(2-methyl-5-(methylsulfonyl)benzoyl)guanidine Na+/H+ antiporter inhibitors

Article abstract of DOI:10.1021/jm960768n

The inhibition of the Na⁺/H⁺ exchanger during cardiac ischemia and reperfusion has been shown to be beneficial for the preservation of the cellular integrity and functional performance. The aim of the present investigation was to come up with potent and selective benzoylguanidines as NHE inhibitors for their use as an adjunctive therapy in the treatment of acute myocardial infarction. During the course of our investigations it became clear that the substitution ortho to the acylguanidine was of crucial importance for the potency of the compounds. 4-Chloro and 4-fluoro-2- methylbenzoic acids 6 and 7 were prepared using the directed ortho metalation technique with the carboxylic acid as the directing group. With the LDA/methyl iodide system the 2-methyl group could be extended to an ethyl group. 4-Alkyl groups were inserted by the palladium-catalyzed cross-coupling reaction into the 4-bromo-2-methylbenzoic acid methyl ester (20). Starting with benzoic acids 6-19, the methylsulfonyl group was introduced by a sequence of standard reactions (sulfochlorination, reduction, and methylation). 4-Aryl derivatives 6875 were synthesized by the palladium- catalyzed Suzuki reaction. A large number of nucleophilic displacement reactions in the 4-position were carried out with S-, O-, and N-nucleophiles as well as with the cyano and trifluoromethyl group. Using the ester method, acid chlorides, or Mukaiyama's procedure, the 5-(methylsulfonyl)benzoic acid derivatives were finally converted to the (5- (methylsulfonyl)benzoyl)guanidines 165-267 with excessive guanidine. In some cases nucleophilic substitutions with pyridinols and piperidine derivatives were carried out at the end of the reaction sequence with the 4-halo-N- (diaminomethylene)-5-(methylsulfonyl)benzamides. Variations in the 4-position were most reasonable, but the volume of the substituents was of crucial importance. Substitution in the 3- and particularly in the 6-position led to considerable worsening of the inhibitory effects of the Na⁺/H⁺ exchanger. The 2-methyl compounds, however, showed without exception higher in vitro activities than their respective demethyl counterparts as they are exemplified by the reference compounds 266 and 267, obviously caused by a conformational restriction of the acylguanidine chain. The development compound (2-methyl-5-(methylsulfonyl)-4-pyrrolobenzoyl)guanidine, methanesulfonate (246) is a NHE-1 subtype specific NHE inhibitor, being 27- fold more potent toward the NHE-1 than the NHE-2 isoform, 246 was found to act cardioprotectively not only when given before an experimentally induced ischemia, but also curatively after the onset of symptoms of acute myocardial infarction when given prior to the induction of reperfusion.

Full text of DOI:10.1021/jm960768n

J . Med. Chem. 1997, 40, 2017-2034
2017
(2-Meth yl-5-(m eth ylsu lfon yl)ben zoyl)gu a n id in e Na +/H+ An tip or ter In h ibitor s†
Manfred Baumgarth, Norbert Beier, and Rolf Gericke*
Preclinical Pharmaceutical Research, Merck KGaA, 64271 Darmstadt, Germany
Received November 6, 1996X
The inhibition of the Na+/H+ exchanger during cardiac ischemia and reperfusion has been
shown to be beneficial for the preservation of the cellular integrity and functional performance.
The aim of the present investigation was to come up with potent and selective benzoylguanidines
as NHE inhibitors for their use as an adjunctive therapy in the treatment of acute myocardial
infarction. During the course of our investigations it became clear that the substitution ortho
to the acylguanidine was of crucial importance for the potency of the compounds. 4-Chloro-
and 4-fluoro-2-methylbenzoic acids 6 and 7 were prepared using the directed ortho metalation
technique with the carboxylic acid as the directing group. With the LDA/methyl iodide system
the 2-methyl group could be extended to an ethyl group. 4-Alkyl groups were inserted by the
palladium-catalyzed cross-coupling reaction into the 4-bromo-2-methylbenzoic acid methyl ester
(20). Starting with benzoic acids 6-19, the methylsulfonyl group was introduced by a sequence
of standard reactions (sulfochlorination, reduction, and methylation). 4-Aryl derivatives 68-
75 were synthesized by the palladium-catalyzed Suzuki reaction. A large number of nucleophilic
displacement reactions in the 4-position were carried out with S-, O-, and N-nucleophiles as
well as with the cyano and trifluoromethyl group. Using the ester method, acid chlorides, or
Mukaiyama’s procedure, the 5-(methylsulfonyl)benzoic acid derivatives were finally converted
to the (5-(methylsulfonyl)benzoyl)guanidines 165-267 with excessive guanidine. In some cases
nucleophilic substitutions with pyridinols and piperidine derivatives were carried out at the
end of the reaction sequence with the 4-halo-N-(diaminomethylene)-5-(methylsulfonyl)-
benzamides. Variations in the 4-position were most reasonable, but the volume of the
substituents was of crucial importance. Substitution in the 3- and particularly in the 6-position
led to considerable worsening of the inhibitory effects of the Na+/H+ exchanger. The 2-methyl
compounds, however, showed without exception higher in vitro activities than their respective
demethyl counterparts as they are exemplified by the reference compounds 266 and 267,
obviously caused by a conformational restriction of the acylguanidine chain. The development
compound (2-methyl-5-(methylsulfonyl)-4-pyrrolobenzoyl)guanidine, methanesulfonate (246)
is a NHE-1 subtype specific NHE inhibitor, being 27-fold more potent toward the NHE-1 than
the NHE-2 isoform. 246 was found to act cardioprotectively not only when given before an
experimentally induced ischemia, but also curatively after the onset of symptoms of acute
myocardial infarction when given prior to the induction of reperfusion.
In tr od u ction
ATPase, ATP consumption is increased. Eventually,
because of decreased energy stores and increased Na+
influx, the intracellular Na+ concentration is markedly
increased. Cellular Na+ overload finally causes cellular
Ca2+ overload due to a coupling of the Na+ and Ca2+
concentrations via the Na+/Ca2+ exchanger. Especially
Ca2+ overload is deleterious, since it causes serious
contractile dysfunction and arrhythmias and may con-
tribute to cellular death.
Na+/H+ antiporters or exchangers are integral mem-
brane proteins which belong to a family of at least four
different protein isoforms, termed NHE-1 to NHE-4.1
They are capable of exchanging intracellular H+ for
extracellular Na+ ions. Metabolic acid is actively ex-
truded from the cells in order to maintain the cytosolic
pH within the physiological range. Biological functions
of this antiport mechanism include the regulation of
intracellular pH and cell volume as well as the trans-
Protocols for reperfusion of ischemic myocardium with
acidic media or agents that inhibit the Na+/H+ exchange
mechanism have shown to protect myocardial function
and structure.3 Commonly applied inhibitors of the
exchanger are amiloride and its 5-N-substituted deriva-
tives (Chart 1), e.g. 5-N-(ethylisopropyl)-amiloride (EIPA).
While all these compounds showed protective effects in
different models of cardiac ischemia and reperfusion,
their specificity and tolerability has been questioned in
recent studies. Nevertheless, it is encouraging that
novel, more specific inhibitors of the NHE-1 isoform
have been discovered. These are the lead compound 266
(HOE 694)4 and its more potent and more NHE-1
specific follow up compound 267 (cariporide, HOE 642).5
They seem to be well tolerated according to reports from
preclinical studies. All compounds have in common an
- 2
cellular transport of Na+ and HCO3
.
Alterations in Na+/H+ exchange have been implicated
in pathophysiological processes such as essential hy-
pertension, postischemic dysfunction, and cellular death.
The regulation of internal myocardial pH is of special
importance to the function of the heart. The resting
intracellular pH, which is typically near 7.2, can drop
dramatically during ischemia. Excessive activation of
the Na+/H+ exchange leads to a significant elevation of
Na+ influx into the endangered tissue. Since an in-
crease in cytosolic Na+ in turn activates the Na+/K+
† Dedicated to Professor Ekkehard Winterfeldt on the occasion of
his 65th birthday.
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(96)00768-6 CCC: $14.00 © 1997 American Chemical Society

2018 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Ch a r t 1. Na+/H+ Antiporter Inhibitors
Baumgarth et al.
tion of 4-bromo-2-methylbenzoic acid10 (17) failed due
to halogen-metal exchange. As expected, 3 was lithi-
ated regioselectively in the 2-fold activated 2-position.
Despite numerous variations of the standard conditions,
no complete conversion could be achieved with the
4-alkylbenzoic acids 4 and 5 and there was no useful
way to separate the mixture of the methylated and
nonmethylated acids. The raw products 6 and 7 ob-
tained were only contaminated slightly with the parent
acids (<5%) and suitable for all further reactions.
In no case did methylation occur adjacent to R4;
nevertheless, the substituent considerably influenced
the reaction. Taking into account the results of Mortier
et al.,7 increasing yields could be observed in the
following sequence: t-Bu < i-Pr < H < OMe < Cl < F.
These results seem to cohere with the strength of the
inductive effect of R4. Thus, the halogens and OMe
exhibit electron-withdrawing (F > Cl > OMe) and the
alkyl compounds (t-Bu > i-Pr) electron-donating induc-
tive effects, which should be responsible for stabilization
or destabilization of the anion in the meta position,
respectively. The strong inductive effects of R4 in the
halogen compounds enable a higher temperature of
metalation as well. In particular, the fluoro compound
7 could already be prepared at -70 °C without any
losses of yield or quality.
Sch em e 1a
Deprotonation of 2-methylbenzoic acids occurs at the
methyl group, leading to an extension of the group by a
C1 unit on quenching the dianion with MeI. 4-Chloro-
2-ethylbenzoic acid (18) was thus obtained using 2.3
equiv of LDA at -70 °C.13 The 4-bromo-2-ethylbenzoic
acid (19) could be prepared from 17 employing the same
protocol. Starting with 4-bromo-2-methylbenzoic acid
methyl ester14 (20), 4-alkyl-2-methylbenzoic acid methyl
esters 21-27 were produced by the palladium-catalyzed
cross-coupling reaction.15 Due to their compatibility
with the ester function, alkylzinc reactants were used,
prepared by transmetalation of the corresponding Grig-
nard compounds with ZnCl2. The dichloro[1,1′-bis-
(diphenylphosphino)ferrocene]palladium(II) [PdCl2(dppf)]
was a suitable catalyst in this reaction, and the esters
21-27 formed were then hydrolyzed to give the acids
10-16. Recently the Hoechst group has found that the
Negishi-Kumada type coupling can also be achieved in
the presence of an o-methylsulfonyl substituent, which
we introduced afterward, when running the reaction
under copper(I) cocatalysis.16
a
(a) s-BuLi/TMEDA, THF, MeI; (b) LDA, THF, MeI; (c) Mg,
THF, ZnBr2, PdCl2(dppf); (d) NaOH, MeOH.
acylguanidine group attached to an aromatic ring. In
the present study we have investigated the influence
of substituents on the activity of the compounds, in
particular of those in the 2- and 4-positions of the
aromatic ring, which were found to have the greatest
effects.
Ch em istr y
Higher substituted 2-alkylbenzoic acids are known,
but their availability leaves much to be desired. In most
cases the benzoic acid is generated in several steps from
the corresponding aniline via the benzonitrile deriva-
tive. However, Cu salts resulting from the Sandmeyer
reaction are unwelcome waste products in the chemical
industry. The use of directed ortho metalation6 opened
up a modern approach to higher substituted benzoic acid
derivatives, particularly when Mortier et al.7 recently
found the carboxylic acid as a suitable group for this
technique. To suppress ketone formation on quenching
with methyl iodide as electrophile, the metalation was
carried out according to the conditions given with 2.2
equiv of sec-butyllithium (s-BuLi)/N,N,N′,N′-tetra-
methyl-1,2-ethylenediamine (TMEDA) at -90 °C in
THF. By this method, which was also found to be
feasible for large-scale preparation, 4-chloro-2-methyl-
benzoic acid8 (6) could be prepared in 73% isolated yield
from 4-chlorobenzoic acid (1, Scheme 1, Table 1). While
4-fluorobenzoic acid (2) and 3,4-dichlorobenzoic acid (3)
could be handled analogously (f79 and 8), the prepara-
Starting with benzoic acids 6-19, the methylsulfonyl
group was introduced by a sequence of standard reac-
tions (Scheme 2).17 Due to potential instabilities and
reduced possibilities for purification, intermediates 28
and 29 were not characterized. Sulfochlorination with
excessive chlorosulfonic acid which required reaction
temperatures of about 140 °C, but 85 °C in the case of
the activated 4-alkylbenzoic acids 10-16, exclusively
took place in the 5-position. 4-tert-Butyl-2-methylben-
zoic acid (9) already decomposed at this temperature.
Sulfinic acids 29 were prepared by Na2SO3 reduction
of the sulfonyl chlorides 28. Using basic reaction
conditions, the following alkylations could be performed
with or without isolation of the more stable sodium
sulfinate intermediates. The methylsulfonyl compounds
could be obtained as acids 30-47 on alkylation under
aqueous/methanolic conditions (method D, Table 2), or
alternatively in the form of the methyl esters 48-60

