Selective angiotensin II AT2 receptor agonists: Benzamide structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2008.05.066

In the investigation of the structure-activity relationship of nonpeptide AT₂ receptor agonists, a series of substituted benzamide analogues of the selective nonpeptide AT₂ receptor agonist M024 have been synthesised. In a second ser

Full text of DOI:10.1016/j.bmc.2008.05.066

Bioorganic & Medicinal Chemistry 16 (2008) 6841–6849
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Selective angiotensin II AT2 receptor agonists:
Benzamide structure–activity relationships
Charlotta Wallinder a, Milad Botros c, Ulrika Rosenström a, Marie-Odile Guimond b, Hélène Beaudry b,
Fred Nyberg c, Nicole Gallo-Payet b, Anders Hallberg a, Mathias Alterman a,
*
a Department of Medicinal Chemistry, BMC, Uppsala University, PO Box 574, SE-751 23 Uppsala, Sweden
b Service of Endocrinology and Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Que., Canada J1H 5N4
c Department of Biological Research on Drug Dependence, BMC, Uppsala University, PO Box 591, SE-751 23 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
In the investigation of the structure–activity relationship of nonpeptide AT2 receptor agonists, a series of
substituted benzamide analogues of the selective nonpeptide AT2 receptor agonist M024 have been syn-
thesised. In a second series, the biphenyl scaffold was compared to the thienylphenyl scaffold and the
impact of the isobutyl substituent and its position on AT1/AT2 receptor selectivity was also investigated.
Both series included several compounds with high affinity and selectivity for the AT2 receptor. Three of
the compounds were also proven to function as agonists at the AT2 receptor, as deduced from a neurite
outgrowth assay, conducted in NG108-15 cells.
Received 23 January 2008
Revised 23 May 2008
Accepted 28 May 2008
Available online 1 July 2008
Keywords:
Angiotensin
AT2
Ó 2008 Elsevier Ltd. All rights reserved.
Agonist
1. Introduction
tain pathological conditions such as myocardial infarction, vascu-
lar injury, brain ischemia, and renal failure as well as in
Angiotensin II (Ang II) is the major component of the rennin–
angiotensin system (RAS), which plays an important role in the
regulation of blood pressure and fluid and electrolyte homeosta-
sis. This octapeptide mediates its effects through stimulation of
two receptor subtypes, the AT1 receptor and the AT2 receptor.
Both receptors are G-protein coupled receptors, but exhibit only
32–34% homology in their amino acid sequences.1,2 The AT1
receptor is associated primarily with classical Ang II effects, such
as vasoconstriction, aldosterone and vasopressin release, reten-
tion of sodium and water, and cellular growth and proliferation.
Activation of the AT2 receptor on the other hand frequently ren-
ders opposing effects and stimulation will, for example, induce
vasodilatation, antiproliferation and apoptosis.1–3 Furthermore,
stimulation of neuronal cell AT2 receptors leads to neurite out-
growth, which is characteristic for neuronal differentiation.1,4,5
Another feature of the AT2 receptor, which is connected to its
involvement in cell differentiation, is the suggested importance
in fetal development. The AT2 receptor is widely expressed in
the fetal tissues, but its expression drops dramatically after birth.6
Notably in the adults the AT2 receptor is up-regulated during cer-
cutaneous wounds.2,7 The RAS is an established target in the
treatment of hypertension and it was recently postulated that
some of the positive effects of AT1 receptor antagonists could
be ascribed to a stimulation of the AT2 receptor.8,9 We have
previously reported the first selective nonpeptide AT2 receptor
agonist, M024,10 and we are now further exploring the struc-
ture–activity relationship for selective nonpeptide AT2 receptor
agonists. M024 was derived from the nonselective agonist
L-162,313 by stepwise simplification of the nitrogen containing
heterocyclic ring system (Fig. 1).10,11
The N-benzylimidazole fragment in M024 was initially believed
to be a strong determinant for AT2 receptor selectivity. Recently,
however, we disclosed two series of compounds, including highly
selective AT2 receptor agonists, where the imidazole has been re-
placed by amides. We investigated analogues with both the nitro-
gen in the same position as the alkylated nitrogen in the imidazole
ring (48, Fig. 2), as well as inverted amide analogues (49).12 In both
series amides with small substituents showed the best affinity to
the AT2 receptor.
Herein, we report a series of benzamides (series A, Fig. 2) with
good AT2 receptor affinity and selectivity including ligands proven
to serve as agonists. Furthermore, we have compared the biphenyl
and thienylphenyl scaffolds and examined the impact of the isobutyl
substituent and its position on the AT1/AT2 receptor selectivity (ser-
ies B, Fig. 2).
* Corresponding author. Tel.: +46 18 4714124; fax: +46 18 4714474.
E-mail address: mathias.alterman@orgfarm.uu.se (M. Alterman).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.066

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C. Wallinder et al. / Bioorg. Med. Chem. 16 (2008) 6841–6849
16 and 17 were then alkylated under Negishi coupling conditions
with freshly prepared isobutylzinc bromide or benzylzinc chloride,
LiBr and with (1,3-diisopropylimidazol-2-ylidene)(3-chloropyr-
idyl)palladium(II) dichloride (PEPPSI)16 as the catalyst to yield
the desired compounds 18–21 in good to excellent yields (77–
95%). Benzenesulfonamide, 4-phenylbenzenesulfonamide, and
phenoxybenzenesulfonamide (22–24) were synthesized from their
corresponding benzenesulfonyl chlorides and isolated in good to
excellent yields (73–99%).
