol) and 20-azido-
3,6,9,12,15,18-hexaoxaeicosan-1-amine (32 mg, 0.09 mmol) in a
mixture of DMF/H2O (1:1; 2 mL) and the reaction mixture was stir-
red at 18 °C for 3 h and then at 60 °C for 16 h. Additional acetylene
13 (30 mg, 0.1 mmol) in DCM (1 mL) was added and the mixture

M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 711–720
717
was heated at 60 °C for 1 h, at which time all the starting azide has
been consumed. The reaction mixture was diluted with MeOH
(15 mL), SiO2 (1 g) was added and the solvent was evaporated.
The residue was purified by column chromatography, eluting with
0.5% aq NH3 (25%)/1.5% MeOH/EtOAc, to give crude amine 16
(35 mg, 66%). The product was further purified by reverse phase
preparative HPLC [gradient (90–2–90%) (H2O/TFA pH 2.56)/(90%
CH3CN/H2O)] to give amine 16 as the trifluoroacetate salt (18 mg,
pension of acetylene 18 (71 mg, 0.22 mmol) and 20-azido-
3,6,9,12,15,18-hexaoxaeicosan-1-amine (80 mg, 0.22 mmol) in a
mixture of DMF, DCM and H2O (1:1:1; 3 mL), and the mixture
was heated at 50 °C for 3 h. The reaction mixture was cooled to
18 °C, diluted with DCM and MeOH (30 mL), SiO2 (1 g) was added
and the solvent was evaporated. The residue was purified by col-
umn chromatography, eluting with 0.5% aq NH3 (25%)/1.5%
MeOH/EtOAc, to afford crude amine 19 (90 mg). The product was
further purified by preparative HPLC [gradient (95–64%) (H2O/
TFA pH 2.56)/(90% CH3CN/H2O)] to give the amine 19 as the trifluo-
roacetate salt (60 mg, 41%) as a tan oil: 1H NMR d 8.81 (s, 1H, H-200),
8.69 (d, J = 5.4 Hz, 1H, H-600), 8.09 (s, 1H, H-50), 7.89 (d, J = 5.4 Hz,
1
26%) as a tan oil: H NMR d 10.19 (s, 1H, NH), 8.84 (s, 1H, H-200),
8.69 (d, J = 5.1 Hz, 1H, H-600), 7.99 (s, 1H, H-50), 7.83 (d, J = 5.2 Hz,
+
1H, H-500), 7.74 (br s, 3H, NH3 ), 7.23–7.26 (m, 2H, H-20000, H-60000),
7.13 (t, J = 7.7 Hz, 1H, H-50000), 6.98 (s, 1H, H-5000), 6.75 (d,
J = 7.4 Hz, 1H, H-40000), 4.48 (t, J = 5.3 Hz, 2H, H-20), 3.70 (t,
J = 5.3 Hz, 2H, H-19), 3.58 (t, J = 5.3 Hz, 2H, H-2), 3.42–3.56 (m,
20 H, 10 ꢂ CH2O), 2.97 (sext, J = 5.5 Hz, 2H, H-1), 2.24 (s, 3H,
CH3); 13C NMR d 162.9, 149.1, 147.9, 146.8, 142.4, 141.9, 140.8,
138.1, 128.7, 124.8, 122.1, 117.3, 114.0, 109.7, 69.69 (8), 69.66
(2), 69.6, 69.55, 69.53, 68.5, 66.6, (2C not observed); MS m/z
642.1 (MH+, 100%); HRMS (ESI) Calcd for C31H44N7O6S (MH+) m/z
642.3068; found m/z 642.3071.
+
1H, H-500), 7.75 (br s, 3H, NH3 ), 7.37 (t, J = 7.7 Hz, 1H, H-50000),
7.25 (br s, 1H, H-20000), 7.22 (br d, J = 7.9 Hz, 1H, H-60000), 7.17 (br d,
J = 7.6 Hz, 1H, H-40000), 6.73 (s, 1H, H-5000), 4.58 (t, J = 5.2 Hz, 2H, H-
20), 3.86 (q, J = 7.1 Hz, 2H, CH2CH3), 3.84 (t, J = 5.2 Hz, 2H, H-19),
3.58 (t, J = 5.3 Hz, 2H, H-2), 3.42–3.56 (m, 20 H, 10 ꢂ CH2O), 2.97
(sext, J = 5.5 Hz, 2H, H-1), 2.34 (s, 3H, CH3), 1.11 (t, J = 7.1 Hz, 3H,
CH2CH3); 13C NMR d 168.5, 158.2 (q, J = 36 Hz), 148.4, 147.2,
146.7, 144.1, 142.8, 142.2, 139.7, 129.8, 128.0, 126.9, 125.2,
124.8, 124.2, 123.6, 115.8 (q, J = 292 Hz), 109.7, 69.69 (4), 69.67
(3), 69.59 (2), 69.56, 68.6, 66.6, 49.5, 47.2, 38.6, 20.8, 12.6; MS m/
z 672.4 (MH+, 100%); HRMS (ESI) Calcd for C33H48N7O6S (MH+)
m/z 670.3381; found m/z 670.3349.
