Identification of false positives in hTS hits to lead : The application of Bayesian models in HTS triage to rapidly deliver a series of selective TRPV4 antagonists

Article abstract of DOI:10.1039/c2md20259j

Herein, we describe the discovery and optimisation of a series of potent and selective TRPV4 antagonists. The application of a variety of computational techniques (including Bayesian modelling) at the HTS triage stage enabled the early deprioritisation of likely frequent hitters. The use of methods to positively prioritise compounds for follow-up screening allowed the rapid identification of a number of interesting TRPV4 antagonist series. The hit-to-lead efforts in one such series, the hydroxypiperidines, will be described. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2md20259j

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CONCISE ARTICLE
Identification of false positives in “HTS hits to lead”: The
application of Bayesian models in HTS triage to rapidly
deliver a series of selective TRPV4 antagonists
a
Cite this: Med. Chem. Commun., 2013,
4, 244
Sarah E. Skerratt, James E. J. Millsb and Jayesh Mistryc
*
Herein, we describe the discovery and optimisation of a series of potent and selective TRPV4 antagonists.
The application of a variety of computational techniques (including Bayesian modelling) at the HTS triage
stage enabled the early deprioritisation of likely frequent hitters. The use of methods to positively prioritise
compounds for follow-up screening allowed the rapid identification of a number of interesting TRPV4
antagonist series. The hit-to-lead efforts in one such series, the hydroxypiperidines, will be described.
Received 29th August 2012
Accepted 22nd October 2012
DOI: 10.1039/c2md20259j
www.rsc.org/medchemcomm
assays can deliver false positives or ‘frequent hitters’ due to a
compound being reactive, autouorescent, cytotoxic or active at
another target in the signal transduction pathway.12,13 A key goal
of the HTS triage effort was therefore to identify and remove
false positives at the outset, allowing efforts to be focussed on
true TRPV4 antagonists.
Introduction
TRPV4 is a member of the Transient Receptor Potential (TRP)
superfamily of mammalian cation channels. It is expressed in a
wide range of tissues1 and is thought to play a role in a number
of physiological responses such as osmoregulation,2,3 thermo-
sensation,4 vascular regulation5,6 and mechanosensation.2,7
Indeed, it has been postulated that TRPV4 plays a crucial part in
the mechanosensory pathway of the bladder by detecting
changes in intravesical pressure.8,9 TRPV4 is activated by a range
of stimuli including small molecules (e.g. anandamide and 5,6-
epoxyeicosatrienoic acid), temperature (27–34 ꢀC), hypotonic
solutions and mechanical stimuli.10,11
At the outset of the TRPV4 project, there were no known
selective TRPV4 antagonists with which to build condence in
mechanism. A high through-put screening (HTS) campaign was
therefore initiated on the Pzer compound le and, in order to
meet the through-put requirements of this campaign, a TRPV4
calcium ux assay using FLIPR Tetra technology was utilised.
The data from a number of prior Pzer FLIPR HTS screening
campaigns were used as a means of identifying likely false
positives from the ꢁ11 K putative actives emerging from the
TRPV4 single-point screen. Firstly, if a compound was active
(>75% inhibition at 10 mM) in more than one of these HTS
screens, it was deemed likely to be a false positive (provided the
prior HTS was not directed at a target from the same gene family
as TRPV4). Secondly, for each previous FLIPR HTS dataset, a
Bayesian activity model14 was built to predict the likelihood of
activity of a compound in that assay. If a compound was scored
highly by more than one such model (Bayesian score >5), it was
deemed likely to be a false positive. This second in silico
approach offers the advantage that it does not require the
compound itself to have been screened in HTS assays, because
evidence of activity of its near neighbours is sufficient to suggest
it could be a false positive. Indeed, this in silico method ltered
out ꢁ5 times as many compounds as the method based on
in vitro data. An example of use of these Bayesian specicity
models is shown in Fig. 1. Putative actives from the TRPV4
single point assay were trellised by molecular cluster, and %
TRPV4 inhibition was plotted against molecular weight.
Compounds were coloured by the number of times they were
deemed “active” (i.e. scored >5) by any of the non-TRP FLIPR
HTS Bayesian models (red ¼ active according to $2 models,
yellow ¼ active according to 1 model, green ¼ active according
to 0 models). Compounds such as those found in Cluster 4 were
retained, whereas compounds such as those found in Cluster 3
were discarded. A selection of compounds such as those found
in Clusters 1 and 2 were also retained.
Results and discussion
HTS screening and triage
Compressed le screening generated a large number of hits
(ꢁ2.5% of compounds had >40% inhibition of TRPV4 at 10 mM)
for conrmatory follow-up in a single-well, single-point TRPV4
assay. The single-point assay conrmed ꢁ11 k of these to be
active in this assay (>40% inhibition at 10 mM), a 14% conr-
mation rate. It is, however, widely recognised that calcium ux
aPzer Neusentis, The Portway Building, Granta Park, Cambridge, UK. E-mail: sarah.
skerratt@pzer.com; Tel: +44 (0)304644073
b45 Queen St., Deal, Kent, UK. E-mail: james.mills@sandexis.co.uk; Tel: +44 (0)771
541 5402
cElsevier Limited (Corporate Office), 32 Jamestown Road, Camden, London, UK.