The benzenesulfonamides (compounds 18–24) were trans-
formed into the corresponding boronic acids by selective ortho
lithiation and subsequent boronation. In the boronation of com-
pounds 19 and 21 (see Scheme 3) only one product was formed
(compounds 27 and 29, respectively), the formation of the di-
ortho-substituted boronic acids was not observed, presumably
due to steric hindrance. Compound 25 was prepared from para-
iodobenzoic acid, through formation of the corresponding acyl
chloride by reaction with oxalyl chloride, and subsequent amida-
tion with diethylamine, which after isolation yielded the desired
compound in 83% yield. The boronic acids (26–32) were coupled
with compound 25 in a Suzuki coupling reaction with Pd(PPh3)4
as the catalyst and Na2CO3 as base under microwave irradiation
(110 °C, 5 min) to form compounds 33–39 in moderate to good
yields (46–92%). Compounds 40–46 were formed by deprotection
of the sulfonamide with borontrichloride and subsequent acylation
with n-butyl chloroformate in dichloromethane and TEA to yield
the final products (40–46) in 28–88% yield after purification by col-
umn chromatography or preparative LC–MS (Scheme 3).
Figure 1.
2. Results
2.1. Chemistry
2.1.1. Binding assays
Compounds 4–15, 40–46 were evaluated in radioligand-binding
assays by displacement of [125I]Ang II from AT1 receptors in rat li-
ver membranes and from AT2 receptors in pig uterus membranes
as described previously.17,18 The natural substrate Ang II and the
selective AT1 receptor antagonist losartan were used as reference
substances.19 The affinity results are presented in Tables 1 and 2.
In series A all compounds exhibited moderate to high affinity for
the AT2 receptor and none of the compounds possessed any affinity
for the AT1 receptor (Table 1). Compound 4 with a primary amide
moiety had a much lower affinity compared to the secondary and
tertiary amides. Introducing methyl groups to the amide (5,
36.9 nM) increased the affinity 2.5 times. The affinity was further
improved 10 times when ethyl groups were applied (6, 3.0 nM).
The isopropyl amide 7 showed a better affinity than the cyclopro-
pyl amide 8. The cyclopropyl amide seems to be equipotent to
the thiazolyl amide, compound 12, while the cyclohexyl amide, 9,
resulted in two times higher affinity. Among the secondary amides
the benzyl substituent, compound 10, exhibited the highest affin-
ity, 1.0 nM. When exchanging the amide hydrogen in compound
10 for a methyl group, rendering compound 11, the affinity was re-
duced 30 times. The cyclic substituents in compounds 13 and 14
resulted in good affinity, very similar to the open N,N-diethylamide,
but changing the piperidino moiety of compound 14 to the mor-
pholino of compound 15 dramatically lowered the affinity.
The synthesis of series A, outlined in Scheme 1, started with the
thiopheneboronic acid 1 which was prepared essentially as de-
scribed by Kevin et al.13,14 The thiopheneboronic acid 1 was cou-
pled with 4-iodobenzoic acid in a Suzuki reaction with Pd(PPh3)4
as catalyst and Na2CO3 as base under microwave irradiation
(110 °C, 5 min), rendering compound 2 in 65% yield. The carboxylic
acid 2 was transformed into the corresponding acyl chloride 3 by
treatment with oxalyl chloride and a catalytic amount of DMF.
The acyl chloride 3 served as a good starting material for prepara-
tion of the first series of compounds (4–15) in a parallel manner.
Compound 3 was dissolved in dry DCM and added to sample vials
loaded with a diverse set of amines. The resulting amides were
then treated with borontrichloride or TFA to remove the tert-butyl
protecting group.15 Finally, the primary sulfonamides were acety-
lated with n-butyl chloroformate in DCM and TEA to yield the final
compounds 4–15, in 15–72% yield after purification by chromato-
tron or preparative LC–MS.
The series B compounds were prepared from a set of different
benzenesulfonamides (Schemes 2 and 3). The synthesis of 3-, 4-
isobutyl- and 3-, 4-benzylbenzenesulfonamide (18–21, Scheme 2)
started with the conversion of 3- and 4-bromobenzenesulfonyl
chloride to the corresponding N-tert-butylsulfonamides, com-
pounds 16 and 17, in excellent yields (95–96%). The compounds
Figure 2.

C. Wallinder et al. / Bioorg. Med. Chem. 16 (2008) 6841–6849
6843
Scheme 1. Reageants: (a) Pd(PPh3)4, Na2CO3, DME, EtOH, H2O; (b) oxalyl chloride, 1,2-dichloroethane, cat. DMF; (c) amine, DCM; (d) TFA or BCl3, DCM; (e) n-butyl
chloroformate, TEA, DCM.
ious concentrations ranging from 1 pM to 1 lM. For all the com-
pounds, it was only at the highest concentration that any
evidence of cell death was observed. Cells were then treated in
the absence or in the presence of Ang II (100 nM), compounds 6
(1 and 10 nM), 10 (1 and 10 nM), and 45 (100 nM) in the absence
or in the presence of PD-123,319 (10 lM), an AT2 receptor antago-
nist introduced daily 30 min prior to Ang II, compounds 6, 10, or
45. After 3 days of treatment, cells were examined under a phase
contrast microscope and micrographs were taken. As can be seen
in Figure 3 compounds 6, 10, and 45 all induced neurite outgrowth
in an extent comparable to the endogenous agonist Ang II, treat-
ment with the higher concentration of compounds 6 and 10
(10 nM) gave a slightly higher induction of neurite outgrowth (data
not shown). Coincubation with the selective AT2 receptor antago-
nist PD 123,319 (10 lM) reduced neurite outgrowth to control lev-
Scheme 2. Reageants: (a) tertbutylamine, DCM (b) isobutylmagnesium bromide or
els, verifying that the effect was mediated through the AT2
receptor. Treatment with PD 123,319 alone did not alter the mor-
phology compared to untreated cells (data not shown).
benzylmagnesium chloride, ZnBr2, LiBr, PEPPSI, THF, NMP.
