Inhibition of insulin-regulated aminopeptidase (IRAP) by arylsulfonamides

Article abstract of DOI:10.1002/open.201402027

The inhibition of insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) by angiotenesin IV is known to improve memory and learning in rats. Screening 10 500 low-molecular-weight compounds in an enzyme inhibition assay with IRAP from Chinese Hamster Ovary (CHO) cells provided an arylsulfonamide (N-(3-(1H-tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-sulfonamide), comprising a tetrazole in the meta position of the aromatic ring, as a hit. Analogues of this hit were synthesized, and their inhibitory capacities were determined. A small structure-activity relationship study revealed that the sulfonamide function and the tetrazole ring are crucial for IRAP inhibition. The inhibitors exhibited a moderate inhibitory potency with an IC₅₀=1.1±0.5 μm for the best inhibitor in the series. Further optimization of this new class of IRAP inhibitors is required to make them attractive as research tools and as potential cognitive enhancers. Hit optimization! The inhibition of insulin-regulated aminopeptidase (IRAP) by angiotenesin IV is known to improve memory and learning in rats. Screening 10 500 low-molecular-weight compounds as IRAP inhibitors provided an arylsulfonamide as a hit. Analogues of this hit were synthesized, and their inhibitory capacity and structure-activity relationships were determined. With further optimization, these new inhibitors can become potential cognitive enhancers.

Full text of DOI:10.1002/open.201402027

DOI: 10.1002/open.201402027
Inhibition of Insulin-Regulated Aminopeptidase (IRAP) by
Arylsulfonamides
Sanjay R. Borhade,[b] Ulrika Rosenstrçm,[b] Jonas Sꢀvmarker,[c] Thomas Lundbꢀck,[d]
Annika Jenmalm-Jensen,[d] Kristmundur Sigmundsson,[d] Hanna Axelsson,[d]
Fredrik Svensson,[b] Vivek Konda,[b] Christian Skçld,[b] Mats Larhed,[e] and Mathias Hallberg*[a]
The inhibition of insulin-regulated aminopeptidase (IRAP, EC
3.4.11.3) by angiotenesin IV is known to improve memory and
learning in rats. Screening 10500 low-molecular-weight com-
pounds in an enzyme inhibition assay with IRAP from Chinese
Hamster Ovary (CHO) cells provided an arylsulfonamide (N-(3-
(1H-tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-sulfona-
mide), comprising a tetrazole in the meta position of the aro-
matic ring, as a hit. Analogues of this hit were synthesized, and
their inhibitory capacities were determined. A small structure–
activity relationship study revealed that the sulfonamide func-
tion and the tetrazole ring are crucial for IRAP inhibition. The
inhibitors exhibited a moderate inhibitory potency with an
IC50 =1.1ꢀ0.5 mm for the best inhibitor in the series. Further
optimization of this new class of IRAP inhibitors is required to
make them attractive as research tools and as potential cogni-
tive enhancers.
Introduction
It was reported 25 years ago that intracerebroventricular (i.c.v.)
injection of angiotensin IV (Ang IV, Val-Tyr-Ile-His-Pro-Phe), an
angiotensin II metabolite, improved memory and learning in
rats.[1] The positive impact of Ang IV on cognition was subse-
quently confirmed in a series of animal models in both rats
and mice.[2–5] Structurally related analogues to Ang IV, such as
the endogenous LVV-hemorphin-7 (Leu-Val-Val-Tyr-Pro-Trp-Thr-
Glu-Arg-Phe), in which a tyrosine residue is attached to lipo-
philic amino acid residues, as in Ang IV, exhibited similar out-
comes.[6] These abilities to improve processes related to
memory and learning have attracted considerable interest in
recent years.[7,8]
A specific binding site for Ang IV was identified in 1992,[9,10]
and high densities of the binding sites were found in areas of
the brain that are associated with cognitive, sensory, and
motor functions, including the hippocampus.[9] Insulin-regulat-
ed aminopeptidase (IRAP), a high-affinity receptor for Ang IV,
was purified in 2001.[11] IRAP has been identified as cystinyl
aminopeptidase (CAP, EC 3.4.11.3), placental leucine aminopep-
tidase (P-LAP, soluble human homologue), oxytocinase, gp160,
or vp165.[12–14] IRAP, a single-spanning transmembrane zinc-
metallopeptidase that belongs to the M1 family of aminopep-
tidases,[15] has attracted awareness as a potential target for
pharmaceuticals aimed at the treatment of cognitive disor-
ders.[8,16–18] It is hypothesized that the inhibition of the catalytic
activity of IRAP by the hexapeptide Ang IV results in longer
half-lives of endogenous substrates to IRAP, such as vasopres-
sin and oxytocin, which are macrocyclic disulfides and are
known to exert favorable influences on cognitive parameters
in the brain,[19 ]although several alternative and relevant hy-
potheses explaining the action of Ang IV have been pro-
posed.[20,21]
[a] Dr. M. Hallberg
Beijer Laboratory, Department of Pharmaceutical Biosciences
Division of Biological Research on Drug Dependence
BMC, Uppsala University, P.O. Box 591, 751 24 Uppsala (Sweden)
E-mail: mathias.hallberg@farmbio.uu.se
[b] Dr. S. R. Borhade, Dr. U. Rosenstrçm, F. Svensson, Dr. V. Konda, Dr. C. Skçld
Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry
BMC, Uppsala University, P.O. Box 574, 751 23 Uppsala (Sweden)
[c] Dr. J. Sꢀvmarker
Beijer Laboratory, Department of Medicinal Chemistry
BMC, Uppsala University, P.O. Box 574, 751 23 Uppsala (Sweden)
[d] Dr. T. Lundbꢀck, Dr. A. Jenmalm-Jensen, Dr. K. Sigmundsson, H. Axelsson
Chemical Biology Consortium Sweden, Science for Life Laboratory
Division of Translational Medicine and Chemical Biology
Department of Medical Biochemistry and Biophysics
We recently reported the syntheses and IRAP inhibition data
from a series of constrained macrocycles derived from Ang IV
and with structural similarities to, for example, oxytocin in the
N terminus.[22–24] Among those, HA-08 (1), which has a b3hTyr
residue in a 13-membered macrocyclic system is 20 times
more potent than Ang IV as an IRAP inhibitor.[24] Although fur-
ther optimization and application of the metathesis reaction
delivered very potent and more metabolically stable macrocy-
clic carba analogues,[22] these inhibitors, designed from struc-
tures of cyclic substrates, are still too peptidic and not likely to
be able to enter the brain.