E-mail: j.mistry@elsevier.com; Tel: +44 (0)7584263708
244 | Med. Chem. Commun., 2013, 4, 244–251
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signicant; however compounds with a Bayesian score of >15
are likely to be enriched in actives.
Finally, compounds with little evidence supporting either
inclusion or exclusion were analysed. These moderately active
compounds (40–75% inhibition of TRPV4 at 10 mM) with an
unmeasurable local hit rate (0/0 active neighbours) were ana-
lysed by trellising TRPV4 activity plots by Library ID. Library
compounds (i.e. compounds synthesised by parallel chemistry)
were deemed synthetically tractable, and therefore prioritised
for selection, along with further close-in library members not
present in the original HTS screening set.
A total of 1050 compounds were selected for TRPV4 IC50
follow-up. The results from the IC50 screen were used to seed a
second round of compound selection. Work focussed on close-
in mining of active compounds with the aim of deriving SAR
around each hit. A number of ngerprint methods (BCI,18
ECFP619 and Atom Pair20 (in-house Tanimoto similarity algo-
rithm)) were used to identify near neighbours around each
selected molecule (the probe). Each neighbour was assessed by
calculating its maximum common substructure (MCSS)21,22 with
the probe and only neighbours that differed from the probe by a
single moiety were retained. For each probe, the number of
neighbours per point of variation was restricted to ten to permit
a diverse exploration of close-in neighbours around each probe.
Tracking the relative occurrence of the attachment point for
each probe also gave an indication of the chemical space
occupied around it, suggesting gaps that could be explored by
synthesis. The triage workow is highlighted in Scheme 1.
Fig. 1 TRPV4 activity vs. molecular weight, trellised by molecular cluster. Red ¼
predicted active by $2 HTS Bayesian models, yellow ¼ predicted active by 1
model, green ¼ predicted active by 0 models.
Compounds were also removed from the screening cascade
if they possessed non-drug-like properties (molecular weight
>600 Da, or clogP >6), or contained undesirable or reactive
substructures e.g. nitro groups, aldehydes, etc.15,16 In addition to
the application of negative lters, a number of positive lters
were applied to prioritise compounds for TRPV4 IC50 follow-up.
Positive lters were utilised in order to select compounds with
either a higher chance of repeating their activity, or that
appeared synthetically tractable for follow-up in the event that
they were active. Compounds with; a high level of inhibition in
the TRPV4 single point assay (>75% inhibition at 10 mM), a high
local hit rate (LHR) value (proportion of near neighbours that
are active) with either a LHR >10% or a LHR greater than the
background hit rate to the extent that the P value, as judged
from a chi-squared test, was less than 10ꢂ20
,
or that scored
17
highly in the TRPV4 Bayesian model built from the TRPV4 HTS
data generated thus far (a Bayesian cut-off score of 15 giving a
kappa value k ¼ 0.09) were selected on the basis of their like-
lihood to retain activity. As can be seen in Fig. 2, the separation
of active and inactive peaks in the Bayesian model is not
Fig. 2 Bayesian plot of single-point TRPV4 antagonist data.
This journal is ª The Royal Society of Chemistry 2013
Scheme 1 TRPV4 HTS triage workflow.
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Table 2 In vitro selectivity data for compounds 2c and 3c
2c
3c
hTRPV4 EC50 (nM)
rTRPV4 IC50 (nM)
rTRPV4 EC50 (nM)
hTRPA1 IC50 (nM)
hTRPA1 EC50 (nM)
hTRPV1 IC50 (nM)
hTRPV1 EC50 (nM)
hTRPM8 IC50 (nM)
>1000
1150
>1000
34.1
>1000
>12 500
>12 500
>3900
>20 000
4800
>1000
>12 500
>12 500
>16 000
>20 000
9580
Fig. 3 Structure and properties of compound 1.
Table 3 In vitro ADME and safety data for compound 3c
HLM
RLM
Dof.
Compd (mL minꢂ1 mgꢂ1
)
(mL minꢂ1 mgꢂ1
)
DDIa Log D IC50 (nM)
3c
<8.1
87
<20% 3.2
9670
a
DDI (Drug–Drug Interaction). Potential for DDI at Cytochrome P450
1A2, 2C9, 2D6 and 3A4 was assessed at 3 mM.
Fig. 4 Structure and properties of compound 3c.