Benzoylguanidine Na+/ H+ Antiporter Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2019
Ta ble 1. Physical Constants for Benzoic Acid Derivatives 6-16 and 21-27
compd
R1
R3
R4
method
yield, %
mp, °C
recryst solvent
PhMe
(i-Pr)2O
PhMe
Et2O
ND
petroleum ether
petroleum ether/Et2O
petroleum ether
petroleum ether/Et2O
petroleum ether
Et2O
formulaa
b
6
7
8
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
Cl
t-Bu
i-Pr
i-Pr
Et
A
A
A
A
A
B
B
B
B
B
B
B
C
C
C
C
C
C
C
73
80
40
18
49e
84
98
83
82
94
99
99
92
98
94
88
97
95
72
169-170.5
168-169
186
C8H7ClO2
C8H7FO2
c
C8H6Cl2O2
d
9
138-139
C12H16O2
C11H14O2
C11H14O2
NDf
g
10
10
11
12
13
14
15
16
21
22
23
24
25
26
27
g
89-90
81-83
93-95
132-135
59-60
131-134
66-68
oil
oil
oil
oil
oil
C10H12O2
C11H14O2
C14H18O2
C13H18O2‚0.6H2O
C12H14O2
C12H16O2
C12H16O2
C11H14O2
C12H16O2
C15H20O2
C14H20O2
C13H16O2
C13H18O2
Pr
c-C6H11
-(CH2)4Me
c-C4H7
s-Bu
petroleum ether
i-Pr
Et
Pr
c-C6H11
-(CH2)4Me
c-C4H7
s-Bu
oil
oil
a
b
d
Analyses for C, H, and Cl are within (0.4% of the expected value for the formula. Lit.8 c Lit.9 Lit.11 e The rate of conversion was
determined by HPLC. f Not done. Lit.12
g
Ta ble 2. Physical Constants for 5-Methylsulfonylbenzoic Acids 30-47
reaction
overall
yield, %
recryst
solvent
compd
R2
R3
R4
method
temperature,a °C
mp, °C
formulab
C9H9ClO4S
C9H9FO4S
C9H8Cl2O4S
C8H7FO4S
C12H16O4S
C11H14O4S‚0.25H2O
C12H16O4S
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47c
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
Et
Me
Et
F
Cl
OMe
Me
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Cl
F
Cl
F
i-Pr
Et
Pr
c-C6H11
-(CH2)4Me
c-C4H7
s-Bu
Br
Br
Cl
F
F
Cl
Cl
D
B
B
D
D
D
D
D
D
D
D
D
D
B
B
D
D
D
135-140
68
90
51
26
64
51
42
30
33
62
39
38
82
89
81
25
18
41
217-218
190-193
180-182
207-209
179-182
194-196
146-149
219-222
152-154
176-179
149-151
204-206
218-220
187-188
193-196
214-216
236-237
188-189
MeOH
EtOAc
MeCN
i-PrOH
H2O
H2O
H2O
H2O
H2O
140
85
85
85
85
85
85
85
C15H20O4S
C14H20O4S
H2O
C13H16O4S‚0.25H2O
EtOAc
i-PrOH
MeOH
H2O
C
C
13H18O4S‚0.25H2O
10H11BrO4S
120-130
140
C9H9BrO4S
C10H11ClO4S
C8H6F2O4S‚0.75H2O
C8H6ClFO4S
C9H9ClO5S
H2O
140
70
140
EtOAc
MeOH
EtOAc
C10H11ClO4S
a
b
Bath temperature of chlorosulfonation. Analyses for C, H, Cl, and S are within (0.4% of the expected value for the formula. c The
starting material was 4-chloro-2,3-dimethylbenzoic acid.18
Sch em e 2a
(method E, Table 3) using DMF as the solvent. In no
case was the formation of isomeric methyl sulfinates
observed. While the latter technique offered the ad-
vantage of a simpler way of working, the former usually
provided the higher yields. Frequently small percent-
ages of methyl benzoates were formed in addition to the
acids in method D. Alkaline hydrolyses of methyl
benzoates (method B) as well as esterifications of
benzoic acids (method F) were performed in some cases.
4-Chloro-2-methyl-3-(methylsulfonyl)benzoic acid,
which is a positional isomer of 30, could not be straight-
forwardly prepared via sulfochlorination of 6, but nitra-
tion led to a mixture of the acids 61 and 62 (Scheme 3).
The wanted benzoic acid 62 was separated as its ester
64 from the mixture with poor yield. After catalytic
a
(a) ClSO3H; (b) Na2SO3; (c) MeI, H2O/MeOH, NaOH; (d) MeI,
K2CO3, DMF; (e) NaOH, MeOH; (f) HCl/MeOH.

2020 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Baumgarth et al.
Ta ble 3. Physical Constants for 5-(Methylsulfonyl)benzoic Acid Methyl Esters 48-60
reaction
overall
yield, %
recryst
solvent
compd
R2
R3
R4
method
temperature,a °C
mp, °C
formulab
48
49
50
51
52
53
54
55
56
57
58
59c
60
Me
Me
Me
H
Et
Me
Et
H
H
Cl
H
H
H
H
H
Me
H
Cl
F
Cl
F
Br
Br
Cl
F
Cl
Me
Cl
Me
F
E
E
E
E
E
E
E
E
F
E
E
E
F
140
125
140
140
120-130
140
140
155
39
54
69
24
45
55
59
11
82
31
43
20
62
152-153
137-139
124-125
108
102-104
146-148
98-100
140
MeOH
i-PrOH
Et2O
C10H11ClO4S
C10H11FO4S‚0.25H2O
C10H10Cl2O4S
C9H9FO4S
i-PrOH
(i-Pr)2O
EtOAc
i-PrOH
EtOAc
EtOAc
MeOH
EtOH
C11H13BrO4S
C
C
10H11BrO4S
11H13ClO4S
F
C9H8F2O4S
Me
Me
Cl
Me
Cl
137
C
C
11H13ClO4S
11H14O4S
140
140
140
141-142
154-155
130
H
Me
H
C9H8Cl2O4S
C12H16O4S
C9H8ClFO4S
EtOAc
EtOAc
137
a
b
Bath temperature of chlorosulfonation. Analyses for C, H, Br, and S are within (0.4% of the expected value for the formula. c The
starting material was 2,3,4-trimethylbenzoic acid.19
Sch em e 3a
Sch em e 4a
a
(a) HNO3, HCl/MeOH; (b) H2, RaNi, MeOH; (c) NaNO2, SO2,
FeSO4, Cu, MeI, DMF.
hydrogenation (f65) the sulfinic acid could be intro-
duced using Wittig and Hoffmann’s method20 by treat-
ment of the diazonium salt with sulfur dioxide in the
presence of FeSO4 and copper metal followed by conver-
sion to the sulfone with methyl iodide.
a
(a) R4B(OH)2 or R4SnBu3; (b) RSH; (c) ROH; (d) HetH; (e)
RNH2; (f) CuCN; (g) CF3CO2K.
Further variations by cross-coupling reactions and by
nucleophilic displacements of 4-halo-5-(methylsulfonyl)-
benzoic acid derivatives of the general formula 67,
included in Tables 2 and 3 and the preparation of which
is described above, are shown in Scheme 4. Starting
with appropriately substituted arylboronic acids, 4-aryl
derivatives 68-75 were synthesized by the Pd-catalyzed
Suzuki reaction15d,21 from methyl 4-bromobenzoates,
and 76 with 2-(tributylstannyl)furan under Stille condi-
tions22 (Table 4). Substitutions were carried out with
S-nucleophiles (alkyl, aryl, hetaryl thiols, f83-93),
O-nucleophiles (alcohols and phenols, f94-111), and
N-nucleophiles (such as piperidines, imidazoles, pyra-
zoles, and anilines, f112-142). The substitution con-
ditions could be widely varied: In most cases the
nucleophile was offered in excess and a base was added
(NaH, NaOR, K2CO3). The reactions were run with or
without additional solvent, usually with free benzoic
acids as starting materials; sometimes methyl benzoates
were used (see the Experimental Section). The reactiv-
ity of halogen in 67 is increased by the presence of
activating groups (SO2Me and CO2R1) and is highest for
fluorine compounds. In some cases the esters thus
obtained were hydrolyzed after cross-coupling or, vice
versa, the benzoic acids were esterified after substitu-
tion, and such raw intermediates were not always
characterized. The cyano group was introduced with
CuCN (f143-145), and the trifluoromethylation (f146
and 147) was performed by reacting a bromo derivative
with CF3CO2K in the presence of CuI using the phase-
transfer technique.23
The important 4-amino intermediate 150 was advan-
tageously prepared in two stages (Scheme 5): Substitu-
tion with benzylamine was followed by hydrogenation.
With the use of 150, the preparation of the amino-
bridged pyridyl compound 151 was possible in the
inverse way as it was carried out in the case of 139-
142. The 4-pyrrolyl compound 153 was built up with
2,5-dimethoxytetrahydrofuran starting with the methyl
4-aminobenzoate 152. This synthesis was also applied
to the 3-pyrrolyl compounds 157 and 159 (Scheme 6).
The 3-amino compounds 156 and 158 required for that
were prepared by nitration of 30, esterification
(154f155), and catalytic hydrogenation. Using Raney
nickel (RaNi) the 4-chloro substituent was preserved
(f156), but under Pd/C catalysis and triethylamine
addition chlorine was simultaneously removed (f158).
Side-chain bromination with N-bromosuccinimide (NBS)

Benzoylguanidine Na+/ H+ Antiporter Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2021
Sch em e 5a
Sch em e 9a
a
(a) BnNH2; (b) H2, Pd/C, MeOH; (c) 2-fluoropyridine, NaH,
NMP; (d) MeOH/HCl; (e) 2,5-dimethoxytetrahydrofuran, 4-chlo-
ropyridine, HCl, dioxane.
Sch em e 6a
a
(a) 3-Hydroxypyridine, 5-chloro-3-hydroxypyridine, or 3-[(tri-
methylsilyl)oxy]pyridine; (b) 4-[(tert-butoxycarbonyl)amino]piperi-
dine, HCl, piperidine, or 3-hydroxypiperidine; (c) 4-[(trimethyl-
silyl)oxy]pyridine.
of Mukaiyama’s procedure25 were successful in cases
where the former methods failed. 4-Amino-1-piperidinyl
compounds 232-234 were prepared from the corre-
sponding Boc substances 119-121 by ester synthesis
followed by deprotection with HCl (method R). Com-
pounds 165-267 of Table 5 were characterized in the
form of the free bases, hydrochlorides, or methane-
sulfonates, respectively.
a
(a) H2SO4/HNO3; (b) HCl/MeOH; (c) H2, RaNi, MeOH; (d) 2,5-
dimethoxytetrahydrofuran, 4-chloropyridine, HCl, dioxane; (e) H2,
Pd/C, MeOH, THF, Et3N.
Sch em e 7a
Nucleophilic substitutions, performed in great num-
bers with the 4-halo-3-(methylsulfonyl)benzoic acid
derivatives 67 (Scheme 4), could also be carried out as
the final step of the reaction sequence with (4-halo-5-
(methylsulfonyl)benzoyl)guanidines 268 (Scheme 9).
Pyridinols and piperidine derivatives were used as
nucleophiles (f269-279). Analogous reaction condi-
tions were employed, but due to the basic nature of the
acylguanidines, alkaline workup was required (Table 6).
Mostly the pyridinols were reacted in the form of their
trimethylsilyl derivatives30 without additional solvents
in the presence of potassium carbonate at high temper-
ature (method T). 4-[(Trimethylsilyl)oxy]pyridine only
gave N-substitution.
a
(a) NBS, CH2Cl2, hν; (b) Et2NH, Na2CO3.
Sch em e 8a
a
(a) Guanidine, HCl, Na, MeOH; (b) guanidine, HCl, Na,
MeOH, SOCl2, 1,2-dimethoxyethane; (c) 2-chloro-1-methylpyri-
dinium iodide, guanidine, HCl, N-ethyldiisopropylamine, NMP.
Resu lts a n d Discu ssion
produced 160, which was subsequently reacted with
diethylamine (f161, Scheme 7).
The Na+/H+ antiport activity was assessed by observ-
ing the uptake of 22Na+ ions into acidified rabbit
erythrocytes; rabbit erythrocytes have been widely
used31 in investigations into the Na+/H+ exchange
activity. The EIPA-sensitive portion of the 22Na+
uptake into acidified erythrocytes was taken as the Na+/
H+ dependent 22Na+ uptake.32 All compounds were
tested as hydrochloride or methanesulfonate salts. On
the basis of the lead compounds 266 and 267 (Chart 1),
we evaluated variations of R2-R6 in the benzoylguani-
dine (Tables 5 and 6). So as not to exceed the scope of
the paper, this was restricted to the 5-methylsulfonyl
compounds. Extensive patent literature to date as well
In the last synthetic step the 5-(methylsulfonyl)-
benzoic acid derivatives, most of which are listed in
Table 4, were converted to benzoylguanidines of the
general formula 164 (Scheme 8, Table 5). Compounds
164 were synthesized by simple heating of the methyl
benzoates 162 with excessive guanidine in methanol
(method N) or from the benzoic acids 163 by reaction of
the acid chlorides with guanidine at room temperature
in good yield (method O). Guanidine base was always
freshly prepared from the hydrochloride using sodium
methoxide in methanol. The mild reaction conditions

2022 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Baumgarth et al.