5.1.9. 4-(3-Bromo-4-pyridinyl)-N-ethyl-N-(3-methylphenyl)-
1,3-thiazol-2-amine (17)
EtI (56 lL, 0.7 mmol) was added to a mixture of bromopyridine
12 (200 mg, 0.58 mmol) and K2CO3 (97 mg, 0.7 mmol) in anhy-
drous DMF and the reaction mixture was stirred at 18 °C for
20 h. The mixture was partitioned between EtOAc (150 mL) and
H2O (50 mL). The organic layer was washed with H2O (50 mL)
washed with brine (50 mL), dried and the solvent was evaporated.
The residue was purified by column chromatography, eluting with
20% EtOAc/pet. ether, to give the amine 17 (130 mg, 60%) as a col-
5.1.12. tert-Butyl 17-hydroxy-3,6,9,12,15-pentaoxaheptadec-1-
ylcarbamate (23)
Mesyl chloride (1.78 mL, 23.0 mmol) was added dropwise to a
stirred suspension of hexaethylene glycol 20 (5.42 g, 19.2 mmol)
and Ag2O (4.67 g, 20.2 mmol) in dry DCM (50 mL) at 20 °C and
the mixture was stirred at 20 °C for 3 days. The mixture was fil-
tered through diatomaceous earth and the solvent evaporated.
The residue was purified by column chromatography, eluting with
1
ourless oil: H NMR d 8.79 (s, 1H, H-200), 8.58 (d, J = 5.0 Hz, 1H,
H-600), 7.87 (br d, J = 5.1 Hz, 1H, H-500), 7.49 (s, 1H, H-5), 7.39 (t,
J = 7.7 Hz, 1H, H-50), 7.30 (br s, 1H, H-20), 7.27 (br d, J = 7.8 Hz,
1H, H-60), 7.19 (br d, J = 7.5 Hz, 1H, H-40), 3.99 (q, J = 7.1 Hz, 2H,
CH2CH3), 2.36 (s, 3H, CH3), 1.22 (t, J = 7.1, 3H, CH2CH3); 13C NMR
d 163.3, 152.7, 148.6, 145.8, 144.2, 141.5, 139.8, 129.9, 128.1,
127.0, 125.1, 123.6, 118.1, 110.0, 47.3, 20.8, 12.8; MS m/z 376.0
(MH+, 100%), HRMS (ESI) Calcd for C17H17BrN3S (MH+) m/z
374.0321; found m/z 374.0324.
a
gradient (0–10%) of MeOH/EtOAc, to give 17-hydroxy-
3,6,9,12,15-pentaoxaheptadec-1-yl methanesulfonate (21) (3.52 g,
51%) as a colourless oil: 1H NMR (CDCl3) d 4.36–4.40 (m, 2H,
CH2OSO2), 3.76–3.78 (m, 2H, CH2O), 3.70–3.74 (m, 2H, CH2O),
3.64–3.67 (m, 16 H, 8 ꢂ CH2O), 3.59–3.62 (m, 2H, CH2O), 3.09 (s,
3H, SO2CH3), 2.80 (br s, 1H, OH); MS m/z 361.6 (MH+, 100%). A mix-
ture of the mesylate 21 (3.52 g, 9.8 mmol) and NaN3 (1.27 g,
19.5 mmol) in dry DMF (20 mL) was stirred at 110 °C for 2 h. The
mixture was cooled to 20 °C and the solvent evaporated. The resi-
due was purified by column chromatography, eluting with 10%
MeOH/EtOAc, to give 17-azido-3,6,9,12,15-pentaoxaheptadecan-
1-ol (22) (2.98 g, 99%) as a colourless oil: 1H NMR (CDCl3) d
3.71–3.74 (m, 2H, CH2O), 3.65–3.69 (m, 18 H, 9 ꢂ CH2O), 3.59–
3.62 (m, 2H, CH2O), 3.39 (br t, J = 5.2 Hz, 2H, CH2N3), 2.82 (br s,
1H, OH); MS m/z 308.5 (MH+, 100%). A mixture of azide 22
(2.98 g, 9.7 mmol) and Pd/C (100 mg) in EtOH (50 mL) was stirred
under H2 (60 psi) for 1 h. The mixture was filtered through diato-
maceous earth and washed with EtOH (3 ꢂ 20 mL) and the solvent
was evaporated. The crude residue was dissolved in DCM (50 mL)
and di-tert-butyl dicarbonate (2.56 g, 11.7 mmol) in DCM (20 mL)
was added dropwise and the solution was stirred at 20 °C for
16 h. The solvent was evaporated and residue was purified by col-
umn chromatography, eluting with 10% MeOH/EtOAc, to give car-
bamate 23 (2.97 g, 80%) as a colourless oil: 1H NMR (CDCl3) d 5.17
(br s, 1H, NHCO2), 3.70–3.74 (m, 2H, CH2O), 3.60–3.68 (m, 18 H,
9 ꢂ CH2O), 3.54 (br t, J = 5.1 Hz, 2H, CH2O), 3.31 (br q, J = 5.1 Hz,
2H, CH2N), 2.81 (br s, 1H, OH), 1.44 [s, 9 H, C(CH3)3]; MS m/z
382.5 (MH+, 100%).