Considering series B, the thienylphenyl scaffold is far superior
to the biphenyl scaffold (Table 2). Biphenyl compounds 40 and
41 are 50 times less potent than the corresponding thienylphenyl
compound 6. Compound 44, lacking substituent on the phenyl ring,
exhibited a Ki of only 402 nM, indicating the importance of the
lipophilic side chain for good binding. The isobutyl substituent re-
sults in better affinities in both positions 4 and 5 as compared to
the benzyl substituent (40, 41 vs 42, 43). With regards to the ben-
zyl substituted compounds (42 and 43), a more pronounced differ-
ence was observed as result of the position of the substituent.
Compound 42, with the benzyl in the 4-position, exhibited twice
as high affinity as compound 43, which contained the benzyl group
in the 5-position. Exchanging the benzyl substituent of 42 into a
phenoxy (46) increased the affinity to 91.7 nM and exchanging it
to a phenyl (45) rendered an affinity of 52.0 nM, which was the
most potent compound in series B.
3. Discussion
The benzamides presented herein exhibit generally a higher
affinity to the AT2 receptor as compared to the previously disclosed,
somewhat larger amides.12 This observation is consistent with the
trends seen in the previous series where smaller substituents im-
proved the AT2 receptor affinity.12 However, the primary benzamide
4, with a Ki value of 105.6 nM was found to be a relatively poor bin-
der while the corresponding primary phenylacetamide exhibited a
Ki of 51 nM.12 Conversion of the benzamide to the N,N-dimethyla-
mide 5 afforded an improved affinity (36.9 nM) but still lower than
the N,N-dimethylphenylacetamide (49, 7.0 nM), the later one being
the most potent in the phenylacetamide series.12 Extending the size
of the benzamide substituent further results in ligands more potent
than 49, for example, compound 6, 7, 10, and 14 exhibiting Ki values
ranging from 1.0 to 3.2 nM.
It has previously been shown that the biphenyl scaffold is
exchangeable to the thienylphenyl scaffold in this type of ligands,
see Figure 1.24,25 However, the N,N-diethylbenzamide thienylphe-
nyl containing compound, 6 (3.0 nM), was 50 times more potent
than the corresponding biphenyl compounds (40 and 41). The posi-
tion of the substituent had little effect on compound potency. Com-
pound 41, with the isobutyl in the 5-position, showed slightly
better affinity compared to compound 40. It appears as though
the thienylphenyl scaffold positions the isobutyl substituent in a
2.1.2. In vitro morphological effects induced by compounds 6,
10, and 45 in NG108-15 cells
We have shown previously that these cells express only the AT2
receptor and that a 3-day treatment with Ang II or the selective
peptidic AT2 receptor agonist CGP-42112 induces neurite out-
growth.20,4 The signaling pathways involve a sustained increase
in Rap1/B-Raf/p42/p44mapk activity and activation of the nitric
oxide/guanylyl cyclase/cGMP pathway.21–23 The cells were plated
as described in Section 5 and adequate test concentrations for
the three compounds 6, 10, and 45 were determine by testing var-

6844
C. Wallinder et al. / Bioorg. Med. Chem. 16 (2008) 6841–6849
Scheme 3. Reageants: (a) n-BuLi, THF, triisopropyl borate, HCl (aq); (b) Pd(PPh3)4, Na2CO3, DME, EtOH, H2O; (c) BCl3, DCM; (d) n-butyl chloroformate, TEA, DCM.
more favorable position than the 4- or the 5-position on the biphe-
nyl scaffold in the N,N-diethylamide series. Accordingly, we believe
that the N,N-diethylbenzamide and the N-benzylimidazole group,
as in M024, are probably binding to the receptor in slightly different
ways. It is, however, interesting to see that both position 4 and 5
render almost equipotent ligands and that the isobutyl substituent
is necessary for good AT2 affinity since compound 44 (402.2 nM),
which lacks the isobutyl substituent, possess significantly lower
affinity than compounds 6 (3.0 nM) and 40 (144.5 nM).
Both the benzyl substituted compounds, 42 and 43, had a lower
affinity toward the AT2 receptor as compared to the isobutyl
substituted analogues and a larger difference between the 4- and
5-positions was observed. Contrary to the previously reported m-
metoxybenzyl analogue of M024, which lacks affinity for the AT2
receptor,25 compound 42 exhibited affinity, although not in the
lower nanomolar range. Compound 46, with a phenoxy substituent
instead of the isobutyl, showed almost twice as high affinity as
compared to compound 42. Ethers in the isobutyl position have
previously been demonstrated to either increase or reduce the
affinity to the AT2 receptor depending on the molecular framework
studied.25 The big difference seen in this particular case is difficult
to explain, but might be accounted for by the higher hydrophilicity
or higher electron density.
The good affinity exhibited by the triphenyl, compound 45,
encouraged us to assess if the compound acts as an AT2 receptor
agonist or antagonist. Although, higher concentrations were
needed to induce the same agonism as with 6 and 10 this very rigid
compound may be a valuable tool in future studies of bioactive
conformations.
Neither of the compounds in series A or B possessed any affinity
toward the AT1 receptor. According to the literature any change in
the size of the substituent in the isobutyl position will diminish the
affinity for the AT2 receptor and enhance the affinity for the AT1
receptor.13,26 However, in series B, where these kinds of alterations
were performed (44, 402.2 nM, 42, 162.5 nM) we observed a de-
crease in AT2 affinity but no AT1 affinity was encountered. The sub-
stitution pattern in this part of the molecules does not seem to
influence the AT1/AT2 selectivity in these smaller compounds. So
far we have not seen any affinity for the AT1 receptor from com-
pounds lacking the bicyclic heteroaromatic substituents in the
benzylic position.10–12 Thus, the smaller heterocyclic and linear
substituents strongly discriminate between the two receptors,
favouring an interaction with the AT2 receptor.