Benzoylguanidine Na+/ H+ Antiporter Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2023

2024 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Baumgarth et al.

Benzoylguanidine Na+/ H+ Antiporter Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2025

2026 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Ta ble 6. (5-(Methylsulfonyl)benzoyl)guanidines of Scheme 9
Baumgarth et al.
compd sma method rt,b °C time, h
solvent
yield,c %
mp, °C
recryst solvent
formulad
IC50,e nM
27
8.8
14
75
160
25
269
270
271
272
274
275
276
277
278
279
168
T
140
140
RFg
RF
3
3
2
2
2
5
3
12
5
4.5
WSf
WS
DMF
DMF
sulfolane
WS
WS
WS
WS
WS
23
19
12
26
19
24
21
1
257-258 MeCN
273-274 H2O
208-210 EtOH
C16H18N4O4S‚0.25H2O
166 T + S
C15H16N4O4S‚2HCl‚1.5H2O
166
263
J
J
C15H15ClN4O4S
233
CH2Cl2/MeOH C14H13ClN4O4S
263 K + Rh 150
239-241 H2O
C14H21N5O3S‚H2O
168 Ki
RF
218-220 Et2O
C16H24N4O3S‚0.25H2O
168
263
168
165
K
T
T
T
100
160
130
135
225
282
MeCN
MeOH
C
C
C
C
16H24N4O4S
41
14H14N4O4S
16H18N4O4S‚0.5H2O
15H16N4O4S‚0.5H2O
710
930
75
49
61
256-258 EtOH
267-268 MeOH
a
b
d
Starting material. Reaction temperature. c Overall yield. Analyses for C, H, Cl, N, and S are within (0.4% of the expected value
for the formula. e Drug concentration to achieve half-maximal inhibition of the EIPA-sensitive 22Na+ uptake into rabbit erythrocytes. The
standard deviation of the IC50 values was 14% on the average. f Without additional solvent. Reflux temperature. The nucleophile used
g
h
was 4-[(tert-butoxycarbonyl)amino]piperidine,24 and the Boc intermediate 273 was not characterized. The free base 274 was prepared by
i
HCl treatment as described in the second part of method R followed by neutralization with NaOH. Due to the basic nature of the
acylguanidine, alkaline workup was required.
as our own research33 has shown that 5-substituents
such as acetyl, trifluoromethyl, and heterocycles can
also be introduced without any substantial loss of
activity.
stituted with 1-pyrrolyl, but the 4-position is left free.
It turned out that pyrrole in the 3-position is not as good
as in the 4-position; however, the occupied 3-position is
better than both left unsubstituted (253 vs 246 and
265).
Mod ifica tion of P osition 2 of 164. The most
striking and surprising result of our investigation is the
higher in vitro activity of the (2-methyl-5-(methylsul-
fonyl)benzoyl)guanidines in comparison with their re-
spective demethyl counterparts. Without exception, all
17 cases tested show marked improvements upon
introduction of a methyl group adjacent to the acylguani-
dine (166 vs 263, 167 vs 264, 179 vs 262, 182 vs 267,
186 vs 261, 190 vs 189, 210 vs 259, 226 vs 266, 227 vs
228, 232 vs 274, 237 vs 245, 244 vs 240, 255 vs 254,
257 vs 258, 270 vs 260, 271 vs 272, 279 vs 277). The
2-methyl compounds are more potent by a factor of at
least 2, but mostly the difference is considerably greater,
e.g. compounds 266 and 226 differ by a factor of 6.2; in
the case of the Hoechst compound 267 and its methyl
analogue 182, the factor is 13.
With one exception (278) the insertion of a 2-ethyl
group into the unsubstituted species also led to an
improvement of activity. However, with respect to the
2-methyl analogues, all ethyl compounds prepared (168,
169, 211, 269, 275, 276, 278) show a reduced inhibitory
effect on the Na+/H+ exchanger. The same is true with
other substituents introduced in the 2-position; they do
not show the inhibitory activity seen with the methyl
compounds. With the (2-halobenzoyl)- (170, 171, 188,
221, 222, 234, 235, 241, 252), (2-aminobenzoyl)- (177,
229, 230), and (2-methoxybenzoyl)guanidines (233),
similar results as with the 2-ethyl compounds were
obtained: with few exceptions the substances are more
effective than the nonmethylated counterparts. Larger
substituents such as (diethylamino)methyl (178) and
piperidinyl (231) give rise to a loss of potency.
Mod ifica tion s of P osition s 3 a n d 6. While intro-
duction of methyl ortho to the acylguanidine residue in
the 2-position leads to an improvement of in vitro
activity, a methyl group in the other ortho position
(position 6) leads to a clear worsening of this. The
activity of the respective isomers differs by factors of
52 and 250 (166 vs 176 and 270 vs 224). Various
3-substituents were introduced in the (5-(methylsulfo-
nyl)benzoyl)guanidine molecule in addition to the exist-
ing 2-methyl group: Cl (172), Me (173, 187, 223, 242,
256), NO2 (174), and 1-pyrrolyl (175). This led to a drop
in activity by on average a factor of 4. Compound 253
represents a special case: Here the 3-position is sub-
Mod ifica tion of P osition 4. Compared to the other
ring positions, the 4-position could be broadly varied.
Only those (5-(methylsulfonyl)benzoyl)guanidines hav-
ing an optimal substitution pattern (R2 ) Me, R3 and
R6 ) H) are discussed in this chapter. A relatively weak
Na+ uptake inhibition (IC50 ) 150 nM) of the unsub-
stituted compound 265 indicates that there must be a
substituent in the 4-position. Halogen substitution
(165-167) improves this value up to 1 order of magni-
tude; the potency increases from 4-fluoro to 4-bromo.
Insertion of alkyl groups (179-186) produced further
improvement of the in vitro activity. The inhibitory
effect rises from methyl to ethyl and culminates in the
isopropyl compound. To our knowledge, (4-isopropyl-
2-methyl-5-(methylsulfonyl)benzoyl)guanidine (182) with
an IC50 value of 2 nM is the most potent Na+/H+
antiporter inhibitor known to date. Larger alkyl groups
lead to a reduction in activity again, which is most
pronounced with the pentyl and cyclohexyl residues.
(4-Arylbenzoyl)guanidines 190-196 as well as the
4-(2-furyl) compound 197 are moderately active. Ring
substitution always led to a reduction in potency.
Nanomolar potencies were established with the sulfur
compounds 198-207, in particular in alkylthio subtypes
with lower alkyl groups (Me, Et, and Pr), but the
arylthio and hetarylthio compounds 203-207 were
found to be somewhat weaker. In the alkoxy series
(210, 212-215, and 208) the potency drops with grow-
ing alkyl residue, as was shown in the sulfur series. The
O-tert-butyl compound 216 does not follow this trend:
It is active in the nanomolar range whereas the hydroxy
compound 209 is only weakly active, on the other hand.
The activity of the aryloxy and hetaryloxy compounds
(217-220, 270, 271) is in the same order as that of the
alkoxy analogues, and again, the unsubstituted species
are the best of these.
In the group of acylguanidines fitted with saturated
nitrogen nucleophiles in the 4-position (225-227, 232,
236), enhanced activity of the 4-amino derivative 232
with respect to the parent 226 is noticeable while, as
expected, a 3-hydroxy group (f227) leads to a reduction
of activity. The relatively good activity of 236 is
remarkable in view of the large 4-pyrimidin-2-yl-1-
piperazinyl group as well. With the 4-(1,4-dihydro-4-

Benzoylguanidine Na+/ H+ Antiporter Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2027
methanesulfonate salt of 246. The benzene lies in the
plane of the paper and the pyrrole ring is twisted by an
angle of 40° by the vicinal methylsulfonyl group. The
acylguanidine is protonated at the amide nitrogen, the
anion of the methanesulfonic acid forming hydrogen
bridges to N2 and N3 of the acylguanidine. Another
hydrogen bridge forms a 6-membered ring between the
amino (N4) and carbonyl group (O3). The planar
acylguanidine group is also twisted by a torsion angle
of 40° with respect to the benzene ring.
Figure 1 also shows the preferential conformation of
the acylguanidine residue in the crystalline state. The
carbonyl oxygen as the smaller group points upward in
the direction of the methyl group whereas the guanidine
is aligned downward, avoiding the sphere of the 2-sub-
stituent. In those compounds bearing a 6-methyl group
(176, 224) the acylguanidine residue should adopt the
inverse orientation with the carbonyl group pointing
downward. According to biological results, this must
be an unfavorable arrangement for an approach to the
receptor site. With respect to the biological activity, the
dissolved drugs are of more relevance than the crystal
structure. Methyl groups in the ortho position consider-
ably restrict rotation of the acylguanidine, which leads
more or less to orientations discussed for the solid state.
In the compounds that are free of ortho methyl groups
(e.g. 258-267) the acylguanidine has freedom of rota-
tion around the C8/C11 bond and both conformations
are of similar probability. Consequently these com-
pounds exhibit medium activities.
Clin ica l Releva n ce. It is well-known that the Na+/
H+ exchanger constitutes a family of at least four
different isoforms (NHE-1, -2, -3, and -4), all of which
are cloned. While the NHE-1 subtype is distributed
ubiquitously throughout the body in all tissues, the
other subtypes are expressed selectively in specific
organs like the kidney or the luminal and contraluminal
wall of the small intestine. This distribution reflects
the specific functions being served by the various
isoforms, namely regulation of the intracellular pH and
cell volume by the NHE-1 subtype and Na+ uptake and
re-uptake in the gut and kidney by the NHE-2 and -3
isoforms, respectively.
F igu r e 1. X-ray crystal structure of the methanesulfonate of
compound 246.
oxo-1-pyridyl) compound 279, only modest activity was
found. The outstanding compound of the unsaturated
5-membered N-heterocycles (237-239, 243, 244, 246)
is the pyrrole 246 having an IC50 of 8 nM. The change
from the imidazole 237 to the benzimidazole 238 causes
drastic reduction in potency. In the series of acyl-
guanidines substituted with N-nucleophiles, last but not
least those types have to be discussed in which the
4-amino function forms a bridge to an aryl or hetaryl
ring (248-251). These substances can be regarded as
N-equivalents to sulfur or oxygen compounds such as
207 and 270, and again it turns out that with the
attachment of a further ring system an enhancement
in potency is possible even to the nanomolar region (247
vs 249 and 251). The cyano and trifluoromethyl com-
pounds 255 and 257 were moderately active.
Con stitu tion a l a n d Con for m a tion a l Requ ir e-
m en ts. The results given in Tables 5 and 6 clearly
show, that only minor structural variations of the parent
structures 266 and 267 are allowed. These variations
seem to be most reasonable in the 4-position. Alkyl
residues in this position show an increasing activity
with respect to their size up to isopropyl (f182). For
larger alkyl substituents a decrease can be observed.
The space-filling nature of the alkyl group is of crucial
importance. This relationship is inherent with other
groups as well. Thus, substituents such as fluoro and
hydroxy (f165 and 209) obviously are too small for
optimal receptor binding. On the other hand, we have
shown that residues in the 4-position with an extended
spacial requirement led to decreased potency, for ex-
ample when introducing additional substituents in
phenyl or heterocyclic rings. As a measure of the size
of the substituents, their molar refraction34 (MR) was
used and a MR range 10-26 was found to give the best
results (f180-183, 185, 198-200, 246, 251, 270).
An interesting result of this study lies in the com-
pletely different effects caused by methyl groups ortho
to the acylguanidine. Whereas small groups in the
2-positionsmethyl being the optimumsalways increase
activity, 6-substitution led to considerable worsening.
This gives rise to the supposition that the conformation
of the acylguanidine chain required at the receptor site
is influenced in a benefical way by the 2-methyl group.
Figure 1 shows the X-ray crystal structure of the
For an NHE inhibitor to be developed as a cardiopro-
tective agent, it is mandatory for it to be a NHE-1
subtype specific one.35 On the one hand, the NHE-1
isoform is the only one to be expressed in the heart;
furthermore, it is this subtype which is activated during
ischemic episodes. On the other hand, it is of interest
to avoid gastrointestinal and renal complications. We
therefore characterized the Na+/H+ antiport inhibitors
with regard to their isoform selectivity by investigating
a selected group of compounds toward the inhibition of
the NHE isoforms 1-3. The various isoforms were
stably expressed in mouse fibroblast cell lines. The
inhibitory potency of the compounds were accessed by
determining the EIPA-sensitive 22Na+ uptake into the
cells after intracellular acidosis.1
We observed for our developmental compound 246
(EMD 96785) half-maximal inhibitory concentrations
(IC50) of 10 nM for the NHE-1, 270 nM for the NHE-2,
and 700 000 nM for the NHE-3 isoforms. Thus, com-
pound 246 was about 27-fold more potent toward the
NHE-1 than the NHE-2 isoform. Moreover, 246 was
also more potent than the reference compound 267, for