5.1.10. N-Ethyl-4-(3-ethynyl-4-pyridinyl)-N-(3-methylphenyl)-
1,3-thiazol-2-amine (18)
PdCl2(PPh3)2 (30 mg, 0.04 mmol) was added to a purged solu-
tion of bromopyridine 17 (130 mg, 0.35 mmol), TMS-acetylene
(0.5 mL, 3.5 mmol), and CuI (8 mg, 0.04 mmol) in a mixture of
DMF/NEt3 (1:1; 4 mL), and the reaction mixture was stirred at
50 °C for 2 h. The mixture was cooled to 18 °C and was partitioned
between EtOAc (200 mL) and H2O (50 mL). The organic phase was
washed with brine (50 mL), dried and the solvent was evaporated.
The residue was dissolved in a mixture of THF/MeOH (1:1; 30 mL),
K2CO3 (50 mg, 0.35 mmol) was added and the mixture was stirred
at 18 °C for 1 h. The solvent was then evaporated and the residue
purified by column chromatography, eluting with 20% EtOAc/pet.
ether, to give acetylene 18 (72 mg, 65%) as an unstable yellow oil
which was used without further purification: 1H NMR d 8.74 (s,
1H, H-200), 8.57 (d, J = 5.1 Hz, 1H, H-600), 8.04 (d, J = 5.3 Hz, 1H,
H-500), 7.76 (s, 1H, H-5), 7.34 (dt, J = 7.4, 1.1 Hz, 1H, H-50), 7.19
(br s, 1H, H-20), 7.14–7.19 (m, 2H, H-40, H-60), 4.07 (q, J = 7.1 Hz,
2H, CH2CH3), 3.47 (s, 1H, C„CH), 2.39 (s, 3H, CH3), 1.31 (t,
J = 7.1 Hz, 3H, CH2CH3); MS m/z 320.7 (MH+, 100%).
5.1.11. 20-(4-{4-[2-(Ethyl-3-methylanilino)-1,3-thiazol-4-yl]-3-
pyridinyl}-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18-hexaoxaicosan-
1-ammonium trifluoroacetate (19)
5.1.13. tert-Butyl 3,6,9,12,15,18-hexaoxahenicos-20-yn-1-
ylcarbamate (24)
CuSO4ꢀ5H2O (6 mg, 0.022 mmol), sodium ascorbate (10 mg,
NaH (343 mg, 8.56 mmol) was added in small portions to a stir-
red solution of alcohol 15 (2.97 g, 7.8 mmol) in THF (50 mL) at 0 °C
0.044 mmol) and TBTA (25 mg, 0.044 mmol) were added to a sus-

718
M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 711–720
and the resulting mixture stirred at 0 °C for 30 min. Propargyl
138.1 (2), 135.8, 133.1, 128.2 (2), 127.7 (2), 127.5 (2), 127.3,
123.5, 100.3, 45.7; MS m/z 494.6 (MH+, 100%). Anal. Calcd for C19-
H16IN3O3S: C, 46.26; H, 3.27; N, 8.52. Found: C, 46.43; H, 3.30; N,
8.52.
bromide (0.87 mL, 7.8 mmol) was added followed by nBu4N+Iꢁ
(29 mg, 78 lmol) and the mixture was stirred at 20 °C for 16 h.
The reaction was quenched with satd aq NH4Cl and extracted with
EtOAc (4 ꢂ 50 mL). The combined organic fraction was washed
with brine (50 mL), dried and the solvent evaporated. The residue
was purified by column chromatography, eluting with 80% EtOAc/
pet. ether, to give the acetylene 24 (2.56 g, 79%) as a colourless oil:
1H NMR (CDCl3) d 5.05 (br s, 1H, NHCO2), 4.20 (d, J = 2.4 Hz, 2H,
CH2C„C), 3.68–3.71 (m, 4H, 2 ꢂ CH2O), 3.64–3.67 (m, 12H,
6 ꢂ CH2O), 3.60–3.63 (m, 4H, 2 ꢂ CH2O), 3.54 (br t, J = 5.2 Hz, 2H,
CH2O), 3.31 (br q, J = 5.2 Hz, 2H, CH2N), 2.42 (t, J = 2.4 Hz, 1H,
CH), 1.44 [s, 9 H, C(CH3)3]; MS m/z 420.7 (MH+, 100%); HRMS
(ESI) Calcd for C20H38NO8 (MH+) m/z 420.2592, found 420.2590
(0.4 ppm).