Karolinska Institutet, Stockholm 171 77 (Sweden)
[e] Prof. M. Larhed
Department of Medicinal Chemistry, Science for Life Laboratory
BMC, Uppsala University, P.O. Box 574, 751 23 Uppsala (Sweden)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/open.201402027.
ꢁ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited and is not used for commercial purposes.
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Recently, Albiston et al. discovered a series of drug-like IRAP
inhibitors encompassing a benzopyran system.[7,25,26] These in-
hibitors were identified after in silico screening of 1.5 million
commercially available compounds against an IRAP model
structure. After i.c.v. administration of one of these inhibitors,
HFI-419 (2), a pronounced enhancement of memory in two
memory paradigms in rat was observed. We were encouraged
by these findings and performed a screening campaign of a li-
brary of 10500 low-molecular-weight compounds and mea-
sured the inhibition of the catalytic activity of IRAP from Chi-
nese Hamster Ovary (CHO) cells. After subsequent hit confirma-
tion experiments, we identified sulfonamide 3 as one of the
most promising compounds. We herein report the syntheses
of a series of analogues of 3, their inhibitory capacity, and
structure–activity relationships of this new class of IRAP inhibi-
tors.
Scheme 1. Synthesis of IRAP inhibitors 3, 7–22. Reagents and conditions:
a) CH2Cl2, pyridine, 258C, 14 h.
Scheme 2. Synthesis of IRAP inhibitor 25. Reagents and conditions: a) 4-
bromo-5-chlorothiophene-2-sulfonyl chloride, CH2Cl2, pyridine, 258C, 14 h;
b) THF, NaH, MeI, 08C to 258C, 3 h, 78%; c) NaN3, Et3N·HCl, toluene reflux,
12 h, 71%.
benzamide (5) reacting with the corresponding aryl/heteroaryl
sulfonyl chloride to give good to excellent yields.[27] N-Methy-
lated arylsulfonamide 25 was synthesized from 3-amino benzo-
nitrile (23) as shown in Scheme 2. First, the sulfonylation of 4-
bromo-5-chlorothiophene-2-sulfonyl chloride with 23 gave the
desired product, 4-bromo-5-chloro-N-(3-cyanophenyl)thio-
phene-2-sulfonamide, which was used without purification for
further treatment with methyl iodide in presence of sodium
hydride to afford compound 24 in 78% overall yield. The tetra-
zole ring formation starting from cyanophenyl sulfonamide
(24) was successfully achieved via the reported method of
Koguro et al.[28] by using sodium azide and triethylammonium
hydrochloride salt in dry toluene to deliver product 25 in 71%
yield. For the synthesis of compound 27, 4-bromo-5-chloro-
thiophene-2-carboxylic acid was coupled with 3-amino phenyl-
tetrazole via standard peptide coupling conditions using ben-
zotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophos-
phate (PyBOP) as a peptide coupling reagent for 18 hours at
258C with dimethylformamide (DMF) as the solvent
(Scheme 3).[29]
Results and Discussion
In the primary high-throughput screen (HTS), analogues of 3,
with a carboxylate group rather than the bioisosteric tetrazole
in the ortho, meta, or para positions of the aromatic ring, were
all found to be devoid of capacity to inhibit or be very weak
inhibitors of IRAP, suggesting that an acidic function is not
a sufficient criterion to achieve inhibition. Hence, the IC50
values for the meta carboxylate and the corresponding ethyl
ester were determined to be greater than 125 mm in the hit
confirmation experiments. Furthermore, the regioisomer of 3,
with the tetrazole ring positioned in the para position rather
than in the meta position, was inactive according to the pre-
liminary data.
To examine the basic structure–activity relationships, com-
pounds 3, 7–22, 25, and 27 were synthesized and evaluated as
inhibitors in an IRAP enzyme assay with a special emphasis to
assess whether the thiophene ring, sulfonamide function, and
the acidic NH of the tetrazole are prerequisites for binding to
IRAP.
The biological data from the evaluation of 3, 7–22, 25, and
27 as inhibitors of IRAP are presented in Table 1. The NH
group on the tetrazole ring seems essential to the inhibitory
activity since methylation of the nitrogen affords inactive com-
pound 7. To find out whether a considerably less acidic NH is
sufficient for activity, compound 8 was evaluated and found to
be inactive as well. Apparently a tetrazole with an acidic hydro-
The target compounds 3, 7–22, 25, and 27 were synthesized
as shown in Schemes 1–3. Compound 3, 7–22 were synthe-
sized from 3-amino phenyltetrazole (4) or 3-amino-N-methyl-
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gen but neither a carboxylate nor amide in the meta
position of the aromatic ring results in IRAP inhibitory
activity.
Classic bacteriostatic arylsulfonamides, such as sul-
famethoxazole and sulfadiazine, which are structural-
ly similar to 3, are weak acids. The presence of the
moderately acidic NH is important to retain activity
since methylation, leading to 25, rendered a consider-
ably less potent inhibitor. Furthermore, displacement
of the sulfonamide linker for an amide provided the
Scheme 3. Synthesis of IRAP inhibitors 27. Reagents and conditions: a) DMF, PyBOP, Et3N,
258C, 18 h, 81%.
Table 1. Biological evaluation of compounds 3, 7–22, 25, and 27 in the IRAP inhibition assay.
Compd
Structure
IC50 [mm][a]
Compd
Structure
IC50 [mm][a]
3
2.1ꢀ1.2 (15)
16
1.3ꢀ0.75 (3)
7
>125[b] (3)
17
39ꢀ3.4[b] (2)
8
9
>125[b] (3)
18
19
16ꢀ4.9 (2)
22ꢀ6.4 (2)
1.8ꢀ1.3 (4)
10
11
12
1.1ꢀ0.46 (4)
2.9ꢀ0.28 (3)
1.6ꢀ0.93 (3)
20
21
22
>125[b] (2)
>125[b] (2)
>125[b] (2)
13
7.9ꢀ2.1 (2)
25
27
58ꢀ23[b] (3)
14
15
3.1ꢀ1.8 (3)
67ꢀ7.8[b] (3)
44ꢀ3.3[b] (3)
[a] IC50 is defined as the compound concentration required to inhibit IRAP activity by 50%. Values represent the mean ꢀstandard deviation of best-fit
values from individual test occasions (carried out on different days). The number of separate test occasions is given in parentheses. [b] Fitting was per-
formed with maximal inhibition fixed to 100%. No values above 125 mm are reported as this was the highest compound concentration tested.