Hit to lead follow-up
Hit-to-lead follow-up on the hydroxypiperidine series largely
focussed on improving TRPV4 potency and efficiency (LIPE,
LE). Any tool compound would need to be suitable for pre-
clinical in vivo assessment so must also have an appropriate
ADME prole15 and orthologue (rat) pharmacology. Initial
targets sought to replace the thio-ether linker with a more
polar group to lower logP24 and increase the probability of
achieving good metabolic stability.25 In addition, replacement
of the 4-pyridyl group was desired to reduce the liability for
drug–drug interactions (DDI).16,26 A small library of aryl ether
hydroxypiperidines was synthesised and assessed in the
hTRPV4 (human TRPV4) assay. Gratifyingly, a number of these
compounds demonstrated improved TRPV4 potency and LIPE/
LE, and were devoid of the pyridyl structural alert.16 Key
compounds 2a–d are outlined in Table 1. Examples within the
hydroxypiperidine series showed no TRPV4 agonist activity,
were moderately potent inhibitors of rat TRPV4 (rTRPV4) and
Table 1 In vitro TRPV4 data for compounds 2a–d and 3a–d
Compd
R1
R2
R3
R4
hTRPV4 IC50(nM)
LE
LIPE
2a
2b
2c
2d
3a
3b
3c
3d
CN
CN
CN
CN
CN
CN
CN
CN
F
F
H
F
F
Cl
F
Cl
Cl
Cl
Cl
CN
CN
CN
Cl
Cl
CN
Cl
CN
Cl
Cl
12.9
49.1
49.7
50.5
3.4
3.6
3.8
4.0
0.37
0.33
0.36
0.32
0.41
0.41
0.44
0.41
4.43
5.13
4.03
6.10
5.39
4.93
4.66
5.18
Cl
CN
were selective over
a number of other TRP channels.
Cl
Compound 2c exemplies the selectivity prole of this series
(Table 2):
In order to further improve compound potency, a pairwise
analysis was conducted on the piperidine linker.27 In this
analysis, a substructure search based method (named SWAP)27
was used to interrogate the Pzer database and identify pairs of
molecules differing only in the replacement of a piperidine
linker group. The biological activity (for a given biological assay)
for each matched pair was recorded to generate a dataset that
showed the performance of each isostere. Performance was
assessed in terms of the likelihood of retaining, improving or
losing potency with respect to the starting point. This analysis
highlighted a number of potential isosteres, of which an azeti-
dine replacement was calculated to have a ꢁ70% chance of
retaining or improving potency (as well as lowering logP).28
The TRPV4 HTS and rapid follow-up screening campaign
generated a number of interesting TRPV4 series. One series of
particular interest was the hydroxypiperidines, exemplied by
compound 1 (Fig. 3). This series demonstrated encouraging
levels of hTRPV4 inhibitory activity and binding efficiency
(LIPE, LE†)15,23 and was known to be straightforward to
synthesise.
† LIPE (Lipophilic Ligand Efficiency) ¼ pKi ꢂ clogP (or log D). LE (Ligand
Efficiency) ¼ 1.3643pKi/number of heavy atoms.
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Table 4 In vivo PK parameters of 3c following IV (intravenous) and PO (oral)
administration (n ¼ 2, Sprague-Dawley rats)
the selectivity prole of the hydroxyazetidine series (Table 2). In
vitro ADME‡ end-points for compound 3c were determined
along with an assessment of hERG liability29 utilising a Dofeti-
lide binding assay30 (Table 3). The in vitro ADME prole of 3c
was very promising, with low turnover in human liver micro-
somes and only a relatively weak activity signal in the Dofetilide
assay. The pharmacokinetic properties of 3c were also assessed.
Compound 3c demonstrated almost complete oral absorption
and a ꢁ2 hour half-life in the rat (Table 4). It was therefore
deemed a suitable tool with which to conduct further rodent in
vivo studies.
3c
1 mg kgꢂ1 IV
2 mg kgꢂ1 PO
Half life (h)
2.2
Cl (mL minꢂ1 kgꢂ1
)
23.5
756
4.3
Clu (mL minꢂ1 kgꢂ1
)
Vdss (L kgꢂ1
)
Cmax (nM free)
Tmax (h)
F%
16
0.75
73
Chemistry
A number of azetidine-linked compounds, utilising the
sulfonamide and aryl-ether SAR established in the hydrox-
ypiperidine series, were synthesised. Pleasingly, a number of
these analogues such as compounds 3a–d demonstrated
improved hTRPV4 potency and an improved LIPE/LE prole
(Table 1).
The hydoxyazetidines displayed similarly high levels of TRP
selectivity as the hydroxypiperidines but gratifyingly delivered
enhanced rTRPV antagonist activity. Compound 3c exemplies
The synthetic route towards hydroxypiperidines 2a–d is out-
lined in Scheme 2. Briey, nucleophilic addition of phenol
derivatives (R1)(R2)Ar–OH to epoxide 4 under basic conditions
furnished N-Boc hydroxypiperdines 5a and b. N-Boc depro-
tection of compounds 5a and b was accomplished with TFA/
DCM to furnish amines 6a and b. Addition of an appropriate
sulfonyl chloride, ((R1)(R2)ArSO2Cl), to amines 6a and b, in tri-
ethylamine and dichloromethane (DCM), gave hydroxypiper-
idines 2a–d.