2028 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Baumgarth et al.
on a Bruker IFS 48 IR spectrophotometer and a Bruker AC
200 or WM 250 (TMS as internal standard), respectively.
Crystal data were collected on a Enraf-Nonius CAD4 diffrac-
tometer with graphite-monochromated Mo KR radiation. Mi-
croanalyses were obtained with an elementar Analysensyste-
me CHN-O-RAPID analyzer. Precoated silica gel 60 F254
plates from Merck KGaA, Darmstadt, Germany, were used for
thin-layer chromatography.
which Counillon et al. reported apparent Ki values of
50 nM for the NHE-1 and 3000 nM for the NHE-2
isoforms.5 In our experiments we found respective IC50
values of 20 and 650 nM for this compound. In addition
to that we could confirm the fact that amiloride does
not differentiate between the NHE-1 and NHE-2 sub-
types.36 Therefore, amiloride-derived NHE inhibitors,
and in particular amiloride itself, do not fulfill the
prerequisite of being selective and specific NHE-1
inhibitors.
246 was able to reduce ischemia-induced arrhythmia,
after both intravenous and oral administration. It was
also found to reduce ischemia-induced myocardial dam-
age in animal studies. Importantly the compound was
not only effective when given prior to the experimentally
induced ischemia but also when infused intravenously
shortly before the re-initialization of the blood flow to
the ischemic area.37
The therapeutic applicability of such a selective
NHE-1 inhibitor appears to be manifold: Compound 246
thus seems to be useful not only preventively when
given before the onset of ischemia, but also curatively
after the onset of symptoms of acute myocardial infarc-
tion, adjunctively to percutaneous transluminal coro-
nary angioplasty (PTCA) or thrombolysis. In addition,
NHE inhibitors are expected to inhibit or diminish
tissue damage and cell necrosis after pathophysiological
hypoxic and ischemic episodes as might occur in the
heart during angina pectoris attacks or in the central
nervous system. Furthermore, NHE inhibitors are
believed to be protective during short-term hypoperfu-
sion of organs during open-chest angioplastic vessel or
cardiac surgery as well as organ transplantation. The
antiproliferative efficacy of NHE inhibitors may also
indicate that the Na+/H+ exchanger plays a role in
various pathologies like arteriosclerosis, pulmonary
hypertension, insulin-dependent diabetes, tumor growth,
fibrotic diseases, and organ/cell hypertrophy or hyper-
plasia. Furthermore, NHE inhibitors might turn out
to be of diagnostic usefulness in diseases which are
characterized by an exaggerated activity of the Na+/H+
exchanger in blood cells, e.g. erythrocytes, platelets, or
leukocytes.
Meth od A. 4-Ch lor o-2-m eth ylben zoic Acid 8 (6). A 2-L,
three-necked, round-bottomed flask was equipped with a
mechanical stirrer, a pressure-equalizing dropping funnel with
drying tube, and a Claisen head fitted with a low-temperature
thermometer and a nitrogen inlet. The flask was charged with
TMEDA (99.6 mL, 660 mmol) and dry THF (600 mL), flushed
with N2, and cooled down to -90 °C using an external bath of
EtOH/liquid N2. Then consecutively s-BuLi (471 mL, 1.4 M
cyclohexane solution, 660 mmol) and 4-chlorobenzoic acid (1;
47.0 g, 300 mmol) dissolved in THF (400 mL) were added
dropwise, maintaining temperature and N2 introduction, and
the orange solution was stirred for an additional 1 h. MeI (80
mL, 1.28 mol) was dropped in at -80 °C, and the mixture was
stirred for an additional 10 min at this temperature. After
removal of the cold bath, H2O (600 mL) was slowly added and
the mixture warmed to room temperature. The layers were
separated, the aqueous phase was washed with Et2O (2 × 500
mL), and the organic phases were discarded. The aqueous
phase was acidified with 25% aqueous HCl (600 mL), and the
crystals were allowed to separate first with stirring in an ice
bath for 2 h and then on standing in a refrigerator overnight.
The precipitate was separated, washed with H2O, dried in a
vacuum oven at 75 °C, and recrystallized from toluene (350
mL) to give compound 6 (37.3 g, 73%). An analytical sample
was prepared by repeated recrystallization from toluene: mp
169-170.5 °C.
Meth od B. 4-Isop r op yl-2-m eth ylben zoic Acid 12 (10). A
mixture of 4-isopropyl-2-methylbenzoic acid methyl ester (21;
30.8 g, 160 mmol), 20% aqueous NaOH (225 mL, 6.75 mol),
and MeOH (150 mL) was stirred at 65 °C for 4 h. The MeOH
portion was evaporated, and the resulting aqueous phase was
acidified with HCl and extracted with EtOAc (2 × 300 mL).
The combined organic phases were washed with H2O (200 mL),
dried, evaporated, and crystallized from Et2O to give the
desired product as white solid (24 g, 84%). An analytical
sample was prepared by recrystallization from petroleum
ether: mp 89-90 °C.
4-Br om o-2-eth ylben zoic Acid (19). A 2-L, three-necked
flask equipped with a mechanical stirrer, dropping funnel,
drying tube, N2 inlet, and a thermometer was charged with
dry THF (700 mL) and diisopropylamine (70 mL, 498 mmol).
After the mixture was cooled to 0 °C, butyllithium (280 mL,
1.6 M hexane solution, 459 mmol) and 4-bromo-2-methylben-
zoic acid10 (17, 43 g, 200 mmol) dissolved in THF (300 mL)
were slowly added, and the mixture was stirred for an
additional 15 min at this temperature. MeI (52 mL, 832 mmol)
was added dropwise at -70 °C, and the mixture was allowed
to warm to 0 °C. After the reaction was quenched with H2O
(150 mL) the mixture was poured into H2O (800 mL). The
organic phase was separated and washed with H2O (250 mL).
The combined aqueous phases were acidified with HCl,
extracted with EtOAc (3 × 200 mL), dried, and evaporated to
dryness to give compound 19 (42.9 g, 94%). An analytical
sample was prepared by recrystallization from (i-Pr)2O: mp
Con clu sion
For the preparation of benzoylguanidine precursors,
the ortho metalation technique was advantageously
used with the carboxylic acid as the directing group. The
2-methyl species of this class of compounds were con-
siderably more active in the Na+/H+ exchange inhibition
in vitro assay than their respective demethyl analogues,
and they were characterized as the most potent inhibi-
tors known to date. The new compounds belong to the
subtype-1 specific NHE inhibitors and are thus predes-
tined for cardiac indications. The development of 246
as a cardioprotective agent has been initiated recently.
The compound is designated for use in acute infarction
as well as for preventive treatment under conditions of
angina pectoris. The action of the Na+/H+ antiporter
inhibitors in the treatment of cardiac ischemia is based
on a novel therapeutic principle. Further applications
of these drugs are within the realm of possibility.
1
106-108 °C; H NMR (DMSO-d6) δ 1.16 (t, 7.5, 3H), 2.92 (q,
7.4, 2H), 7.48 (dd, 8.4, 2.0, 1H), 7.54 (d, 1.8, 1H), 7.71 (d, 8.5,
1H), 13.0 (s br, 1H). Anal. (C9H9BrO2) C, H, Br.
Meth od C. 4-Isop r op yl-2-m eth ylben zoic Acid Meth yl
Ester (21). A 250-mL, three-necked flask was charged with
Mg (4.25 g, 175 mmol) and THF (10 mL). Under an N2
atmosphere, the Grignard reaction was started by adding a
first portion of i-PrBr (16.4 mL, 175 mmol) in THF (75 mL)
and a few drops of CH2Br2 with stirring and gentle heating.
The heating bath was removed, and the rest of i-PrBr was
dropped in at such a rate that the internal temperature
remained at 45 °C. The reaction mixture was stirred for an
additional 1 h at room temperature. In a 500-mL three-necked
flask was dissolved ZnBr2 (39.1 g, 174 mmol) in dry THF (130
Exp er im en ta l Section
Melting points were determined with a Bu¨chi 535 melting
point apparatus and are uncorrected. IR and NMR spectra
are in agreement with the structures cited and were recorded

