Selective angiotensin II AT2 receptor agonists devoid of the imidazole ring system

Article abstract of DOI:10.1016/j.bmc.2007.07.026

A versatile parallel synthetic method to obtain three series of non-cyclic analogues of the first drug-like selective angiotensin II AT₂ receptor agonist (1) has been developed. In analogy with the transformation of losartan to valsartan it was

Full text of DOI:10.1016/j.bmc.2007.07.026

Bioorganic & Medicinal Chemistry 15 (2007) 7166–7183
Selective angiotensin II AT2 receptor agonists devoid
of the imidazole ring system
A. M. S. Murugaiah,a Chalotta Wallinder,a A. K. Mahalingam,a Xiongyu Wu,a
a
Yiqian Wan,a Bianca Plouffe,b Milad Botros,c,d Anders Karlen,
´
Mathias Hallberg,c,d Nicole Gallo-Payetb, and Mathias Altermana,*
aDepartment of Medicinal Chemistry, BMC, Uppsala University, PO Box 574, SE-751 23 Uppsala, Sweden
bService of Endocrinology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Que., Canada J1H 5N4
cDepartment of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Que., Canada J1H 5N4
dDepartment of Biological Research on Drug Dependence, BMC, Uppsala University, PO Box 591, SE-751 23 Uppsala, Sweden
Received 19 February 2007; revised 28 June 2007; accepted 6 July 2007
Available online 22 August 2007
Abstract—A versatile parallel synthetic method to obtain three series of non-cyclic analogues of the first drug-like selective angio-
tensin II AT2 receptor agonist (1) has been developed. In analogy with the transformation of losartan to valsartan it was demon-
strated that a non-cyclic moiety could be employed as an imidazole replacement to obtain AT2 selective compounds. In all the three
series, AT2 receptor ligands with affinities in the lower nanomolar range were found. None of the analogues exhibited any affinity for
the AT1 receptor. Four compounds, 17, 22, 39 and 51, were examined in a neurite outgrowth cell assay. All four compounds were
found to exert a high agonistic effect as deduced from their capacity to induce neurite elongation in neuronal cells, as does angio-
tensin II.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
adults, activation of the AT2 receptor exhibits effects
that could be considered more ‘anti AT1’, for example,
activation lowers blood pressure, inhibits cell prolifera-
tion, induces programmed cell death and extracellular
matrix remodelling, as well as axonal regeneration.6–11
Moreover, AT2 receptor expression is up-regulated in
pathological conditions such as heart failure, renal fail-
ure, myocardial infarction, brain lesions, vascular injury
and wound healing.9,12–14 In addition, it has been dem-
onstrated that activation of the AT2 receptor stimulates
alkaline secretion by the duodenal mucosa in rats.15
Angiotensin II (Ang II), recognized as the most impor-
tant bioactive peptide of the renin-angiotensin system,
is the endogenous ligand to the AT1 and the AT2 recep-
tors where it acts as an agonist. The AT1 receptor is clo-
sely associated with the regulation of blood pressure,
fluid and electrolyte balance, while the role of the AT2
receptor has been less clear due to the low level of
AT2 expression in healthy adults.1 One interesting fea-
ture of the AT2 receptor is the high level of expression
in most foetal tissues, including the brain. Notably,
the AT2/AT1 receptor ratio decreases dramatically after
birth,2,3 which suggests that the AT2 receptor may be in-
volved in foetal development. This is further supported
by the observed induction of neurite outgrowth, elonga-
tion and the modulated neuronal excitability upon AT2
receptor activation in cells of neuronal origin.4,5 In
The AT1 selective antagonist losartan (Fig. 1)16 was the
first drug to be registered for the treatment of high blood
pressure via blockage of the AT1 receptor. The hydroxy-
methyl group of the losartan is metabolized in vivo to
the active corresponding carboxylic acid. Valsartan is
one of the successors of losartan.17 An interesting fea-
ture of valsartan is that it does not contain the com-
monly used nitrogen containing heterocycle, found in
many of the ‘sartans’.18 In valsartan the imidazole ring
is replaced by a tertiary amide and remarkably both
the AT1 affinity and the selectivity were maintained with
this open chain structural element.
Keywords: Angiotensin; AT2; Agonist.
*
Corresponding author. Tel.: +46 18 4714905; fax: +46 18 4714474;
e-mail addresses: nicole.gallo-payet@usherbrooke.ca; mathias.
alterman@ orgfarm.uu.se
Tel.: +1 819 5645243; fax: +1 819 5645292.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.026

A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
7167
O
We have recently reported the first drug-like selective AT2
receptor agonist (1, Fig. 1).19 We have also demonstrated
that alterations in the 2- and 5-positions of the thiophene
moiety modulate the affinity for the AT2 receptor, but did
not render any compounds with affinity for the AT1 recep-
tor.20 Thus, we postulated that the imidazole was a strong
determinant for the AT2 selectivity.
N
Cl
N
N
OH
OH
N
O
N
N
NH
N
N
N
NH
The successful conversion of losartan to valsartan (Fig. 1)
encouraged us to explore the impact of a tertiary amide as
a replacement for the imidazole structure. The question
arose whether the imidazole ring, in some system associ-
ated with CYP-P450 inhibition, could be favourably dis-
placed and whether AT2/AT1 selectivity, AT2 receptor
agonism and AT2 receptor affinity could be achieved with
open chain amide analogues (General structure A,
Fig. 1). Herein, we report a synthetic strategy that conve-
niently gives access to a series of tertiary amide analogues.
In addition, a number of these analogues act as high
affinity selective AT2 receptor ligands as deduced from
the structure–activity relationship data enclosed.
Losartan
IC50 (nM)
AT1: 12.1
AT2: >10000
antagonist (AT1)
Valsartan
Ki (nM)
AT1: 2.4
AT2: >10000
antagonist (AT1)
O
N
N
R2
R1
N
O
O
O
S
O
S
O
O
O
O
HN
HN
S
S
2. Results
1
A
2.1. Chemistry
Ki (nM)
AT1: >10000
AT2: 0.4
agonist (AT2)
The thiopheneboronic acid 2 (Scheme 1) was prepared,
in essence, as described by Kevin et al.19–23 Thus,
thiophene-2-sulfonyl chloride was first converted to the
Figure 1.
c
R2
N
H
N
O
R1
O
R1
O
O
(HO)2B
O
S
O
a
b
d
O
HN
O
O
O
S
S
S
S
HN
HN
HN
S
Br
S
S
2
3
4 a-e
5 a-p
R2
N
R2
R1
O
N
R1
f
e
O
S
O
O
S
O
O
S
HN
NH2
S
6 a-p
7-22
Scheme 1. Reagents: (a) Pd(OAc)2, P(Ph)3, K2CO3, THF/DME/EtOH/H2O; (b) primary amine, NaBH4, MeOH; (c) secondary amine, NaB(OAc)3H,
MeOH; (d) acid chloride, N(Et)3, DMAP, CH2Cl2; (e) TFA, anisole; (f) n-butyl chloroformate, pyrrolidinopyridine, Et3N, CH2Cl2. 4a: benzylamine,
b: 3-picolylamine, c: 4-aminotoluene, d: ethylamine, e: methylamine. 5a: benzylamine, valeroyl chloride, b: benzylamine, acetyl chloride, c: 3-
picolylamine, benzoyl chloride, d: 3-picolylamine, acetyl chloride, e: 4-aminotoluene, acetyl chloride, f: 4-aminotoluene, benzoyl chloride, g:
ethylamine, acetyl chloride, h: ethylamine, thiophene-2-carbonyl chloride, i: methylamine, valeroyl chloride, j: methylamine, ethyl chloroformate, k:
methylamine, acetyl chloride, l: methylamine, methansulfonyl chloride, m: methylamine, ammonium formiate, n: dimethylamine, o: methylami-
noacetonitrile, p: N-methoxymethylamine.

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A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
corresponding N-tert-butylsulfonamide. Subsequent
alkylation followed by selective 3-lithiation/boronation
delivered the boronic acid 2. The boronic acid 2 was
then reacted with 4-bromobenzaldehyde under Suzuki
coupling conditions with in situ prepared palladium tet-
rakis as catalyst and K2CO3 as base to give the aldehyde
3. This aldehyde (3) was used as a key intermediate for
the first series (compounds 7–22, Scheme 1).
(4a–e) were dissolved in dichloromethane and triethyl
amine and DMAP were added. Acid chlorides were
added to the reaction vials and the reaction mixtures
were stirred overnight at ambient temperature to achieve
the tertiary amides 5a–p (to synthesise the formyl com-
pound 5m ammonium formate was refluxed with the
secondary amine 4e). The same workup as in the previ-
ous step was also applied in this step to obtain the ter-
tiary amides (5a–p) in high yields and with high purity.
The tert-butyl protecting group was removed from the
sulfonamide with TFA and n-butyl chloroformate was
added to accomplish the final products 7–22, in 45–
92% yields after purification on preparative HPLC. To
synthesise compound 25, N-(4-bromo-benzyl)-acetam-
ide was coupled directly to the boronic acid 2 under Su-
zuki conditions. The product was then deprotected and
reacted with n-butyl chloroformate, as above, to obtain
25 (Scheme 2). This procedure was applied to avoid the
reductive amination with ammonia.
In the preparation of the first series, the aldehyde 3 was
dissolved in methanol and added to sample vials. A di-
verse set of amines were dispensed to the vials and
NaBH4 was added as reducing agent to achieve the
reductive amination, with full conversion. In the cases
where secondary amines were used, NaB(OAc)3H was
applied as the reducing agent (5n–p, Scheme 1). The
workup was conveniently performed with a diatoma-
ceous earth plug (solid–liquid extraction) in a polypro-
pylene column and eluted with ethyl acetate. The
secondary amines (4a–e, Scheme 1) were obtained in
high purity and were therefore used in the second step,
or third step for the secondary amines, without further
purification. In the second step, the secondary amines
To obtain the amides in the second series (compound
30–40) the thiopheneboronic acid 2 was coupled to
ethyl-4-bromophenyletanoate using Pd(OAc)2 and
O
O
O
NH
O
NH
NH
NH
(HO)2B
O
S
O
a
c
b
O
S
O
S
O
S
HN
O
O
O
S
O
O
HN
NH2
HN
S
S
S
Br
2
23
24
25
Scheme 2. Reagents: (a) Pd(OAc)2, P(Ph)3, K2CO3, THF/DME/EtOH/H2O; (b) TFA, anisole; (c) n-butyl chloroformate, pyrrolidinopyridine, Et3N,
CH2Cl2.
O
O
O
R2
O
OH
O
N
O
R1
(HO)2B
O
O
S
O
a
c
b
O
O
S
HN
O
O
O
S
S
S
HN
HN
HN
S
Br
S
S
2
26
27
28 a-k
O
O
R2
R2
N
N
R1
R1
d
e
O
S
O
S
O
O
O
HN
NH2
S
S
O
29 a-k
30-40
Scheme 3. Reagents: (a) Pd(OAc)2, P(Ph)3, K2CO3, CsF, DME; (b) LiOH, THF/H2O/MeOH; (c) amine, EDC, HOBT, N(Et)3, DMF; (d) TFA,
anisole; (e) n-butyl chloroformate, pyrrolidinopyridine, Et3N, CH2Cl2. 28a: methylbutylamine, b: benzylamine, c: ethylbenzylamine, d:
dibenzylamine, e: diphenylamine, f: morpholine, g: thiazolidine, h: 4-tolylamine, i: hexylamine, j: dimethylamine, k: methylamine.

A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
7169
DPPF as catalyst. The mild base CsF was employed in
the Suzuki reaction to avoid the hydrolysis of the ester
functionality. Compound 26 was isolated in 95% yield
(Scheme 3). The ester was then hydrolyzed with LiOH
to afford the carboxylic acid 27 that was used as a key
intermediate (Scheme 3). The carboxylic acid 27 was
thereafter coupled to a selection of amines using EDC
and HOBT as coupling reagents. Though the reaction
was run in parallel, we did not yield products of suffi-
cient purity after solid phase extraction work up as in
the aforesaid step, presumably due to the use of DMF
as solvent. Hence, liquid extraction was applied to ob-
tain the compounds pure enough to carry forward to
the next step. The subsequent deprotection with TFA
and final reaction of the sulfonamides with n-butyl chlo-
roformate afforded the compounds 30–40 in 46–77%
yields after purification on preparative HPLC. For sim-
ilar reasons as those for compound 25, compound 43
was synthesised by directly coupling 2-(4-bromo-phe-
nyl)-acetamide with the boronic acid 2 (Scheme 4).
The resulting compound 41 was thereafter deprotected
and reacted with n-butyl chloroformate, as above, to ob-
tain 43.
In the third series, the central phenyl ring was exchanged
with five- and six-membered heterocycles. The thiophen-
eboronic acid 2 was coupled with 6-bromo-pyridine-3-
carbaldehyde, 5-bromofuran-2-carboxaldehyde and
4-bromothiophene-2-carboxaldehyde using the same
Suzuki coupling conditions as in the second series
(Scheme 5). This protocol gave the three aldehydes
44–46 in modest yields (51–63%). The aldehydes were
then reacted with methylamine and NaBH4 followed
by acetyl chloride as described for series one. TFA treat-
ment and reaction with n-butyl chloroformate delivered
the compounds 50–52 in 51–68% yields.
2.2. Binding assays
Compounds 7–22, 25, 30–40, 43 and 50–52 were evalu-
ated in radioligand-binding assays by displacement of
[
125I]Ang II from AT1 receptors in rat liver membranes
and from AT2 receptors in pig uterus membranes as de-
scribed previously (Tables 1–3).24,25 The natural sub-
strate Ang II, the selective AT1 receptor antagonist
losartan,16 and the selective AT2 receptor antagonist
PD 123,31926 were used as reference substances.
O
O
O
NH2
NH2
NH2
O
(HO)2B
NH2
O
S
O
a
c
b
O
S
O
S
O
S
HN
O
O
O
S
O
O
HN
NH2
HN
S
S
S
Br
2
42
43
41
Scheme 4. Reagents: (a) Pd(OAc)2, P(Ph)3, K2CO3, DME; (b) TFA, anisole; (c) n-butyl chloroformate, pyrrolidinopyridine, Et3N, CH2Cl2.
O
N
O
O
O
H
N
N
S
O
O
(HO)2B
O
S
O
Br
Br
Br
c
b
O
S
O
O
HN
O
O
O
S
S
S
a
HN
HN
HN
S
S
S
2
44-46
47 a-c
48 a-c
O
O
O
O
N
N
N
N
S
N
O
e
d
O
S
HN
O
O
O
O
O
O
O
O
O
S
O
O
S
O
O
S
HN
HN
NH2
S
S
S
S
49 a-c
50
51
52
Scheme 5. Reagents: (a) Pd(OAc)2, DPPF, K2CO3, DME/EtOH/H2O; (b) MeNH2, NaBH4, MeOH; (c) acetyl chloride, Et3N, DMAP, CH2Cl2; (d)
TFA, anisole; (e) n-butyl chloroformate, pyrrolidinopyridine, Et3N, CH2Cl2.