4. Conclusion
In conclusion, we have demonstrated that substituted benzam-
ides function well in producing high affinity nonpeptide AT2 recep-
tor selective agonists, for example, 6 and 10. We have also shown
that the thienylphenyl scaffold is not generally exchangeable to the
biphenyl scaffold. Interestingly, substituents in both positions 4 or
5 of this scaffold result in active and selective AT2 ligands. Further-
more, the triphenyl analogue 45 possesses a higher affinity com-
pared to both the corresponding isobutyl and benzyl compounds
and it acts as an agonist at the AT2 receptor. This rigid compound
will serve as a very valuable tool in future modeling of the bioac-
tive conformation of selective nonpeptide AT2 receptor agonists.
Furthermore, these small ligands strongly discriminate between
the AT1 and the AT2 receptors, favouring an interaction with the
AT2 receptor.
5. Experimental
5.1. Chemistry: general considerations
1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded
on a Varian Mercury 400 at ambient temperature. Chemical shifts
are given as d values (ppm) downfield from tetramethylsilane and
referenced to d 7.26 and d 77.16 for CDCl3 and d 3.31 and d 49.00
for CD3OD. Analytical GC–MS with EI ionization was performed
on a Varian 3800 or 3900 equipped with a CP-SIL 5 CB Low Bleed
(30 m  0.25 mm) or CP-SIL 8 CB Low Bleed (30 m  0.25 mm)
and using He as carrier gas. Analytical RP-LC–MS was performed
on a Gilson HPLC system equipped with a Zorbax SB C8, 5 lm
(4.6 Â 50 mm) column, and a Finnigan AQA quadrupole mass spec-
trometer at a flow rate of 4 mL/min. The mobile phase consisted of
H2O/CH3CN/0.05% HCOOH. Preparative RP-LC–MS was performed
on a Gilson-Finnigan Thermo Quest AQA system equipped with a
Zorbax SB-C8, 5
lm 21.2 Â 150 mm (Agilent technologies) column
at a flow rate of 15 mL/min. The mobile phase consisted of H2O/
CH3CN/0.05% HCOOH. Elemental analyses were performed by Ana-
lytische Laboratorien, Lindlar, Germany. Exact molecular masses
(HRMS) were determined on a Micromass Q-Tof2 mass spectrom-
eter equipped with an electrospray ion source. Dry CH2Cl2 was dis-
tilled over calcium hydride and dry THF over sodium. Other
chemicals and solvents used were of analytical grade and pur-

C. Wallinder et al. / Bioorg. Med. Chem. 16 (2008) 6841–6849
6845
Table 1
Table 2
Series A
Series B
Compound
R
Ki (nM)
Compound
R1
R2
H
Ki (nM)
AT2
AT1
AT2
AT1
4
5
105.6 0.7
>10,000
40
41
42
43
144.5 3.4
>10,000
>10,000
>10,000
>10,000
36.9 1.8
3.0 0.3
2.6 0.2
10.5 0.5
6.4 0.5
1.0 0.08
31.5 0.7
10.9 0.4
6.4 0.3
3.2 0.2
348 5.4
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
H
121.8 1.8
162.5 1.0
299.8 2.9
6
7
H
H
H
8
44
45
H
H
402.2 10.1
52.0 4.3
>10,000
>10,000
9
10
11
12
13
14
15
46
H
91.7 2.3
>10,000
were combined and the organic solvents were removed under vac-
uum. The residue was dissolved in slightly basic water and washed
with EtOAc. The aqueous phase was acidified and extracted with
diethylether. The combined organic phase was washed with water
and brine, dried with MgSO4, filtered, and concentrated in vacuo.
The same extraction procedure was repeated three times to isolate
compound 2, without further purification, as white crystals in 65%
yield (672 mg, 1.7 mmol). 1H NMR (CDCl3 + CD3OD), d, ppm: 8.10–
8.06 (m, 2H), 7.67–7.64 (m, 2H), 6.76 (t, J = 0.8 Hz, 1H), 2.62 (dd,
J = 7.2, 0.8 Hz, 2H), 1.96–1.86 (m, 1H), 0.99 (s, 9H), 0.95 (d,
J = 6.6 Hz, 6H). 13C NMR (CD3OD), d, ppm: 168.4, 148.8, 142.4,
139.4, 137.1, 130.2, 129.8, 129.2, 129.0, 54.5, 39.2, 30.6, 29.4,
22.1. Anal. (C19H25NO4S2) C, H, N.
chased from commercial suppliers, and used without further puri-
fication unless stated. Thin-layer chromatography was performed
on aluminium plates precoated with silica gel 60 F254 (Merck)
and visualized in UV light. Flash chromatography was performed
on silica gel 60 (0.040–0.063 mm, Merck).
5.1.2. N-tert-Butyl-3-(4-chloroformylphenyl)-5-
isobutylthiophene-2-sulfonamide (3)
Compound 2 (740 mg, 1.9 mmol), 60 mL 1,2-dichloroethane,
three drops DMF and oxalylchloride (10 mL, 120 mmol) were
mixed together under a nitrogen atmosphere. The reaction mixture
was heated to 40 °C over night. The reaction mixture was then
cooled and the solvents were removed under vacuum and the res-
idue was co-evaporated with dry CH2Cl2four times and left under
vacuum. Compound 3 was used without further purification.
5.1.1. N-tert-Butyl-3-(4-carboxyphenyl)-5-isobutylthiophene-
2-sulfonamide (2)
Eight Smith Process VialsTM (2–5 mL) were charged with com-
pound 1 (total amount 1.82 g, 5.7 mmol) and para-iodobenzoic
acid (total amount 1.28 g, 5.2 mmol). 2.8 mL of DME, 0.7 mL of eth-
anol, 0.8 mL of water, Na2CO3 (2 M, 0.6 mL, 1.2 mmol), and
Pd(PPh3)4 (20 mg, 0.017 mmol) were added to each tube. The reac-
tion mixtures were flushed with nitrogen, sealed, and heated by
microwave irradiation to 110 °C for 5 min. The reaction mixtures
5.2. General procedure of compounds 4–15
Compound 3 (65 mg, 016 mmol) was dissolved in 2 mL dry
CH2Cl2. An excess of the respective amine or in combination with

6846
C. Wallinder et al. / Bioorg. Med. Chem. 16 (2008) 6841–6849
1
1
1
1
1
88
6
4
2
0
8
6
4
2
0
Ang II
PD 123,319
6
10
45
Figure 3. Effect of compounds 6, 10, and 45 on neurite outgrowth in NG108-15 cells. The cells were plated at a cell density of 3.6 Â 104 cells/35 mm Petri dishes and were
cultured for 3 days in the absence or presence of 100 nM Ang II, 1 nM compound 6, 1 nM compound 10 or 100 nM compound 45 alone or in combination with 1 lM PD
123,319. Cells with at least one neurite longer than a cell body were counted as positive for neurite outgrowth. The number of cells with neurites was expressed as the
percentage of the total number of cells in the micrographs (at least 290 cells according to the experiment).