Benzoylguanidine Na+/ H+ Antiporter Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2029
mL) with stirring and under N2 protection. On the mixture
cooling below 0 °C, the Grignard solution was added dropwise.
A precipitate formed on exothermic reaction, and stirring was
continued for an additional 15 min at 0 °C. Then the reaction
mixture was cooled down to -78 °C, and PdCl2(dppf) (584 mg,
0.8 mmol) and 4-bromo-2-methylbenzoic acid methyl ester14
(20 g, 87.3 mmol) were added consecutively. The cooling bath
was removed, and the reaction mixture was stirred overnight.
After the reaction was quenched with 10% aqueous HCl (200
mL), the THF portion of the mixture was evaporated. The
remaining aqueous phase was extracted with Et2O (3 × 250
mL), and the combined organic phases were washed with H2O
(4 × 200 mL), dried, and evaporated. The residue was purified
by means of a Kugelrohr distillation (2 mbar, 105 °C bath
temperature) to give 21 (15.5 g, 92%) as a colorless oil: NMR
(DMSO-d6) δ 1.20 (d, 7.0, 3H), 2.51 (s, 3H), 2.90 (sept, 6.9, 1H),
3.80 (s, 3H), 7.17 (m, 1H), 7.19 (m, 1H), 7.77 (m, 1H). Anal.
(C12H16O2) C, H.
Meth od D. 4-Ch lor o-2-m eth yl-5-(m eth ylsu lfon yl)ben -
zoic Acid (30). On cooling with an ice bath, 6 (165 g, 967
mmol) was added portionwise to chlorosulfonic acid (488 mL,
7.3 mol) at such a rate that the internal temperature remained
at 20 °C. The resultant mixture was heated at 135-140 °C
bath temperature for 6 h. After cooling, the reaction mixture
was added dropwise to stirred ice water (3.5 L), and stirring
was continued for an additional 30 min at 10 °C. The
precipitate was collected by filtration and washed with ice
water (100 mL) to give crude 4-chloro-5-(chlorosulfonyl)-2-
methylbenzoic acid which was used directly in the next
reaction.
The moist chlorosulfonyl compound was added in portions
to a solution of Na2SO3 (308 g, 2.44 mol) in H2O (600 mL) at
15-20 °C. By addition of 32% aqueous NaOH (∼230 mL) the
pH was adjusted to 9.0. Stirring was continued for an
additional 3 h, and then the mixture was left to stand
overnight at room temperature. In an ice bath the mixture
was acidified to pH 1 using 25% aqueous HCl. The 2-chloro-
4-methyl-5-carboxybenzenesulfinic acid which precipitated was
filtered, washed with ice water (100 mL), and used in the next
step without further purification.
The moist sulfinic acid was placed in a 4-L, three-necked
flask to which was added H2O (600 mL), MeOH (800 mL), MeI
(400 mL, 6.4 mol), and sufficient 32% aqueous NaOH to attain
a pH of 9. The reaction mixture was refluxed for 30 h with
occasional addition of NaOH to maintain pH 9. The MeOH
was distilled at reduced pressure, and ice water (2 L) was
added to give a precipitate of 4-chloro-2-methyl-5-(methylsul-
fonyl)benzoic acid methyl ester (48, 18 g, 7% overall): mp 152-
153 °C (MeOH); NMR (DMSO-d6) δ 2.61 (s, 3H), 3.37 (s, 3H),
3.88 (s, 3H), 7.79 (s, 1H), 8.42 (s, 1H). Anal. (C10H11ClO4S)
C, H, Cl, S.
white solid: mp 137-139 °C; NMR (DMSO-d6) δ 2.63 (s, 3H),
3.34 (s, 3H), 3.87 (s, 3H), 7.58 (d, 11.2, 1H), 8.28 (d, 7.3, 1H).
Anal. (C10H11FO4S‚0.25H2O) C, H, S.
Meth od E. 4-Br om o-2-m eth yl-5-(m eth ylsu lfon yl)ben -
zoic Acid Meth yl Ester (53). Analogously to method D,
4-bromo-2-methylbenzoic acid10 (17, 1.0 kg, 4.65 mol) was
sulfochlorinated with ClSO3H (930 mL, 14.0 mol) at 140 °C
bath temperature for 3 h, and then reduced with Na2SO3 (1.32
kg, 10.5 mol) to yield the crude 2-bromo-4-methyl-5-carboxy-
benzenesulfinic acid. On cooling, this was suspended in DMF
(6 L), and K2CO3 (1.59 kg, 11.5 mol) was added within 15 min,
followed by MeI (656 mL, 10.5 mol) over a period of 1 h with
stirring, and stirring was continued overnight at room tem-
perature. DMF was removed under reduced pressure, and the
residue was treated with H2O (10 L), filtered off, and washed
with H2O (2 L). After this procedure was repeated, the
obtained crystals were air-dried and recrystallized from EtOAc
(4 L) to give 53 (606 g, 55% overall): mp 146-148 °C; NMR
(DMSO-d6) δ 2.60 (s, 3H), 3.78 (s, 3H), 3.88 (s, 3H), 7.98 (s,
1H), 8.44 (s, 1H). Anal. (C10H11BrO4S) C, H, Br, S.
Meth od F . 4-Ch lor o-2,3-d im eth yl-5-(m eth ylsu lfon yl)-
ben zoic Acid Meth yl Ester (56). 4-Chloro-2,3-dimethyl-5-
(methylsulfonyl)benzoic acid (47, 20 g, 76.1 mmol) was treated
with saturated HCl/MeOH (150 mL) overnight at room tem-
perature and heated under reflux for an additional 20 h. The
solution was concentrated, and the residue was taken up in
EtOAc (150 mL), washed with saturated NaHCO3 solution (2
× 75 mL) and H2O (75 mL), dried, filtered, and concentrated
to a small volume. The desired product was collected by
filtration as yellow crystals and dried at 90 °C (17.3 g, 82%):
mp 137 °C; NMR (DMSO-d6) δ 2.45 (s, 3H), 2.54 (s, 3H), 3.38
(s, 3H), 3.89 (s, 3H), 8.21 (s, 1H). Anal. (C11H13ClO4S) C, H,
Cl, S. Esterifications of compounds 93 and 138 were carried
out with MeI and K2CO3 in DMF as described in method E.
4-Ch lor o-2-m eth yl-3-n itr oben zoic Acid Meth yl Ester
(64). To stirred HNO3 (100%, 80 mL) was added 4-chloro-2-
methylbenzoic acid (6, 20 g, 117 mmol) in portions at 5-10
°C. After an additional 1-h period of stirring at 0-5 °C, the
mixture was poured onto ice, and the yellow precipitate was
collected and washed with H2O (100 mL). The moist substance
was taken up with EtOAc (250 mL) and dried with Na2SO4.
After concentration of the filtered solution to half of the
volume, a precipitate (9 g) consisting mainly of 61 was
separated and discarded. The evaporated mother liquor (15
g) consisting of a mixture of acids 61 and 62 was treated with
saturated HCl/MeOH (150 mL) overnight at room temperature
and heated under reflux for an additional 6 h. The solution
was concentrated, mixed with NaHCO3 solution (250 mL),
extracted with EtOAc (2 × 150 mL), dried, and filtered, and
the solvent was removed to yield a yellowish crystalline crop
(15.2 g). This was chromatographed on silica gel (400 g,
petroleum ether/t-BuOMe 97.5 : 2.5). The homogeneous
nonpolar fractions were combined to give 4-chloro-2-methyl-
3-nitrobenzoic acid methyl ester (64, 1.4 g, 5.2% overall): mp
76-77 °C; NMR (DMSO-d6) δ 2.43 (s, 3H), 3.88 (s, 3H), 7.76
(dq, 8.5, 0.5, 1H), 8.01 (d, 8.5, 1H); IR (KBr) 1729, 1538, 1287,
1260, 1126 cm-1. Anal. (C9H8ClNO4) C, H, Cl, N.
The cold aqueous mother liquor was acidified with HCl (pH
1), and the product 30 was filtered, washed with H2O (100
mL), and dried (163 g, 68% overall): mp 217-218 °C (MeOH);
NMR (DMSO-d6) δ 2.62 (s, 3H), 3.37 (s, 3H), 7.76 (s, 1H), 8.44
(s, 1H), 13.50 (s br, 1H); IR (KBr) 1686, 1316, 1295, 1273, 1155,
968, 769, 517, 500 cm-1. Anal. (C9H9ClO4S) C, H, Cl, S.
Meth od E. 4-F lu or o-2-m eth yl-5-(m eth ylsu lfon yl)ben -
zoic Acid Meth yl Ester (49). In analogy to method D, 7 (98
g, 636 mmol) was sulfochlorinated with ClSO3H (240 mL, 3.60
mol) at 125 °C bath temperature for 1.5 h and then reduced
with Na2SO3 (78 g, 619 mmol) to give the crude sulfinic acid.
This was dissolved in H2O (600 mL), and sufficient 32% NaOH
was added to attain pH 10. The H2O was distilled off, and
the resinous residue was triturated with Me2CO (1.5 L),
filtered, and dried to give 2-fluoro-4-methyl-5-carboxyben-
zenesulfinic acid sodium salt as a light brown amorphous
powder.
After the mixed fractions (9.6 g) were separated, the
homogeneous polar fractions yielded 4-chloro-2-methyl-5-ni-
trobenzoic acid methyl ester (63, 4.2 g, 16% overall): mp 75-
76 °C; NMR (DMSO-d6) δ 2.60 (s, 3H), 3.89 (s, 3H), 7.79 (s,
1H), 8.42 (s, 1H); IR (KBr) 1727, 1562, 1529, 1343, 1307, 1250,
1099 cm-1. Anal. (C9H8ClNO4) C, H, Cl, N.
3-Am in o-4-ch lor o-2-m eth ylben zoic Acid Meth yl Ester
(65). A solution of compound 64 (44.1 g, 192 mmol) and RaNi
(20 g) in MeOH was shaken in the presence of H2 at
atmospheric pressure for 3 h, while the mixture was allowed
to warm to 45 °C. The solvent was removed, and the resulting
dark oil was then purified by flash column chromatography
on silica gel with t-BuOMe (10%) in PhMe as the eluent to
give 65 (26.8 g, 70%) as white crystals: mp 53-56 °C (EtOH/
H2O); NMR (DMSO-d6) δ 2.28 (s, 3H), 3.80 (s, 3H), 5.27 (s,
2H), 6.92 (d, 8.6, 1H), 7.18 (d, 8.3, 1H); IR (KBr) 3396, 1720,
1621, 1442, 1285, 1256 cm-1. Anal. (C9H10ClNO2) C, H, Cl,
N.
A stirred mixture of the crude, air-dried sodium salt (160
g) and MeI (160 mL, 2.56 mol) in DMF was refluxed for 5 h.
During this time K2CO3 (25 g, 181 mmol) was added in
portions to maintain
a pH >7. The MeI was removed,
employing reduced pressure, the resultant mixture was poured
in H2O (1.7 L), and the crystals were allowed to separate with
stirring in an ice bath. The product was collected, washed with
H2O (100 mL), and dried to obtain 49 (86 g, 54% overall) as a