7170
A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
Table 1.
R2
R1
N
O
O
S
O
O
HN
S
Entry
Compound
R1
R2
Kia (nM)
AT2
84
AT1
O
1
2
7
8
>10,000
O
200
404
>10,000
>10,000
O
3
9
N
O
O
4
5
6
10
11
12
59
60
46
>10,000
>10,000
>10,000
N
O
O
7
8
13
14
15
16
17
18
126
138
12
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
O
S
O
9
O
10
11
12
9.7
8.8
32
O
O
O O
S
O
13
14
15
16
17
19
20
21
22
25
3.1
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2.8
NC
O
O
5.8
H
a Ki values are an average from three determinations. Standard deviations are less than 15% in all cases.

A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
7171
Table 2.
Table 3.
O
O
R
S
N
O
S
HN
O
O
S
O
O
O
O
O
HN
S
Entry
Compound
R
Kia (nM)
Entry
Compound
Aromatic
structure
Kia (nM)
AT1
AT2
AT1
AT2
1
2
30
31
367
>10,000
N
1
2
50
51
>10,000
>10,000
>10,000
N
O
NH
221
322
>10,000
>10,000
9.1
N
3
4
32
33
S
3
52
>10,000
>10,000
185
>10,000
N
N
a Ki values are an average from three determinations. Standard devi-
ations are less than 15% in all cases.
5
6
34
35
219
124
>10,000
>10,000
the smallest substituents gave the highest affinity for the
AT2 receptor. This trend is corroborating with our ear-
lier observations regarding the nitrogen containing het-
erocycles.19 Although the trend was not linear and
fully consistent; for example, compound 11 has a larger
substituent in R1 than compound 13, but is twice as ac-
tive. By removing a methyl from compound 17, either
compound 19 or 25 could be obtained depending if R1
or R2 lacks a methyl group. Independently of which side
that lacks the methyl group the affinity was improved,
but with a slight advantage in affinity for compound
19. The introduction of a nitrogen into the phenyl ring
gave a fourfold increased affinity for the AT2 receptor
(cf. 8 and 10), although the 3-pyridyl moiety in combina-
tion with benzoyl in R2 gave the least potent compound
in the series (compound 9).
N
N
O
7
8
36
37
133
58
>10,000
>10,000
S
N
H
9
38
39
158
7.0
>10,000
>10,000
NH
N
10
NH
11
12
40
43
80
51
>10,000
>10,000
In the first series, four compounds lacked an amide as
structural element. Compound 20 encompassing the ter-
tiary amine in the sidechains was found to be inactive
against the AT2 receptor. This was also the case for
the acetonitrile compound 21. However, the sulfon-
amide compound 18 showed good affinity for the AT2
receptor (32 nM), although not as high as for the com-
parable amide compound (17, 8.8 nM). The N-meth-
oxymethylamide compound (22, 2.8 nM) exhibited the
highest affinity in the first series.
NH2
a Ki values are an average from three determinations. Standard devi-
ations are less than 15% in all cases.
In the first series, the imidazole ring of compound 1 was
substituted with a tertiary amide, sulfonamide or amine.
As seen in Table 1 none of the compounds in the first
series showed any AT1 affinity, which is notable consid-
ering the high AT1 selectivity that valsartan exhibits. All
of the compounds except the tertiary amines exhibited
moderate to high affinity towards the AT2 receptor. A
pattern that could be seen from the first series was that
In the second series, the amide moiety is reversed. This
series was prepared to allow an assessment of impact
of the position of the carbonyl and nitrogen atom. Inter-

7172
A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
12.00
estingly, the dimethyl compound 39 (Table 2) was found
to display a slightly inferior Ki value than the corre-
sponding compound in the first series (compound 17,
Table 1). However, deletion of the methyl groups, that
is, compound 43, leads to a substantial decrease in the
affinity. This was also the case when only one of the
methyl groups was replaced with a hydrogen (40). The
introduction of larger or heterocyclic amine substituents
did not render any highly potent compounds (com-
pounds 30–38, Table 2).
10.00
8.00
6.00
4.00
2.00
0.00
Ang II
PD123.,319
17
-
+
+
+
-
-
-
+
-
-
+
+
-
-
-
-
-
7
1
2
3
4
5
6
-
-
-
-
-
-
+
-
+
By modifying the central phenyl ring, not only are the
electrostatic properties of the compounds altered, but
also the angles of which the benzylic amide substituents
are presented to the AT2 receptor. As seen in Table 3
only the furanyl compound 51 showed any affinity for
the AT2 receptor. It is notable that this compound
exhibits similar affinity as the related compound 17.
22
-
+
Figure 3. Effect of 51 and 39 on neurite outgrowth in NG108-15 cells.
NG108-15 cells were plated at a density of 3.6 · 104 cells per dish in
35 mm Petri dishes and were cultured for 3 days in the absence or in
the presence of 0.1 lM Ang II, compound 51 (1 nM) or compound 39
(0.1 lM), alone or in the presence of 10 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 represents the
percentage of the total number of cells in the micrographs (at least 290
cells according to the experiment).
2.3. In vitro morphological effects induced by 17, 22, 39
and 51 in NG108-15 cells
To study the effects of compounds 17, 22, 39 and 51 on
AT2 induced differentiation, NG108-15 cells were used.
In their undifferentiated state, neuroblastoma · glioma
hybrid NG108-15 cells have a rounded shape and divide
actively. We have shown previously that these cells ex-
press only the AT2 receptor27,28 and that a 3-day treat-
ment with Ang II or the selective peptidic AT2
receptor agonist CGP-42112 induces neurite out-
growth.28 The mechanisms involve a sustained increase
in p42/p44mapk activity5 and activation of the nitric
oxide/guanylyl cyclase/cGMP pathway (for a review
see Ref. 11).
occasion. After 3 days of culture, cells were examined
under a phase contrast microscope and micrographs
were taken. The compounds 17, 22, 39 and 51 were first
examined at concentrations ranging from 1 pM to 1 lM.
Except for the higher concentration of 1 lM, none of
the other doses induced cell death. As shown in Figures
2 and 3, treatment for 3 days with compounds 17
(1 nM), 22 (1 nM), 39 (0.1 lM) and 51 (1 nM) induced
neurite outgrowth, comparable with Ang II. This effect
was mediated through the AT2 receptor, since co-incu-
bation of 17, 22, 39 and 51 with the AT2 receptor anta-
gonist, PD 123,319,21 virtually abolished neurite
elongation (Figs. 2 and 3), while alone, PD 123,319
did not alter the morphology compared to the untreated
cells (data not shown).
The cells were plated at the same initial density
(3.6 · 104 cells/35 mm Petri dish) and were treated in
with or without of Ang II, compound 17, 22, 39 or 51.
Ang II, compounds 17 and 22 were tested at one
occasion and Ang II, compounds 39 and 51 on a second
3. Discussion
20.00
18.00
16.00
14.00
12.00
10.00
8.00
Replacement of the imidazole in compound 1 with a ter-
tiary amide rendered analogues with high affinity for the
AT2 receptor. Thus, a modification resembling the losar-
tan to valsartan transformation was also allowed with
AT2 ligands. Our previous results suggested that the
imidazole might be a strong determinant for the AT2
selectivity.20 The fact that the nitrogen and oxygen of
the amide could occupy the same positions as the two
nitrogens in the imidazole ring provides a rationale for
the high AT2 receptor affinities found with analogues
bearing small substituents. However, when considering
the balanced nature of the compound L-162,31323,29,30
(Ki AT1: 3.9 nM, AT2: 2.8 nM), which was used as a
template for compound 1, it was unexpected to find that
none of the compounds in the first series possessed any
AT1 receptor affinity, especially compounds with larger
substituents, for example, 7–12. The non-cyclic amide
structure cannot fully explain the lack of AT1 affinity
since valsartan, with its non-cyclic structure in the ben-
zylic position, is an AT1 selective antagonist. It seems
more likely that a combination of a substituent in the
6.00
4.00
2.00
0.00
Ang II
PD123.,319
51
-
+
+
+
-
-
-
+
-
-
-
-
-
-
7
1
2
3
4
5
6
-
-
-
-
-
-
+
+
-
+
-
+
39
-
+
Figure 2. Effect of 17 and 22 on neurite outgrowth in NG108-15 cells.
NG108-15 cells were plated at a density of 3.6 · 104 cells per dish in
35 mm Petri dishes and were cultured for 3 days in the absence or in
the presence of 0.1 lM Ang II, compound 17 (1 nM) or compound 22
(1 nM), alone or in the presence of 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 represents the
percentage of the total number of cells in the micrographs (at least 290
cells according to the experiment).

A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
7173
5-position of the thiophene and the structure of the ben-
zylic moiety is responsible for the AT2 selectivity of the
structures.
gands. In addition, we demonstrated that the imidazole
heterocycle can be displaced with a small non-cyclic
moiety and that high AT2 receptor selectivity and agon-
ism is retained after these modifications. In all the three
series, AT2 receptor ligands with affinity in the lower
nanomolar range were identified. None of the ana-
logues, regardless of the substituents, exhibited any
affinity for the AT1 receptor. This finding is notable con-
sidering the structural relationship to the AT1 selective
antagonist valsartan. The presence as well as the dis-
tance of a hydrogen bond accepting atom, in the amine
substituents of the first series, appears to be important
for the binding to the AT2 receptor as deduced from
the compounds 20 and 21, that lack affinity for the
AT2 receptor. Furthermore, all of the four compounds
17, 22, 39 and 51 were found to induce neurite elonga-
tion in NG108-15 cells and served as potent AT2 selec-
tive agonists.
Considering AT2 receptor affinities, the trend was that
smaller substituents were more favorable than larger
lipophilic structures. In addition, a hydrogen bond-
acceptor atom has to be present in the structure and at
the right distance from the benzylic nitrogen to obtain
any AT2 receptor affinity (cf. compounds 20 and 21).
It was therefore interesting to find that the N-methoxy-
methyl amine analogue (22, 2.8 nM) showed the highest
affinity for the AT2 receptor of the compounds in the
first series. It seems that the position of the oxygen in
the amide is not the optimal distance for the AT2
receptor.
To further study the effect of the position of the heteroa-
toms the second series, with reversed amide bonds, were
examined. Our hypothesis was that the reversed amide
might adopt the position of the oxygen in the compound
22. As seen from the resulta high affinity analogue was ob-
tained in this series (39, 7.0 nM), which displayed a slight
improvement in affinity compared to the non-switched
compound 17 (8.8 nM), although the affinity was not as
good as for the compound 22. As in the first series, the
preference in the second series was for smaller substitu-
ents, although when one or two methyl groups were re-
moved from compound 39 (compounds 40 and 43) a
large drop in affinity was encountered.
5. Experimental
5.1. Chemistry
1
5.1.1. General considerations. H and 13C NMR spectra
were recorded on a JEOL JNM-EX 270 spectrometer at
270.2 and 67.8 MHz, respectively. Chemical shifts are
given as d values (ppm) downfield from tetramethylsil-
ane. Elemental analyses were performed by Mikro Kemi
AB, Uppsala, Sweden or Analytische Laboratorien,
Lindlar, Germany. Flash chromatography was per-
formed on silica gel 60 (0.04–0.063 mm, E. Merck).
Thin-layer chromatography was performed on pre-
coated silica gel F-254 plates (0.25 mm, E. Merck) and
was visualized with UV light. Analytical RP-LC/MS
was performed on a Gilson HPLC system with a Zorbax
SB-C8, 5 lm 4.6 · 50 mm (Agilent Technologies) col-
umn, with a Finnigan AQA quadropole mass spectrom-
eter at a flow rate of 1.5 mL/min (H2O/CH3CN/0.05%
HCOOH). All the organic phases were dried over
MgSO4, unless otherwise stated. All chemicals were pur-
chased from commercial suppliers and used directly
without further purification.
With the introduction of a heterocycle in the central
phenyl position, as exemplified in the third series, the
angle of which the amide is presented to the receptor
was changed slightly and the electrostatic properties
of the aromatic ring were also altered. Interestingly,
the furanyl compound 51 (9.1 nM) was found to exert
a high affinity for the AT2 receptor whereas both the
thiophene and the pyridine were inactive. The affinity
of the furanyl compound was similar to that of the cor-
responding phenyl compound 17. This suggests that a
thienylfuran bicyclic system could provide an alterna-
tive core structure to be considered in further optimiza-
tion processes.
5.1.1.1. 3-(4-Formylphenyl)-5-iso-butyl-N-tert-butyl-
thiophene-2-sulfonamide (3). Palladium acetate (69.6 mg,
0.31 mmol) and triphenylphosphine (0.33 g, 1.24 mmol)
in THF (5 mL) were stirred for 30 min under N2(g). The
solvent was removed in vacuo and the residue was dis-
solved in DME (5 mL). The catalyst was then transferred
into a nitrogen-flushed mixture of 2 (1.11 g, 3.13 mmol), 4-
bromobenzaldehyde (1.45 g, 7.84 mmol), and K2CO3
(1.73 g, 12.5 mmol) in a solvent mixture of DME
(10 mL), ethanol (3 mL), and water (2 mL). After stirring
for 20 h at reflux under N2 atmosphere, the reaction mix-
ture was diluted with 1 M NaOH solution (20 mL) fol-
lowed by ethyl acetate (70 mL). The organic layer was
washed with water, and brine, dried over anhydrous
MgSO4, concentrated in vacuo, and the residue subjected
to flash chromatographic purification (20% ethyl acetate
in pet. ether) to afford 3 as colourless solid (0.76 g, 64%).
1H NMR (CDCl3), d: 0.97 (d, J = 6.6 Hz, 6H), 1.01 (s,
9H), 1.94 (m, 1H), 2.68 (d, J = 6.6 Hz, 2H), 4.24 (s, 1H),
To study the effect of the structural modifications on the
agonistic properties, the most potent AT2 ligands in the
three series, compounds 22, 39 and 51, and as a refer-
ence to the compounds 39 and 51, that is, compound
17, were selected for further in vitro studies to determine
if the compounds acted as AT2 agonists. Our results
demonstrate that all four compounds induce neurite
outgrowth (Figs. 2 and 3), one of the first steps of neu-
ronal differentiation, as do Ang II and the AT2 selective
peptide CGP-42112.28 Hence, as deduced from these
data the determinant for the agonistic property appears
not to be located in benzylic region of the structure.
4. Conclusion
In summary, we have presented a versatile parallel syn-
thetic route to obtain series of selective AT2 receptor li-