triethylamine was added under nitrogen. The reaction mixture was
stirred at ambient temperature over night and diluted with CH2Cl2
and washed with 10% citric acid, water and brine. The organic layer
was dried with MgSO4, filtered, and evaporated. The residue was dis-
7.36–7.29 (m, 4H), 7.26–7.21 (m, 1H), 6.84 (t, J = 0.8 Hz, 1H), 4.60
(s, 2H), 3.99 (t, 2H, J = 6.5 Hz), 2.74 (dd, J = 7.1, 0.8 Hz, 2H), 2.00–
1.90 (m, 1H), 1.53–1.46 (m, 2H), 1.29–1.14 (m, 2H), 0.99 (d,
J = 6.6 Hz, 6H), 0.87 (t, J = 7.3 Hz, 3H). 13C NMR (CDCl3), d, ppm:
167.2, 152.0 150.6, 145.6 138.3, 137.4, 134.3, 131.4, 129.3, 129.2,
128.8, 128.3, 127.7, 127.3, 67.0, 44.3, 39.4, 30.7, 30.6, 22.4, 18.9,
13.7. HRMS (MÀH+): 527.1700, C27H31N2O5S2 requires 527.1674.
Anal. (C27H32N2O5S2) C, H, N.
solved in CH2Cl2 and reacted with an excess of 1.0 M BCl3 (500
lL) in
hexane fractions under nitrogen for 1 h. More BCl3 (500 L) was
l
added if the reaction had not reached full conversion after this time.
The reaction mixture was diluted with CH2Cl2 and washed with
water and brine. The organic layer was dried with MgSO4, filtered,
and evaporated. Compounds 1 and 12 was deprotected using triflu-
oroacetic acid and anisole instead of BCl3. The residue was dissolved
5.3. General procedure for compound 16–17 and 22–24
in dry CH2Cl2 (5 mL) and triethylamine (100
otherwise stated) and n-butyl chloroformate (30
l
L, 0.72 mmol, if not
The respective sulfonylchloride was dissolved in dry CH2Cl2.
The mixture was cooled on an ice bath and tert-butylamine was
added dropwise. After completion of addition, the reaction mixture
was left stirring at ambient temperature for 1 h to over night. The
reaction mixture was diluted with CH2Cl2 and washed with water
and brine. The organic layer was dried with MgSO4, filtered, and
evaporated to achieve the pure compounds 16–17 and 22–20 with-
out further purification.
lL, 0.24 mmol, if
not otherwise stated) were added under nitrogen and the reaction
was left for 2 h. The reaction mixture was diluted with CH2Cl2 and
washed with water and brine. The organic layer was dried with Mg
SO4, filtered, evaporated and purified by chromatotron or prepara-
tive HPLC to give the pure compounds 4–15.
5.2.1. N-Butoxycarbonyl-3-[4-(N,N-diethylcarbamoyl)phenyl]-
5-isobutylthiophene-2-sulfonamide (6)
5.3.1. N-tert-Butyl-4-phenylbenzenesulfonamide (23)
4-Phenylbenzenesulfonylchloride (1.00 g, 4.6 mmol) dissolved
in 50 mL dry CH2Cl2 was used according to the general procedure
and reacted with tert-butylamine (2.40 mL, 23 mmol) for 3 h. Com-
pound 23 was achieved as white crystals in 77% yield (1.02 g,
3.5 mmol). 1H NMR (CDCl3), d, ppm: 7.96–7.93 (m, 2H), 7.71–
7.68 (m, 2H), 7.63–7.60 (m, 2H), 7.50–7.40 (m, 3H), 4.44 (br s,
1H), 1.27 (s, 9H). 13C NMR (CDCl3), d, ppm: 145.2, 142.2, 139.6,
129.2, 128.5, 127.64, 127.62, 127.4, 54.9, 30.4. Anal. (C16H19NO2S)
C, H, N.
Compound 3 was used according to the general procedure and
reacted with triethylamine (42
lL, 0.30 mmol) and diethylamine
(24 L, 0.23 mmol). Compound 6 was purified by chromatotron
l
(100% CH2Cl2 to 3% MeOH in CH2Cl2) to give the pure product in
45% yield (36 mg, 0.072 mmol). 1H NMR (CDCl3), d, ppm: 7.53–
7.50 (m, 2H), 7.43–7.40 (m, 2H), 6.77 (t, J = 0.8 Hz, 1H), 4.04 (t,
J = 6.6 Hz, 2H), 3.62–3.48 (m, 2H), 3.36–3.22 (m, 2H), 2.71 (dd,
J = 7.1, 0.8 Hz, 2H), 2.00–1.90 (m, 1H), 1.54–1.47 (m, 2H), 1.32–
1.08 (m, 8H), 1.00 (d, J = 6.6 Hz, 6H), 0.88 (t, J = 7.3 Hz, 3H). 13C
NMR (CDCl3), d, ppm: 171.0, 151.5, 150.5, 145.5, 137.1, 135.1,
131.6, 129.4, 129.2, 126.5, 66.8, 43.5, 39.5, 39.4, 30.7, 30.5, 22.4,
18.9, 14.4, 13.7, 13.0. HRMS (M+H+): 495.1964, C24H36N2O5S2 re-
quires 495.1987. Anal. (C24H35N2O5S2) C, H, N.