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weak inhibitor 27. The loss of most of the inhibitory potency
of 27 could be attributed to 1) the restricted rotation of the
amide bond not allowing adoption of an optimal binding to
IRAP, 2) the loss of an acidic NH, or alternatively 3) the sulfona-
mide oxygen(s) being crucial for binding. Substitution of the 4-
bromo for a 4-chloro group to provide 9 did not result in an
improvement in the inhibitory activity, but substitution of the
thiophene for a benzene ring resulted in a small improvement
(c.f., compounds 9 and 10). Furthermore, an additional chloro
substituent in the ortho position rendered an inhibitor with
a good inhibitory capacity (11). A fluoro group in the ortho po-
sition of a para bromo derivative (12) provided a potent inhibi-
tor while with two ortho substituents, as in compound 13, a de-
cline in potency was observed. Compound 14 with two methyl
groups located in the meta and para positions exhibited good
potency, but biphenyl compound 15 was found to be more
than ten times less active (IC50 =3.1ꢀ1.8 vs 44ꢀ3.3 mm). The
observation that a chloro or fluoro substituent was accepted in
the ortho position by the enzyme prompted us to make the
more bulky annelated benzooxadiazole derivative (16), which
acted as a potent IRAP inhibitor. Benzothiophenes 17 and 18
and methylindole derivative 19 were approximately 10 times
less active as inhibitors.
Figure 1. Potential binding mode of 3 shown in the homologue aminopepti-
dase N (APN) active site (PDB code 4FYS[30]). The catalytic zinc is depicted as
a grey bullet, and amino acids discussed in the text are highlighted; Gln213
and Phe896 are displayed in pink sticks, and Phe472 is displayed in light
grey sticks.
It is notable that the nonsubstituted thiophene, benzene,
and pyridine derivatives 20, 21, and 22, respectively, exhibited
all very poor abilities to inhibit the protease. Furthermore, IRAP
inhibitors 10, 14, and 16 exhibited a more than 10-fold prefer-
ence for IRAP than for the protein homologue aminopepti-
dase N (APN) (unpublished data).
between Phe472 (IRAP: Phe544) and Phe896 (IRAP: Tyr961) in
the active site. The stacking interaction with Phe544 in IRAP
has previously been reported as a key interaction for ligand
and substrate binding.[31,32] Two of the amino acids in contact
with compound 3 differ between APN and IRAP. Phe896 is re-
placed by Tyr in IRAP, and Gln213 is replaced by Glu. We antici-
pate that the difference between the protein structures should
not have a significant impact on the present binding mode
model of 3 as the aromatic stacking interaction should remain
largely the same changing from Phe to Tyr and since Gln213 is
not involved in any specific interaction with the ligand.
In an attempt to rationalize the observed activities of the
synthesized compounds, a docking study of the series was
conducted using Glide (version 5.8; for details, see Experimen-
tal Section). To date, no crystal structure of IRAP has been re-
ported. In order to model the binding of the inhibitors, we uti-
lized APN for which several high-resolution protein–ligand co-
crystal structures have been reported.[30] Twelve of the sixteen
amino acids that are found in the catalytic site of APN are con-
served in IRAP, where the catalytic site is defined as within 3 ꢂ
of Val and Tyr in Ang IV when co-crystallized in APN (PDB code
4FYS[30]); see Supporting Information for sequence alignment.
Since APN and IRAP have a high sequence identity in proximity
to the catalytic zinc, where we hypothesize that the modeled
ligands are binding, we find it reasonable to assume that
models of the binding modes found in the catalytic region of
APN can be extended to IRAP.
This binding mode model fits nicely with the inactivity of
the methylated tetrazole found in 7, which cannot produce
a complimentary negative charge to the catalytic zinc. The
model can possibly also account for the preference for tetra-
zole over carboxylic acid by the different directionality of their
zinc binding moiety. Replacing the tetrazole with a carboxylic
acid, the carboxylate interacts with the catalytic zinc in the
least common anti interaction.[33] Furthermore, N-methylation
of the sulfonamide in 25 would, in this binding mode, gener-
ate a steric clash with the enzyme, thus possibly explaining the
decrease in activity caused by this structural modification.
However, the decreased activity of compounds extended in
the phenyl para position is not accounted for properly by this
model. In order to fully conclude the binding mode of this
series of compounds, more experimental information needs to
be obtained. It is possible that a crystal structure of IRAP
would reveal differences in structure that are hard to predict
using the approach applied herein, and thus fully account for
the observed structure–activity relationships.
The docking produced several possible binding modes but
all with rather poor Glide docking scores. However, by visual
inspection, we identified a potential binding mode of the
series that to some extent accounts for the observed struc-
ture–activity relationships. Figure 1 shows this binding mode il-
lustrated using compound 3. In the proposed binding mode,
the negatively charged tetrazole of 3 is involved in zinc bind-
ing and, in addition, is stabilized in the catalytic site by a hydro-
gen bond to Tyr477 (IRAP: Tyr549). This Tyr residue is highly
conserved in the M1 family of metalloproteases and is indicat-
ed to be important for binding and stabilization of the catalyt-
ic transition state.[30] Furthermore, the compound is stacked
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rated aq NH4Cl (20 mL); the pH was adjusted to around 4 by addi-
tion of 1m aq HCl. The aqueous phase was extracted with CH2Cl2
(3ꢃ10 mL). The combined organic phase was dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue ob-
tained was purified by silica gel flash column chromatography
(CH2Cl2/MeOH, 98:2!90:10) to give the corresponding product.
Conclusions
New improved enhancers of cognitive functions are persistent-
ly desired for the treatment of the cognitive decline associated
with Alzheimer’s disease, brain trauma, and cerebral ischemia,
since the clinical studies of the cholinesterase inhibitors and N-
methyl-d-aspartate (NMDA) receptor antagonists used today
have been mostly disappointing.[34,35] It is therefore not surpris-
ing that the receptor(s) involved in the beneficial effects of
Ang IV has emerged as a relevant new target for drug inter-
vention. High-throughput screening of 10500 low-molecular-
weight compounds resulted in a series of hits, one of which
was sulfonamide 3. As deduced from an analysis of analogues
to 3, for example the corresponding carboxylic acid that was
assayed in the primary screen and found to be inactive, and
from a small structure–activity relationship study involving
compounds 3, 7–22, 25, and 27, it is concluded that the sulfo-
namide function and a tetrazole ring in the meta position of
the aromatic ring are crucial for IRAP inhibition. Further optimi-
zation, together with characterization of their mechanism of
action and modes of binding are required to make these com-
pounds attractive as research tools for in vivo studies and as
potential cognitive enhancers.