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A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
6.78 (s, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.95 (d, J = 6.6 Hz,
2H), 10.04 (s, 1H); 13C NMR (CDCl3), d: 22.1, 29.6, 30.5,
39.1, 54.7, 128.7, 129.6, 129.8, 135.7, 137.5, 141.0, 141.8,
149.0, 191.8; IR (compression cell), cmÀ1: 3284, 2963,
1702, 1606; Anal. Calcd for C19H25NO3S2: C, 60.13; H,
6.64, N, 3.69; O, 12.65; S, 16.9. Found: C, 59.9; H, 6.6;
N, 3.6; O, 12.4; S, 16.8.
Spectroscopic data of step 1: 1H NMR (CDCl3), d: 0.82–
1.08 (m, 15H), 1.82–2.1 (m, 2H), 2.69 (d, J= 6.9 Hz, 2H),
3.76 (d, J = 11.9 Hz, 4H), 4.08 (s, 1H), 6.77 (s, 1H),
7.23–7.48 (m, 5H), 7.44 (d, J = 7.9 Hz, 2H), 7.59 (d,
J = 8.3 Hz, 2H); 13C NMR (CDCl3), d: 22.1, 29.4,
30.5, 39.1, 52.6, 53.0, 54.4, 125.7, 127.0, 128.1, 128.3,
128.4, 128.8, 129.0, 133.6, 136.1, 139.8, 140.5, 143.0,
148.2; IR (compression cell, cmÀ1) m: 3294, 2962, 1465,
1391, 1367, 1312, 1051. Anal. (C26H34N2O2S2): C, H, N.
5.1.2. General procedure to compound 7–22
Step 1. To a solution of 3 (30 mg, 0.08 mmol) in meth-
anol (1.5 mL) taken in a sample vial (5 mL
size), amine (1.1 equiv, 0.09 mmol) was added.
After being stirred for 2 h, NaBH4 (6.1 mg,
0.16 mmol, or NaB(OAc)3H for the secondary
amines) was added and the stirring continued
for 2 h. The mixture was acidified with dilute
HCl (5 M, 0.1 mL), stirred for 10 min, neutra-
lised with saturated NaHCO3 solution
(ꢀ0.5 mL) and diluted with ethyl acetate
(10 mL). The contents were poured into diato-
maceous earth (solid–liquid extraction
catridge) in a polypropylene column (packed
for 1.5 cm, 24 mL size) and eluted with ethyl
acetate (30 mL). Concentration under vacuum
afforded the crude product.
Step 2. The preceding product was dissolved in dry
CH2Cl2 (1.5 mL) in a sample vial (5 mL size).
Triethylamine (0.033 mL, 0.24 mmol), N,N-
dimethylaminopyridine (1 mg, 0.008 mmol)
and acid chloride (2 equiv, 0.16 mmol) were
then added sequentially. The sample vial was
tightly closed. The mixture was stirred over-
night, quenched with aqueous saturated NaH-
CO3 solution (0.5 mL), stirred for 30 min, and
filtered through diatomaceous earth (packed
for 1.5 cm in the column of 24 mL capacity)
on elution with CH2Cl2 (30 mL). Concentra-
tion in vacuo afforded the crude product.
Spectroscopic data of step 2 after column chromatogra-
phy purification (pet. ether/EtOAc 7:3): 1H NMR
(CDCl3), d: 0.8–1.08 (m, 18H), 1.28–1.44 (m, 2H), 1.62–
2.0 (m, 3H), 2.36–2.50 (m, 2H), 2.63–2.73 (m, 2H), 4.25
(s, 1H), 4.47 (d, J = 3.6 Hz, 2H), 4.61 (d, J = 6.3 Hz,
2H), 6.75 (d, J = 4.3 Hz, 1H), 7.12–7.42 (m, 7H), 7.52–
7.65 (m, 2H); 13C NMR (CDCl3), d: 13.9, 22.1, 22.5,
27.5, 29.4, 30.5, 32.9, 39.1, 47.8, 48.1, 49.6, 50.1, 54.4,
126.3, 126.5, 127.4, 127.6, 128.2, 128.6, 128.9, 128.9,
129.2, 129.6, 133.9, 134.3, 136.4, 137.0, 137.3, 137.9,
142.6, 142.8, 148.3, 148.5, 173.6, 173.8; IR (compression
cell, cmÀ1) m: 3292, 2958, 1650, 1434, 1312, 1206, 1143,
1051. Anal. (C31H42N2O3S2): C, H, N.
Spectroscopic data of step 3 after column chromatogra-
phy purification (pet. ether/EtOAc 7:3): 1H NMR
(CDCl3), d: 0.91 (t, J = 7.3 Hz, 3H), 0.98 (d,
J = 6.6 Hz, 6H), 1.28–1.44 (m, 2H), 1.62–1.76 (m, 2H),
1.84–2.0 (m, 1H), 2.38–2.48 (m, 1H), 2.63–2.72 (m,
2H), 4.45–4.80 (m, 6H), 6.77 (d, J = 6.3 Hz, 1H), 7.13–
7.42 (m, 8H), 7.50–7.61 (m, 2H); 13C NMR (CDCl3),
d: 13.9, 22.2, 22.5, 27.5, 30.5, 32.9, 39.1, 48.2, 49.8,
50.5, 126.3, 126.6, 128.2, 128.6, 128.9, 129.1, 129.3,
129.5, 133.4, 133.7, 134.8, 136.4, 137.2, 137.3, 138.1,
143.3, 143.4, 148.3, 148.5, 173.7, 174.0; IR (compression
cell, cmÀ1) m: 2958, 1634, 1466, 1432, 1341, 1161. Anal.
(C27H34N2O3S2 · 1/3 H2O): C, H, N.
1
Spectroscopic data of compound 7: H NMR (CDCl3),
Step 3. The mixture of the above product and anisole
(ꢀ2 drops) in trifluoroacetic acid (1.5 mL) in a
sample vial (5 mL size) was stirred at 30 ꢁC
overnight. After the removal of the solvent in
vacuo, the residue was dissolved in acetonitrile
(2 mL) and evaporated (2·).
Step 4. To a mixture of the preceding product in
dry CH2Cl2 (1.5 mL), pyrrolidinopyridine
(17.8 mg, 0.12 mmol) and triethylamine
(0.5 mL, 0.36 mmol), n-butyl chloroformate
(0.04 mL, 0.3 mmol) were sequentially added.
The solution was stirred for 12 h, concentrated
in vacuo and the crude product was purified by
preparativeLC–MStoaffordtheproducts 7–22.
d: 0.83–1.03 (m, 12H), 1.17–1.44 (m, 6H), 1.68 (m,
2H), 1.95 (m, 1H), 2.44 (dt, J = 1.25, 8.3 Hz, 2H),
2.66–2.74 (m, 2H), 4.05 (q, J = 5.3 Hz, 2H), 4.43–4.68
(m, 4H), 6.76 (d, J = 7.9 Hz, 1H), 7.1–7.50 (m, 9H),
7.60–7.95 (br s, 1H); 13C NMR (CDCl3), d: 13.6, 13.9,
18.7, 22.2, 22.5, 27.5, 30.4, 30.5, 33.0, 39.3, 48.1, 49.7,
50.4, 66.8, 66.9, 126.4, 127.4, 127.7, 128.0, 128.3,
128.6, 128.9, 129.1, 129.5, 130.6, 133.0, 133.3, 136.3,
137.2, 137.3, 138.0, 146.1, 146.2, 150.2, 151.4, 151.6,
173.9, 174.1; IR (compression cell), cmÀ1: 2959, 2871,
1748, 1626, 1453; Anal. Calcd for C32H42N2O5S2: C,
64.18; H, 7.07; N, 4.68. Found: C, 63.8; H, 7.0; N, 4.70.
5.1.2.2. N-Butyloxycarbonyl-3-[4-(N-acetyl-N-benzyl-
aminomethyl)phenyl]-5-iso-butylthiophene-2-sulfonamide
(8). The compound 8 was synthesised from 3 in the same
fashion following the above procedure with the appro-
priate choice of amine and acid chloride (benzylamine
and acetyl chloride). The crude product in the final step
was purified by LC–MS (25% aqueous acetonitrile to
pure acetonitrile, reverse phase) to afford 8 as a colour-
5.1.2.1. N-Butyloxycarbonyl-3-[4-(N-benzyl-pentylam-
idomethyl)phenyl]-5-iso-butylthiophene-2-sulfonamide (7).
The compound 7 was synthesised from 3 in the same
fashion following the above procedure with the appro-
priate choice of amine and acid chloride (benzylamine
and valeroyl chloride). The crude product in the final
step was purified by LC–MS (30% aqueous acetonitrile
to pure acetonitrile, reverse phase) to afford 7 as a col-
ourless solid (26 mg, 55%).
1
less solid (30 mg, 68%). H NMR (CDCl3), d: 0.86 (t,
J = 7.3 Hz, 3H), 0.98 (d, J = 7.4 Hz, 6H), 1.25 (m,
2H), 1.49 (m, 2H), 1.94 (m, 1H), 2.2 (d, 3 H,