5.4. General procedure for compound 18–21
A solution of ZnBr2 in dry THF was prepared under inert condi-
tions and cooled on an ice bath. The respective Grignard reagent
was added dropwise and a white precipitate was formed. The reac-
tion was left under nitrogen at ambient temperature for 1 h. A dry
vial was charged with LiBr and the respective bromide and thor-
oughly flushed with nitrogen. Dry NMP was added to the solids
which dissolved upon exposure to an ultrasonic bath. A dry vial
was charged with (1,3-diisopropylimidazol-2-ylidene)(3-chloro-
pyridyl)palladium(II) dichloride and thoroughly flushed with nitro-
5.2.2. N-Butoxycarbonyl-3-[4-(N-benzylcarbamoyl)phenyl]-5-
isobutylthiophene-2-sulfonamide (10)
Compound 3 was used according to the general procedure and
reacted with benzylamine (36 lL, 0.33 mmol). Compound 10 was
purified by chromatotron (100% CH2Cl2 to 4% MeOH in CH2Cl2) to
give the pure product in 46% yield (39 mg, 0.074 mmol). 1H NMR
(CD3OD + CDCl3), d, ppm: 7.90–7.86 (m, 2H), 7.59–7.56 (m, 2H),

C. Wallinder et al. / Bioorg. Med. Chem. 16 (2008) 6841–6849
6847
gen. The catalyst was dissolved in dry THF and transferred to the
NMP solution. The three component solution was added dropwise
under nitrogen to the zinc reagent. The reaction mixture was left un-
der nitrogen at ambient temperature over night. The next day the
reaction was cooled on an ice bath and 1 M HCl was added until a
cleardark solutionwith acidic pH was achieved. Themixturewas ex-
tracted with diethylether and the combined organic phase was
washedwaterand brine. Theorganiclayerwasdriedwith MgSO4, fil-
tered, and evaporated. The crude product was purified by column
chromatography to achieve the pure compounds 18–21.
0.20 mmol). 1H NMR (CDCl3), d, ppm: 8.24 (d, J = 8.3 Hz, 1H), 7.72
(dd, J = 8.3 Hz, J = 2.0 Hz, 1H), 7.65–7.61 (m, 4H), 7.52 (d,
J = 2.0 Hz, 1H), 7.50–7.39 (m, 5H), 3.65–3.48 (m, 3H), 3.40–3.26
(m, 2H), 1.33–1.09 (m, 6H), 1.05 (s, 9H). 13C NMR (CDCl3), d,
ppm: 170.9, 144.9, 141.0, 140.8, 139.9, 139.2, 137.4, 131.1, 130.2,
129.3, 129.3, 128.7, 127.5, 126.6, 126.4, 54.7, 43.6, 39.7, 30.1,
14.5, 13.1. Anal. (C27H32N2O3S) C, H, N.
5.5. General procedure for compound 40–46
Compound 33–39 were dissolved in dry CH2Cl2 (5–10 mL) and
cooled to 0 °C and reacted with an excess of 1.0 M BCl3 in hexane
fractions under nitrogen. The reaction was left at room tempera-
ture for 1 h. More BCl3 was added if the reaction had not reached
full conversion after this time. The reaction mixture was then
evaporated and co-evaporated several times with CHCl3. The resi-
due was dissolved in dry CH2Cl2 (5 mL) and triethylamine and n-
butyl chloroformate were added under nitrogen and the reaction
was left for 2 h at ambient temperature. The reaction mixture
was diluted with CH2Cl2 and washed with water and brine. The or-
ganic layer was dried with MgSO4, filtered, evaporated, and puri-
fied by chromatotrone or preparative HPLC to give the pure
products 40–46.
5.4.1. 4-Iodo-(N,N-diethyl)benzenamide (25)
Para-iodobenzoic acid (3.46 g, 14 mmol) was dissolved in
200 mL dry CH2Cl2 under a nitrogen atmosphere and 15 drops
DMF and oxalylchloride (22 mL, 28 mmol) were subsequently
added. The reaction mixture was heated to 40 °C and stirred over
night. The reaction mixture was then cooled and the solvents were
removed under vacuum and the residue was co-evaporated with
CHCl3 three times and left under vacuum. The crude product was
dissolved in 250 mL dry CH2Cl2 under a nitrogen atmosphere and
triethylamine (15.6 mL, 113 mmol) and diethylamine (7.30 mL,
71 mmol) were added and the reaction was left under nitrogen
at ambient temperature over night. The reaction was washed with
1 M HCl, water, and brine. The organic layer was dried with MgSO4,
filtered, and evaporated. The crude product was purified by column
chromatography (isohexane/EtOAc 20:1 first then 3:1) to isolate
compound 25 as white crystals in 83% yield (3.52 g, 11.62 mmol).
1H NMR (CDCl3), d, ppm: 7.76–7.73 (m, 2H), 7.13–7.10 (m, 2H),
3.64–3.41 (m, 2H), 3.33–3.15 (m, 2H), 1.32–1.04 (m, 6H). 13C
NMR (CDCl3), d, ppm: 170.4, 137.7, 136.8, 128.2, 95.2, 43.4, 39.5,
14.4, 13.0. Anal. (C11H14INO) C, H, N.