N-(3-(1H-Tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-sul-
1
fonamide (3): White solid (358 mg, 85%); mp: 211–2138C; H NMR
(400 MHz, [D6]DMSO): d=7.89 (t, J=1.9 Hz, 1H), 7.83–7.75 (m, 1H),
7.69 (s, 1H), 7.56 (t, J=8.0 Hz, 1H), 7.35 ppm (ddd, J=8.2, 2.2,
1.0 Hz, 1H); 13C NMR (101 MHz, [D6]DMSO): d=155.93, 138.47,
138.11, 134.13, 132.85, 131.11, 126.18, 123.89, 123.47, 119.31,
112.03 ppm; HRMS (ESI+): m/z [M+H]+ calcd for C11H8BrClN5O2S:
419.8991, found: 419.8981; IR (neat): n˜ =3247, 1624, 1583, 1572,
1479, 1403, 1372, 1336, 1314, 1158, 1040, 1022, 935 cmꢁ1
.
4-Bromo-5-chloro-N-(3-(1-methyl-1H-tetrazol-5-yl)phenyl)thio-
phene-2-sulfonamide (7): White solid (387 mg, 89%); mp: 89–
1
918C; H NMR (400 MHz, [D6]DMSO): d=11.08 (s, 1H), 7.73 (s, 1H),
7.64 (dt, J=6.7, 1.6 Hz, 2H), 7.61–7.55 (m, 1H), 7.43–7.39 (m, 1H),
4.13 ppm (s, 3H); 13C NMR (101 MHz, [D6]acetone): d=154.42,
139.22, 138.50, 134.76, 133.96, 131.31, 126.63, 126.51, 124.40,
122.20, 112.18, 35.64 ppm; HRMS (ESI+): m/z [M+H]+ calcd for
C12H10BrClN5O2S2: 433.9148, found: 433.9150; IR (neat): n˜ =3057,
1738, 1592, 1485, 1404, 1342, 1214, 1154, 1023, 941 cmꢁ1
.
3-(4-Bromo-5-chlorothiophene-2-sulfonamido)-N-methylbenza-
mide (8): White solid (344 mg, 84%); mp: 195–1978C; 1H NMR
(400 MHz, [D6]acetone): d=7.86 (td, J=1.8, 0.6 Hz, 1H), 7.81 (s,
1H), 7.67 (dt, J=7.3, 1.5 Hz, 1H), 7.48 (s, 1H), 7.48–7.39 (m, 2H),
2.90 ppm (d, J=4.6 Hz, 3H); 13C NMR (101 MHz, [D6]acetone): d=
167.13, 139.58, 138.20, 137.20, 134.41, 133.59, 130.28, 124.64,
124.48, 121.59, 112.00, 26.81 ppm; HRMS (ESI+): m/z [M+H]+
calcd for C12H9BrClN2O2S2: 408.9083, found: 408.9093; IR (neat): n˜ =
3424, 2929, 1739, 1641, 1550, 1472, 1401, 1336, 1220, 1147,
Experimental Section
Chemistry
General information and materials: All chemicals were purchased
from Sigma–Aldrich/Apollo scientific/Acros and used as received.
Purification was performed on column chromatography using silica
gel (60–120 mesh size) and then preparative thin-layer chromatog-
raphy (TLC) or preparative reversed-phase high-performance liquid
chromatography (RP-HPLC) (UV-triggered (254 nm) fraction collec-
tion with a Dionex UltiMate 3000 HPLC system, using an Agilent
PrepHT Zorbax SB-C8 column (21.2ꢃ150 mm, 5 mm particle size) or
a Macherey–Nagel Nucleodur C18 column (21ꢃ125 mm, 5 mm par-
ticle size), and H2O/CH3CN/0.05% HCOOH as eluents in a gradient
(30–90%, 10 mLminꢁ1 over 10 min). Analytical HPLC-mass spec-
trometry (HPLC-MS) was performed on a Dionex UltiMate 3000
HPLC system with a Bruker amaZon SL ion trap mass spectrometer
and detection by UV (diode array detector) and MS (electrospray
ionization, ESIꢁ or ESI+), using a Phenomenex Kinetex C18
column (50ꢃ3.0 mm, 2.6 mm particle size, 100 ꢂ pore size) and
a flow rate of 1.5 mLminꢁ1. A gradient of H2O/CH3CN/0.05%
HCOOH was used. Melting points were determined on an electro-
thermal melting point apparatus and are uncorrected. Infrared (IR)
spectra were obtained on a Varian 1000 FT-IR spectrometer; absor-
1009 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-4,5-dichlorothiophene-2-sulfona-
mide (9): White solid (312 mg, 89%); mp: 212–2148C; 1H NMR
(400 MHz, [D6]acetone): d=8.10 (ddd, J=2.3, 1.6, 0.5 Hz, 1H), 7.95
(ddd, J=7.7, 1.6, 1.1 Hz, 1H), 7.58 (t, J=7.8 Hz, 1H), 7.55 (s, 1H),
7.50 ppm (ddd, J=8.1, 2.3, 1.1 Hz, 1H); 13C NMR (101 MHz,
[D6]acetone): d=157.54, 138.67, 138.04, 132.25, 131.80, 131.44,
127.40, 125.40, 124.89, 124.51, 120.55 ppm; HRMS (ESI+): m/z [M+
H]+ calcd for C11H10Cl2N5O2S2: 375.9496, found: 375.9498; IR (neat):
n˜ =3237, 1740, 1573, 1478, 1402, 1324, 1230, 1151, 1024, 933,
882 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-3,4-dichlorobenzenesulfonamide
(10): White solid (300 mg, 81%); mp: 184–1868C; 1H NMR
(400 MHz, [D6]acetone): d=9.49 (s, 1H), 8.05 (ddd, J=2.2, 1.7,
0.5 Hz, 1H), 7.98 (dd, J=2.0, 0.5 Hz, 1H), 7.89 (ddd, J=7.7, 1.6,
1.1 Hz, 1H), 7.82–7.72 (m, 2H), 7.52 (td, J=7.9, 0.5 Hz, 1H),
7.43 ppm (ddd, J=8.2, 2.3, 1.1 Hz, 1H); 13C NMR (101 MHz,
[D6]acetone): d=157.48, 140.64, 139.09, 137.85, 133.77, 132.43,
131.36, 129.82, 127.82, 127.27, 124.44, 124.23, 120.22 ppm; HRMS
(ESI+): m/z [M+H]+ calcd for C13H10Cl2N5O2S: 369.9932, found:
369.9930; IR (neat): n˜ =3178, 1740, 1564, 1484, 1373, 1329, 1156,
bances are reported in cmꢁ1 1H and 13C NMR spectra were ob-
.