A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
7175
J = 3.6 Hz), 2.66–2.75 (m, 2H), 3.98–4.08 (m, 2H), 4.46–
4.60 (m, 4H), 6.75 (d, 1 H, J = 8.6 Hz), 7.14–7.51 (m,
9H), 8.25 (br d, 1H); 13C NMR (CDCl3), d: 13.6, 18.7,
21.5, 22.2, 29.6, 30.4, 30.5, 39.3, 48.0, 50.5, 51.1, 66.7,
66.8, 126.3, 127.4, 127.7, 128.0, 128.3, 128.6, 129.0,
129.1, 129.4, 129.5, 130.7, 133.2, 133.4, 136.0, 136.9,
137.0, 137.6, 145.9, 146.1, 150.3, 151.3, 151.5, 171.4,
171.6; IR (compression cell), cmÀ1: 2955, 1747, 1629,
1451; Anal. Calcd for C29H36N2O5S2: C, 62.56; H,
6.52; N, 5.03. Found: C, 62.8; H, 6.7; N, 5.0.
acetonitrile to pure acetonitrile, reverse phase) to afford
11 as a colourless solid (30 mg, 68%). 1H NMR
(CDCl3), d: 0.86 (t, J = 7.3 Hz, 3H), 0.98 (d,
J = 6.6 Hz, 6H), 1.24 (m, 2H), 1.48 (m, 2H), 1.82–12.1
(m, 4H), 2.33 (s, 3H), 2.69 (d, J = 7.3 Hz, 2H), 4.02 (t,
J = 6.6 Hz, 2H), 4.83 (s, 2H), 6.76 (s, 1H), 6.88 (d,
J = 8.3 Hz, 2H), 7.13 (d, J = 8.3 Hz, 2H), 7.23 (d,
J = 7.9 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 8.36 (s, 1H);
13C NMR (CDCl3), d: 13.6, 18.8, 21.1, 22.2, 22.5, 29.7,
30.5, 39.3, 52.6, 66.7, 127.8, 128.7, 128.9, 129.3, 130.2,
130.9, 133.1, 138.0, 140.0, 146.0, 150.3, 151.2, 171.1;
IR (compression cell), cmÀ1: 2960, 1747, 1636, 1513,
1465; Anal. Calcd for C29H36N2O5S2 · 1/2H2O: C,
61.6; H, 6.6; N, 4.9. Found: C, 61.7; H, 6.5; N, 4.8.
5.1.2.3.
N-Butyloxycarbonyl-3-{4-[N-(pyridin-3-yl-
methyl)-benzylamidomethyl]phenyl}-5-iso-butylthiophene-
2-sulfonamide (9). The compound 9 was synthesised
from 3 in the same fashion following the above proce-
dure with the appropriate choice of amine and acid chlo-
ride (3-picolylamine, benzoyl chloride). The crude
product in the final step was purified by LC–MS (45%
aqueous acetonitrile to pure acetonitrile, reverse phase)
to afford 9 as a colourless solid (46 mg, 92%). 1H
NMR (CDCl3), d: 0.89 (t, J=7.3 Hz, 3H), 0.97 (d,
J = 6.6 Hz, 6H), 1.30 (m, 2H), 1.55 (m, 2H), 1.92 (m,
1H), 2.68 (d, J = 6.9 Hz, 2H), 4.08 (t, J = 6.6 Hz, 2H),
4.58–4.82 (m, 4H), 6.6 (br s, 1H), 6.9–7.54 (m, 11H),
8.23–8.43 (m, 2H); 13C NMR (CDCl3), d: 13.7, 18.9,
22.2, 30.5, 30.7, 39.2, 48.1, 50.9, 53.5, 55.9, 65.9, 123.5,
123.9, 126.1, 126.5, 128.3, 128.8, 129.3, 129.7, 133.0,
134.1, 134.8, 135.9, 136.7, 139.9, 144.4, 146.5, 147.5,
147.3, 148.3, 148.4, 150.3, 152.0, 172.1; IR (compression
cell), cmÀ1: 2959, 1740, 1635, 1457, 1410; Anal. Calcd
for C33H37N3O5S2: C, 63.95; H, 6.02; N, 6.78. Found:
C, 63.6; H, 6.2; N, 6.4.
5.1.2.6. N-Butyloxycarbonyl-3-[4-(N-p-tolyl-benzyl-
amidomethyl)phenyl]-5-iso-butylthiophene-2-sulfonamide
(12). The compound 12 was synthesised from 3 in the
same fashion following the above procedure with the
appropriate choice of amine and acid chloride (p-toly-
amine, benzoyl chloride). The crude product in the final
step was purified by LC–MS (45% aqueous acetonitrile
to pure acetonitrile, reverse phase) to afford 12as a col-
ourless solid (46 mg, 92%). 1H NMR (CDCl3), d: 0.85 (t,
J = 7.6 Hz, 3H), 0.98 (d, J = 6.6 Hz, 6H), 1.24 (m, 2H),
1.48 (m, 2H), 1.93 (m, 1H), 2.23 (s, 3H), 2.69 (d,
J = 6.9 Hz, 2H), 3.99 (t, J = 6.6 Hz, 2H), 5.12 (s, 2H),
6.76 (s, 1H), 6.82 (d, J = 8.6 Hz, 2H), 6.95 (d,
J = 7.9 Hz, 2H), 7.1–7.24 (m, 3H), 7.31–7.44 (m, 6H);
13C NMR (CDCl3), d: 13.6, 18.7, 20.9, 22.3, 30.4, 39.5,
53.8, 66.8, 127.4, 127.8, 128.2, 128.8, 129.0, 129.3,
129.7, 133.0, 135.7, 136.7, 138.2, 140.8, 146.1, 146.9,
151.4, 170.6; IR (compression cell), cmÀ1: 2960, 1748,
1628, 1511, 1447; Anal. Calcd for C34H38N2O5S2: C,
65.99; H, 6.19; N, 4.53. Found: C, 65.8; H, 6.4; N, 4.3.
5.1.2.4. N-Butyloxycarbonyl-3-{4-[N-acetyl-N-(pyridin-
3-ylmethyl)-aminomethyl]phenyl}-5-iso-butylthiophene-2-
sulfonamide (10). The compound 10 was synthesised
from 3 in the same fashion following the above proce-
dure with the appropriate choice of amine and acid chlo-
ride (3-picolylamine, benzoyl chloride). The crude
product in the final step was purified by LC–MS (45%
aqueous acetonitrile to 90% acetonitrile, reverse phase)
to afford 10 as a colourless syrup (38 mg, 86%). 1H
NMR (CDCl3), d: 0.89 (t, J = 7.6 Hz, 3H), 0.97 (d,
J = 6.6 Hz, 6H), 1.26 (m, 2H), 1.53 (m, 2H), 1.93 (m,
1H), 2.27 (m, 3H), 2.68 (d, J = 6.9 Hz, 2H), 4.07 (m,
2H), 4.49 (s, 2H), 4.65 (t, J = 6.9 Hz, 2H), 6.68 (m,
1H), 6.93–7.36 (m, 6H), 7.61–8.45 (m, 2H); 13C NMR
(CDCl3), d: 13.6, 18.8, 22.2, 30.4, 30.6, 39.2, 48.2, 50.8,
52.2, 54.9, 65.8, 123.6, 127.2, 128.0, 128.5, 129.3,
129.7, 132.0, 132.5, 133.1, 133.6, 133.9, 134.7, 136.1,
136.4, 138.7, 139.9, 144.3, 145.0, 146.2, 147.4, 147.9,
148.2, 150.3, 151.6, 152.0, 170.6, 170.8, IR (compression
cell), cmÀ1: 2960, 1742, 1650, 1429; Anal. Calcd for
C28H35N3O5S2 · 1/2H2O: C, 59.30; H, 6.4; N, 7.4.
Found: C, 59.5; H, 6.1; N, 7.4.
5.1.2.7. N-Butyloxycarbonyl-3-[4-(N-acetyl-N-ethyl-
aminomethyl)phenyl]-5-iso-butylthiophene-2-sulfonamide
(13). The compound 13 was synthesised from 3 in the
same fashion following the above procedure with the
appropriate choice of amine and acid chloride (ethyla-
mine, acetyl chloride). The crude product in the final
step was purified by LC–MS (25% aqueous acetonitrile
to 90% acetonitrile, reverse phase) to afford 13 as a col-
ourless solid (18 mg, 46%). 1H NMR (CDCl3), d: 0.87 (t,
J = 7.3 Hz, 3H), 0.98 (d, 6.6 Hz, 6H), 1.08–1.34 (m, 5H),
1.50 (m, 2H), 1.94 (m, 1H), 2.14 (m, 3H), 2.69 (m, 2H),
3.38 (m, 2H), 4.04 (m, 2H), 4.58 (m, 2H), 6.75 (m, 1H),
7.14–7.60 (m, 4H); 13C NMR (CDCl3), d: 12.7, 13.6,
18.8, 21.2, 21.8, 22.2, 30.5, 39.3, 40.9, 42.9, 47.7, 51.3,
66.7, 66.8, 126.2, 127.7, 127.9, 129.2, 129.4, 130.9,
133.1, 133.4, 137.6, 138.4, 146.1, 150.4, 151.3, 151.5,
170.7; IR (compression cell), cmÀ1: 2960, 1747, 1627,
1463; Anal. Calcd for C24H34N2O5S2: C, 58.27; H,
6.93; N, 5.66. Found: C, 58.7; H, 7.1; N, 5.8.
5.1.2.5. N-Butyloxycarbonyl-3-[4-(N-acetyl-N-p-tolyl-
aminomethyl)phenyl]-5-iso-butylthiophene-2-sulfonamide
(11). The compound 11 was synthesised from 3 in the
same fashion following the above procedure with the
appropriate choice of amine and acid chloride (p-tolyl-
amine and acetyl chloride). The crude product in the fi-
nal step was purified by LC–MS (40% aqueous
5.1.2.8. N-Butyloxycarbonyl-3-[4-(N-ethyl-thiophene-
carboylaminomethyl)phenyl]-5-iso-butylthiophene-2-sul-
fonamide (14). The compound 14 was synthesised from 3
in the same fashion following the above procedure with
the appropriate choice of amine and acid chloride (eth-
ylamine, thiophenecarboxyl chloride). The crude prod-
uct in the final step was purified by LC–MS (45%

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A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
aqueous acetonitrile to 90% acetonitrile, reverse phase)
to afford 14as a colourless syrup (20 mg, 45%). 1H
NMR (CDCl3), d: 0.87 (t, J = 7.3 Hz, 3H), 0.99 (d,
J = 6.6 Hz, 6 H), 1.24 (m, 5H), 1.50 (m, 2H), 1.95 (m,
1H), 2.71 (d, J = 7.3 Hz, 2H), 3.58 (q, J = 6.9 Hz, 2H),
4.03 (t, J = 6.6 Hz, 2H), 6.78 (s, 1H), 7.02 (t,
J = 4.1 Hz, 1H), 7.29–7.50 (m, 6H); 13C NMR (CDCl3),
d: 13.6, 18.7, 22.2, 30.5, 39.3, 66.9, 126.9, 127.1, 128.6,
129.1, 129.3, 130.6, 133.2, 137.8, 146.1, 150.0, 151.6,
164.6; IR (compression cell), cmÀ1: 2960, 1748, 1604,
1436; Anal. Calcd for C27H34N2O5S3: C, 57.62; H,
6.09; N, 4.98. Found: C, 58.1; H, 6.5; N, 4.6.
to 85% acetonitrile, reverse phase) to afford 17 as a col-
ourless syrup (19 mg, 50%). H NMR (CDCl3), d: 0.81
1
(t, J = 7.3 Hz, 3H), 0.92 (d, J = 6.6 Hz, 6H), 1.18 (m,
2H), 1.44 (m, 2H), 1.87 (m, 1H), 2.09 (m, 3H), 2.65
(m, 2H), 2.90 (m, 3H), 3.97 (m, 2H), 4.52 (m, 2H),
6.69 (m, 1H), 7.17 (m, 2H), 7.39 (m, 2H); 13C NMR
(CDCl3), d: 13.6, 18.8, 21.4, 21.7, 22.2, 30.4, 30.5, 33.8,
35.8, 39.3, 50.5, 54.0, 66.7, 66.8, 126.2, 127.8, 129.2,
129.4, 129.5, 130.9, 133.2, 133.5, 137.0, 137.7, 146.0,
150.5, 151.2, 151.5, 171.2; IR (neat, cmÀ1) m 2960,
1746, 1628, 1466; Anal. Calcd for C23H32N3O5S2: C,
57.47; H, 6.71; N, 5.83. Found: C, 57.0; H, 6.7; N, 5.8.
5.1.2.9. N-Butyloxycarbonyl-3-[4-(N-methyl-pentyl-
amidomethyl)phenyl]-5-iso-butylthiophene-2-sulfonamide
(15). The compound 15 was synthesised from 3 in the
same fashion following the above procedure with the
appropriate choice of amine and acid chloride (methyl-
amine, valeroyl chloride). The crude product in the fi-
nal step was purified by LC–MS (30% aqueous
acetonitrile to 85% acetonitrile, reverse phase) to afford
15 as a colourless syrup (26 mg, 63%). 1H NMR
(CDCl3), d: 0.82–1.02 (m, 12H), 1.17–1.56 (m, 6H),
1.66 (m, 2H), 1.93 (m, 1H), 2.38 (m, 2H), 2.65–2.74
(d, J = 6.9 Hz, 2H), 2.95 (m, 3H), 4.02 (t, J = 6.6 Hz,
2H), 4.57 (s, 2H), 6.75 (m, 1H), 7.22 (m, 2H), 7.44
(m, 2H); 13C NMR (CDCl3), d: 13.6, 13.9, 18.7, 22.2,
22.6, 27.2, 27.5, 30.4, 30.5, 32.9, 33.2, 33.9, 35.2,
39.3, 50.6, 53.1, 66.7, 66.8, 126.2, 127.7, 129.1, 129.4,
130.7, 133.0, 133.3, 137.3, 138.0, 146.1, 150.3, 151.3,
151.5, 173.6, 173.7; IR (neat, cmÀ1) m 2959, 1747,
1628, 1466; Anal. Calcd for C26H38N2O5S2: C, 59.74;
H, 7.33; N, 5.36. Found: C, 59.8; H, 7.5; N, 5.5.
5.1.2.12. N-Butyloxycarbonyl-3-[4-(N-methanesulfonyl-
N-methyl-aminomethyl)phenyl]-5-iso-butylthiophene-2-sul-
fonamide (18). The compound 18 was synthesised from 3
in the same fashion following the above procedure with
the appropriate choice of amine and acid chloride
(methylamine, methansulfonyl chloride). The crude
product in the final step was purified by LC–MS (35%
aqueous acetonitrile to 70% acetonitrile, reverse phase)
to afford 18 as a colourless syrup (24 mg, 59%). 1H
NMR (CDCl3), d: 0.87 (t, J = 7.3 Hz, 3H), 0.99 (d,
J = 6.6 Hz, 6H), 1.25 (m, 2H), 1.50 (m, 2H), 1.94 (m,
1H), 2.71 (d, J = 7.3 Hz, 2H), 2.80 (s, 3H), 2.88 (s,
3H), 4.04 (t, J = 6.6 Hz, 2H), 4.34 (s, 2H), 6.77 (s,
1H), 7.35 (s, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.47 (d,
J = 8.5 Hz, 2H); 13C NMR (CDCl3), d: 13.6, 18.7,
22.2, 30.4, 30.5, 34.5, 36.0, 39.3, 53.5, 66.9, 128.2,
129.3, 129.4, 130.6, 133.8, 136.3, 146.0, 150.0, 151.7;
IR (compression cell), cmÀ1: 3208, 2960, 1749, 1458;
Anal. Calcd for C22H32N2O6S3: C, 51.14; H, 6.24; N,
5.42. Found: C, 51.0; H, 5.8; N, 5.7.
5.1.2.10. N-Butyloxycarbonyl-3-[4-(N-(ethyloxy-car-
bonyl)-N-methyl-aminomethyl)phenyl]-5-iso-butylthioph-
ene-2-sulfonamide (16). The compound 16 was
synthesised from 3 in the same fashion following the
above procedure with the appropriate choice of amine
and acid chloride (methylamine, ethyl chloroformate).
The crude product in the final step was purified by
LC–MS (45% aqueous acetonitrile to 75% acetonitrile,
reverse phase) to afford 16 as a colourless syrup
(30 mg, 74%). 1H NMR (CDCl3), d: 0.87 (t,
J = 7.3 Hz, 3H), 0.98 (d, J = 6.6 Hz, 6H), 1.17–1.30
(m, 5H), 1.49 (m, 2H), 1.94 (m, 1H), 2.70 (d,
J = 7.3 Hz, 2H), 2.87 (s, 3H), 4.02 (t, J = 6.6 Hz, 2H),
4.18 (q, J = 7.3 Hz, 2H), 4.49 (s, 2H), 6.76 (s, 1H),
7.26 (m, 2H), 7.44 (d, J = 8.3 Hz, 2H); 13C NMR
(CDCl3), d: 13.6, 14.7, 18.7, 22.2, 30.4, 30.5, 33.7, 34.3,
39.3, 52.1, 61.6, 66.8, 127.2, 127.6, 129.1, 129.4, 130.5,
133.0, 138.2, 146.2, 150.1, 151.5, 156.5, 157.0; IR (com-
pression cell), cmÀ1: 2960, 1750, 1678, 1465, 1348, 1222,
1158; Anal. Calcd for C24H34N2O6S2: C, 56.5; H, 6.7; N,
5.5. Found: C, 56.9; H, 7.1; N, 5.4.
5.1.2.13. N-Butyloxycarbonyl-3-[4-(N-formyl-N-methyl-
aminomethyl)phenyl]-5-iso-butylthiophene-2-sulfonamide
(19). The compound 19 was synthesised from 3 in the
same fashion following step 1 in the above procedure
with the appropriate choice of amine (methylamine).
To the preceding product from step
1 (32 mg,
0.08 mmol) was added ammonium formate (120 mg,
0.57 mmol) in CH3CN (1.5 mL). The reaction mixture
was refluxed for 12 h, cooled and evaporated. Work
up as in step 2 was applied to the crude product.
The preceding product was then treated as in steps 3
and 4 in the above procedure. The crude product in
the final step was purified by LC–MS (35% aqueous
acetonitrile to 75% acetonitrile, reverse phase) to afford
19 as a colourless solid (29 mg, 77%) 1H NMR
(CDCl3), d: 0.87 (t, J = 7.3 Hz, 3H), 0.98 (d,
J = 6.6 Hz, 6H), 1.24 (m, 2H), 1.50 (m, 2H), 1.94 (m,
1H), 2.69 (d, J = 6.9 Hz, 2H), 2.78 and 2.86 (s, 3H),
4.04 (td, J = 2.3 Hz, J = 6.6 Hz, 2H), 4.42 (s, 1H),
4.53 (s, 1H), 6.76 (d, J = 3.0 Hz, 1H), 7.26 (t,
J = 7.9 Hz, 2H), 7.47 (t, J = 7.9 Hz, 2H), 8.11 and
8.23 (s, 1H), 8.6–8.9 (br m, 1H); 13C NMR (CDCl3),
d: 13.6, 18.7, 22.2, 29.6, 30.4, 30.5, 34.3, 39.3, 47.6,
53.3, 66.7, 126.8, 127.3, 128.0, 129.3, 129.5, 131.0,
133.5, 133.9, 136.1, 136.3, 145.6, 145.7, 150.4, 151.3,
151.5, 163.1; IR (compression cell), cmÀ1: 2960, 1746,
1662, 1466, 1346, 1158; Anal. Calcd for C22H30N2O5S2:
C, 56.63; H, 6.48; N, 6.00. Found: C, 56.4; H, 6.59; N,
5.88.
5.1.2.11. N-Butyloxycarbonyl-3-[4-(N-acetyl-N-methyl-
aminomethyl)phenyl]-5-iso-butylthiophene-2-sulfonamide
(17). The compound 17 was synthesised from 3 in the
same fashion following the above procedure with the
appropriate choice of amine and acid chloride (methyl-
amine, acetyl chloride). The crude product in the final
step was purified by LC–MS (35% aqueous acetonitrile