5.1.17. tert-Butyl 21-{4-[({4-[(3-pyridinylamino)carbonyl]
benzyl}amino)sulfonyl]phenyl}-3,6,9,12,15,18-hexaoxahenicos-
20-yn-1-ylcarbamate (29)
PdCl2(PPh3)2 (36 mg, 51
solution of iodide 29 (250 mg, 510
770 mol) and CuI (10 mg, 51 mol) in Et3N (3 mL) and DMF
lmol) was added to a stirred, degassed
lmol), acetylene 24 (320 mg,
l
l
(3 mL), and the mixture was stirred in a sealed pressure vessel at
50 °C for 3 h. The mixture was cooled to 20 °C, diluted with EtOAc
(150 mL) and washed with water (3 ꢂ 50 mL), washed with brine
(50 mL) and dried. The solvent was evaporated and the residue
purified by column chromatography, eluting with a gradient (0–
5%) of MeOH/EtOAc, to give carbamate 29 (367 mg, 92%) as a tan
5.1.14. Benzyl 4-(pyridine-3-ylcarbamoyl)benzylcarbamate (26)
Oxalyl chloride (4.58 mL, 52.5 mmol) was added dropwise to a
solution of 4-(benzyloxycarbonylamino)methyl)benzoic acid 2520
(10.0 g, 35.0 mmol) and DMF (4 drops) in dry THF (150 mL), and
the mixture stirred at 50 °C for 4 h. The solvent was evaporated
and the residue dissolved in pyridine (80 mL). 3-Aminopyridine
(3.62 g, 38.5 mmol) was added and the solution stirred at 20 °C
for 48 h. Water (150 mL) was added, the mixture stirred for an-
other 2 h, the precipitate filtered off, washed with water and dried
to give the carbamate 26 (7.82 g, 62%) as a white solid: mp (EtOH)
207–210 °C; 1H NMR d 10.37 (s, 1H, NHCO), 8.92 (d, J = 2.3 Hz, 1H,
H-20), 8.31 (dd, J = 4.7, 1.5 Hz, 1H, H-60), 8.18 (ddd, J = 8.3, 2.5,
1.5 Hz, 1H, H-40), 7.93 (br d, J = 8.3 Hz, 2H, H-2, H-6), 7.89 (br t,
J = 6.0 Hz, 1H, NHCO2), 7.41 (br d, J = 8.3 Hz, 2H, H-3, H-5),
7.31–7.39 (m, 6 H, H-50, H-200, H-300, H-400, H-500, H-600), 5.06 (s, 2H,
CH2O), 4.30 (d, J = 6.2 Hz, 2H, CH2N). Anal. Calcd for C21H19N3O3:
C, 69.79; H, 5.30; N, 11.63. Found: C, 69.60; H, 5.40; N, 11.63.
1
oil: H NMR (CDCl3) d 8.61 (br s, 1H, H-20), 8.41 (br s, 1H, NHSO2),
8.31 (d, J = 4.6 Hz, 1H, H-60), 8.25 (ddd, J = 8.3, 2.4, 1.4 Hz, 1H, H-40),
7.79–7.84 (m, 4H, H-2, H-6, H-200, H-600), 7.55 (dd, J = 8.6, 1.8 Hz, 2H,
H-300, H-500), 7.28–7.34 (m, 3H, H-3, H-5, H-50), 5.78 (br s, 1H,
NHCO), 5.07 (br s, 1H, NHCO2), 4.42 (s, 2H, CH2O), 4.22 (br d,
J = 6.0 Hz, 2H, CH2N), 3.74–3.77 (m, 2H CH2O), 3.68–3.71 (m, 2H
CH2O), 3.55–3.66 (m, 18H, 9 ꢂ CH2O), 3.25 (br dd, J = 5.4, 5.2 Hz,
2H, CH2N), 1.43 [s, 9 H, C(CH3)3]; MS m/z 786.0 (MH+, 100%); HRMS
(ESI) Calcd for C39H53N4O11S (MH+) m/z 785.3426, Found 785.3410
(2.5 ppm).
5.1.18. 4-[({[4-(21-Amino-4,7,10,13,16,19-hexaoxahenicos-1-
yn-1-yl)phenyl]sulfonyl}amino)methyl]-N-(3-pyridinyl)
benzamide (30)
A solution of carbamate 29 (360 mg, 0.46 mmol) in HCl satu-
rated MeOH (10 mL) was stood at 20 °C for 16 h. The solvent was
evaporated and the crude oil purified by preparative HPLC [gradi-
ent elution 5–55% of (90%MeCN/H2O)/(0.02% v/v aqueous CF3CO2-
H)] to give the amine 30 as the trifluoracetate salt (270 mg, 73%)
as a brown gum: 1H NMR d 11.34 (s, 1H, NHCO), 9.42 (d,
J = 2.2 Hz, 1H, H-20), 8.88 (d, J = 8.8 Hz, 1H, H-40), 8.64 (d,
J = 4.9 Hz, 1H, H-40), 8.47 (br s, 1H, NHSO2), 8.04 (br d, J = 8.3 Hz,
2H, H-2, H-6), 7.96 (m, 4H, NH2ꢀCF3CO2H, H-50), 7.82 (dd, J = 8.5,
1.8 Hz, 2H, H-200, H-600), 7.65 (dd, J = 8.5, 1.8 Hz, 2H, H-300, H-500),
7.44 (br d, J = 8.3 Hz, 2H, H-3, H-5), 4.43 (s, 2H, CH2O), 4.12 (br d,
J = 6.2 Hz, 2H, CH2N), 3.51–3.65 (m, 22H, 11 ꢂ CH2O), 2.95 (br q,
J = 5.5 Hz, 2H, CH2N); 13C NMR d 165.9, 142.5, 140.5, 138.6,
137.0, 134.7, 133.5, 132.0 (2), 131.9, 129.1, 128.1 (2), 127.7,
127.5 (2), 127.1, 126.8 (2), 125.8, 89.2, 84.3, 69.7 (3), 69.6 (2),
69.5, 68.8, 66.6, 58.0, 48.5, 45.7, 38.8; HRMS (ESI) Calcd for C34H45-
N4O9S (MH+) m/z 685.2902, Found 685.2898 (1.2 ppm).