5.5.1. N-Butoxycarbonyl-2-[4-(N,N-diethylcarbamoyl)phenyl]-
4- phenylbenzenesulfonamide (45)
According to the general procedure compound 38 (92 mg,
0.20 mmol) was reacted with BCl3 (1.0 mL + 0.50 mL, 1.0 M in
hexane) and afterwards with triethylamine (110
lL, 0.80 mmol)
and n-butyl chloroformate (31 L, 0.24 mmol). The crude product
l
was purified by preparative HPLC to isolate compound 45 in 28%
yield (28 mg, 0.056 mmol). 1H NMR (CDCl3), d, ppm: 8.34 (d,
J = 8.3 Hz, 1H), 7.78 (dd, J = 8.3 Hz, J = 1.9 Hz, 1H), 7.64–7.61 (m,
2H), 7.54 (d, J = 1.9 Hz, 1H), 7.52–7.41 (m, 7H), 7.10–6.94 (br s,
1H), 4.03 (t, J = 6.5 Hz, 2H), 3.66–3.49 (m, 2H), 3.42–3.24 (m,
2H), 1.53–1.46 (m, 2H), 1.35–1.10 (m, 8H), 0.86 (t, J = 7.3 Hz,
3H). 13C NMR (CDCl3), d, ppm: 171.0, 150.4, 146.3, 141.0, 139.6,
138.8, 137.3, 136.0, 131.4, 130.9, 129.5, 129.2, 129.0, 127.6,
126.6, 126.2, 66.9, 43.6, 39.6, 30.6, 18.9, 14.5, 13.7, 13.1. Anal.
(C28H32N2O5S) C, H, N.
5.4.2. General procedure for compound 33–39
To a cooled (À78 °C) solution of compounds 18–24 in dry THF
was added n-BuLi (1.6 M in hexane) under nitrogen and the reac-
tion was stirred for 1 h. The temperature was raised to À30 °C
and kept for 3 h and subsequently decreased to À40 °C. Triisopro-
pyl borate was then added. The reaction mixure was stirred over
night at room temperature. The reaction mixture was cooled
(0 °C) and treated with an excess of 2 M HCl solution. The mix-
ture was extracted with EtOAc and the combined organic phase
was washed with water and brine. The organic layer was dried
with MgSO4, filtered and evaporated. The crude product (26–
32) was then used in the next step without further purification.
A Smith Process VialTM (2–5 mL) was charged with the crude
boronic acid (26–32), compound 25, DME, ethanol, water, 2 M
Na2CO3, and Pd(PPh3)4. The reaction mixture was flushed with
nitrogen, sealed, and heated by microwave irradiation to 110 °C
for 5 min. The reaction mixture was diluted with EtOAc, washed
with water and brine, dried with MgSO4, filtered, and concen-
trated under vacuum. The crude product was purified by column
chromatography or preparative HPLC to isolate the pure com-
pounds 33–39.
5.6. Rat liver membrane AT1 receptor-binding assay
Rat liver membranes were prepared according to the method
of Dudley et al.17 Binding of [125I]Ang II to membranes was con-
ducted in a final volume of 0.5 mL containing 50 mM Tris–HCl
(pH 7.4), 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 0.025% bac-
itracin, 0.2% BSA (bovine serum albumin), liver homogenate cor-
responding to 5 mg of the original tissue weight, [125I]Ang II
(80,000–85,000 cpm, 0.03nM ) and variable concentrations of
test substance. Samples were incubated at 25°C for 2 h, and
binding was terminated by filtration through Whatman GF/B
glass-fiber filter sheets, which had been pre-soaked overnight
with 0.3% polyethylamine, using a Brandel cell harvester. The fil-
ters were washed with 3Â 3 mL of Tris–HCl (pH 7.4) and trans-
5.4.3. N-tert-Butyl-2-[4-(N,N-diethylcarbamoyl)phenyl]-4-
phenylbenzenesulfonamide (38)
ferred to tubes. The radioactivity was measured in a
c-counter.
According to the general procedure compound 23 (0.997 g,
3.45 mmol) was dissolved in dry THF (20 mL) and reacted with
n-BuLi (5.4 mL, 1.6 M in hexane, 8.62 mmol) and triisopropyl bo-
rate (1.2 mL, 5.18 mmol). Two Smith Process VialsTM (2–5 mL) were
charged with the crude boronic acid 31 (total amount 240 mg,
<0.72 mmol) and compound 25 (total amount 133 mg, 0.43 mmol).
2.0 mL of DME, 0.5 mL of ethanol, 0.5 mL of water, Na2CO3 (2 M,
0.4 mL, 0.88 mmol) and Pd(PPh3)4 (15 mg, 0.013 mmol) were
added to each tube. The crude product was purified by preparative
HPLC to isolate compound 38 as white crystals in 46% yield (92 mg,
The characteristics of the Ang II-binding AT1 receptor were
determined by using six different concentrations (0.03–5 nmol/
L) of the labeled [125I]Ang II. Nonspecific binding was determined
in the presence of 1 lM Ang II. The specific binding was deter-
mined by subtracting the nonspecific binding from the total
bound [125I]Ang II. The apparent dissociation constant Ki values
were calculated from IC50 values using the Cheng–Prusoff equa-
tion (Kd = 1.7 0.1 nM, [L] = 0.057 nM). The binding data were
best fitted with a one-site fit. All determinations were performed
in triplicate.

6848
C. Wallinder et al. / Bioorg. Med. Chem. 16 (2008) 6841–6849
5.7. Porcine (pig) myometrial membrane AT2 receptor-binding
assay
antagonist introduced daily 30 min prior to Ang II, compounds 6,
10 or 45. During the three days treatment the transfected cell line
was cultured without Geneticin.