A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
7177
5.1.2.14.
N-Butyloxycarbonyl-3-[4-(dimethylamino-
was added. The solution was flushed with nitrogen again
and stirred under N2 atmosphere for 30 min. The
brownish suspension was transferred to the nitrogen-
flushed mixture of 2 (0.44 g 1.25 mmol), N-(4-bromob-
enzyl)acetamide (0.71 g, 3.12 mmol) and K2CO3
(0.86 g, 6.2 mmol) in DME/H2O/EtOH (3.5–1.5–1 mL).
The mixture was refluxed overnight under N2 atmo-
sphere, washed with aqueous NaOH solution (1 M),
water and brine, dried over anhydrous MgSO4 and con-
centrated to afford the residue ,which was purified by
circular chromatography (50% acetone in pet. ether) to
afford the colourless solid as the pure product (0.43 g,
methyl)phenyl]-5-iso-butylthiophene-2-sulfonamide (20).
The compound 20 was synthesised from 3 in the same
fashion following the above procedure with the appro-
priate choice of amine (dimethylamine). The crude prod-
uct in the final step was purified by LC–MS (40%
aqueous acetonitrile to 80% acetonitrile, reverse phase)
to afford 20 as a colourless syrup (30 mg, 80%). 1H
NMR (CD3CN + D2O), d: 0.84 (t, J = 7.3 Hz, 3H),
0.97 (d, J = 6.6 Hz, 6H), 1.20 (m, 2H), 1.37 (m, 2H),
1.98 (m, 1H), 2.71 (m, 8H), 3.79 (t, J = 6.3 Hz, 2H),
4.14 (s, 2H), 6.78 (s, 1H), 7.39 (d, J = 8.3 Hz, 2H),
7.67 (d, J = 8.3 Hz, 2H); 13C NMR (CD3CN + D2O),
d: 13.2, 18.9, 21.5, 30.4, 30.9, 38.5, 42.1, 60.5, 64.4,
128.7, 129.2, 130.0, 130.4, 137.1, 139.0, 140.7, 146.7,
158.4; IR (compression cell), cmÀ1: 2958, 2476, 2159,
2025, 1663, 1465, 1273, 1091, 880, 756; Anal. Calcd
for C22H32N2O4S2 · 1/3H2O: C, 57.6; H, 7.2; N, 6.1.
Found: C, 57.6; H, 7.0; N, 6.1Anal.
65%): 1H NMR (270 MHz, CDCl3):
d 0.97 (d,
J = 6.6 Hz, 6H), 1.0 (s, 9H), 1.91 (m, 1H), 2.07 (s,
3H), 2.67 (d, J = 6.9 Hz, 2H), 4.47 (d, J = 5.6 Hz, 2H),
6.20 (br s, 1H), 6.73 (s, 1H), 7.37 (d, J = 7.9 Hz, 2H),
7.53 (d, J = 7.9 Hz, 2H); 13C NMR (67.5 MHz, CDCl3):
d 22.1, 23.2, 29.5, 30.5, 39.2, 43.3, 54.5, 127.8, 128.9,
129.3, 133.9, 136.1, 138.6, 143.1, 148.5, 170.2; IR (neat,
cmÀ1): m 3314, 2963, 1650, 1535, 1432, 1308, 1136. Anal.
Calcd for C21H30N2O3S2: C, 59.68; H, 7.16; N, 6.63.
Found: C, 59.4; H, 7.16; N, 6.0.
5.1.2.15. N-Butyloxycarbonyl-3-{4-[N-(cyanomethyl)-
N-methyl-aminomethyl]phenyl}-5-iso-butylthiophene-2-sul-
fonamide (21). The compound 21 was synthesised from 3
in the same fashion following the above procedure with
the appropriate choice of amine (methylamino-acetoni-
trile). The crude product in the final step was purified
by LC–MS (45% aqueous acetonitrile to 85% acetoni-
trile, reverse phase) to afford 21 as a colourless syrup
(20 mg, 51%). 1H NMR (CDCl3), d: 0.88 (t,
J = 7.3 Hz, 3H), 0.99 (d, J = 6.6 Hz, 6H), 1.25 (m,
2H), 1.49 (m, 2H), 1.94 (m, 1H), 2.45 (s, 3H), 2.71 (d,
J = 7.3 Hz, 2H), 3.46 (s, 2H), 3.67 (s, 2H), 4.03 (t,
J = 6.6 Hz, 2H), 6.77 (s, 1H), 7.35 (d, J = 8.3 Hz, 2H),
7.44 (d, J = 8.3 Hz, 2H); 13C NMR (CDCl3), d: 13.6,
18.7, 22.2, 30.4, 30.5, 39.3, 42.2, 44.0, 59.8, 66.8, 114.3,
129.1, 129.2, 129.3, 130.9, 133.8, 136.9, 145.9, 150.1,
151.5; IR (compression cell), cmÀ1: 2960, 1749, 1465,
1347, 1221, 1158; Anal. Calcd for C23H31N3O4S2: C,
57.84; H, 6.54; N, 8.80. Found: C, 57.8; H, 6.6; N, 8.6.
5.1.2.18. N-Butyloxycarbonyl-3-[4-(N-acetyl-amino-
methyl)phenyl]-5-iso-butylthiophene-2-sulfonamide (25).
A solution of 23 (40 mg, 0.09 mmol) and anisole
(0.1 mL) in trifluoroacetic acid (2.5 mL) was stirred for
12 h at room temperature, evaporated and the residue
reevaporated with acetonitrile (2· 6 mL). The crude
product was dissolved in CH2Cl2 (2 mL). Triethylamine
(0.037 mL,
(0.023 mL,
0.27 mmol),
0.18 mmol)
n-butyl chloroformate
and pyrrolidinopyridine
(1.5 mg, 0.009 mmol) were successively added. The mix-
ture was stirred at room temperature for 30 min, and
evaporated. The residue was purified by preparative
LC–MS on gradual elution from 40% aqueous acetoni-
trile over 30 min duration to afford the pure product as a
colourless
solid
(31 mg,
70%).
1H
NMR
(CD3COCD3 + D2O), d: 0.84 (t, J = 7.3 Hz, 3H), 0.95
(d, J = 6.6 Hz, 6H), 1.24 (m, 2H), 1.46 (m, 2H), 1.87–
2.02 (m, 4H), 2.75 (d, J = 7.3 Hz, 2H), 3.56 (br s, 2H),
3.97 (t, J = 6.3 Hz, 2H), 4.38 (s, 2H), 6.92 (s, 1H), 7.32
(d, J = 8.3 Hz, 2H), 7.47 (d, J = 8.3 Hz, 2H); 13C
NMR (CD3COCD3 + D2O), d: 12.5, 18.0, 21.0, 21.4,
29.9, 38.0, 41.8, 65.2, 126.2, 126.7, 128.5, 129.2, 131.3,
132.2, 139.2, 145.0, 149.8, 150.4, 169.7; IR (compression
cell), cmÀ1: 2954, 2496, 1748, 1612, 1458; Anal. Calcd
for C22H30N2O5S2 · 1/3H2O: C, 55.91; H, 6.54; N,
5.93. Found: C, 55.9; H, 6.6; N, 5.9Anal.
5.1.2.16.
N-Butyloxycarbonyl-3-[4-(N-methoxy-N-
methylaminomethyl)phenyl]-5-iso-butylthiophene-2-sulfon-
amide (22). The compound 22 was synthesised from 3 in
the same fashion following the above procedure with the
appropriate choice of amine (N-methoxymethylamine).
The crude product in the final step was purified by
LC–MS (45% aqueous acetonitrile to 85% acetonitrile,
reverse phase) to afford 22 as a colourless syrup
(30 mg, 79%). 1H NMR (CDCl3), d: 0.87 (t,
J = 7.3 Hz, 3H), 0.98 (d, J = 6.6 Hz, 6H), 1.25 (m,
2H), 1.49 (m, 2H), 1.95 (m, 1H), 2.64 (s, 3H), 2.71 (d,
J = 6.9 Hz, 2H), 3.40 (s, 3H), 3.81 (s, 2H), 4.02 (t,
J = 6.6 Hz, 2H), 6.78 (s, 1H), 7.43 (m, 4H); 13C NMR
(CDCl3), d: 13.6, 18.7, 22.2, 30.4, 30.5, 39.3, 44.8, 59.9,
64.2, 66.9, 128.7, 129.5, 130.5, 133.0, 138.1, 146.3,
149.9, 151.5; IR (compression cell), cmÀ1: 2958, 1751,
1436, 1347, 1159, 1047; Anal. Calcd for C22H32N2O5S2:
C, 56.4; H, 6.9; N, 6.0. Found: C, 56.8; H, 7.1; N, 5.7.
5.1.2.19. {4-[2-(tert-Butylsulfamoyl)-5-isobutyl-thio-
phen-3-yl]-phenyl}-acetic acid ethyl ester (26). Pd(OAc)2
(120 mg, 0.25 mmol) and DPPF (280 mg, 0.5 mmol)
were dissolved in DME (5 mL) under N2-bubbling.
After stirring for 20 min, the brownish suspension was
cannulated into a pre-N2-flushed mixture of 2 (2.0 g,
6.27 mmol), ethyl-4-bromophenylethanoate (1.52 g,
6.27 mmol) and CsF (2.86 g, 18.9 mmol) in DME
(20 mL). The mixture was heated to reflux under N2
atmosphere for 12 h, cooled to room temperature, and
partitioned between ethyl acetate (75 mL) and water
(25 mL). The organic layer was washed with water,
and brine, dried over anhydrous MgSO4, concentrated
5.1.2.17. 3-[4-(N-acetyl-aminomethyl)phenyl]-5-iso-butyl-
N-tert-butylthiophene-2-sulfonamide (23). To a nitrogen-
flushed solution of Pd(OAc)2 (17.5 mg, 0.078 mmol) in
DME (2 mL), triphenylphosphine (82 mg, 0.31 mmol)