The synthesis of hydroxyazetidines 3a–d is described in
Scheme 3. Ketone 7 was converted to alkene 8 via addition of
Ph3PCH2Br and potassium tert-butoxide. Alkene 8 was further
converted to a regioisomeric mixture of hydroxybromides (9a
and 9b) through addition of NBS in DMSO and water. Addition
of sodium hydride to a solution of 9a and 9b furnished epoxide
10 in good yield. Nucleophilic ring-opening of epoxide 10 with
the appropriate phenol derivative, ((R1)(R2)Ar–OH), afforded N-
Boc-hydroxypiperdines 11a and b, which were subsequently
deprotected to their respective amines (12a and b) under TFA/
DCM conditions. A nal sulfonylation step on amines 12a and
bwith the appropriate sulfonyl chloride ((R1)(R2)SO2Cl), in tri-
ethylamine and DCM, furnished hydroxyazetidines 3a–d.
Scheme 2 Synthesis of hydroxypiperidines 2a–d. Reagents and conditions: (i)
(R1)(R2)Ar–OH, Cs2CO3, IPA/DMSO/H20, 80 ꢀC, 49–60%; (ii) TFA, DCM, 25 ꢀC, 75–
89%; (iii) (R1)(R2)ArSO2Cl, NEt3, DCM, 0–25 ꢀC, 8–79%.
Conclusions
The application of computational techniques (including
Bayesian modelling) in the triage of TRPV4 HTS data enabled
the early deprioritisation of putative “actives” that were, in
fact, likely to be “frequent hitters”. In addition, the use of
methods to positively prioritise compounds for follow-up
screening allowed the rapid identication of a number of
interesting TRPV4 series. Hit-to-lead work on the hydrox-
ypiperidine series included the replacement of the sulde
linker and pyridyl group to generate potent and selective
compounds such as 2a–d. Further optimisation, utilising a
pairwise analysis protocol (SWAP) delivered a series of
hydroxyazetine compounds. Compounds within this series,
such as compound 3c, full the TRPV4 potency (rat and
human), selectivity and ADME criteria required for use as in
vitro/in vivo tools. Pre-clinical in vivo TRPV4 pharmacology
and safety data for compound 3c will be reported in due
course.
Scheme 3 Synthesis of hydroxyazetidines 3a–d. Reagents and conditions: (i)
Ph3PCH2Br, KOt-Bu, Et2O, RT, 61%; (ii) NBS, DMSO/H2O, RT, 0–25 ꢀC, 83%; (iii)
NaH, THF, 0–25 ꢀC, 62%; (iv) (R1)(R2)Ar–OH, Cs2CO3, IPA/DMSO/H20, 80 ꢀC, 55–
71%; (v) TFA, DCM, 25 ꢀC, 85–97%; (vi) (R1)(R2)SO2Cl, NEt3, DCM, 0–25 ꢀC, 16–
90%.
‡ ADME (Absorption Distribution Metabolism Excretion).
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General procedure for synthesis of N-Boc hydroxypiperidines
5a and b and N-Boc hydroxyazetidines 11a and b. To a stirred
solution of epoxide (4 or 10) (0.47 mmol) and cesium carbonate
(0.56 mmol) in IPA (3 mL), DMSO (0.5 mL) and water (0.5 mL),
was added (R1)(R2)Ar–OH (0.52 mmol) and the resulting mixture
Experimental section
In vitro TRPV4 assay
Receptor-evoked changes in intracellular calcium were
measured using calcium-selective uorescent Ca2+ NW dye
(Molecular Devices, Sunnyvale, CA, USA) quantitated with a
uorometric imaging plate reader (FLIPR; Molecular Devices).
Stably transfected CHOK1 cells expressing human and rat
TRPV4 receptors have been used for this assay. On the day
before the FLIPR experiment, frozen cryovials of cells were
thawed, centrifuged and resuspended in DMEM medium
(Invitrogen Cat. # 21063) containing glutamax, sodium pyru-
vate, non-essential amino acids and 10% FBS (Invitrogen Cat. #
10082-147). The cells were plated into black walled, clear
bottom Greiner 384 poly-D-lysine plates at a density of ꢀ10 000
cells per well and incubated overnight in 5% CO2 at 37 C. On
the day of the experiment, media was removed and cells were
loaded with dye loading buffer (0.5ꢃ Ca2+ NW-dye in HBSS with
Ca++, Mg++, 2.5 mM Probenecid, 0.025% pluronic, 20 mM
HEPES, pH 7.4) and incubated for 45 minutes at 37 ꢀC. The test
compounds and the reference compound (ruthenium red) were
serially diluted in 100% DMSO followed by dilution in assay
buffer (HBSS without Ca++, Mg++, 20 mM HEPES, 5.4 mM CaCl2,
1 mM MgCl2, 0.12% P104, pH 7.4) in order to achieve a nal
highest concentration of 10 mM in the assay plate. The plates
were then placed in the FLIPR, and a baseline uorescence
measurement (excitation @ 488 nm and emission @ 510–570
nm) was obtained for 10 s before compound or vehicle addition.