tained on a Varian Mercury spectrometer (1H: 400 MHz, 13C:
101 MHz) using CDCl3, CD3OD, [D6]acetone, or [D6]DMSO as the sol-
vent. High-resolution mass spectra (HRMS) were recorded on a Mi-
cromass Q-Tof2 mass spectrometer equipped with an electrospray
ion source. Compounds 11, 12, 14, 16, and 18 are previously re-
ported.[27]
1093, 1031, 949 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-2,4,5-trichlorobenzenesulfona-
mide (11): White solid (312 mg, 77%); mp: 250–2548C; 1H NMR
(400 MHz, CD3OD): d=10.08 (br s, 1H), 8.33 (m (s), 1H), 8.16 (m,
1H), 8.0 (m (s), 1H), 7.94 (m, 1H), 7.64–7.56 ppm (m, 2H); 13C NMR
(101 MHz, acetone): d=159.19, 137.79, 137.66, 136.87, 133.38,
General procedure for the synthesis of 3, 7–22: To a solution of
appropriate amine (4 or 5) (1.0 mmol) in anhydrous CH2Cl2 (5 mL),
under an atmosphere of nitrogen, was added appropriate sulfonyl
chloride (1.2 mmol) and pyridine (3.0 mmol). The resulting reaction
mixture was stirred at RT overnight and then quenched with satu-
ꢁ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemistryOpen 2014, 3, 256 – 263 260

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132.93, 131.45, 130.75, 130.61, 123.51, 122.80, 118.84, 118.78 ppm;
HRMS (ESI+): m/z [M+MeCN+H]+ calcd for C15H12Cl3N6O2S:
444.9808, found: 444.9832; IR (neat): n˜ =3237, 3093, 2360, 1891,
MeCN+H]+ calcd for C17H15N6O2S2: 399.0698, found: 399.0710; IR
(neat): n˜ =3422, 3096, 2814, 1569, 1481, 1150, 935, 744, 587 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)benzo[b]thiophene-2-sulfonamide
1566, 1173, 930, 687 cmꢁ1
.
(18): White solid (243 mg, 68%); mp: 205–2088C; 1H NMR
(400 MHz, CD3OD): d=7.93 (ddd, J=2.2, 1.7, 0.5 Hz, 1H), 7.89–7.83
(m, 3H), 7.72 (ddd, J=7.7, 1.7, 1.1 Hz, 1H), 7.50–7.35 ppm (m, 3H);
13C NMR (101 MHz, CD3OD): d=156.26, 141.61, 140.09, 138.50,
137.48, 129.98, 129.63, 127.08, 125.49, 125.35, 125.17, 123.21,
123.07, 122.26, 119.17 ppm; HRMS (ESI+): m/z [M+MeCN+H]+
calcd for C17H15N6O2S2: 399.0698, found: 399.0698; IR (neat): n˜ =
3602, 3520, 3249, 2912, 2762, 2642, 2428, 1592, 1562, 1484, 1421,
N-(3-(1H-Tetrazol-5-yl)phenyl)-4-bromo-2-fluorobenzenesulfona-
mide (12): White solid (283 mg, 71%); mp: 196–1988C; 1H NMR
(400 MHz, [D6]acetone): d=8.07 (ddd, J=2.2, 1.6, 0.6 Hz, 1H), 7.90–
7.79 (m, 2H), 7.63 (dd, J=9.7, 1.8 Hz, 1H), 7.57 (ddd, J=8.4, 1.9,
0.8 Hz, 1H), 7.50 (ddd, J=8.1, 7.4, 0.6 Hz, 1H), 7.45 ppm (ddd, J=
8.2, 2.2, 1.3 Hz, 1H); 13C NMR (101 MHz, [D6]acetone): d=160.63,
158.05, 138.89, 132.94, 131.27, 129.13, 129.09, 127.30, 124.23,
123.64, 121.74, 121.50, 119.67 ppm; HRMS (ESI+): m/z [M+H]+
calcd for C13H10BrFN5O2S: 397.9723, found: 397.9725; IR (neat): n˜ =
3235, 1705, 1563, 1468, 1409, 1368, 1331, 1268, 1231, 1170, 1059,
1358, 1152, 873 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-1-methyl-1H-indole-6-sulfonamide
(19): White solid (269 mg, 76%); mp: 235–2408C; 1H NMR
(400 MHz, CD3OD): d=8.21 (m, 1H), 7.95 (m, 1H), 7.74–7.70 (m,
2H), 7.52 (m, 1H), 7.46 (m, 1H), 7.39 (ddd, J=8.2, 2.2, 1.1 Hz, 1H),
7.36 (d, J=3.2 Hz, 1H), 6.63 (dd, J=3.2, 0.9 Hz, 1H), 3.87 ppm (s,
3H); 13C NMR (101 MHz, CD3OD): d=156.22, 139.52, 138.57, 131.48,
129.87, 129.59, 127.81, 125.15, 122.86, 122.30, 121.08, 119.44,
118.74, 109.49, 102.02, 31.79 ppm; HRMS (ESI+): m/z [M+H]+
calcd for C16H15N6O2S: 355.0977, found: 355.0981; IR (neat): n˜ =
915 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-4-bromo-2,6-difluorobenzenesul-
fonamide (13): White solid (325 mg, 78%); mp: 210–2138C;
1H NMR (400 MHz, CD3OD): d=7.89 (td, J=1.7, 0.8 Hz, 1H), 7.71
(ddd, J=7.7, 1.7, 1.1 Hz, 1H), 7.46 (ddd, J=8.2, 7.6, 0.5 Hz, 1H),
7.40–7.34 ppm (m, 3H); 13C NMR (101 MHz, CD3OD): d=159.40 (dd,
3
3
1JCꢁF =261.6, JCꢁF =5.0 Hz), 156.27, 137.90, 130.15, 127.83 (t, JCꢁF