7178
A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
in vacuo and the residue purified by flash chromatogra-
phy (ethyl acetate/petroleum ether, 1:7) to afford com-
pound 26 as colourless solid (2.6 g, 95%). 1H NMR
(CDCl3), d: 0.97 (m, 15H), 1.25 (t, J = 6.9 Hz, 3H),
1.91 (m, 1H), 2.67 (d, J = 7.3 Hz, 2H), 3.65 (s, 2H),
4.10–4.20 (m, 3H), 6.75 (s, 1H), 7.35 (d, J = 8.3 Hz,
2H), 7.57 (d, J = 7.9 Hz, 2H); 13C NMR (CDCl3), d:
14.1, 22.1, 29.4, 30.5, 39.2, 41.1, 54.4, 60.9, 128.8,
129.1, 129.4, 133.7, 134.5, 136.3, 142.7, 148.3, 171.2;
IR (compression cell), cmÀ1: 3290, 2961, 1738, 1513,
1368, 1312, 1144, 1050; Anal. Calcd for C22H31NO4S2:
C, 60.38; H, 7.14; N, 3.20. Found: C, 60.38; H, 7.14;
N, 3.20.
5.1.3.1.
N-Butyloxycarbonyl-3-[4-(N-butyl-N-meth-
ylcarbamoylmethyl)phenyl]-5-iso-butylthiophene-2-sulfon-
amide (30). The compound 30 was synthesised as stated
in the above general procedure using methylbutylamine.
The crude product in the final step was purified by LC–
MS (50–80% aqueous acetonitrile, reverse phase) to af-
1
ford 30 as a colourless solid (45 mg, 71%). H NMR
(CDCl3), d: 0.84–0.98 (m, 12H), 1.17–1.58 (m, 8H),
1.93 (m, 1H), 2.69 (d, J = 6.9 Hz, 2H), 2.93 and 2.99
(s, 3H), 3.29 and 3.37 (t, J = 7.9 Hz, J = 7.6 Hz, 2H),
3.68 (d, J = 3.6 Hz, 2H), 4.02 (t, J = 6.6 Hz, 2H), 6.73,
6.74 (s, 1H), 7.25 (d, J = 6.6 Hz, 2H), 7.40 (d,
J = 7.9 Hz, 2H); 13C NMR (CDCl3), d: 48.5, 48.7,
53.6, 54.8, 54.9, 57.1, 64.1, 65.3, 68.5, 70.6, 74.1, 74.9,
75.6, 82.7, 85.0, 101.4, 163.4, 163.8, 164.0, 164.2,
165.9, 167.4, 170.5, 170.7, 180.9, 185.5, 185.9; IR (com-
pression cell), cmÀ1: 2959, 1747, 1627, 1466, 1346, 1222,
1158; Anal. Calcd for C26H38N2O5S2: C, 59.7; H, 7.33;
N, 5.36. Found: C, 59.6; H, 7.1; N, 5.4.
5.1.2.20. 3-(4-Carboxymethyl-phenyl)-5-iso-butyl-N-
tert-butylthiophene-2-sulfonamide (27). A mixture of 26
(1.0 g, 2.3 mmol) and LiOH (140 mg, 5.72 mmol) in a
solvent mixture of THF/H2O/MeOH (4.5 mL, 1:1:1)
was refluxed for 3 h, acidified with dilute hydrochloric
acid (2 mL) and diluted with cold water (30 mL). The
precipitated solid was filtered and dried to afford the
pure compound 27 as colourless solid (0.93 g, 99%).
1H NMR (CD3OD), d: 0.91–1.01 (m, 15H), 1.91 (m,
1H), 2.70 (d, J = 7.3 Hz, 2H), 3.65 (s, 2H), 6.83 (s,
1H), 7.34 (d, J = 8.3 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H);
13C NMR (CD3OD), d: 22.5, 29.8, 31.8, 39.9, 41.8,
54.8, 129.8, 130.3, 130.4, 130.6, 134.8, 136.3, 137.9,
144.7, 149.4, 175.5; IR (compression cell), cmÀ1: 3283,
2963, 1709, 1310, 1144, 756; Anal. Calcd for
C20H27NO4S2: C, 58.65; H, 6.64; N, 3.42. Found: C,
58.65; H, 6.64; N, 3.42.
5.1.3.2. N-Butyloxycarbonyl-3-[4-(N-benzylcarbamoyl-
methyl)phenyl]-5-iso-butylthiophene-2-sulfonamide (31).
The compound 31 was synthesised as stated in the above
general procedure using benzylamine. The crude prod-
uct in the final step was purified by LC–MS (45–75%
aqueous acetonitrile, reverse phase) to afford 31 as a col-
ourless solid (30 mg, 46%). 1H NMR (CDCl3), d: 0.86 (t,
J = 7.3 Hz, 3H), 0.99 (d, J = 6.6 Hz, 6H), 1.24 (m, 2H),
1.47 (m, 2H), 1.93 (m, 1H), 2.69 (d, J = 7.3 Hz, 2H), 3.56
(s, 2H), 3.99 (d, J = 6.6 Hz, 2H), 4.38 (d, J = 5.9 Hz,
2H), 6.1 (br s, 1H), 6.74 (s, 1H), 7.18–7.32 (m, 7H),
7.41 (d, J = 7.9 Hz, 2H); 13C NMR (CDCl3), d: 13.6,
18.7, 22.2, 30.4, 39.2, 43.2, 66.7, 127.4, 127.5, 128.6,
129.3, 130.9, 132.9, 135.3, 138.1, 145.9, 150.4, 151.4,
170.8; IR (compression cell), cmÀ1: 2960, 1746, 1650,
1454, 1345, 1158; Anal. Calcd for C28H34N2O5S2: C,
62.0; H, 6.3; N, 5.2. Found: C, 62.06; H, 6.42; N, 5.32.
5.1.3. General procedure for compound 30–40
Step 1. To a mixture of 27 (50 mg, 0.12 mmol), EDC
(29.3 mg,
0.15 mmol),
HOBT
(26 mg,
0.15 mmol) and triethylamine (150 lL,
0.021 mmol) in DMF (2 mL), amine
(0.15 mmol) was added. The mixture was stir-
red at room temperature overnight. The reac-
tion mixture was partitioned between ethyl
acetate and ether (2.5 mL + 7.5 mL) and water
(10 mL). The organic layer was washed with
dilute hydrochloric acid, aqueous sodium
hydroxide (1 M), water and brine, dried over
anhydrous MgSO4 and evaporated to afford
the crude product, which was directly used in
the next step.
5.1.3.3. N-Butyloxycarbonyl-3-[4-(N-benzyl-N-ethyl-
carbamoylmethyl)phenyl]-5-iso-butylthiophene-2-sulfon-
amide (32). The compound 32 was synthesised as stated
in the above general procedure using ethylbenzylamine.
The crude product in the final step was purified by LC–
MS (48–77% aqueous acetonitrile, reverse phase) to af-
1
ford 32 as a colourless solid (53 mg, 77%). H NMR
(CDCl3), d: 0.86 (t, J = 7.3 Hz, 3H), 0.98 (d,
J = 6.6 Hz, 6H), 1.12 (dt, J = 1.3 Hz, J = 7.3 Hz, 3H),
1.24 (m, 2H), 1.50 (m, 2H), 1.93 (m, 1H), 2.70 (d,
J = 7.3 Hz, 2H), 3.31 and 3.45 (q, J = 7.3 Hz, 2H),
3.68 and 3.77 (s, 2H), 4.02 (t, J = 6.6 Hz, 2H), 4.55
and 4.61 (s, 2H), 6.72 and 6.74 (s, 1H), 7.14–7.44 (m,
9H); 13C NMR (CDCl3), d: 12.5, 13.6, 18.7, 22.2, 30.4,
39.2, 40.0, 40.4, 41.3, 41.8, 47.9, 50.9, 66.5, 126.2,
127.3, 127.6, 128.0, 128.5, 128.9, 129.0, 129.1, 129.4,
131.1, 132.6, 135.5, 135.6, 137.5, 145.9, 150.6, 151.0,
170.7, 171.0; IR (compression cell), cmÀ1: 2960, 1747,
1628, 1452, 1347, 1158; Anal. Calcd for C30H38N2O5S2:
C, 63.1; H, 6.71; N, 4.91. Found: C, 62.91; H, 6.73; N,
5.01.
Step 2. To a solution of the product from step 1 in tri-
fluoroacetic acid (2.5 mL) was added anisole
(0.05 mL) and the solution was stirred for
12 h at room temperature. The residue was
evaporated and reevaporated with acetonitrile
(2· 5 mL) to give the crude product.
Step 3. The crude product from step 2 was dissolved in
CH2Cl2 (2 mL). Triethylamine (0.5 mL,
0.33 mmol), n-butyl chloroformate (0.04 mL,
0.3 mmol) and pyrrolidinopyridine (1.5 mg,
0.009 mmol) were successively added. The
mixture was stirred at room temperature for
12 h and evaporated. The residue was purified
by preperative LC–MS to afford the products
30–40.
5.1.3.4. N-Butyloxycarbonyl-3-[4-(N,N-dibenzylcarbamo-
ylmethyl)phenyl]-5-iso-butylthiophene-2-sulfonamide (33).
The compound 33 was synthesised as stated in the above

A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
7179
general procedure using dibenzylamine. The crude prod-
uct in the final step was purified by LC–MS (55–85%
aqueous acetonitrile, reverse phase) to afford 33 as a col-
ourless solid (54 mg, 70%). 1H NMR (CDCl3), d: 0.84 (t,
J = 7.3 Hz, 3H), 0.97 (d, J = 6.6 Hz, 6H), 1.21 (m, 2H),
1.47 (m, 2H), 1.92 (m, 1H), 2.68 (d, J = 6.9 Hz, 2H), 3.75
(s, 2H), 3.99 (t, J = 6.6 Hz, 2H), 4.45 (s, 2H), 4.59 (s,
2H), 6.71 (s, 1H), 7.1–7.4 (m, 14H); 13C NMR (CDCl3),
d: 13.6, 18.7, 22.2, 30.37, 30.42, 39.2, 40.3, 48.4, 50.1,
66.5, 126.3, 127.4, 127.7, 128.3, 128.6, 129.0, 129.2,
129.3, 131.0, 132.7, 135.3, 136.0, 137.0, 145.9, 150.5,
151.0, 171.5; IR (compression cell), cmÀ1: 2959, 1747,
1628, 1452, 1348, 1222, 1158; Anal. Calcd for
C35H40N2O5S2: C, 66.4; H, 6.37; N, 4.43. Found: C,
66.2; H, 6.2; N, 4.4.
1.27–1.31 (m, 2H), 1.4–1.52 (m, 2H), 1.86–2.04 (m,
1H), 2.7 (d, J = 6.9 Hz, 2H), 2.9 (t, J = 5.6 Hz, 2H),
3.08 (t, J = 5.9 Hz, 2H), 3.58 (s, 2H), 3.95–4.07 (m,
4H), 6.76 (s, 1H), 7.35 (d, J = 8.3 Hz, 2H), 7.43 (d,
J = 8.3 Hz, 2H); 13C NMR (CDCl3), d: 13.6, 18.7,
22.2, 29.4, 30.4, 30.5, 39.2, 56.2, 57.3, 59.6, 66.7, 128.0,
129.0, 129.4, 130.9, 133.3, 138.5, 145.9, 150.4, 151.2;
IR (compression cell), cmÀ1: 2958, 1748, 1444, 1345,
1289, 1223, 1157; Anal. Calcd for C24H32N2O5S3: C,
54.9; H, 6.2; N, 5.3. Found: C, 55.0; H, 6.4; N, 5.2.
5.1.3.8. N-Butyloxycarbonyl-3-{4-[N-(4-methylphenyl)-
carbamoylmethyl]phenyl}-5-iso-butylthiophene-2-sulfon-
amide (37). The compound 37 was synthesised as stated
in the above general procedure using 4-tolylamine. The
crude product in the final step was purified by LC–MS
(40–70% aqueous acetonitrile, reverse phase) to afford
37 as a colourless solid (43 mg, 65%). 1H NMR
(CDCl3), d: 0.71 (t, J = 7.3 Hz, 3H), 0.86 (d, J =
6.6 Hz, 6H), 1.08 (m, 2H), 1.32 (m, 2H), 1.81 (m, 1H),
2.14 (s, 3H), 2.57 (d, J = 6.9 Hz, 2H), 3.55 (s, 2H),
3.86 (t, J = 6.6 Hz, 2H), 6.63 (s, 1H), 6.93 (d,
J = 8.3 Hz, 2H), 7.13–7.29 (m, 6H), 7.48 (br s, 1H);
13C NMR (CDCl3), d: 13.5, 18.7, 22.2, 29.8, 30.3, 30.5,
39.2, 44.1, 66.8, 120.0, 128.5, 129.3, 130.6, 132.9,
133.9, 135.2, 135.3, 146.2, 150.4, 151.6, 169.0; IR (com-
pression cell), cmÀ1: 3367, 2960, 1748, 1664, 1464, 1353,
1222, 1158; Anal. Calcd for C28H34N2O5S2: C, 62.0; H,
6.3; N, 5.2. Found: C, 61.71; H, 6.38; N, 5.09.
5.1.3.5. N-Butyloxycarbonyl-3-{4-[N-(diphenylmethyl)-
carbamoylmethyl]phenyl}-5-iso-butylthiophene-2-sulfon-
amide (34). The compound 34 was synthesised as stated in
the above general procedure using diphenylmethylamine.
The crude product in the final step was purified by LC–
MS (55–85% aqueous acetonitrile, reverse phase) to af-
1
ford 34 as a colourless solid (39 mg, 54%). H NMR
(CDCl3), d: 0.84 (t, J = 7.3 Hz, 3H), 0.95 (d, J = 6.6 Hz,
6H), 1.20 (m, 2H), 1.44 (m, 2H), 1.90 (m, 1H), 2.67 (d,
J = 7.1 Hz, 2H), 3.60–3.74 (m, 3H), 3.97 (t, J = 6.6 Hz,
2H), 6.22 (s, 2H), 6.71–6.80 (m, 1H), 6.96–7.30 (m,
12H), 7.34–7.40 (m, 2H); 13C NMR (CDCl3), d: 13.6,
18.7, 22.2, 30.4, 30.5, 39.3, 43.3, 55.2, 55.9, 56.9, 66.8,
113.6, 126.2, 127.3, 127.5, 128.2, 128.6, 129.2, 129.4,
130.3, 130.7, 133.0, 135.3, 141.2, 146.1, 150.1, 151.6,
169.7; IR (compression cell), cmÀ1: 2958, 1751, 1654,
1509,1458, 1345, 1157; Anal. Calcd for C34H38N2O5S2:
C, 65.99; H, 6.19; N, 4.53. Found: C, 66.1; H, 6.2; N, 4.5.
5.1.3.9. N-Butyloxycarbonyl-3-[4-(N-hexylcarbamoyl-
methyl)phenyl]-5-iso-butylthiophene-2-sulfonamide (38).
The compound 38 was synthesised as stated in the above
general procedure using hexylamine. The crude product
in the final step was purified by LC–MS (38–68% aque-
ous acetonitrile, reverse phase) to afford 38 as a colour-
5.1.3.6. N-Butyloxycarbonyl-3-[4-(2-morpholin-4-yl-2-
oxo-ethyl)-phenyl]-5-iso-butylthiophene-2-sulfonamide
(35). The compound 35 was synthesised as stated in the
above general procedure using morpholine. The crude
product in the final step was purified by LC–MS (38–
68% aqueous acetonitrile, reverse phase) to afford 35
1
less solid (35 mg, 54%). H NMR (CDCl3), d: 0.83–0.90
(m, 6H), 0.99 (d, J = 6.6 Hz, 6H), 1.18–1.32 (m, 8H),
1.38–1.55 (m, 4H), 1.94 (m, 1H), 2.70 (d, J = 7.3 Hz,
2H), 3.19 (q, J = 6.9 Hz, 2H), 3.54 (s, 2H), 4.03 (t,
J = 6.6 Hz, 2H), 5.65 (s, 1H), 6.75 (s, 1H), 7.28 (d,
J = 8.9 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H); 13C NMR
(CDCl3), d: 13.6, 14.0, 18.7, 22.2, 22.5, 26.5, 29.4, 30.4,
30.5, 32.38, 39.29, 39.81, 43.33, 66.8, 129.32, 130.9,
132.9, 135.6, 146.0, 150.3, 151.44, 170.7; IR (compres-
sion cell), cmÀ1: 2958, 1747, 1650, 1543, 1465, 1346,
1221, 1158; Anal. Calcd for C27H40N2O5S2: C, 60.4;
H, 7.5; N, 5.2. Found: C, 60.5; H, 7.4; N, 5.2.
as
a
colourless solid (37 mg, 59%). 1H NMR
(CDCl3 + CD3OD), d: 0.73 (t, J = 7.3 Hz, 3H), 0.85 (d,
J = 6.6 Hz, 6H), 1.04–1.18 (m, 2H), 1.29–1.41 (m, 2H),
1.72–1.88 (m, 1H), 2.48 (t, J = 4.3 Hz, 4H), 2.56 (d,
J = 6.6 Hz, 2H), 3.45 (s, 2H), 3.61 (t, J = 4.6 Hz, 4H),
3.84 (s, 2H), 6.60 (s, 1H), 7.21(d, J = 8.3 Hz, 2H), 7.33
(d, J = 8.3 Hz, 2H); 13C NMR (DMSO-d6), d: 13.6,
18.5, 22.0, 30.0, 30.5, 52.3, 60.8, 64.3, 65.0, 129.0,
129.2, 129.5, 129.8, 133.6, 134.2, 134.3, 141.7, 146.1,
146.9, 154.0, 154.3; IR (compression cell), cmÀ1: 2964,
1657, 1461, 1253, 1138, 1089, 1033; Anal. Calcd for
C25H34N2O6S2: C, 57.5; H, 6.6; N, 5.4. Found: C,
57.2; H, 6.8 ; N, 5.5.
5.1.3.10. N-Butyloxycarbonyl-3-[4-(N,N-dimethyl-
carbamoylmethyl)phenyl]-5-iso-butylthiophene-2-sulfon-
amide (39). The compound 39 was synthesised as stated
in the above general procedure using dimethylamine.
The crude product in the final step was purified by
LC–MS (45–80% aqueous acetonitrile, reverse phase)
to afford 39 as a colourless solid (38 mg, 65%). 1H
NMR (CDCl3), d: 0.87 (t, J = 7.3 Hz, 3H), 0.96 (d,
J = 6.6 Hz, 6H), 1.22 (m, 2H), 1.47 (m, 2H), 1.91 (m,
1H), 267 (d, J = 7.3 Hz, 2H), 2.93 (s, 3H), 3.01 (s, 3H),
3.67 (s, 2H), 4.0 (t, J = 6.6 Hz, 2H), 6.72 (s, 1H), 7.24
(d, J = 7.6 Hz, 2H), 7.39 (d, J = 7.9 Hz, 2H); 13C
NMR (CDCl3), d: 13.5, 18.6, 22.1, 29.7, 30.3, 35.6,
37.7, 39.1, 40.2, 66.4, 129.8, 129.1, 129.3, 130.8, 132.4,
5.1.3.7. N-Butyloxycarbonyl-3-[4-(2-thiazolidine-3-yl-
2-oxo-ethyl)-phenyl]-5-iso-butylthiophene-2-sulfonamide
(36). The compound 36 was synthesised as stated in the
above general procedure using thiazolidine. The crude
product in the final step was purified by LC–MS (38–
68% aqueous acetonitrile, reverse phase) to afford 36
1
as a colourless solid (39 mg, 62%). H NMR (CDCl3),
d: 0.87 (t, J = 7.3 Hz, 3H), 0.99 (d, J = 6.6 Hz, 6H),