2030 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Baumgarth et al.
4-Ch lor o-2-m et h yl-3-(m et h ylsu lfon yl)b en zoic Acid
Meth yl Ester (66). Compound 65 (32 g, 160 mmol) was
dissolved in a mixture of concentrated H2SO4 (120 mL),
H3PO4 (89%, 160 mL), and H2O (80 mL). A solution of NaNO2
(13.44 g, 195 mmol) in H2O (40 mL) was added to the well-
stirred mixture at such a rate that the temperature was
maintained at 5-10 °C, and stirring was continued overnight
at this temperature. The mixture was cooled to -15 °C, and
condensed SO2 (128 mL) was poured into the reaction. This
was immediately poured onto a cooled mixture of FeSO4‚7H2O
(89 g, 320 mmol) and Cu powder (1.6 g) with much foaming.
After removal of the ice/salt bath, stirring was continued for
4 h. The obtained suspension was extracted with EtOAc (3 ×
250 mL), and the organic layers were combined, extracted with
2 N NaOH (3 × 100 mL), and discarded. The combined
aqueous phases were cooled, acidified with HCl, and extracted
with EtOAc (200 mL), and the organic phase was dried,
filtered, and evaporated to give the crude sulfinic acid (22 g).
This was added to a cooled solution of dry DMF (92 mL),
consecutively followed by K2CO3 (38.6 g, 279 mmol) and MeI
(17.4 mL, 278 mmol), and the mixture was stirred for 24 h
under an N2 atmosphere at room temperature. H2O was
added, the reaction mixture was extracted with EtOAc (3 ×
75 mL), and the combined organic layers were washed with
H2O (5 × 50 mL), dried, filtered, and concentrated. Silica gel
chromatography (EtOAc/petroleum ether, 1:2, f EtOAc) gave
crystalline 66 (12.7 g, 30% overall): mp 71 °C [CH2Cl2/(i-
Pr)2O]; NMR (DMSO-d6) δ 2.70 (s, 3H), 3.43 (s, 3H), 3.87 (s,
3H), 7.67 (d, 8.3, 1H), 7.83 (d, 8.3, 1H); IR (KBr) 1726, 1310,
1281, 1156, 1097 cm-1. Anal. (C10H11ClO4S) C, H, Cl, S.
Meth od G. 2-Meth yl-5-(m eth ylsu lfon yl)-4-p h en ylben -
zoic Acid Meth yl Ester (75). A suspension of powdered
NaOH (400 mg, 10.0 mmol) in diglyme (20 mL) was heated to
90 °C with stirring. After addition of benzeneboronic acid (1.02
g, 8.37 mmol), Pd(PPh3)4 [99%; 150 mg, 0.13 mmol], and
4-bromo-2-methyl-5-(methylsulfonyl)benzoic acid methyl ester
(53; 2.25 g, 7.32 mmol), the reaction mixture was stirred for 6
h at 130 °C. The solution was then diluted with H2O (100
mL) at room temperature and extracted with EtOAc (2 × 100
mL). The combined organic layers were dried and concen-
trated with the aid of an oil pump to give a viscous residue,
which was triturated with Et2O/petroleum ether. Recrystal-
lization from (i-Pr)2O yielded 75 (1.3 g, 58%): mp 110-112
°C; NMR (DMSO-d6) δ 2.63 (s, 3H), 2.80 (s, 3H), 3.91 (s, 3H),
7.46 (m, 6H), 8.51 (s, 1H); IR (KBr) 1719, 1308, 1246, 1143,
1100, 770, 525 cm-1. Anal. (C16H16O4S‚0.25H2O) C, H, S.
4-(2-F u r yl)-2-m eth yl-5-(m eth ylsu lfon yl)ben zoic Acid
Meth yl Ester (76). Pd(PPh3)4 [99%; 7.27 g, 6.28 mmol] was
added to a stirred solution of 2-(tributylstannyl)furan (20.5 mL,
62.8 mmol) and 4-bromo-2-methyl-5-(methylsulfonyl)benzoic
acid methyl ester (53, 19.3 g, 62.8 mmol) in dry dioxane (500
mL), and the mixture was heated at reflux temperature under
an argon atmosphere for 3 h. The reaction mixture was
filtered through Celite, the filtrate was evaporated in vacuo,
and the residue was extracted with boiling t-BuOMe. Evapo-
ration of the solution and recrystallization of the residue from
t-BuOMe/CH2Cl2 yielded 76 (16.9 g, 91%): mp 142-143 °C;
NMR (DMSO-d6) δ 2.66 (s, 3H), 3.31 (s, 3H), 3.90 (s, 3H), 6.71
(dd, 3.5, 1.8, 1H), 7.17 (d, 3.4, 1H), 7.80 (s, 1H), 7.96 (d, 1.9,
1H), 8.54 (s, 1H); IR (KBr) 1721, 1603, 1541, 1499, 1436, 1304,
1141 cm-1. Anal. (C14H14O5S) C, H, S.
1693, 1307, 1264, 1150 cm-1. Anal. (C12H16O4S2‚0.25H2O) C,
H, S. For 83, 84, and 87, commercially available Na thiolates
were used instead of the NaH addition. Compound 91 was
prepared without DMF, and K2CO3 was used as the base. The
analytical sample of 87 was prepared by esterification (f93),
silica gel chromatography, and saponification.
Meth od I. 4-Eth oxy-2-m eth yl-5-(m eth ylsu lfon yl)ben -
zoic Acid (94). Under an N2 atmosphere Na (420 mg, 18.3
mmol) was dissolved in EtOH (50 mL). 4-Chloro-2-methyl-5-
(methylsulfonyl)benzoic acid (30, 600 mg, 2.41 mmol) was
added, and the mixture was heated under reflux for 72 h. The
EtOH was evaporated, and the residue was taken up in H2O
(75 mL), acidified with HCl, and extracted twice with EtOAc
(75 mL). The combined organic layers were dried, concen-
trated, and triturated with Et2O/EtOAc to give crystalline 94
(475 mg, 75%): mp 221-223 °C; NMR (DMSO-d6) δ 1.41 (t,
7.0, 3H), 2.63 (s, 3H), 3.24 (s, 3H), 4.30 (q, 7.0, 2H), 7.21 (s,
1H), 8.31 (s, 1H); IR (KBr) 1691, 1599, 1381, 1323, 1252, 1147,
1045 cm-1. Anal. (C11H14O5S‚0.25H2O) C, H, S.
Meth od J . 2,3-Dim eth yl-5-(m eth ylsu lfon yl)-4-(3-p yr -
id yloxy)ben zoic Acid Meth yl Ester (103). A mixture of
4-chloro-2,3-dimethyl-5-(methylsulfonyl)benzoic acid methyl
ester (56, 6.2 g, 22.4 mmol), 3-hydroxypyridine (2.13 g, 22.4
mmol), and K2CO3 (9.3 g, 67.3 mmol) in dry DMF (125 mL)
was stirred at 130 °C for 20 h. Saturated NaHCO3 solution
(300 mL) was added, and the mixture was extracted with
EtOAc (3 × 100 mL). The combined organic layers were
washed with saturated NaCl solution (4 × 100 mL) and dried,
and the solvent was removed to leave brownish crystals of 103
(3.1 g, 41%). An analytical sample was prepared by recrys-
tallization from EtOAc: mp 144 °C; NMR (DMSO-d6) δ 2.04
(s, 3H), 2.53 (s, 3H), 3.31 (s, 3H), 3.90 (s, 3H), 7.14 (ddd, 8.4,
2.9, 1.2, 1H), 7.34 (dd, 8.6, 4.6, 1H), 8.18 (s, 1H), 8.28 (m, 2H);
IR (KBr) 1728, 1578, 1480, 1430, 1312, 1254 cm-1
. Anal.
(C16H17NO5S) C, H, N, S. Preparation of 105-109 was carried
out without DMF but with a large excess (4 equiv) of the
appropriate OH reagent instead. The raw ester was not
characterized in the case of 109 but directly hydrolyzed using
method B. The yield given is the overall yield.
Meth od K. 2-Meth yl-5-(m eth ylsu lfon yl)-4-(p h en yla m i-
n o)b en zoic Acid (139). A mixture of 4-fluoro-2-methyl-5-
(methylsulfonyl)benzoic acid (31, 1.0 g, 4.30 mmol) and PhNH2
(5 mL, 475 mmol) was heated at 140 °C for 5 h. The mixture
was dissolved in 1 N NaOH (30 mL) and washed with EtOAc
(2 × 50 mL). After acidification with HCl the aqueous phase
was extracted with EtOAc (2 × 50 mL) and the combined
organic layers were dried, evaporated, and triturated with
EtOAc/Et2
O to give a white solid of 139 (500 mg, 37%): mp
182-184 °C; NMR (CDCl3) δ 2.57 (s, 3H), 3.14 (s, 3H), 7.07 (s,
1H), 7.23 (m, 3H), 7.43 (t, 7.6, 2H), 8.26 (s, 1H), 8.61 (s, 1H).
Anal. (C15H15NO4S‚0.5H2O) C, H, N, S. Experiments 120-
122 were run with an excess of 3 equiv of the nucleophile only
but with additional solvent instead. In the case of 120 this
was PhMe, in 121 and 122 sulfolane, and in 123 N-methylpyr-
rolidin-2-one (NMP). The formed benzoic acids were not
characterized for 119-123 but in the following ester stage.
Esterifications were carried out in the case of compounds 122
and 123 according to method F, in 119 and 120 with MeI/
K2
CO3 in DMF, and in 121 with CH2N2. Yields given in Table
4 are overall yields.
Meth od L. 2,3-Dim eth yl-4-(1-im id a zolyl)-5-(m eth yl-
su lfon yl)ben zoic Acid Meth yl Ester (129). Under N2
4-chloro-2,3-dimethyl-5-(methylsulfonyl)benzoic acid (47, 10.0
g, 38.1 mmol) was added in portions within 30 min at 0 °C to
a well-stirred suspension of NaH (60% in mineral oil, 1.52 g,
38.1 mmol) in NMP (100 mL), and stirring was continued for
1 h at room temperature. A solution of imidazole sodium in
NMP (100 mL) was analogously prepared from imidazole (2.6
g, 38.1 mmol) and NaH (60% in mineral oil, 1.52 g, 38.1 mmol).
Both solutions were combined and heated at 160 °C for 90 h.
NMP was removed under reduced pressure, and the residue
of the crude benzoic acid was esterified with saturated HCl/
MeOH (150 mL) according to method F to give 129 (5.8 g, 49%
overall) upon recrystallization from EtOAc: mp 184 °C; NMR
(DMSO-d6) δ 1.82 (s, 3H), 2.52 (s, 3H), 3.30 (s, 3H), 3.92 (s,
3H), 7.17 (s, 1H), 7.36 (s, 1H), 7.77 (s, 1H), 8.23 (s, 1H); IR
Met h od H . 2-Met h yl-5-(m et h ylsu lfon yl)-4-p r op yl-
th ioben zoic Acid (86). To a solution of DMF (50 mL) under
N2 was added PrSH (3.6 g, 47.3 mmol) followed by NaH (60%
in mineral oil, 1.5 g, 37.5 mmol), and the temperature was
allowed to rise to 40 °C. After 30 min of stirring, 4-fluoro-2-
methyl-5-(methylsulfonyl)benzoic acid (31, 2 g, 8.6 mmol) was
added, and the mixture was heated for 1 h at 80 °C. The
solution was poured into ice water (300 mL), washed with
EtOAc (3 × 100 mL), acidified, and extracted with EtOAc (2
× 50 mL). The combined organic phases were dried, concen-
trated, and triturated with Et2O to obtain the desired product
(1.5 g, 60%). Recrystallization of a sample from i-PrOH gave
86 as white crystals: mp 177-178 °C; NMR (DMSO-d6) δ 1.03
(t, 7.3, 3H), 1.70 (sext, 7.3, 2H), 2.64 (s, 3H), 3.15 (t, 7.2, 2H),
3.29 (s, 3H), 7.50 (s, 1H), 8.37 (s, 1H), 13.12 (s br, 1H); IR (KBr)