The assay was performed in dual mode: rstly, upon addition of
compounds to the cells, any possible agonistic effect of the test
compounds were monitored for 5 minutes aer which the assay
plate was incubated for further 15 minutes at room tempera-
ture. At the second step, EC75 of the agonist PF-04674114/
GSK1016790A was added and uorescence was measured for an
additional 5 minutes. Change in uorescence values in
response to the addition of PF-04674114 (in the absence and
presence of test compounds) was measured and inhibition
curves generated using nonlinear regression (Prism v.4,
GraphPad Soware, San Diego, CA, USA) employing a four
parameter logistic equation (Y ¼ bottom + (top ꢂ bottom)/(1 +
10^(logEC50 ꢂ X)hillslope).
ꢀ
reuxed at 80 C for 2.5 h. The reaction mixture was concen-
trated in vacuo and the residue partitioned between water and
ethyl acetate. The organics were further washed with brine,
dried over sodium sulphate, concentrated in vacuo and puried
by silica-gel column chromatography.
4-(4-Cyano-3-uoro-phenoxymethyl)-4-hydroxy-piperidine-1-
carboxylic acid tert-butyl ester (5a). Following the general
procedure for the synthesis of N-Boc hydroxypiperidines 5a and
b and N-Boc hydroxyazetidines 11a and b, and using 2-uoro-4-
hydroxybenzonitrile and epoxide 4, the title compound was
afforded as a white solid (80 mg, 49%). 1H NMR (400 MHz,
CDCl3) d: 1.45 (s, 9H), 1.60–1.64 (m, 2H), 1.73 (m, 2H), 2.60 (m,
3H), 3.18 (m, 2H), 3.83 (s, 1H), 3.95 (m, 1H), 6.71–6.78 (m, 2H),
7.53 (t, 1H); m/z 351 [M + H]+.
4-(4-Cyano-phenoxymethyl)-4-hydroxy-piperidine-1-carbox-
ylic acid tert-butyl ester (5b). Following the general procedure
for the synthesis of N-Boc hydroxypiperidines 5a and b and N-
Boc hydroxyazetidines 11a and b, and using 4-hydrox-
ybenzonitrile and epoxide 4, the title compound was prepared
as a white solid (9.25 g, 85%). 1H NMR (400 MHz, DMSO-d6) d:
1.39 (s, 9H), 1.52 (m, 4H), 3.08 (d, 2H), 3.73 (d, 2H), 3.86 (d, 2H),
7.11 (d, 2H), 7.76 (d, 2H); m/z 333 [M + H]+.
General procedure for Boc deprotection of N-Boc hydrox-
ypiperidines (5a and b) and N-Boc hydroxyazetidines (11a and
b). To a stirred solution of 5a and b or 11a and b (0.92 mmol)
in DCM (5 mL) was added TFA (1 mL) and the
ꢀ
resulting mixture stirred at 25 C for 2 h. The reaction mixture
was then concentrated in vacuo, triturated with pentane–ether
(5 mL) and used in the next step without further purication.
2-Fluoro-4-(4-hydroxy-piperidin-4-ylmethoxy)-benzonitrile
(6a). Following the general procedure for Boc deprotection of N-
Boc hydroxypiperdines (5a and b) and N-Boc hydroxyazetidines
(11a and b), and using N-Boc hydroxypiperdine 5a, the title
compound was a white solid (TFA salt) (251 mg, 75%). 1H NMR
(400 MHz, DMSO-d6) d: 1.74 (m, 3H), 3.09 (m, 2H), 3.17 (m, 2H),
3.95 (s, 2H), 5.20 (s, 1H), 6.90 (d, 1H), 7.15 (d, 1H), 7.84 (t, 1H),
8.23 (bs, 1H), 8.44 (bs, 1H); m/z 251 [M + H]+.
4-(4-Hydroxy-piperidin-4-ylmethoxy)-benzonitrile
(6b).
Chemistry
Following the general procedure for Boc deprotection of N-Boc
hydroxypiperdines (5a and b) and N-Boc hydroxyazetidines (11a
and b), and using N-Boc hydroxypiperdine 6a, the title compound
was a white solid (TFA salt) (300 mg, 79%). m/z 233 [M + H]+.
General procedure for sulfonamide formation (2a–e, 3a–d).