5.1.15. 4-(Aminomethyl)-N-(3-pyridinyl)benzamide
dihydrobromide (27)
A suspension of carbamate 26 (2.2 g, 6.09 mmol) in HBr/AcOH
(30%, 30 mL) was stirred at 20 °C for 3 h. Et2O (200 mL) was added,
the mixture was stirred for another 30 min, the precipitate filtered
off, washed with Et2O and dried to give benzamide 27 (2.35 g, 99%)
as a white solid: mp (EtOAc) 292–296 °C; 1H NMR d 11.06 (s, 1H,
NHCO), 9.35 (d, J = 2.2 Hz, 1H, H-20), 8.70 (ddd, J = 8.5, 2.2, 1.1 Hz,
1H, H-40), 8.64 (br d, J = 5.4 Hz, 1H, H-60), 8.31 (br s, 3H, NH2ꢀHBr),
8.09 (br d, J = 8.2 Hz, 2H, H-2, H-6), 7.96 (dd, J = 8.6, 5.4 Hz, 1H, H-
50), 7.67 (d, J = 8.4 Hz, 2H, H-3, H-5), 5.95 (br s, 1H, pyrNꢀHBr), 4.16
(q, J = 5.8 Hz, 2H, CH2N). Anal. Calcd for C13H15Br2N3O: C, 40.13; H,
3.89; N, 10.80. Found: C, 39.99; H, 3.94; N, 10.36.
5.1.19. tert-Butyl 21-{4-[({4-[(3-pyridinylamino)carbonyl]
benzyl}amino)sulfonyl]phenyl}-3,6,9,12,15,18-hexaoxahenicos-
1-ylcarbamate (31)
5.1.16. 4-((4-Iodophenylsulfonamido)methyl)-N-(pyridin-3-
yl)benzamide (28)
A
mixture of amine 27 (727 mg, 3.2 mmol) and
A mixture of alkyne 29 (743 mg, 0.95 mmol) and 10% Pd/C
(250 mg, 0.1 mmol) in absolute EtOH (30 mL) was stirred at 20 °C
under of H2 (60 psi) for 16 h. The mixture was filtered through dia-
tomaceous earth, washed with EtOH (100 mL), and the solvent was
evaporated. The residue was purified by column chromatography,
eluting with a gradient (3–5%) of MeOH/DCM, to give carbamate
31 (650 mg, 87%) as a pale yellow oil: 1H NMR d 10.35 (s, 1H,
CONH), 8.92 (d, J = 2.4 Hz, 1H, H-20), 8.31 (dd, J = 4.7, 1.4 Hz, 1H,
H-60), 8.17–8.20 (m, 2H, NHSO2, H-40), 7.88 (br d, J = 8.3 Hz, 2H,
H-2, H-6), 7.88 (br d, J = 8.3 Hz, 2H, H-200, H-600), 7.37–7.40 (m, 5
H, H-50, H-3, H-5, H-300, H-500), 6.69 (br s, 1H, NHCO2), 4.08 (d,
J = 5.0 Hz, 2H, CH2N), 3.45–3.52 (m, 20 H, 10 ꢂ CH2O), 3.37 (t,
J = 6.2 Hz, 4H, H-3000, H-20000), 3.05 (q, J = 6.0 Hz, 2H, H-21000), 2.69
(t, J = 7.7 Hz, 2H, H-1000), 1.77–1.84 (m, 2H, H-2000), 1.37 [s, 9 H,
4-iodobenzenesulfonyl chloride (970 mg, 3.2 mmol) in dry pyri-
dine (10 mL) was stirred at 20 °C for 16 h. The solvent was evapo-
rated and the residue stirred in water (20 mL) for 1 h. The
precipitate was filtered, washed with water (5 mL) and dried. The
crude solid was purified by column chromatography, eluting with
a gradient (0–20%) of MeOH/EtOAc, to give benzamide 28 (1.47 g,
93%) as a cream powder: mp (MeOH/EtOAc) 249–251 °C; 1H
NMR d 10.36 (s, 1H, NHCO), 8.93 (d, J = 2.3 Hz, 1H, H-20), 8.34 (br
s, 1H, NHSO2), 8.31 (dd, J = 4.7, 1.5 Hz, 1H, H-60), 8.18 (ddd,
J = 8.3, 2.5, 1.5 Hz, 1H, H-40), 7.97 (ddd, J = 8.6, 2.2, 1.9 Hz, 2H,
H-200, H-600), 7.90 (br d, J = 8.3 Hz, 2H, H-2, H-6), 7.56 (ddd, J = 8.6,
2.2, 1.9 Hz, 2H, H-300, H-500), 7.37–7.42 (m, 3H, H-3, H-5, H-50),
4.09 (s, 2H, CH2N); 13C NMR d 165.5, 144.5, 142.0, 141.6, 140.3,

M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 711–720
719
C(CH3)3]; HRMS (ESI) Calcd for C39H56N4O11S (MH+) m/z 789.3739;
found 789.3723 (1.6 ppm).