Benzoylguanidine Na+/ H+ Antiporter Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2031
(KBr) 1723, 1499, 1440, 1309, 1203, 1140 cm-1
(C14H16N2O4S‚0.5H2O) C, H, N, S. For 130-132 esterifications
were omitted and the compounds were isolated in the form of
the benzoic acids.
.
Anal.
was prepared according to method F in a 93% yield: mp 150-
151 °C (EtOAc); NMR (DMSO-d6) δ 2.47 (s, 3H), 3.11 (s, 3H),
3.76 (s, 3H), 6.62 (s br, 2H), 6.73 (s, 1H), 8.15 (s, 1H); IR (KBr)
3466, 3366, 1717, 1300, 1284, 1133 cm-1. Anal. (C10H13NO4S)
C, H, N, S.
Meth od M. 4-Cya n o-2-m eth yl-5-(m eth ylsu lfon yl)ben -
zoic Acid (144). A mixture of 4-chloro-2-methyl-5-(methyl-
sulfonyl)benzoic acid (30, 50 g, 200 mmol), CuCN (44.6 g, 500
mmol), and NMP (400 mL) was stirred under an N2 atmo-
sphere at 150 °C for 72 h. Afterward this was poured into
H2O (2 L), EtOAc (2 L) was added, and the mixture was stirred
for an additional 30 min. After filtration through Celite, the
aqueous layer was separated and extracted with EtOAc (3 ×
500 mL). The combined organic layers were washed with H2O
(6 × 750 mL), dried, and evaporated to give compound 144
(22.5 g, 55%): mp 249-250 °C (MeOH); NMR (DMSO-d6) δ
2.65 (s, 3H), 3.39 (s, 3H), 8.21 (s, 1H), 8.39 (s, 1H); IR (KBr)
2-Meth yl-5-(m eth ylsu lfon yl)-4-(1-pyr r olyl)ben zoic Acid
Meth yl Ester (153). Compound 152 (89.6 g, 368 mmol), 2,5-
dimethoxytetrahydrofuran (60.45 mL, 467 mmol), and 4-chlo-
ropyridine hydrochloride (5.53 g, 36.9 mmol) were heated
under reflux in 1,4-dioxane (2.2 L) for 2.5 h. The mixture was
concentrated to a small volume, and the residue was taken
up in EtOAc (2 L), washed with H2O (3 × 500 mL), dried, and
filtered. After addition of charcoal (5 g) the solution was
refluxed for 45 min, filtered, evaporated, and recrystallized
from MeOH to give 153 (94 g, 87%): mp 161-162 °C; NMR
(DMSO-d6) δ 2.64 (s, 3H), 2.67 (s, 3H), 3.91 (s, 3H), 6.33 (t,
2.1, 2H), 7.01 (t, 2.2, 2H), 7.57 (s, 1H), 8.49 (s, 1H); IR (KBr)
2235, 1703, 1681, 1597, 1443, 1416, 1312, 1265, 1149 cm-1
Anal. (C10H9NO4S) C, H, N, S.
.
1726, 1298, 1259, 1147, 1101 cm-1
H, N, S.
. Anal. (C14H15NO4S) C,
2-Met h yl-5-(m et h ylsu lfon yl)-4-(t r iflu or om et h yl)b en -
zoic Acid Meth yl Ester (146). A mixture of 4-bromo-2-
methyl-5-(methylsulfonyl)benzoic acid methyl ester (53, 10.0
g, 33 mmol), CF3CO2K (7.53 g, 49.5 mmol), CuI (13.0 g, 69.3
mmol), and tetramethylammonium bromide (660 mg, 3.3
mmol) was stirred into PhMe (300 mL), after which part of
the PhMe (∼100 mL) was stripped. NMP (100 mL) was added,
and PhMe and NMP were stripped until the temperature
reached about 155 °C. The reaction mixture was maintained
at this temperature for 4 h. After cooling, the mixture was
diluted with EtOAc (1 L) and H2O (1 L) and filtered through
Celite. The organic phase was separated, washed with brine
(3 × 500 mL), dried, and evaporated. The resulting dark
residue was purified by column chromatography on silica gel
with EtOAc/petroleum ether (1:1) as the solvent to give 146
(3.7 g, 28%) as white crystals: mp 135-136 °C (CH2Cl2/
EtOAc); NMR (DMSO-d6) δ 2.69 (s, 3H), 3.31 (s, 3H), 3.92 (s,
3H), 8.06 (s, 1H), 8.56 (s, 1H); IR (KBr) 1724, 1599, 1452, 1434,
1303, 1251, 1151, 1106, 1078 cm-1. Anal. (C11H11F3O4S) C,
H, S. Analogous treatment of 4-bromo-3-(methylsulfonyl)-
benzoic acid methyl ester15d yielded compound 147.
4-Ch lor o-2-m et h yl-5-(m et h ylsu lfon yl)-3-n it r ob en zoic
Acid (154). The title compound was similarly prepared as
described for 61 and 62 by treatment of compound 30 with a
5:1 H2SO4 (98%)/HNO3 (100%) mixture at 70-80 °C for 6 h as
a white powder in a 79% yield: mp 244-247 °C (EtOAc/
MeOH); NMR (DMSO-d6) δ 2.55 (s, 3H), 3.47 (s, 3H), 8.57 (s,
1H), 14.10 (s br, 1H); IR (KBr) 1703, 1550, 1324, 1298, 1142
cm-1. Anal. (C9H8ClNO6S) C, H, Cl, N, S.
4-Ch lor o-2-m et h yl-5-(m et h ylsu lfon yl)-3-n it r ob en zoic
Acid Meth yl Ester (155). Starting from 154 method F gave
pale yellow crystals in a 95% yield: mp 156-157 °C (EtOAc);
NMR (DMSO-d6) δ 2.54 (s, 3H), 3.46 (s, 3H), 3.93 (s, 3H), 8.56
(s, 1H); IR (KBr) 1737, 1549, 1319, 1293, 1265, 1148, 1132
cm-1. Anal. (C10H10ClNO6S) C, H, Cl, N, S.
3-Am in o-4-ch lor o-2-m et h yl-5-(m et h ylsu lfon yl)b en zo-
ic Acid Meth yl Ester (156). Hydrogenation of 155 in analogy
to the method described for 65 gave pale yellow crystals of
the title compound in a 34% yield: mp 188 °C (EtOAc); NMR
(DMSO-d6) δ 2.38 (s, 3H), 3.33 (s, 3H), 3.85 (s, 3H), 5.89 (s br,
2H), 7.59 (s, 1H); IR (KBr) 3447, 3367, 1725, 1289, 1210, 1135
cm-1. Anal. (C10H12ClNO4S) C, H, Cl, N, S.
4-Ch lor o-2-m et h yl-5-(m et h ylsu lfon yl)-3-(1-p yr r olyl)-
ben zoic Acid Meth yl Ester (157). This compound was
prepared from 156 in a manner analogous to that described
for 153 above to give a 76% yield of 157 as pale yellow
crystals: mp 136-137 °C (t-BuOMe/CH2Cl2); NMR (DMSO-
d6) δ 2.19 (s, 3H), 3.43 (s, 3H), 3.91 (s, 3H), 6.32 (t, 2.1, 2H),
6.85 (t, 2.1, 2H), 8.49 (s, 1H); IR (KBr) 1711, 1315, 1237, 1147,
735 cm-1. Anal. (C14H14ClNO4S) C, H, N.
4-(Ben zyla m in o)-2-m eth yl-5-(m eth ylsu lfon yl)ben zoic
Acid (149). The title compound was prepared according to
method K by treatment of compound 30 with BnNH2 at 160
°C for 4 h in an 88% yield: mp 225-226 °C (H2O); NMR
(DMSO-d6) δ 2.46 (s, 3H), 3.19 (s, 3H), 4.55 (d, 5.8, 2H), 6.67
(s, 1H), 7.06 (t, 5.8, 1H), 7.24-7.40 (m, 5H), 8.22 (s, 1H), 12.41
(s br, 1H); IR (KBr) 3369, 1684, 1603, 1561, 1307, 1258 cm-1
Anal. (C16H17NO4S) C, H, N, S.
.
4-Am in o-2-m eth yl-5-(m eth ylsu lfon yl)ben zoic Acid (150).
In an analogous manner as described for 65 the title compound
was prepared by hydrogenation of 149 using a Pd/C (10%)
catalyst as an off-white solid in a 92% yield: mp 268-269 °C
(CH2Cl2/MeOH); NMR (DMSO-d6) δ 2.48 (s, 3H), 3.12 (s, 3H),
6.54 (s, 2H), 6.72 (s, 1H), 8.16 (s, 1H), 12.39 (s br, 1H); IR (KBr)
3453, 3357, 1686, 1301, 1288 cm-1. Anal. (C9H11NO4S) C, H,
N, S.
3-Am in o-2-m et h yl-5-(m et h ylsu lfon yl)b en zoic Acid
Meth yl Ester (158). Hydrogenation of 155 in a similar
manner as described for 65 using a Pd/C (10%) catalyst in a
MeOH/THF/Et3N mixture (30:20:1) gave a 67% yield of the
title compound as yellow crystals: mp 121 °C (EtOAc/t-
BuOMe); NMR (DMSO-d6) δ 2.25 (s, 3H), 3.12 (s, 3H), 3.84 (s,
3H), 5.70 (s, 2H), 7.30 (d, 2.0, 1H), 7.34 (d, 2.0, 1H); IR (KBr)
3485, 3378, 1711, 1263, 1130 cm-1
H, N, S.
. Anal. (C10H13NO4S) C,
2-Met h yl-5-(m et h ylsu lfon yl)-4-(2-p yr id yla m in o)b en -
zoic Acid Meth yl Ester (151). Compound 150 (10.0 g, 43.6
mmol) was added in portions to a cooled suspension of NaH
(60% in mineral oil, 4.2 g, 105 mmol) in NMP (130 mL). Under
N2 protection the suspension was stirred at room temperature
for 1 h. 2-Fluoropyridine (12.5 mL, 145 mmol) was added, and
the mixture was heated at 100 °C for 48 h. The solution was
cooled down, mixed with some drops of H2O, and evaporated
with the help of an oil pump, leaving a dark gum. The crude
benzoic acid was refluxed with saturated MeOH/HCl (150 mL)
for 6 h and the MeOH removed to a great extent under reduced
pressure. The residue was taken up in EtOAc (250 mL),
washed with 2 N NaOH (100 mL) and H2O (100 mL), dried,
and evaporated to give 151 (4.6 g, 32%) on crystallization from
MeOH: mp 141-142 °C; NMR (DMSO-d6) δ 2.59 (s, 3H), 3.27
(s, 3H), 3.84 (s, 3H), 7.05 (ddd, 7.2, 5, 0.8, 1H), 7.14 (d, 8.2,
1H), 7.76 (ddd, 8.2, 7.3, 1.8, 1H), 8.31 (s, 1H), 8.34 (s, 1H),
2-Meth yl-5-(m eth ylsu lfon yl)-3-(1-pyr r olyl)ben zoic Acid
Meth yl Ester (159). This compound was prepared from 158
in a manner analogous to that described for 153 to give a 79%
yield of 159: mp 116-117 °C (t-BuOMe); NMR (DMSO-d6) δ
2.34 (s, 3H), 3.32 (s, 3H), 3.91 (s, 3H), 6.30 (t, 2.1, 2H), 7.04 (t,
2.2, 2H), 7.95 (d, 2.0, 1H), 8.27 (d, 2.0, 1H); IR (KBr) 1722,
1329, 1150, 1121 cm-1. Anal. (C14H15NO4S) C, H, N, S.
4-Br om o-2-(b r om om et h yl)-5-(m et h ylsu lfon yl)b en zo-
ic Acid Meth yl Ester (160). To a solution of 4-bromo-2-
methyl-5-(methylsulfonyl)benzoic acid methyl ester (53, 40 g,
130 mmol) in dry CH2Cl2 (280 mL) was added NBS (27.2 g,
152 mmol) in portions over a period of 5 h. During this time
the mixture was refluxed and irradiated by means of an
ordinary 400-W UV lamp. Afterward the reaction mixture was
washed with H2O (3 × 100 mL) and the organic layer dried
and evaporated, yielding a yellow crystalline crop. This was
chromatographed on silica gel with hexane/EtOAc (4:1). The
polar fractions were combined to give 160 (30.4 g, 61%). An
analytical sample of white crystals was prepared by recrys-
8.89 (s, 1H); IR (KBr) 3353, 1703, 1478, 1420, 1296, 1127 cm-1
.
Anal. (C15H16N2O4S‚0.25H2O) C, H, N, S.
4-Am in o-2-m et h yl-5-(m et h ylsu lfon yl)b en zoic Acid
Meth yl Ester (152). Starting with 150 the title compound