To a stirred solution of amine (6a and b or 12a and b, 0.61
All commercially available chemicals and solvents were used
without further purication. All temperatures are in C. Flash
ꢀ
column chromatography was carried out using Merck silica gel
60 (9385) or Redisep silica. NMR spectra were obtained on a
Varian Mercury (400 MHz) or a Bruker Avance (400 MHz) using
the residual signal of the deuterated NMR solvent as the
internal reference. Chemical shis are expressed in parts per
million (ppm), multiplicity of the signals are indicated by lower-
case letters (singlet s, doublet d, triplet t, quadruplet q, multiple
m, broad singlet br s), and deuterated solvents are dime-
thylsulphoxide d6, methanol d4, and chloroform d1. Mass
spectral date were obtained using Waters ZQ ESCI or Applied
Biosystem’s API-2000.
ꢀ
mmol) and triethylamine (1.55 mmol) in DCM (5 mL) at 0 C
was added the appropriate benzenesulfonyl chloride (0.61
mmol). The reaction mixture was allowed to warm to 25 ꢀC and
stir for 18 h. The reaction mixture was partitioned between
water and DCM. The organics were further washed with brine,
dried over sodium sulphate, evaporated in vacuo and puried
via silica-gel column chromatography.
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chromatography (eluting with ethyl acetate : hexane, 10 : 90) to
4-[1-(2,4-Dichloro-benzenesulfonyl)-3-hydroxy-azetidin-3-
ylmethoxy]-2-uoro-benzonitrile (2a). Following the general
procedure for sulfonamide formation (2a–d, 3a–d), and using
amine 6a and 2,4-dichlorobenzene-1-sulfonyl chloride, the title
compound was prepared as a white solid (750 mg, 79%). 1H
NMR (400 MHz, CDCl3) d: 1.74–1.79 (m, 4H), 3.15–3.22 (m,
2H), 3.73–3.76 (m, 2H), 3.84 (s, 2H), 6.70–6.77 (m, 2H), 7.36–
7.38 (dd, 1H), 7.53 (t, 1H), 7.90 (d, 1H), 7.98 (d, 1H); m/z 459
[M + H]+.
afford the isomeric mixture of title compounds as a pale yellow
1
oil (7.65 g, 83%). H NMR (400 MHz, CDCl3) d: 1.43 (s, 18H),
2.03–2.06 (t, 1H), 2.68 (s, 1H), 3.68 (s, 2H), 3.87–3.91 (m, 8H),
4.26–4.27 (dd, 2H).
1-Oxa-5-aza-spiro[2.3]hexane-5-carboxylic acid tert-butyl
ester (10). To a stirred solution of isomeric mixture of 9a and 9b
ꢀ
(4.6 g, 17.29 mmol) in THF (80 mL) at 0 C was added sodium
hydride (760 mg, 19.02 mmol, 60%) in one portion. The
resulting mixture was allowed to warm to 25 ꢀC and stirred for 7
h. The reaction mixture was quenched with saturated ammo-
4-((1-((2-Chloro-4-cyanophenyl)sulfonyl)-4-hydroxypiperidin-
4-yl)methoxy)-2-uorobenzonitrile (2b). Following the general
procedure for sulfonamide formation (2a–d, 3a–d), and using
amine 6a and 2-chloro-4-cyano benzenesulfonyl chloride, the
title compounds was prepared as a white solid (47 mg, 38%
ꢀ
nium chloride (50 mL) and stirred at 25 C for a further 18 h.
The reaction mixture was extracted with ethyl acetate (100 mL)
and the organic phase dried over sodium sulphate, concen-
trated in vacuo, and puried by silica-gel column chromatog-
raphy (eluting with ethyl acetate : hexane, 10 : 90) to afford the
1
yield). H NMR (400 MHz, DMSO-d6) d: 1.64 (m, 4H), 3.06 (m,
2H), 3.58 (m, 2H), 3.91 (s, 2H), 4.92 (s, 1H), 6.95 (d, 1H), 7.13 (d,
1H), 7.81 (t, 1H), 8.04 (d, 1H), 8.11 (d, 1H), 8.35 (s, 1H); m/z 450
[M + H]+.
1
title compound as a pale yellow oil (2.0 g, 62%). H NMR (400
MHz, CDCl3) d: 1.44 (s, 9H), 2.84 (s, 2H), 4.17–4.26 (m, 4H).
3-(4-Cyano-3-uoro-phenoxymethyl)-3-hydroxy-azetidine-1-
carboxylic acid tert-butyl ester (11a). Following the general
procedure for the synthesis of N-Boc hydroxypiperidines 5a and
b and N-Boc hydroxyazetidines 11a and b, and using 2-uoro-4-
hydroxybenzonitrile and epoxide 10, the title compound was
afforded as a white solid (250 mg, 57%). 1H NMR (400 MHz,
DMSO-d6) d: 1.38 (s, 9H), 3.71 (d, 2H), 3.87 (d, 2H), 4.15 (s,
2H), 6.11 (s, 1H), 6.98 (d, 1H), 7.16 (d, 1H), 7.83 (t, 1H); m/z 323
[M + H]+.