=
12.8 Hz), 125.63, 123.11, 122.41, 118.48, 116.82 (dd, 2JCꢁF =26.9,
4JCꢁF =3.9 Hz), 116.34 ppm (t, 2JCꢁF =16.1 Hz); HRMS (ESI+): m/z
[M+MeCN+H]+ calcd for C15H12BrF2N6O2S: 456.9894, found:
456.9904; IR (neat): n˜ =3251, 3087, 2993, 2910, 2844, 2613, 2552,
3245, 3014, 2360, 1575, 1480, 1151, 1049, 934 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)thiophene-2-sulfonamide
(20):
White solid (243 mg, 79%); mp: 175–1808C; 1H NMR (400m,
CD3OD): d=7.99 (m, 1H), 7.87–7.78 (m, 2H), 7.67 (dd, J=3.8,
1.4 Hz, 1H), 7.57 (m, 1H), 7.45 (ddd, J=8.1, 2.2, 1.1 Hz, 1H),
7.15 ppm (dd, J=5.0, 3.8 Hz, 1H); 13C NMR (101 MHz, CD3OD): d=
156.23, 139.99, 138.83, 132.53, 132.51, 130.06, 127.08, 125.38,
123.38, 123.02, 119.20 ppm; HRMS (ESI+): m/z [M+MeCN+H]+
calcd for C13H13N6O2S2: 349.0541, found: 349.0538; IR (neat): n˜ =
2481, 1877, 1595, 1558, 1471, 1420, 1174, 1031, 918, 854 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-3,4-dimethylbenzenesulfonamide
(14): White solid (273 mg, 83%); mp: 221–2238C; 1H NMR
(400 MHz, CD3OD): d=7.94 (m, 1H), 7.77 (m, 1H), 7.63 (m, 1H),
7.52 (m, 1H), 7.39 (m, 1H), 7.32 (m, 1H), 2.35 ppm (br s, 6H);
13C NMR (101 MHz, CD3OD): d=156.23, 142.61, 139.20, 137.81,
136.75, 129.99, 129.79, 127.62, 125.24, 124.55, 122.94, 122.51,
118.76, 18.52, 18.44 ppm; HRMS (ESI+): m/z [M+MeCN+H]+ calcd
for C17H19N6O2S: 371.1290, found: 371.1290; IR (neat): n˜ =3246,
3237, 2360, 1574, 1480, 1326, 1156, 1039, 931, 717 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)benzenesulfonamide (21): White
solid (214 mg, 71%); mp: 179–1838C; 1H NMR (400 MHz, CD3OD):
d=7.99–7.88 (m, 3H), 7.78 (ddd, J=7.7, 1.7, 1.0 Hz, 1H), 7.65 (m,
1H), 7.61–7.54 (m, 2H), 7.51 (ddd, J=8.2, 7.7, 0.5 Hz, 1H), 7.39 ppm
(ddd, J=8.2, 2.2, 1.0 Hz, 1H); 13C NMR (101 MHz, CD3OD): d=
156.24, 139.55, 139.00, 132.79, 130.04, 128.86, 126.88, 125.31,
123.15, 122.72, 118.97 ppm; HRMS (ESI+): m/z [M+MeCN+H]+
calcd for C15H15N6O2S 343.0977, found: 343.0974; IR (neat): n˜ =
3239, 2898, 2360, 1576, 1481, 1233, 1161, 1089, 1040, 932,
2360, 1576, 1481, 1326, 1151, 932 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-[1,1’-biphenyl]-4-sulfonamide (15):
White solid (306 mg, 81%); mp: 206–2108C; 1H NMR (400 MHz,
CD3OD): d=8.03–7.99 (m, 3H), 7.86–7.79 (m, 3H), 7.72–7.68 (m,
2H), 7.58–7.51 (m, 3H), 7.49–7.44 ppm (m, 2H); 13C NMR (101 MHz,
CD3OD): d=156.14, 145.81, 139.10, 139.06, 138.20, 130.11, 128.77,
128.23, 127.53, 127.30, 126.96, 125.47, 123.07, 122.75, 118.95 ppm;
HRMS (ESI+): m/z [M+MeCN+H]+ calcd for C21H19N6O2S:
419.1290, found: 419.1295; IR (neat): n˜ =3239, 2857, 2717, 2466,
683 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)pyridine-3-sulfonamide (22): White
solid (154 mg, 51%); mp: 219–2238C; 1H NMR (400 MHz, CD3OD):
d=8.92 (m, 1H), 8.70 (m, 1H), 8.19 (m, 1H), 7.86 (m, 1H), 7.74 (m,
1H), 7.54 (m,1H), 7.46 (ddd, J=8.2, 7.7, 0.5 Hz, 1H), 7.31 ppm (m,
1H); 13C NMR (101 MHz, CD3OD): d=156.31, 152.83, 147.12, 138.22,
136.46, 135.24, 130.16, 125.73, 124.11, 123.32, 123.19, 119.19 ppm;
HRMS (ESI+): m/z [M+H]+ calcd for C12H11N6O2S: 303.0664, found:
303.0670; IR (neat): n˜ =2896, 2746, 2360, 1581, 1470, 1358, 1166,
1588, 1480, 1400, 1368, 1325, 1157, 933, 680 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-7-chlorobenzo[c][1,2,5]oxadiazole-
4-sulfonamide (16): Yellow solid (287 mg, 76%); mp: 168–1708C;
1H NMR (400 MHz, [D6]acetone): d=8.20 (d, J=7.4 Hz, 1H), 8.05
(td, J=1.8, 0.8 Hz, 1H), 7.89–7.77 (m, 2H), 7.53–7.37 ppm (m, 2H);
13C NMR (101 MHz, [D6]acetone): d=157.46, 149.94, 146.03, 138.54,
136.80, 131.20, 131.06, 128.11, 127.87, 127.15, 124.32, 123.93,
119.86 ppm; HRMS (ESI+): m/z [M+H]+ calcd for C13H9ClN7O3S:
378.0176, found: 378.0174; IR (neat): n˜ =3289, 1692, 1597, 1493,
1112 cmꢁ1
.