7180
A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
135.3, 145.9, 150.5, 150.9, 171.1; IR (compression cell),
cmÀ1: 2959, 1638, 1500, 1393, 1313, 1144; Anal. Calcd
for C23H32N2O5S2 · 2/3H2O: C, 56.07; H, 6.82; N, 5.7.
Found: C, 55.9; H, 6.7; N, 5.8.
(0.05% HCOOH, 35 min duration) to afford the pure
product as a colourless solid (37 mg, 74%). H NMR
1
(CDCl3), d: 0.86 (t, J = 7.3 Hz, 3H), 0.98 (d,
J = 6.6 Hz, 6H), 1.26 (m, 2H), 1.51 (m, 2H), 1.94 (m,
1H), 2.70 (d, J = 6.9 Hz, 2H), 3.60 (s, 2H), 4.03 (t,
J = 6.9 Hz, 2H), 5.85 (br s, 1H), 6.1 (br s, 1H), 6.74 (s,
1H), 7.22 (d, J = 7.9 Hz, 2H), 7.42 (d, J = 7.9 Hz, 2H);
13C NMR (CDCl3), d: 13.6, 18.7, 22.2, 30.4, 39.3, 42.4,
66.6, 129.0, 129.3, 129.5, 131.3, 133.3, 134.7, 145.5,
151.1, 151.3, 174.6; IR (compression cell), cmÀ1: 3456,
3360, 2960, 1745, 1663, 1466, 1345, 1158; Anal. Calcd
for C21H28N2O5S2: C, 55.73; H, 6.24; N, 6.19. Found:
C, 55.6; H, 6.2; N, 6.0.
5.1.3.11. N-Butyloxycarbonyl-3-[4-(N-methylcarbamoyl-
(40).
methyl)henyl]-5-iso-butylthiophene-2-sulfonamide
The compound 40 was synthesised as stated in the above
general procedure using methylamine. The crude product
in the final step was purified by LC–MS (40–70% aqueous
acetonitrile, reverse phase) to afford 40 as a colourless so-
lid (37 mg, 65%). 1H NMR (CDCl3), d: 0.87 (t, J = 7.3 Hz,
3H), 0.99 (d, J = 6.6 Hz, 6H), 1.24 (m, 2H), 1.50 (m, 2H),
1.93 (m, 1H), 2.69–2.73 (m, 5H), 3.55 (s, 2H), 4.02 (t,
J = 6.6 Hz, 2H), 5.81 (br s, 1H), 6.75 (m, 1H), 7.27 (m,
2H), 7.43 (m, 2H); 13C NMR (CDCl3), d: 13.6, 18.7,
22.2, 26.5, 30.4, 30.5, 39.3, 43.1, 66.7, 129.3, 129.4,
130.9, 133.0, 135.4, 145.9, 150.4,151.4, 171.6; IR (com-
pression cell), cmÀ1: 3401, 2960, 1746, 1650, 1465, 1345,
1158; Anal. Calcd for C22H30N2O5S2: C, 56.63; H, 6.48;
N, 6.00. Found: C, 56.4; H, 6.59; N, 5.88.
5.1.4. General procedure for compound 44–46. Palladium
acetate (69.6 mg, 0.31 mmol) and DPPF (349 mg,
0.63 mmol) in DME (5 mL) were stirred for 30 min un-
der N2(g). The suspension was then cannulated into a
nitrogen-flushed mixture of
2 (1.0 g, 3.13 mmol),
K2CO3 (1.29 mg, 9.36 mmol) and bromo-aryl-carbalde-
hyde (2 equiv, 6.26 mmol) in a solvent mixture of
DME/H2O/EtOH (7:3:2 ratio, 12 mL). The reaction
mixture was refluxed for overnight, diluted with ethyl
acetate (25 mL), washed with aqueous NaOH solution
(1 M), brine, dried over anhydrous MgSO4 solution
and concentrated.
5.1.3.12. 3-[4-(Carbamoylmethyl)phenyl]-5-iso-butyl-
N-tert-butylthiophene-2-sulfonamide (41). A suspension
of Pd(0) catalyst was generated in situ by stirring a mix-
ture of Pd(OAc)2 (20.0 mg, 0.09 mmol) and triphenyl-
phosphine (95 mg, 0.36 mmol) in DME (2 mL) under
N2 atmosphere (3· vacuum and 3· nitrogen flush). After
stirring for 30 min, the suspension was introduced via
syringe into a nitrogen-flushed mixture of 2 (0.57 g,
1.8 mmol), 4-bromophenylacetamide (0.39 g, 1.8 mmol)
and K2CO3 (0.96 g, 7.2 mmol) in a solvent mixture of
DME, ethanol and water (7 + 2 + 3 mL). After stirring
for 12 h at reflux under N2 atmosphere, the reaction
mixture was diluted with 1 M NaOH solution (50 mL)
followed by ethyl acetate (150 mL). The organic layer
was washed with water, and brine, dried over anhydrous
MgSO4, concentrated in vacuo and the residue was puri-
fied by preparative LC–MS (40–80% aqueous acetoni-
trile, 35 min duration) to afford the pure product as a
5.1.4.1. 3-(5-Formylpyridin-2-yl)-5-iso-butyl-N-tert-
butylthiophene-2-sulfonamide (44). The compound 44
was synthesised from 2 in the same fashion following
the above procedure with 6-bromo-pyridine-3-carbalde-
hyde. The crude product was purified by column chro-
matography (15% EtOAc/petroleum ether) to afford 44
1
as a colourless syrup (0.78 g, 63%). H NMR (CDCl3),
d: 0.98 (d, J = 6.6 Hz, 6H), 1.33 (s, 9H), 1.93 (s, 1H),
2.71 (d, J = 6.9 Hz, 2H), 7.09 (s, 1H), 7.40 (br s, 1H),
7.76 (d, 8.3 Hz, 1H), 8.25 (dd, J = 2.0 Hz, J = 8.3 Hz,
1H), 9.07 (d, J = 2.3 Hz, 1H), 10.12 (s, 1H); 13C NMR
(CDCl3), d: 22.1, 30.0, 30.5, 39.1, 55.0, 123.7, 127.7,
129.9, 137.2, 137.8, 142.2, 148.3, 150.7, 157.2, 189.7;
IR (compression cell), cmÀ1: 2962, 1701, 1594, 1443,
1324, 1198. Anal. Calcd for C18H24N2O3S2: C, 56.82;
H, 6.36; N, 7.36. Found: C, 57.2; H, 6.5; N, 7.5.
1
colourless solid (0.1 g, 14%). H NMR (CDCl3): d 0.97
(d, 6H), 1.00 (s, 9H), 1.91 (m, 1H), 2.68 (d, J = 6.9 Hz,
2H), 3.61 (s, 2H), 4.72 (br s, 1H), 5.70 (br d, 2H), 6.74
(s, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.58 (d, J = 8.3 Hz,
2H); 13C NMR (CDCl3): d 22.1, 29.5, 30.5, 39.2, 42.9,
54.5, 128.1, 129.0, 129.5, 134.0, 135.1, 136.4, 142.8,
148.5, 173.1; IR (neat, cmÀ1): m 3296, 2960, 1670,
1311, 1142; Anal. Calcd for C20H28N2O3S2: C, H, N.
58.79; H, 6.91; N, 6.86. Found: C, 58.8; H, 7.0; N, 6.6
5.1.4.2.
3-(5-Formyl-furan-2-yl)-5-iso-butyl-N-tert-
butylthiophene-2-sulfonamide (45). The compound 45
was synthesised from 2 in the same fashion following
the above procedure with 5-bromo-furan-2-carbalde-
hyde. The crude product was purified by column chro-
matography (10% EtOAc/petroleum ether) to afford 45
1
5.1.3.13. N-Butyloxycarbonyl-3-[4-(carbamoylmeth-
yl)phenyl]-5-iso-butylthiophene-2-sulfonamide (43).
as a colourless syrup (0.73 g, 51%). H NMR (CDCl3),
A
d: 0.96 (d, J = 6.6 Hz, 6H), 1.28 (s, 9H), 1.84–1.99 (m,
1H), 2.67 (d, J = 7.3 Hz, 2H), 5.82 (s, 1H), 6.91 (d,
J = 3.8 Hz, 1H), 7.05 (s, 1H), 7.29 (d, J = 4.0 Hz, 1H),
9.61 (s, 1H); 13C NMR (CDCl3), d: 22.1, 29.9, 30.5,
39.0, 54.9, 111.3, 123.4, 126.1, 128.7, 139.7, 149.2,
151.7, 153.0, 176.9; IR (compression cell), cmÀ1: 3326,
2963, 1664, 1509, 1331, 1148, 1046. Anal. Calcd for
C17H23NO4S2: C, 55.26; H, 6.27; N, 3.79. Found: C,
55.0; H, 6.1; N, 3.7.
solution of 41 (0.045 g, 0.11 mmol) and anisole
(0.05 mL) in trifluoroacetic acid (2.5 mL) was stirred
for 12 h at room temperature, evaporated and the resi-
due reevaporated with acetonitrile (2· 5 mL). The crude
product was dissolved in CH2Cl2 (2 mL). Triethylamine
(0.05 mL, 0.33 mmol), n-butyl chloroformate (0.028 mL,
0.22 mmol)
and
pyrrolidinopyridine
(1.5 mg,
0.009 mmol) were successively added. The mixture was
stirred at room temperature for 30 min and evaporated.
The residue was purified by preparative LC–MS on
gradual elution from 40% to 80% aqueous acetonitrile
5.1.4.3. 3-(2-Formyl-thiophen-4-yl)-5-iso-butyl-N-tert-
butylthiophene-2-sulfonamide (46). The compound 46