3-(3-Chloro-4-cyano-phenoxymethyl)-3-hydroxy-azetidine-1-
carboxylic acid tert-butyl ester (11b). Following the general
procedure for the synthesis of N-Boc hydroxypiperidines 5a and
b and N-Boc hydroxyazetidines 11a and b, and using 2-chloro-4-
hydroxybenzonitrile and epoxide 10, the title compound was
afforded as a white solid (250 mg, 55%). 1H NMR (400 MHz,
DMSO-d6) d: 1.38 (s, 9H), 3.71 (d, 2H), 3.87 (d, 2H), 4.17 (s,
2H), 6.10 (s, 1H), 7.10 (m, 1H), 7.36 (d, 1H), 7.87 (d, 1H); m/z 339
[M + H]+.
2-Fluoro-4-(3-hydroxy-azetidin-3-ylmethoxy)-benzonitrile
(12a). Following the general procedure for Boc deprotection of
N-Boc hydroxypiperdines (5a and b) and N-Boc hydrox-
yazetidines (11a and b), and using N-Boc hydroxypiperdine 11a,
the title compound was a white solid (TFA salt) (200 mg, 79%).
1H NMR (400 MHz, DMSO-d6) d: 3.94 (d, 2H), 4.03 (d, 2H), 4.19
(s, 2H), 6.59 (s, 1H), 7.05 (d, 1H), 7.22 (s, 1H), 7.88 (t, 1H), 8.74–
8.86 (brs, 1H); m/z 223 [M + H]+.
2-Chloro-4-(3-hydroxy-azetidin-3-ylmethoxy)-benzonitrile
(12b). Following the general procedure for Boc deprotection of
N-Boc hydroxypiperdines (5a and b) and N-Boc hydrox-
yazetidines (11a and b), and using N-Boc hydroxypiperdine 11b,
the title compound was a white solid (TFA salt) (200 mg, 77%).
1H NMR (400 MHz, DMSO-d6) d: 3.92 (d, 2H), 4.03 (d, 2H), 4.20
(s, 2H), 6.58 (s, 1H), 7.17 (d, 1H), 7.44 (s, 1H), 7.93 (d, 1H), 8.71–
8.82 (brs, 1H); m/z 239 [M + H]+.
4-[1-(2,4-Dichloro-benzenesulfonyl)-4-hydroxy-piperidin-4-
ylmethoxy]-benzonitrile (2c). Following the general procedure
for sulfonamide formation (2a–d, 3a–d), and using amine 6b
and 2,4-dichlorobenzene-1-sulfonyl chloride, the title
compound was prepared as a white solid (579 mg, 56%). 1H
NMR (400 MHz, CDCl3) d: 1.61–1.70 (m, 4H), 3.01 (m, 2H), 3.56
(m, 2H), 3.86 (s, 2H), 4.88 (s, 1H), 7.08 (d, 1H), 7.65 (d, 1H),
7.74–7.80 (m, 3H), 7.93 (d, 1H), 7.96 (dd, 1H); m/z 441 [M + H]+.
4-[4-(4-Cyano-3-uoro-phenoxymethyl)-4-hydroxy-piperi-
dine-1-sulfonyl]-isophthalonitrile (2d). Following the general
procedure for sulfonamide formation (2a–d, 3a–d), and using
amine 6a and 2,4-dicyano benzenesulfonyl chloride, the title
compound was prepared as a white solid (22 mg, 8%). 1H NMR
(400 MHz, DMSO-d6) d: 1.66 (m, 4H), 2.94 (m, 2H), 3.60 (m, 2H),
3.90 (s, 2H), 4.90 (s, 1H), 6.95 (d, 1H), 7.13 (d, 1H), 7.81 (t, 1H),
8.18 (d, 1H), 8.41 (d, 1H), 8.80 (s, 1H); m/z 441 [M + H]+.
3-Methylene-azetidine-1-carboxylic acid tert-butyl ester (8).
To a stirred solution of Ph3PCH2Br (88.7 g, 248.4 mmol) in dry
ether (400 mL) was added potassium tert-butoxide (27.9 g, 248.4
mmol) and the mixture stirred at 25 ꢀC for 1 h. Ketone 7 (42.5 g,
248.4 mmol) in dry ether (400 mL) was added and the resulting
mixture stirred at 25 ꢀC for 18 h. The reaction mixture was
ltered through Celiteꢀ. The ltrate was washed with water
(100 mL), brine (100 mL), dried over sodium sulphate and
concentrated in vacuo to afford the crude compound as a yellow
oil (10.8 g, 61% yield), which was used without further puri-
1
cation. H NMR (400 MHz, CDCl3) d: 1.44 (s, 9H), 4.46 (s, 4H),
4.97 (s, 2H).