128.5 (2), 128.2 (2), 127.6 (2), 127.4 (2), 123.6, 120.4 (2), 100.3,
45.7; MS m/z 493.6 (MH+, 100%). Anal. Calcd for C20H17IN2O3S: C,
48.79; H, 3.48; N, 5.69. Found: C, 49.06; H, 3.56; N, 5.84.
5.1.20. 4-[({[4-(21-Amino-4,7,10,13,16,19-hexaoxahenicos-1-
yl)phenyl]sulfonyl}amino) methyl]-N-(3-pyridinyl)benzamide
(32)
5.1.23. tert-Butyl 21-[4-({[4-
(anilinocarbonyl)benzyl]amino}sulfonyl)phenyl]-
Trifluoroacetic acid (0.74 mL, 10 mmol) was added to a solution
of carbamate 31 (313 mg, 0.4 mmol) in anhydrous DCM (5 mL) and
the reaction mixture was stirred at 20 °C for 2 h. The solvent was
evaporated and the residue was purified by column chromatogra-
phy, eluting with 8% MeOH/DCM containing 1% aqueous NH3, to
give the amine 32 (195 mg, 71%) as a colourless oil: 1H NMR d
10.35 (br s, 1H, CONH), 8.92 (d, J = 2.2 Hz, 1H, H-20), 8.31 (dd,
J = 4.7, 1.4 Hz, 1H, H-60), 8.18 (ddd, J = 8.3, 2.5, 1.5 Hz, 1H, H-40),
7.88 (br d, J = 8.3 Hz, 2H, H-2, H-6), 7.70 (br d, J = 8.3 Hz, 2H,
H-200, H-600), 7.37–7.40 (m, 5 H, H-50, H-3, H-5, H-300, H-500), 4.08
(s, 2H, CH2NHSO2), 3.45–3.52 (m, 20 H, 10 ꢂ CH2O), 3.37 (t,
J = 6.3 Hz, 2H, H-3000), 3.34 (t, J = 5.8 Hz, 2H, H-20000), 2.70 (t,
J = 7.9 Hz, 2H, H-1000), 2.63 (t, J = 5.7 Hz, 2H, H-21000), 1.80 (tt,
J = 6.4, 7.9 Hz, 2H, H-2000), NHSO2 and NH2 not observed; 13C NMR
d 165.4, 146.6, 144.4, 141.9, 141.7, 138.0, 135.7, 132.8, 128.9 (2),
127.5 (2), 127.3 (2), 127.2, 126.4 (2), 123.3, 72.2, 69.69 (2), 69.66
(4), 69.62 (2), 69.4, 69.3, 69.1, 45.6, 40.9, 31.3, 30.4; HRMS (ESI)
Calcd for C34H48N4O9S (MH+) m/z 689.3215; Found 389.3224
(ꢁ1.0 ppm).
3,6,9,12,15,18-hexaoxahenicos-20-yn-1-ylcarbamate (36)
PdCl2(PPh3)2 (85 mg, 0.12 mmol) was added to a stirred, de-
gassed solution of iodide 35 (600 mg, 1.22 mmol), acetylene 19
(765 mg, 1.82 mmol) and CuI (23 mg, 0.12 mmol) in Et3N (5 mL)
and DMF (5 mL), and the mixture was stirred in a sealed pressure
vessel at 50 °C for 2 h. It was cooled to 20 °C, diluted with EtOAc
(200 mL), washed with water (3 ꢂ 50 mL), washed with brine
(50 mL) and dried. The solvent was evaporated and the residue
purified by column chromatography, eluting with a gradient
(0–5%) of MeOH/DCM, to give carbamate 36 (1.0 g, 100%) as a tan
oil: 1H NMR d 10.15 (s, 1H, NHCO), 8.34 (br s, 1H, NHSO2), 7.88
(br d, J = 8.3 Hz, 2H, H-300, H-500), 7.80 (br d, J = 8.5 Hz, 2H, H-30,
H-50), 7.76 (br d, J = 7.6 Hz, 2H, H-2000, H-6000), 7.65 (br d, J = 8.5 Hz,
2H, H-20, H-60), 7.38 (br d, J = 8.5 Hz, 2H, H-200, H-600), 7.34 (br d,
J = 8.3 Hz, 2H, H-3000, H-5000), 7.10 (br t, J = 7.4 Hz, 1H, H-4000), 6.70
(br s, 1H, NHCO2), 4.43 (s, 2H, C„CCH2O), 4.10 (s, 2H, CH2NHSO2),
3.62–3.65 (m, 2H, CH2O), 3.56–3.59 (m, 2H, CH2O), 3.49–3.53 (m,
16 H, 8 ꢂ CH2O), 3.37 (br t, J = 6.1 Hz, 2H, H-20), 3.06 (br q,
J = 5.9 Hz, 2H, H-21), 1.37 [s, 9H, C(CH3)3]; HRMS (ESI) Calcd for
C
40H54N3O11S (MH+) m/z 784.3474; Found 784.3460.