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7181
was synthesised from 2 in the same fashion following the
above procedure with 5-bromo-furan-2-carbaldehyde.
The crude product was purified by column chromatog-
raphy (5% EtOAc/petroleum ether) to afford 46 as a col-
in the same fashion following the above procedure.
The crude product in the final step was purified by
LC–MS (35% to 70% aqueous acetonitrile) to afford
50 as a colourless syrup (26 mg, 68%). 1H NMR
(CDCl3), d: 0.87 (t, J = 7.3 Hz, 3H), 0.98 (d,
J = 6.6 Hz, 6H), 1.33 (m, 2H), 1.58 (m, 2H), 1.93 (m,
1H), 2.16 (s, 3H), 2.70 (d, J = 6.9 Hz, 2H), 2.91–2.99
(m, 3H), 4.11 (t, J = 5.3 Hz, 2H), 4.60 (s, 2H), 7.04 (m,
1H), 7.54–7.74 (m, 2H), 8.55 (m, 1H); 13C NMR
(CDCl3), d: 13.6, 18.8, 21.4, 21.7, 22.1, 30.4, 33.7, 35.9,
39.2, 48.1, 51.6, 66.7, 123.1, 123.5, 123.7, 127.7, 128.1,
131.9, 132.9, 133.5, 135.3, 137.3, 141.6, 147.2, 148.3,
150.7, 151.0, 151.2, 151.5, 170.8, 171.0; IR (compression
cell), cmÀ1: 2960, 1746, 1643, 14566, 1348, 1159, 1047;
Anal. Calcd for C22H31N3O5S2: C, 54.86; H, 6.49; N,
8.72. Found: C, 54.8; H, 6.1; N, 8.7.
1
ourless syrup (0.68 g, 61%). H NMR (CDCl3), d: 0.97
(d, J = 6.6 Hz, 6H), 2.08 (s, 9H), 1.84–2.0 (m, 1H),
2.69 (d, J = 6.9 Hz, 2H), 4.4 (s, 1H), 6.83 (s, 1H), 8.8
(d, J = 9.9 Hz, 2H), 9.96 (s, 1H); 13C NMR (CDCl3),
d: 22.1, 29.7, 30.6, 39.1, 54.9, 128.4, 133.9, 136.1,
136.2, 136.4, 136.9, 143.8, 149.2, 182.9; IR (compression
cell), cmÀ1: 3283, 2961, 1671, 1466, 1391, 1315, 1142.
Anal. Calcd for C17H23NO3S3: C, 52.96; H, 6.01; N,
3.63. Found: C, 53.1; H, 6.1; N, 3.5.
5.1.5. General procedure to compound 50–52
Step 1. To a solution of 44, 45 or 46 (30 mg,
0.08 mmol) in methanol (1.5 mL) taken in a
sample vial (5 mL size), methylamine (2 M in
methanol, 0.09 mmol) was added. After being
stirred for 2 h, sodium borohydride (6.1 mg,
0.16 mmol) was added and the stirring contin-
ued for 2 h. The mixture was acidified with
dilute HCl (5 M, 0.1 mL), stirred for 10 min,
neutralised with saturated NaHCO3 solution
(ꢀ0.5 mL) and diluted with ethyl acetate
(10 mL). The contents were poured into diato-
maceous earth (liquid–liquid extraction
catridge) in a polypropylene column (packed
for 1.5 cm, 24 mL size) and eluted with ethyl
acetate (30 mL). Concentration under vacuum
afforded the crude product.
Step 2. The preceding product was dissolved in dry
CH2Cl2 (1.5 mL) in a sample vial (5 mL size).
Triethylamine (0.033 mL, 0.24 mmol), N,N-
dimethylaminopyridine (1 mg, 0.008 mmol)
and acetyl chloride (2 equiv, 0.16 mmol) were
then added sequentially. The sample vial was
tightly closed. The mixture was stirred over-
night, quenched with aqueous saturated NaH-
CO3 solution (0.5 mL), stirred for 30 min and
filtered through diatomaceous earth (packed
for 1.5 cm in the column of 24 mL capacity)
on elution with CH2Cl2 (30 mL). Concentra-
tion in vacuo afforded the crude product.
5.1.5.2. N-Butyloxycarbonyl-3-[5-(N-acetyl-N-methyl-
aminomethyl)furan-2-yl]-5-iso-butylthiophene-2-sulfon-
amide (51). The compound 51 was synthesised from 45
in the same fashion following the above procedure.
The crude product in the final step was purified by
LC–MS (35% aqueous acetonitrile to 70% acetonitrile,
reverse phase) to afford 51 as a colourless syrup
(22 mg, 58%). 1H NMR (CDCl3), d: 0.86 (t,
J = 7.3 Hz, 3H), 0.96 (d, J = 6.6 Hz, 6H), 1.22–1.37
(m, 2H), 1.53–1.64 (m, 2H), 1.84–1.98 (m, 1H), 2.19 (s,
3H), 2.63 (d, J = 6.9 Hz, 2H), 2.99 (s, 3H), 4.10 (t,
J = 6.6 Hz, 2H), 4.58 (s, 2H), 6.33 (d, J = 3.3 Hz, 2H),
6.54 (d, J = 3.3 Hz, 1H), 6.89 (s, 1H); 13C NMR
(CDCl3), d: 13.6, 18.8, 22.2, 30.5, 35.6, 39.2, 43.9, 66.3,
109.8, 110.2, 124.9, 129.8, 131.8, 148.7, 151.2, 151.4,
173.8; IR (compression cell), cmÀ1: 2945, 1742, 1618,
1460, 1343, 1219, 1152, 1039; Anal. Calcd for
C21H30N2O6S2: C, 53.60; H, 6.43; N, 5.95. Found: C,
53.3; H, 6.5; N, 5.8.
5.1.5.3. N-Butyloxycarbonyl-3-[2-(N-acetyl-N-methyl-
aminomethyl)thiophen-4-yl]-5-iso-butylthiophene-2-sul-
fonamide (52). The compound 52 was synthesised from 46
in the same fashion following the above procedure. The
crude product in the final step was purified by LCMS
(35% aqueous acetonitrile to 70% acetonitrile, reverse
phase) to afford 52 as a colourless syrup (20 mg, 51%).
1H NMR (CDCl3), d: 0.85 (t, J = 7.6 Hz, 3H), 0.98 (d,
J = 6.6 Hz, 6H), 1.14–1.29 (m, 2H), 1.40–1.52 (m, 2H),
1.85–1.98 (m, 1H), 2.08–2.24 (m, 3H), 2.66 (d,
J = 7.3 Hz, 2H), 2.95–3.13 (m, 3H), 3.98 (t,
J = 6.27 Hz), 4.63–4.69 (m, 2H), 6.84 (s, 1H), 7.37 (s,
1H), 7.43 (s, 1H), 8.88 (s, 1H); 13C NMR (CDCl3), d:
13.6, 18.7, 21.4, 21.8, 22.2, 30.4, 30.5, 36.6, 39.3, 46.8,
49.6, 66.8, 124.5, 125.3, 126.8, 128.2, 128.9, 130.5, 133.4,
139.3, 139.6, 140.1, 150.5, 151.1, 151.5, 171.7; IR (com-
pression cell), cmÀ1: 2959, 1747, 1620, 1466, 1344, 1221,
1146, 1045; Anal. Calcd for C21H30N2O5S3: C, 51.83; H,
6.21; N, 5.76. Found: C, 51.5; H, 6.1; N, 5.5.
Step 3. The mixture of the above product and anisole
(ꢀ2 drops) in trifluoroacetic acid (1.5 mL) in a
sample vial (5 mL size) was stirred at 30 ꢁC
overnight. After the removal of the solvent in
vacuo, the residue was dissolved in acetonitrile
(2 mL) and evaporated (2·).
Step 4. To a mixture of the preceding product in dry
CH2Cl2
(1.5 mL),
pyrrolidinopyridine
(17.8 mg, 0.12 mmol) and triethylamine
(0.5 mL, 0.36 mmol), n-butyl chloroformate
(0.04 mL, 0.3 mmol) were sequentially added.
The solution was stirred for 12 h, concentrated
in vacuo and the crude purified by LC–MS to
afford the products 50–52.
5.2. Rat liver membrane AT1 receptor binding assay
Rat liver membranes were prepared according to the
method of Dudley et al.24 Binding of [125I]Ang II to
membranes was conducted in a final volume of 0.5 mL
containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl,
5.1.5.1. N-Butyloxycarbonyl-3-[5-(N-acetyl-N-methyl-
aminomethyl)pyridin-2-yl]-5-iso-butylthiophene-2-sulfon-
amide (50). The compound 50 was synthesised from 44

7182
A. M. S. Murugaiah et al. / Bioorg. Med. Chem. 15 (2007) 7166–7183
10 mM MgCl2, 1 mM EDTA, 0.025% bacitracin, 0.2%
BSA (bovine serum albumin), liver homogenate corre-
sponding to 5 mg of the original tissue weight,
Ont., Canada); [Val5]angiotensin II from Bachem
(Marina Delphen, CA, USA). Compound 61 was from
RBI (Natick, MA, USA). All other chemicals were of
grade A purity.
[
125I]Ang II (80,000–85,000 cpm, 0.03 nM ) and variable
concentrations of test substance. Samples were incu-
bated at 25 ꢁC for 2 h, and binding was terminated by
filtration through Whatman GF/B glass-fibre filter
sheets, which had been pre-soaked overnight with
0.3% polyethylamine, using a Brandel cell harvester.
The filters were washed with 3· 3 mL of Tris–HCl (pH
7.4) and transferred to tubes. The radioactivity was mea-
sured in a c-counter. The characteristics of the Ang II
binding AT1 receptor were determined by using six dif-
ferent concentrations (0.03–5 nmol/L) of the labelled
5.5. Cell culture
NG108-15 cells (provided by Drs. M. Emerit and M.
Hamon; INSERM, U. 238, Paris, France) were cultured
(passage 7–30) in DMEM with 10% foetal bovine serum
(FBS, Gibco BRL, Burlington, Ont., Canada), 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.27 Sub-
cultures were performed at subconfluency. Under these
conditions, cells express only the AT2 receptor sub-
type.27,28 Cells were stimulated once a day for 3 days
(first stimulation 24 h after plating). Cells were cultured
for three subsequent days under these conditions. For
all experiments, cells were plated at the same initial den-
sity of 4 · 104 cells/35 mm Petri dish. Cells were treated
without (control cells), with [Val5]Ang II (0.1 lM) or 50,
56 (0.1 lM), in the absence or in the presence of the
inhibitor, 61 (1 lM), an AT2 receptor antagonist (each
introduced daily with inhibitors applied 30 min prior
to Ang II or 50, 56).
[
125I]Ang II. Non-specific binding was determined in
the presence of 1 lM Ang II. The specific binding was
determined by subtracting the non-specific 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 = 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.
5.3. Porcine (pig) myometrial membrane AT2 receptor
binding assay
Myometrial membranes were prepared from porcine uteri
according to the method by Nielsen et al.25 A presumable
interference by binding to AT1 receptors was blocked by
addition of 1 lM losartan.16 Binding of [125I]Ang II to
membranes was conducted 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% bacitracin, 0.2%
BSA, homogenate corresponding to 10 mg of the original
tissue weight, [125I]Ang II (80,000–85,000 cpm, 0.03 nM)
and variable concentrations of test substance. Samples
were incubated at 25 ꢁC for 1.5 h, and binding was termi-
nated by filtration through Whatman GF/B glass-fibre fil-
ter sheets, which had been pre-soaked overnight with
0.3% polyethylamine, using a Brandel cell harvester.
The filters were washed with 3· 3 mL of Tris–HCl (pH
7.4) and transferred to tubes. The radioactivity was mea-
sured in a c-counter. The characteristics of the Ang II
binding AT2 receptor was determined by using six differ-
ent concentrations (0.03–5 nmol/L) of the labelled
[125I]Ang II. Non-specific binding was determined in
the presence of 1 lM Ang II. The specific binding was
determined by subtracting the non-specific 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 0.6 nM,
[L] = 0.057 nM). The binding data were best fitted with
a one-site fit. All determinations were performed in
triplicate.
5.6. Determination of cells with neurites
Cells were examined daily under a phase contrast micro-
scope and micrographs were taken after 3 days under
the various experimental conditions. Cells with at least
one neurite longer than a cell body were counted as po-
sitive for neurite outgrowth. At least 140 cells were
counted in three independent experiments.31
5.7. Data analysis
The data were presented as means SEM of the number
of experiments indicated in the text, each performed in
duplicate or triplicate. Statistical analyses of the data
were performed using the one-way analysis of variance
(ANOVA) test. Homogeneity of variance was assessed
by Bartlett’s test and p values were obtained from Dun-
nett’s tables.
Acknowledgments
We gratefully acknowledge support from the Swedish
Research Council (VR), the Swedish Foundation for
Strategic Research (SSF), Knut and Alice Wallenberg
Foundation (VRmedicine 8663), the Canadian Institutes
of Health Research to N.G.P. and M.D.P. (MOP
37891), and Vicore Pharma AB. N.G.P. is a holder of
a Canada Research Chair in Endocrinology of the Adre-
nal Gland.
5.4. In vitro morphological effects: General
The chemicals used in the present study were obtained
from the following sources: Dulbecco’s modified Ea-
gle’s medium (DMEM), foetal bovine serum (FBS),
HAT supplement (hypoxanthine, aminopterin, thymi-
dine), gentamycin from Gibco BRL (Burlington,
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