4-Bromo-5-chloro-N-(3-cyanophenyl)-N-methylthiophene-2-sulfo-
namide (24): To a solution of 3-aminobenzonitrile (23) (1.0 mmol)
in anhydrous CH2Cl2 (5 mL), under an atmosphere of nitrogen, was
added 4-bromo-5-chlorothiophene-2-sulfonyl chloride (1.2 mmol)
and pyridine (3.0 mmol). The resulting reaction mixture was stirred
at RT overnight and then quenched with saturated aq NH4Cl
(20 mL). The aqueous phase was extracted with CH2Cl2 (3ꢃ10 mL).
The combined organic phase was dried over Na2SO4, filtered and
evaporated under reduced pressure. The crude product (91%
purity determined by LCMS) obtained was dissolved in dry tetrahy-
1427, 1346, 1275, 1228, 1153, 1040, 939 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)benzo[c]thiophene-1-sulfonamide
(17): White solid (261 mg, 73%); mp: 238–2408C; 1H NMR
(400 MHz, [D6]acetone): d=8.56 (s, 1H), 8.31 (ddd, J=8.1, 1.4,
0.7 Hz, 1H), 8.06–7.99 (m, 2H), 7.78 (dt, J=7.4, 1.5 Hz, 1H), 7.60–
7.33 ppm (m, 4H); 13C NMR (101 MHz, acetone): d=156.41, 140.34,
138.62, 136.37, 133.49, 132.87, 130.21, 126.09, 125.73, 125.58,
123.16, 123.00, 122.74, 122.24, 118.27 ppm; HRMS (ESI+): m/z [M+
ꢁ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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drofuran (THF) and cooled to 08C. NaH (60% dispersion in mineral
oil) (1.2 mmol) was added under nitrogen atmosphere. The solu-
tion was stirred for 30 min. CH3I (1.5 mmol) was then added drop-
wise, and the solution was stirred at 258C for 3 h. The reaction
mixture was quenched with saturated aq NH4Cl (20 mL), and the
aqueous phase was extracted with CH2Cl2 (3ꢃ10 mL). The com-
bined organic phase was dried over Na2SO4, filtered and evaporat-
ed under reduced pressure to afford crude product, which was pu-
rified by silica gel flash column chromatography (isohexane/EtOAc,
90:10!80:20) to give product 24 as a white solid (306 mg, 78%);
activity. These cells are known to abundantly express IRAP and
contain minimal amounts of contaminating APN activities.[36] The
CHO cell membrane preparation was conducted based on the pro-
cedure described by Demaegt et al.[37] with lysis of washed cells
being done by means of ultrasonication followed by multiple
strokes (>20) with a Dounze homogenizer and a centrifugation
(30000ꢃg for 30 min at 48C) procedure for pelleting and washing
of the membranes.
The extent of enzymatic activity in each sample was quantified in
a microtiter-based screening assay using the peptide-like substrate
l-leucine-p-nitroanilide (l-Leu-pNA; Sigma–Aldrich, product no.
L9125), which upon IRAP-mediated cleavage produces para-nitroa-
niline that absorbs at 405 nm.[37] The screening campaign was per-
formed at Chemical Biology Consortium Sweden (CBCS) using a pri-
mary screening set of 10500 compounds[38] (majority of com-
pounds donated by Biovitrum AB, Stockholm, Sweden). The screen
was conducted in a 384-well format, whereas follow-up dose–re-
sponse experiments were conducted in a transparent 96-well plate
(Nunc, product no. 269620).
1
mp: 138–1408C; H NMR (400 MHz, [D6]DMSO): d=7.84 (dd, J=6.0,
1.9 Hz, 2H), 7.71 (d, J=0.8 Hz, 1H), 7.69–7.58 (m, 2H), 3.26 ppm (s,
3H); 13C NMR (101 MHz, [D6]DMSO): d=141.48, 135.29, 133.89,
133.59, 132.38, 132.12, 130.93, 130.50, 118.40, 112.84, 112.53,
38.19 ppm; HRMS (ESI+): m/z [M+H]+ calcd for C12H9BrClN2O2S2:
390.8977, found: 390.8976; IR (neat): n˜ =3088, 3070, 2223, 1603,
1580, 1482, 1455, 1436, 1401, 1355, 1311, 1270, 1196, 1158, 1140,
1070, 1018, 933 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-4-bromo-5-chloro-N-methylthio-
phene-2-sulfonamide (25): The mixture of nitrile 24 (1.0 mmol),
NaN3 (2.0 mmol), and Et3N·HCl (2.0 mmol) in dry toluene (5 mL)
was heated at reflux for 12 h with constant stirring. After cooling,
the reaction mixture was acidified (pH 2) using 2n aq HCl and ex-
tracted with CH2Cl2 (3ꢃ20 mL). The combined organic phase was
dried over Na2SO4, filtered and evaporated under reduced pressure
to afford the crude product, which was purified by silica gel flash
column chromatography (CH2Cl2/MeOH, 95:05!90:10) to give
product 25 as a white solid (309 mg, 71%); mp: 152–1548C;
1H NMR (400 MHz, [D6]acetone): d=8.14 (ddd, J=7.8, 1.7, 1.1 Hz,
1H), 8.06 (ddd, J=2.2, 1.7, 0.5 Hz, 1H), 7.65 (ddd, J=8.2, 7.8,
0.5 Hz, 1H), 7.53 (ddd, J=8.1, 2.3, 1.1 Hz, 1H), 7.49 (s, 1H),
3.42 ppm (s, 3H); 13C NMR (101 MHz, [D6]acetone): d=156.40,
141.79, 134.76, 134.29, 133.34, 130.22, 129.01, 126.37, 126.20,
125.32, 111.72, 37.79 ppm; HRMS (ESI+): m/z [M+H]+ calcd for
C12H10BrClN5O2S2: 433.9148, found: 433.9145; IR (neat): n˜ =1712,
In this format, the final assay volume was 200 mL in an assay buffer
consisting of 50 mm Tris-HCl, 150 mm NaCl, and 0.1 mm phenylme-
thanesulfonylfluoride (PMSF) at pH 7.4. The assays were conducted
in the presence of a final concentration of 1 mm l-Leu-pNA and
homogenized CHO cell membranes from 50000–200000 cells per
well, depending on test occasion and membrane batch. The proto-
col for running the dose–response assay started with a serial dilu-
tion of the compound stock solutions at 10 mm in 99.9 % anhy-
drous DMSO (Sigma–Aldrich) by a factor of 1/3 in columns 1–11 of
the 96-well plates. Column 12 was reserved for controls: the equiv-
alent amount of DMSO was added to wells A12–D12 (negative
controls representing 0% inhibition of the enzyme), whereas
a 10 mm stock solution of 3 was placed in wells E12–H12 serving
as controls representing 100% inhibition of the enzyme activity.