5.1.21. 4-(Aminomethyl)-N-phenylbenzamide hydrobromide
(34)
5.1.24. tert-Butyl 21-[4-({[4-(anilinocarbonyl)benzyl]
Oxalyl chloride (0.92 mL, 10.5 mmol) was added dropwise to a
stirred suspension of benzoic acid 2520 (2.0 g, 7.0 mmol) and
DMF (3 drops) in dry THF (50 mL) and the solution was stirred at
20 °C for 4 h. The solvent was evaporated and the residue dissolved
in pyridine (10 mL). Aniline (0.70 mL, 7.7 mmol) was added and
the solution stirred at 20 °C for 16 h. The solvent was evaporated
and the residue suspended in ice/water (100 mL) for 1 h. The pre-
cipitate was filtered, washed with water (5 mL) and dried to give
crude benzyl 4-(anilinocarbonyl)benzyl carbamate (33) (1.45 g,
57%) as a white powder: mp (H2O) 276–279 °C; 1H NMR d 10.16
(s, 1H, CONH), 7.86–7.93 (m, 3H, NHCO2, Haryl), 7.77 (d, J = 8.6 Hz,
2H, Haryl), 7.30–7.41 (m, 9H, Haryl), 7.10 (br t, J = 7.3 Hz, 1Haryl),
4.14 (br q, J = 5.4 Hz, 2H, CH2N). A suspension of carbamate 33
(1.45 g, 4.0 mmol) in HBr/AcOH (30%, 30 mL) was stirred at 20 °C
for 6 h. Et2O (150 mL) was added, the mixture was stirred at 5 °C
for 30 min, the precipitate filtered off, washed with Et2O and dried
to give benzamide hydrobromide 34 (1.15 g, 93%) as a white solid:
mp (Et2O) 276–279 °C; 1H NMR d 10.25 (s, 1H, CONH), 8.27 (br s,
3H, NH2ꢀHBr), 8.30 (br d, J = 8.2 Hz, 2H, H-2, H-6), 7.79 (dd,
J = 8.5, 7.5 Hz, 2H, H-20, H-60), 7.61 (d, J = 8.4 Hz, 2H, H-3, H-5),
7.36 (dd, J = 8.5, 7.5 Hz, 2H, H-30, H-50), 7.11 (br t, J = 7.5 Hz, 1H,
H-40), 4.14 (q, J = 5.4 Hz, 2H, CH2N). Anal. Calcd for C14H15BrN2O:
C, 54.74; H, 4.92; N, 9.12. Found: C, 54.73; H, 4.76; N, 8.96.
amino}sulfonyl)phenyl]-3,6,9,12,15,18-hexaoxahenicos-1-
ylcarbamate (37)
A mixture of carbamate 36 (923 mg, 1.17 mmol) and 10% Pd/C
(300 mg, 0.12 mmol) in absolute EtOH (50 mL) was stirred at
20 °C under H2 (60 psi) for 16 h. The mixture was filtered through
a pad of diatomaceous earth, washed with EtOH (200 mL), the sol-
vent was evaporated and the residue was purified by column chro-
matography, eluting with a gradient (0–5%) of MeOH/DCM to give
carbamate 37 (742 mg, 80%) as a tan oil: 1H NMR d 10.14 (s, 1H,
NHCO), 7.86 (br d, J = 8.3 Hz, 2H, H-300, H-500), 7.76 (br dd, J = 8.7,
1.0 Hz, 2H, H-30, H-50), 7.71 (br d, J = 8.3 Hz, 2H, H-2000, H-6000),
7.32–7.40 (m, 6 H, H-20, H-60, H-200, H-600, H-3000, H-5000), 7.16 (t,
J = 6.2 Hz, 1H, NHSO2), 7.09 (br t, J = 7.4 Hz, 1H, H-4000), 6.69 [br s,
1H, NHC(CH3)3], 4.07 (d, J = 5.9 Hz, 2H, CH2NHSO2), 3.45–3.52 (m,
20 H, 10 ꢂ CH2O), 3.37 (br t, J = 6.2 Hz, 4H, H-19, H-2), 3.05 (br q,
J = 5.9 Hz, 2H, H-1), 2.69 (br t, J = 7.9 Hz, 2H, H-21), 1.80 (tt,
J = 7.9, 6.3 Hz, 2H, H-20), 1.37 [s, 9 H, C(CH3)3]; HRMS (ESI) Calcd
for C40H58N3O11S (MH+) m/z: 788.3787; Found 788.3796.