Myometrial membranes were prepared from porcine uteri
according to the method by Nielsen et al.18 A presumable interfer-
5.10. Determination of cells with neurites
ence by binding to AT1 receptors was blocked by addition of 1 lM
losartan. Binding of [125I]Ang II to membranes was conducted in a fi-
nal volume of 0.5 mL containing 50 mM Tris–HCl (pH 7.4), 100 mM
NaCl, 10 mM MgCl2, 1 mM EDTA, 0.025% bacitracin, 0.2% BSA,
homogenate corresponding to 10 mg of the original tissue weight,
Cells were examined under a phase contrast microscope and
micrographs were taken after 3 days under the various experimen-
tal conditions. Cells with at least one neurite longer than a cell
body were counted as positive for neurite outgrowth. The number
of cells with neurites was reported as the percentage of the total
amount of cells in the micrographs and at least 290 cells were
counted in three independent experiments and each condition
was performed in duplicate.23 The data are represented as mean-
[
125I]Ang II (80,000–85,000 cpm, 0.03 nM) and variable concentra-
tions of test substance. Samples were incubated at 25 °C for 1.5 h,
and binding was terminated by filtration through Whatman GF/B
glass-fiber filter sheets, which had been presoaked overnight with
0.3% polyethylamine, using a Brandel cell harvester. The filters were
washed with 3Â 3 mL of Tris–HCl (pH 7.4) and transferred to tubes.
s
SEM of the average number of cells on a micrograph.
Theradioactivitywas measuredin a c-counter. The characteristics of
Acknowledgments
the Ang II-binding AT2 receptor were determined by using six differ-
ent concentrations (0.03–5 nmol/L) of the labeled [125I]Ang II. Non-
specific binding was determined in the presence of 1 lM Ang II.
We gratefully acknowledge support from the Swedish Research
(VR), the Swedish Foundation for Strategic Research (SSF), Knut
and Alice Wallenberg Foundation (VRmedicine 8663), the Cana-
dian Institutes of Health Research to N.G.P. and M.D. Payet (MOP
27912), and Vicore Pharma AB. N.G.P. is a holder of a Canada Re-
search Chair in Endocrinology of the Adrenal Gland. We also thank
Luke Odell for linguistic advice and Johanna Lindholm for labora-
tory assistance.
The specific binding was determined by subtracting the nonspecific
binding from the total bound [125I]Ang II. The apparent dissociation
constant Ki values were calculated from IC50 values using the Cheng–
Prusoff equation (Kd = 0.73 0.06 nM, [L] = 0.057 nM). The binding
data were best fitted with a one-site fit. All determinations were per-
formed in triplicate.
5.8. In vitro morphological effects: general
Supplementary data
The chemicals used in the present study were obtained from the
following sources: Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum (FBS), HAT supplement (Hypoxanthine, Ami-
nopterin, Thymidine), gentamycin from Gibco–BRL (Burlington,
Ont, Canada); [Val5]angiotensin II from Bachem (Marina Delphen,
CA, USA). PD-123,319 was obtained from RBI (Natick, MA, USA).
All other chemicals were of grade A purity.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.05.066.
References and notes
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To study the in vitro morphological effects NG108-15 cells (pro-
vided by Drs. M. Emerit and M. Hamon; INSERM, U. 238, Paris,
France) were used as well as transfected NG108-15/pcDNA3 cells.
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same behavior as the native cell line.21 In their undifferentiated
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rounded shape and divide actively. Both cell lines were cultured
(NG108-15 passage 18–28, NG108-15/pcDNA3 passage 12–18) in
Dulbecco’s modified Eagle’s medium (DMEM, Gibco–BRL, Burling-
ton, Ont., Canada) with 10% fetal bovine serum (FBS, Gibco), HAT
supplement and 50 mg/L gentamycin at 37 °C in 75 cm2 Nunclon
Delta flasks in a humidified atmosphere of 93% air and 7% CO2, as
previously described.4,20 The transfected cell line was kept stable
by addition of Geneticin (G-418, 200 l
g/mL) to the media.21 Sub-
cultures were performed at subconfluency. Under these conditions,
cells express only the AT2 receptor subtype.4,20 Cells were treated
during three days, once a day (first treatment 24 h after plating),
and micrographs were taken the fourth day. For all experiments,
cells were plated at the same initial density of 3.6 Â 104 cells/
35 mm Petri dish. To determine a good test concentration com-
pounds 6, 10, and 45 were tested at various concentrations ranging
from 1 pM to 1 lM. For all the compounds it was only in the high-
est concentration that a tendency of cell death was observed. Cells
were treated without (control cells), or with [Val5]angiotensin II
(100 nM) or compound 6 (1 and 10 nM), compound 10 (1 and
10 nM) and compound 45 (100 nM) in the absence or in the pres-
19. Carini, D. J.; Duncia, J. V.; Aldrich, P. E.; Chiu, A. T.; Johnson, A. L.; Pierce, M. E.;
Price, W. A.; Santella, J. B., III; Wells, G. J.; Wexler, R. R.; Wong, P. C.; Yoo, S.-E.;
Timmermans, P. B. M. W. M. J. Med. Chem. 1991, 34, 2525.
ence of PD 123,319 (RBI Natick, MA, USA) (10 lM), an AT2 receptor

C. Wallinder et al. / Bioorg. Med. Chem. 16 (2008) 6841–6849
6849
20. Buisson, B.; Bottari, S. P.; de Gasparo, M.; Gallo-Payet, N.; Payet, M. D. FEBS Lett.
24. Perlman, S.; Costa-Neto, C. M.; Miyakawa, A. A.; Schambye, H. T.; Hjorth, S. A.;
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Karlén, A.; Hallberg, M.; Gallo-Payet, N.; Alterman, M. J. Med. Chem. 2006, 49, 7160.
26. Ashton, W. T.; Chang, L. L.; Flanagan, K. L.; Mantlo, N. B.; Ondeyka, D. L.; Kim,
D.; de Laszlo, S. E.; Glinka, T. W.; Rivero, R. A.; Kevin, N. J.; Chang, R. S. L.; Siegl,
P. K. S.; Kivlighn, S. D.; Greenlee, W. J. Eur. J. Med. Chem. 1995, 30, 255s–266s.
22. Gendron, L.; Laflamme, L.; Rivard, N.; Asselin, C.; Payet, M. D.; Gallo-Payet, N.
Mol. Endocrinol. 1999, 13, 1615.
23. Gendron, L.; Coté, F.; Payet, M. D.; Gallo-Payet, N. Neuroendocrinology 2002, 75,
70.