3-Bromomethyl-3-hydroxy-azetidine-1-carboxylic acid tert-
butyl ester and 3-bromo-3-hydroxymethyl-azetidine-1-carboxylic
acid tert-butyl ester (9a and 9b). To a stirred solution of alkene 8
ꢀ
(2.9 g, 17.13 mmol) in DMSO (15 mL) at 0 C was added NBS
(6.10 g, 34.2 mmol) and water (617 mL, 34.2 mmol). The
resulting mixture was allowed to warm to 25 ꢀC and stirred for a
further 18 h. The reaction mixture was partitioned between
water (25 mL) and ethyl acetate (25 mL). The organics were
washed with brine (10 mL), dried over sodium sulphate,
concentrated in vacuo and puried by silica-gel column
4-({1-[4-Chloro-2-cyano-phenylsulfonyl]-3-hydroxyazetidine-
3-yl}methoxy)-2-uorobenzonitrile (3a). Following the general
procedure for sulfonamide formation (2a–d, 3a–d), and using
amine 12a and 4-chloro-2-cyano-benzenesulfonyl chloride, the
title compound was prepared as a white solid (50 mg, 16%). 1H
This journal is ª The Royal Society of Chemistry 2013
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NMR (400 MHz, DMSO-d6) d: 3.77 (d, 2H), 3.96 (d, 2H), 4.12 (s,
2H), 6.34 (s, 1H), 6.90 (dd, 1H), 7.11 (dd, 1H), 7.83 (t, 1H), 8.05
(s, 2H), 8.47 (s, 1H); m/z 422 [M + H]+.
endothelial failure and circulatory collapse: part 2, J.
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2-Chloro-4-({1-[4-chloro-2-cyano-phenylsulfonyl]-3-hydrox-
yazetidine-3-yl}methoxy) benzonitrile (3b). Following the
general procedure for sulfonamide formation (2a–d, 3a–d), and
using amine 12b and 4-chloro-2-cyano-benzenesulfonyl chlo-
ride, the title compound was prepared as an off-white solid (52
mg, 16%). 1H NMR (400 MHz, DMSO-d6) d: 3.78 (d, 2H), 3.97 (d,
2H), 4.14 (s, 2H), 6.33 (s, 1H), 7.03–7.05 (dd, 1H), 7.27 (d, 1H),
7.89 (d, 1H), 8.05 (s, 2H), 8.46 (d, 1H); m/z 438 [M + H]+.
4-[1-(2,4-Dichloro-benzenesulfonyl)-3-hydroxy-azetidin-3-ylme-
thoxy]-2-uoro-benzonitrile (3c). Following the general proce-
dure for sulfonamide formation (2a–d, 3a–d), and using amine
12a and 4-chloro-2-chloro-benzenesulfonyl chloride, the title 10 S. F. Pedersen, G. Owsianik and B. Nilius, TRP channels: an
compound was prepared as a white solid (110 mg, 63%). 1H
overview, Cell Calcium, 2005, 38(3–4), 233–252.
NMR (400 MHz, CDCl3) d: 4.04 (d, 2H), 4.12 (d, 2H), 4.20 (s, 2H), 11 Transient Receptor Potential (TRP) Channels, in Handbook
6.77–6.81 (m, 2H), 7.38–7.41 (dd, 1H), 7.57 (t, 1H), 7.89 (d, 1H),
7.97 (d, 1H); m/z 431 [M + H]+.
of Experimental Pharmacology, ed. V. Flockerzi and B.
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2-Chloro-4-((1-((2-chloro-4-cyanophenyl)sulfonyl)-3-hydroxy- 12 S. D. Pratt, et al., Identication and characterization of
azetidin-3-yl)methoxy)benzonitrile (3d). Following the general
procedure for sulfonamide formation (2a–d, 3a–d), and using
amine 12b and 4-cyano-2-chloro-benzenesulfonyl chloride, the
title compound was prepared as a white solid (12 mg, 22%). 1H
mGlu3 ligands using a high throughput FLIPR assay for
detection of agonists, antagonists, and allosteric
modulators, Comb. Chem. High Throughput Screening, 2011,
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(s, 2H), 6.90 (s, 1H), 7.15 (d, 1H), 7.75 (t, 1H), 8.09 (d, 1H), 8.16
for identication of “frequent hitters” in compound
libraries, J. Med. Chem., 2002, 45(1), 137–142.
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14 M. Glick, et al., Enrichment of extremely noisy high-
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Acknowledgements
The authors would like to thank Francois Bertelli and Chris 15 P. D. Leeson and B. Springthorpe, The inuence of drug-like
Chambers for generating the TRPV HTS data, Florian Wakenhut
for assistance in the HTS triage and TCG Lifesciences for syn-
concepts on decision-making in medicinal chemistry, Nat.
Rev. Drug Discovery, 2007, 6(11), 881–890.
thesising and generating TRPV4 data on compounds 2a–d and 16 A. F. Stepan, et al., Structural alert/reactive metabolite
3a–d.
concept as applied in medicinal chemistry to mitigate the
risk of idiosyncratic drug toxicity: a perspective based on
the critical examination of trends in the top 200 drugs
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