2032 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13
Baumgarth et al.
tallization from EtOAc: mp 136-137 °C; NMR (DMSO-d6) δ
3.43 (s, 3H), 3.92 (s, 3H), 5.04 (s, 2H), 8.24 (s, 1H), 8.47 (s,
1H); IR (KBr) 1730, 1545, 1432, 1310, 1292, 1259, 1150, 1095
cm-1. Anal. (C10H10Br2O4S) C, H, S.
Meth od R. [4-(4-Am in o-1-p ip er id yl)-2-m eth yl-5-(m eth -
ylsu lfon yl)ben zoyl]gu a n id in e Dih yd r och lor id e (232). To
a solution of guanidine in MeOH (15 mL) prepared from
guanidine hydrochloride (4 g, 41.9 mmol) in an analogous
manner to that described in method N 4-[4-[(tert-butoxycar-
bonyl)amino]-1-piperidyl]-2-methyl-5-(methylsulfonyl)benzo-
ic acid methyl ester (119, 3.5 g, 8.2 mmol) was added and the
mixture was stirred for 3 h at 50 °C. After addition of H2O
(30 mL) the precipitate was collected and recrystallized from
MeCN, yielding [4-[4-[(tert-butoxycarbonyl)amino]-1-piperidyl]-
2-methyl-5-(methylsulfonyl)benzoyl]guanidine (1.4 g, 38%) as
white crystals: mp 176 °C dec; NMR (DMSO-d6) δ 1.40 (s, 9H),
1.58 (q br, 10, 2H), 1.82 (d br, 9.5, 2H), 2.53 (s, 3H), 2.79 (t br,
10.3, 2H), 3.14 (d br, 11.2, 2H), 3.32 (s, 3H), 3.40 (s br, 1H),
6.85 (d br, 7.1, 1H), 7.33 (s, 1H), 8.26 (s, 1H); IR (KBr) 1713,
4-Br om o-2-((d ieth yla m in o)m eth yl)-5-(m eth ylsu lfon yl)-
ben zoic Acid Meth yl Ester (161). A mixture of 4-bromo-
2-(bromomethyl)-5-(methylsulfonyl)benzoic acid methyl ester
(160, 5 g, 13 mmol) and Na2CO3 (8.7 g, 82 mmol) was stirred
in Et2NH (35 mL, 335 mmol) under an N2 atmosphere at room
temperature for 24 h. The reaction mixture was then taken
up in EtOAc (150 mL) and washed with H2O (3 × 75 mL).
The organic layer was dried and evaporated, and the residue
was recrystallized from (i-Pr)2O to give 161 (3 g, 61%): mp 94
°C; NMR (DMSO-d6) δ 0.94 (t, 7.1, 6H), 2.46 (q, 7.2, 4H), 3.40
(s, 3H), 3.85 (s, 5H), 8.14 (s, 1H), 8.27 (s, 1H); IR (KBr) 1725,
1323, 1313, 1249, 1149, 1094 cm-1. Anal. (C14H20BrNO4S) C,
H, Br, N, S.
Meth od N. (4-Ch lor o-2-m eth yl-5-(m eth ylsu lfon yl)ben -
zoyl)gu a n id in e (166). Free guanidine base was prepared by
consecutive addition of Na (6.56 g, 285.5 mmol) and guanidine
hydrochloride (30 g, 314 mmol) to dry MeOH (80 mL). After
being stirred for 30 min at room temperature the mixture was
filtered under N2 protection. 4-Chloro-2-methyl-5-(methylsul-
fonyl)benzoic acid methyl ester (48, 15 g, 57.1 mmol) was added
to the filtrate, and the mixture was stirred for 2.5 h at 50 °C.
After the mixture was cooled to room temperature, H2O (250
mL) was added, and the solution was stirred for 30 min and
an additional 30 min with ice cooling while crystallization took
place. The product was collected and recrystallized from
MeOH, yielding 166 as white crystals (9.5 g, 57%): mp 207-
208 °C; NMR (DMSO-d6) δ 2.56 (s, 3H), 3.32 (s, 3H), 7.57 (s,
1H), 8.38 (s, 1H); IR (KBr) 1660, 1615, 1589, 1522, 1377, 1317,
1293, 1135 cm-1. Anal. (C10H12ClN3O3S) C, H, Cl, N, S.
Meth od O. (4-Isop r op yl-2-m eth yl-5-(m eth ylsu lfon yl)-
ben zoyl)gu a n id in e (182). 4-Isopropyl-2-methyl-5-(methyl-
sulfonyl)benzoic acid (34, 10.0 g, 39 mmol) was chlorinated
with SOCl2 (50 mL, 689 mmol) at 120 °C for 2 h. Excessive
SOCl2 was removed by the aid of a water-jet pump, and the
remaining acid chloride was used without further purification.
The preparation of guanidine base (180 mmol) in MeOH was
carried out as described in the preceding instruction. The
MeOH was removed in vacuo and the residue taken up in 1,2-
dimethoxyethane (200 mL). The acid chloride was also taken
up in 1,2-dimethoxyethane (200 mL) and then added to the
guanidine solution. After the mixture was stirred for 1 h at
room temperature, the inorganic precipitate was removed and
the filtrate was evaporated. The residue was purified by silica
gel chromatography (EtOAc f MeOH) to give compound 182
(9.0 g, 79%) after recrystallization from EtOAc: mp 220-223
°C; NMR (DMSO-d6) δ 1.26 (d, 6.7, 6H), 2.56 (s, 3H), 3.17 (s,
3H), 3.75 (sept, 6.6, 1H), 7.42 (s, 1H), 8.26 (s, 1H). Anal.
(C13H19N3O3S‚H2O) C, H, N, S.
1600, 1525, 1449, 1347, 1291, 1160, 1124 cm-1
.
A suspension of the foregoing Boc compound (1.3 g, 2.9
mmol) was stirred in 2 N HCl/dioxane (30 mL) at room
temperature for 3 h. After dilution with Et2O (30 mL) the
precipitate was collected by filtration. Recrystallization from
MeOH yielded 232 (800 mg, 65%) in the form of the white
dihydrochloride: mp 305-310 °C dec; NMR (DMSO-d6) δ 1.81
(q br, 10.5, 2H), 2.04 (d br, 8.8, 2H), 2.52 (s, 3H), 2.87 (t br,
10.4, 2H), 3.36 (s, 3H), 3.1-3.4 (m, 3H), 7.49 (s, 1H), 8.13 (s,
1H), 8.31 (s br, 2H), 8.49 (s br, 2H), 8.68 (s br, 2H); IR (KBr)
1714, 1691, 1596, 1307, 1293, 1255, 1142 cm-1
.
Anal.
(C15H23N5O3S‚2HCl) C, H, Cl, N, S.
Meth od S. (2-Meth yl-5-(m eth ylsu lfon yl)-4-(p h en yla m i-
n o)ben zoyl)gu a n id in e Hyd r och lor id e (248). HCl (1 N,
1.15 mL) was dropped into a stirred suspension of (2-methyl-
5-(methylsulfonyl)-4-(phenylamino)benzoyl)guanidine (400 mg,
1.15 mmol) in H2O (100 mL), which was prepared according
to method O from compound 139. The filtered solution was
frozen and then lyophilized to give the title compound (420
mg, 95%): mp 260 °C; NMR of the base (DMSO-d6) δ 2.47 (s,
3H), 3.30 (s, 3H), 7.01 (s, 1H), 7.11 (t, 7.1, 1H), 7.25 (d, 8.1,
2H), 7.38 (t, 7.7, 2H), 7.90 (s, 1H), 8.36 (s, 1H). Anal.
(C16H18N4O3S‚HCl) C, H, Cl, N, S.
Meth od T. [4-(1,4-Dih yd r o-4-oxo-1-p yr id yl)-2-m eth yl-
5-(m eth ylsu lfon yl)ben zoyl]gu a n id in e (279). In a small,
sealed, round-bottomed flask were heated (4-fluoro-2-methyl-
5-(methylsulfonyl)benzoyl)guanidine (165, 1 g, 3.66 mmol),
4-[(trimethylsilyl)oxy]pyridine30 (10 g, 59.8 mmol) and K2CO3
(2 g, 14.5 mmol) at 135 °C for 4.5 h. The liquid silyl compound
was decanted after cooling. The residue was washed with Et2O
and chromatographed (silica gel, gradient elution, EtOAc f
MeOH). The homogeneous fractions were combined and
recrystallized from MeOH to give 279 (800 mg, 61%): mp 267-
268 °C; NMR (DMSO-d6) δ 2.59 (s, 3H), 3.11 (s, 3H), 6.17 (d,
7.7, 2H), 7.54 (s, 1H), 7.73 (d, 7.7, 2H), 8.36 (s, 1H); IR (KBr)
3559, 3382, 1643, 1600, 1551, 1350, 1303 cm-1
.
Anal.
(C15H16N4O4S‚0.5H2O) C, H, N, S.
Cr ysta l Da ta of 246, Meth a n esu lfon a te: C15H20N4O6S2;
M ) 416.51; triclinic; P1h; a ) 8.559(1) Å; b ) 9.597(3) Å; c )
11.580(7) Å; R ) 105.84(4)°; â ) 98.29(6)°; γ ) 91.97(4)°; V )
Met h od s
P a n d Q. (4-(ter t-Bu t ylt h io)-2-m et h yl-5-
(m eth ylsu lfon yl)ben zoyl)gu a n id in e Meth a n esu lfon a te
(202). 4-(tert-Butylthio)-2-methyl-5-(methylsulfonyl)benzoic
acid (87, 800 mg, 2.65 mmol) and 2-chloro-1-methylpyridinium
iodide (750 mg, 2.94 mmol) in NMP (7.3 mL) were stirred for
20 min. After addition of guanidine hydrochloride (1.15 g, 12
mmol), N-ethyldiisopropylamine (3.3 mL, 19.4 mmol) was
dropped in with slight cooling, and stirring was continued for
an additional 1 h. The mixture was poured onto ice water (50
mL), acidified, washed with EtOAc (2 × 20 mL), alkalified,
and extracted with EtOAc (2 × 40 mL). The combined organic
extracts were dried, evaporated, and triturated with Et2O to
give 202 (400 mg, 44%) as the free base: mp 112-115 °C; IR
902.8(6) Å3; Z ) 2; Fx ) 1.532 g cm-3; µ(Cu KR) ) 0.322 mm-1
;
F(000) ) 436; no. of reflections with I g 3σ(I) ) 3912; no. of
refinement parameters ) 264; final R values, R ) 0.0398; Rw
) 0.0482.
Na +/H+ Exch a n ge In h ibition Assa y: P r ep a r a tion a n d
Wa sh in g of Red Blood Cells. The red blood cells prepara-
tion as well as the internal acidification of the red blood cells
follows closely the procedures as outlined by Morgan and
Canessa.31b Blood was obtained from rabbits (e.g. New Zealand
White) which were sacrificed and exsanguined. The blood was
collected into 50-mL Falcon centrifuge tubes which contained
heparin-Na solution (5 mL, 250 units/mL). The blood was
mixed well with the heparin solution. The red blood cells were
collected by centrifugation at 2000g at 4 °C; the plasma and
buffy coat was removed. The remaining solution was filtered
through 200 µm gaze. The filtrate was resuspended to the
original volume with wash buffer [140 nM KCl, 0.15 mM
MgCl2, 10 mM tris(hydroxymethyl)aminomethane (Tris)/3-
morpholinopropanesulfonic acid (MOPS), pH 7.4]. The red
blood cells were again collected by centrifugation and the
washing was repeated (2×).
(KBr) 3427, 3370, 2966, 1662, 1598, 1527, 1302 cm-1
.
To the solution of the above free base (350 mg, 1.02 mmol)
in Me2CO (5 mL) MeSO3H (0.075 mL, 1.05 mmol) was added
with a suitable pipet. Then Et2O was added until the solution
became cloudy and crystals of the methanesulfonate of 202
(350 mg, 78%), separated on cooling, were collected: mp 200-
202 °C; NMR (DMSO-d6) δ 1.46 (s, 9H), 2.42 (s, 3H), 2.54 (s,
3H), 3.40 (s, 3H), 7.75 (s, 1H), 8.20 (s, 1H), 8.40 (s br, 2H),
8.56 (s br, 2H), 11.69 (s, 1H). Anal. (C14H21N3O3S2‚CH4-
O3S‚0.5H2O) C, H, N, S.

Benzoylguanidine Na+/ H+ Antiporter Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 13 2033
In tr a er yth r ocyte Acid ifica tion . For the intracellular
acidification the packed, collected red blood cells (5 mL) were
resuspended in the acidification buffer [45 mL; 170 mM KCl,
0.15 mM MgCl2, 0.1 mM ouabain, 10 mM glucose, 10 mM
sucrose, 20 mM Tris/2-morpholinoethanesulfonic acid (MES),
pH 6.2]. The red blood cell suspension was incubated for 10
min at 37 °C with occasional mixing. To clamp the internal
pH, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)
and DIAMOX (acetazolamide) were added up to 200 µM and
1 mM, respectively. The incubation was continued for a
further 30 min at 37 °C. Thereafter the red blood cells were
collected by centrifugation for 4 min; they were resuspended
in ice-cold unbuffered wash solution (170 mM KCl, 40 mM
sucrose, 0.15 mM MgCl2) and washed therein (4×).
sions, for 246 (9 pages). Ordering information is given on any
current masthead page.
Refer en ces
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Na +/H+ Exch a n ge Assa y. The incubation was carried out
in Macrowell-Tube strips in an 8 × 12 format. The incubation
was started by adding prewarmed, acidified red blood cell
solution (20 µL) to the incubation buffer (200 µL; 160 mM KCl,
22NaCl, 0.16 µCi/well, 10 mM NaCl, 0.15 mM MgCl2, 0.1 mM
ouabain, 10 mM glucose, 40 mM sucrose, 10 mM Tris/MOPS,
pH 8.0, 0.5 mM DIAMOX, 1% DMSO). Substances to be tested
were dissolved in DMSO and were subsequently diluted to the
appropriate concentrations in the incubation buffer. The
incubation was carried out at 37 °C for 5 min. The incubation
was stopped by adding ice-cold stop solution (800 µL; 112 mM
MgCl2, 0.1 mM ouabain). The red blood cells were collected
by centrifugation for 7 min. The supernatant was sucked off
by using an aspirator which allowed for the simultaneous
aspiration of four adjacent tubes. The red blood cells were
washed (3×) with ice-cold stop solution (900 µL) by repeating
the resuspension/centrifugation step as described before. After
the last wash H2O (200 µL) was added to the red blood cell
pellet. The tubes were then sonified for 2 × 30 min. There-
after the Macrowell-tube strips were taken apart; each tube
was added top to bottom to a separate scintillation vial; and a
slight shaking caused the hemolyzed red blood cell solution
to empty into the scintillation vial. To each vial the scintillant
Aquasafe 300 PS (3 mL, Zinsser Analytic, Frankfurt/M,
Germany) was added; the vials were capped and well mixed.
The radioactivity taken up into the red blood cells was
determined in a Packard scintillation counter by following the
â-decay.
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Eva lu a tion of Resu lts. Each substance concentration was
determined in triplicate. From each value the mean of the
count determination in the presence of EIPA (10 µM) was
subtracted, accounting for the non-Na+/H+-dependent 22Na+
uptake into the erythrocytes. The mean of the remaining
counts in the absence of a substance was taken as 100%
control; the mean values in the presence of the compounds to
be tested were expressed as percent of that control value. The
percent uptake data were plotted in a semilogarithmic plot;
IC50 values were obtained by subjecting the data to a nonlinear
curve fitting procedure using the equation E ) (Emax - Emin)/
(1 + IC50/x), where x corresponds to the concentrations of the
compound to be tested. The standard deviation of the IC50
values was 14% on the average.
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Linz, W.; Weichert, A.; Scho¨lkens, B. A. Protective effects of
HOE642,
a selective sodium-hydrogen exchange subtype 1
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Ack n ow led gm en t. We extend our thanks to Dr.
Dieter Dorsch for helpful discussions, Prof. Dr. H. Fuess,
TH Darmstadt, for providing us with X-ray data, Dr.
Michael Krug for computer-aided drug design, Dr.
Sophie Marquais and Dr. Eike Poetsch for their help in
metalorganic chemistry, and Dr. Klaus Pachler for the
measurement and interpretation of NMR spectra. We
also express our thanks to Heike Ebert, Sylvia Heiss,
Gu¨nther Kritzinger, Horst May, Horst Schiefer, Thomas
Weitzel, and Reinhold Zissel for their skillful experi-
mental work, and Beate Opelt and Corinna Rettig for
their assistance with the preparation of the tables and
schemes included in this paper.
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