The DMSO solutions were then diluted in assay buffer and trans-
ferred to the transparent assay plates in triplicate (50 mL to each
well both for samples and controls), followed by addition of dilut-
ed homogenized CHO cell membranes (50 mL) and substrate
(100 mL) to initiate the enzymatic reaction. The plates were covered
and then incubated at RT until a statistically significant difference
was observed (6–12 h) between the controls (Z’ >0.8). Care was
taken to ensure the readings were taken at a time point when the
absorbance signal still increased linearly with time. Raw data were
then imported into Microsoft Excel for conversion to % inhibition
data based on the controls on each plate. After data from triplicate
samples were averaged, the curves were fitted to a four-parameter
dose–response model within XLfit (model 205) to obtain best-fit
values for the IC50, Hill slope, and the upper and lower limits of the
dose–response curve.
1558, 1454, 1398, 1354, 1154, 1067, 1018, 909 cmꢁ1
.
N-(3-(1H-Tetrazol-5-yl)phenyl)-4-bromo-5-chlorothiophene-2-car-
boxamide (27): 4-Bromo-5-chlorothiophene-2-carboxylic acid (26)
(1.0 mmol) was dissolved in dry DMF, and PyBOP (1.5 mmol) was
added. The solution was stirred at 258C for 30 min. 3-(1H-Tetrazol-
5-yl)aniline (4) (2.0 mmol) and Et3N (2.0 mmol) were then added,
and the stirring continued for an additional 18 h. The reaction mix-
ture was then diluted with water (10 mL) and extracted with EtOAc
(3ꢃ10 mL). The combined organic phase was dried over Na2SO4,
filtered and evaporated under reduced pressure to afford the
crude product, which was purified by silica gel flash column chro-
matography (CH2Cl2/MeOH, 97:03!90:10) to give product 27 as
a white solid (312 mg, 81%); mp: 263–2658C; 1H NMR (400 MHz,
[D6]DMSO): d=10.61 (s, 1H), 8.49 (t, J=1.9 Hz, 1H), 8.15 (d, J=
0.6 Hz, 1H), 7.91 (ddd, J=8.2, 2.2, 1.0 Hz, 1H), 7.77 (ddd, J=7.7,
1.7, 1.0 Hz, 1H), 7.60 ppm (t, J=8.0 Hz, 1H); 13C NMR (101 MHz,
[D6]DMSO): d=158.12, 155.68, 139.09, 138.06, 131.52, 131.05,
130.02, 124.94, 122.55, 122.49, 118.47, 110.96 ppm; HRMS (ESI+):
m/z [M+H]+ calcd for C12H8BrClN5OS: 383.9321, found: 383.9319;
IR (neat): n˜ =3284, 2883, 1739, 1631, 1591, 1539, 1407, 1303, 1178,
Computational methods
Docking was conducted using Glide (version 5.8, Schrçdinger, LLC,
New York, NY, 2012) SP mode.[39–41] The crystal structure 4FYT was
downloaded from the Protein Data Bank (PDB)[42] and prepared
using the Protein Preparation Wizard (Schrçdinger Suite 2012 Pro-
tein Preparation Wizard; Epik version 2.2, Schrçdinger, LLC, New
York, NY, 2012; Impact version 5.7, Schrçdinger, LLC, New York, NY,
2012; Prime version 2.3, Schrçdinger, LLC, New York, NY, 2012) in
Maestro (version 9.3, Schrçdinger, LLC, New York, NY, 2012). All
water molecules were deleted, hydrogen bond assignments were
optimized, and a minimization of the hydrogens was carried out.
The structures were prepared for docking using LigPrep (version
1079, 1028 cmꢁ1
.
Biology
The enzymatic assay applied for screening purposes as well as
follow-up dose–response characterization was based on the use of
membrane preparations from CHO cells as a source of enzymatic
ꢁ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemistryOpen 2014, 3, 256 – 263 262

www.chemistryopen.org
[17] H. Andersson, M. Hallberg, Int. J. Hypertens. 2012, 2012, 1.
[18] P. R. Gard, BMC Neurosci. 2008, 9, S15.
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tonation states.
[19] B. Stragier, D. De Bundel, S. Sarre, I. Smolders, G. Vauquelin, A. Dupont,
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Acknowledgements
[21] P. M. Vanderheyden, Mol. Cell. Endocrinol. 2009, 302, 159–66.
[22] H. Andersson, H. Demaegdt, A. Johnsson, G. Vauquelin, G. Lindeberg,
M. Hallberg, M. Erdelyi, A. Karlen, A. Hallberg, J. Med. Chem. 2011, 54,
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[23] H. Andersson, H. Demaegdt, G. Vauquelin, G. Lindeberg, A. Karlen, M.
Hallberg, Bioorg. Med. Chem. 2008, 16, 6924–6935.
[24] H. Andersson, H. Demaegdt, G. Vauquelin, G. Lindeberg, A. Karlen, M.
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[26] A. L. Albiston, C. J. Morton, H. L. Ng, V. Pham, H. R. Yeatman, S. Ye, R. N.
Fernando, D. De Bundel, D. B. Ascher, F. A. Mendelsohn, M. W. Parker,
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Funding for the work carried out at Chemical Biology Consortium
Sweden (CBCS) was provided by the Swedish Research Council
and Karolinska Institutet, Sweden (A.J.J., H.A., K.S,. and T.L). The
Karolinska High Throughput Center (KHTC) led by Prof. Jussi Tai-
pale is acknowledged for kindly providing access to an automat-
ed screening line. Grants from the Swedish Research Council and
the Kjell and Mꢀrta Beijer Foundation to the Department of Phar-
maceutical Biosciences and to the Department of Medicinal
Chemistry at Uppsala University are acknowledged.
Keywords: aminopeptidases
regulated aminopeptidase
relationships · cognitive enhancers
·
arylsulfonamides
· insulin-
[27] S. Bystrçm, K. Fꢀrnegꢅrdh, C. Hedgecock, E. Homan, M. Jçnsson, T.
Lundbꢀck, J. Martinsson, M. Sari, (KANCERA AB, Solna, Sweden), Int. PCT
Pub. No. WO/2012/035171 A3, 2012.
(IRAP) structure–activity
·
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Received: August 12, 2014
Published online on November 21, 2014
ꢁ 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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