5.1.25. 4-[({[4-(21-Amino-4,7,10,13,16,19-hexaoxahenicos-1-
yl)phenyl]sulfonyl}amino)methyl]-N-phenylbenzamide (38)
A solution of carbamate 37 (245 mg, 0.31 mmol) in anhydrous
DCM (5 mL) was treated with TFA (0.6 mL, 7.8 mmol) and the reac-
tion mixture was stirred at 20 °C for 2 h. The solvent was evapo-
rated and the residue was purified by column chromatography,
eluting with a gradient (92:7:0.8–92:8.5:1) of DCM/MeOH/aq
NH3 to give the amine 38 (164 mg, 77%) as a pale yellow oil: 1H
NMR d 10.14 (s, 1H, NHCO), 7.86 (br d, J = 8.3 Hz, 2H, H-2, H-6),
7.76 (br dd, J = 8.6, 1.0 Hz, 2H, H-200, H-600), 7.71 (br d, J = 8.3 Hz,
2H, H-20, H-60), 7.32–7.41 (m, 6 H, H-3, H-5, H-30, H-50, H300,
H-500), 7.10 (br t, J = 7.4 Hz, 1H, H-40), 4.07 (s, 2H, CH2NHSO2),
3.46–3.52 (m, 20H, 10 ꢂ CH2O), 3.37 (t, J = 6.3 Hz, 2H, H-3000), 3.35
(t, J = 5.8 Hz, 2H, H-20000), 2.69 (t, J = 8.0 Hz, 2H, H-1000), 2.64 (br s,
2H, H-21000), 1.80 (tt, J = 7.9, 6.3 Hz, 2H, H-2000), NHSO2 and NH2
not observed; 13C NMR d 165.0, 146.6, 141.3, 139.0, 138.0, 133.5,
128.9 (2), 128.4 (2), 127.4 (2), 127.2 (2), 126.4 (2), 123.5, 120.2
(2), 69.7 (9), 69.6, 69.4, 69.3, 69.1, 45.6, 31.3, 30.4; HRMS (ESI)
Calcd for C35H50N3O9S (MH+) m/z 688.3262; Found: 688.3252.
5.1.22. 4-({[(4-Iodophenyl)sulfonyl]amino}methyl)-N-
phenylbenzamide (35)
A
mixture of amine 34 (408 mg, 1.8 mmol) and
4-iodobenzenesulfonyl chloride (551 mg, 1.8 mmol) in dry pyridine
(10 mL) was stirred at 20 °C for 16 h. The solvent was evaporated
and the residue stirred in water (20 mL) for 1 h. The precipitate
was filtered, washed with water (5 mL) and dried, to give benzam-
ide 35 (710 g, 80%) as a white powder: mp (EtOAc) 252–254 °C; 1H
NMR d 10.15 (s, 1H, CONH), 8.32 (br t, J = 6.2 Hz, 1H, NHSO2),
7.97(ddd, J = 8.6, 2.2, 1.8 Hz, 2H, H-300, H-500), 7.88 (d, J = 8.3 Hz,
2H, H-2, H-6), 7.77 (dd, J = 8.4, 1.0 Hz, 2H, H-20, H-60), 7.56 (ddd,
J = 8.6, 2.2, 1.8 Hz, 2H, H-200, H-600), 7.38–7.40 (m, 4H, H-3, H-5, H-
30, H-50), 7.10 (tt, J = 7.4, 1.0 Hz, 1H, H-4’), 4.08 (d, J = 6.3 Hz, 2H,
CH2N); 13C NMR d 165.1, 141.2, 140.3, 139.1, 138.0 (2), 133.7,

720
M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 711–720
5.1.26. Preparation of affinity reagents
by measuring the absorbance at 450 nm. IC50 values were calcu-
lated using interpolation.
A slurry of Affi-Gel 10 (5 mL) was washed with cold iPrOH
(5 ꢂ 5 mL) in a sintered funnel and was transferred to a sealed
pressure vessel. A solution of amine 32 or 38 (61 mg, 0.08 mmol)
in MeOH (5 mL) was added, followed by iPr2OEt (0.14 mL,
0.8 mmol). The sealed tube was placed on a rotary shaker and
gently agitated, monitoring the reaction by HPLC. After 6 h, etha-
Acknowledgments
The authors thank Drs. Shannon Black and Sisira Kumara for
technical assistance and acknowledge the Association for Interna-
tional 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.),
and NCI-CA-123823 (D.A.C.).
nolamine (9
lL, 0.15 mmol) was added and the mixture was agi-
tated for another 16 h. The mixture was filtered, and the gel
washed with MeOH (5 ꢂ 5 mL) and iPrOH (5 ꢂ 5 mL), to give an
off-white slurry which was used for affinity chromatography
experiments.
Supplementary data
5.2. Molecular modeling
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.028.
5.2.1. Molecular modeling for PAT chemotype
A base CoMFA 3D-QSAR model was created as previously
described.14
References and notes
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The likely protonation state of the test compounds at pH 7.4
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5.3. Cell viability assays
RCC4 parental cells and RCC4 cells with VHL reintroduced
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the media was aspirated, 2,3-bis[2-methoxy-4-nitro-5-sulfophe-
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(Sigma), 2.65 mg/ml N-methyl dibenxopyrazine methyl sulfate
(Sigma) in phenol red-free media was added, and the plates were
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