Arylcyclopropanecarboxyl guanidines as novel, potent, and selective inhibitors of the sodium hydrogen exchanger isoform-1

Article abstract of DOI:10.1021/jm010100v

A novel series of arylcyclopropanecarboxyl guanidines was synthesized and evaluated for activity against the sodium hydrogen exchanger isoform-1 (NHE-1). In biological assays conducted in an AP1 cell line expressing the human NHE-1 isoform, the starting cyclopropane 3a (IC₅₀ = 3.5 μM) shows inhibitory activity comparable to cariporide (IC₅₀ = 3.4 μM). Structure-activity relationships are used to optimize the affinity of various acyl guanidines for NHE-1 by screening the effect of substituents at both aryl and cyclopropyl rings. It is demonstrated that introduction of appropriate hydrophobic groups at the phenyl ring and a gem-dimethyl group at the cyclopropane ring enhances the NHE-1 inhibitory activity by up to 3 orders of magnitude (compound 7f, IC₅₀ = 0.003 μM). In addition, the gem-dimethyl series of analogues seem to display improved oral bioavailability and longer plasma half-life in rats. Furthermore, the lead benzodihydrofuranyl analogue 1 (BMS-284640) shows over 380-fold increased NHE-1 inhibitory activity as well as improved selectivity for NHE-1 over NHE-2 compared to cariporide.

Full text of DOI:10.1021/jm010100v

3302
J . Med. Chem. 2001, 44, 3302-3310
Ar ylcyclop r op a n eca r boxyl Gu a n id in es a s Novel, P oten t, a n d Selective
In h ibitor s of th e Sod iu m Hyd r ogen Exch a n ger Isofor m -1
Saleem Ahmad,*,† Lidia M. Doweyko,† Sundeep Dugar,† Nyeemah Grazier,† Khehyong Ngu,† Shung C. Wu,†
Kenneth J . Yost,† Bang-Chi Chen,† J ack Z. Gougoutas, J ohn D. DiMarco, Shih-J ung Lan,§ Brian J . Gavin,‡
Alice Y. Chen,‡ Charles R. Dorso,‡ Randy Serafino,‡ Mark Kirby,‡ and Karnail S. Atwal†
Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New J ersey 08543
Received March 5, 2001
A novel series of arylcyclopropanecarboxyl guanidines was synthesized and evaluated for activity
against the sodium hydrogen exchanger isoform-1 (NHE-1). In biological assays conducted in
an AP1 cell line expressing the human NHE-1 isoform, the starting cyclopropane 3a (IC50
)
3.5 µM) shows inhibitory activity comparable to cariporide (IC50 ) 3.4 µM). Structure-activity
relationships are used to optimize the affinity of various acyl guanidines for NHE-1 by screening
the effect of substituents at both aryl and cyclopropyl rings. It is demonstrated that introduction
of appropriate hydrophobic groups at the phenyl ring and a gem-dimethyl group at the
cyclopropane ring enhances the NHE-1 inhibitory activity by up to 3 orders of magnitude
(compound 7f, IC50 ) 0.003 µM). In addition, the gem-dimethyl series of analogues seem to
display improved oral bioavailability and longer plasma half-life in rats. Furthermore, the lead
benzodihydrofuranyl analogue 1 (BMS-284640) shows over 380-fold increased NHE-1 inhibitory
activity as well as improved selectivity for NHE-1 over NHE-2 compared to cariporide.
In tr od u ction
The sodium hydrogen exchanger (NHE) isoforms are
integral plasma membrane proteins which transport
sodium ions in exchange for protons.1 Currently there
are six known isoforms of NHE. NHE-1 is ubiquitous
and plays a role in maintaining cellular pH, intra-
cellular sodium ion concentration, and cell volume.
NHE-2 is present in all three major gastric epithelial
cell types and is expressed in the small intestine, colon,
and kidney.2 It is believed to be involved in the regula-
tion of acid secretion by the stomach, because an NHE-2
knockout transgenic mouse shows decreased acid secre-
tion linked to reduced viability of gastric parietal cells.2
NHE-3 is primarily found in renal epithelia, localized
to the apical membrane, where it has been implicated
in the absorption of sodium.4 NHE-4 is found in the
stomach5 and the collecting tubule of the renal inner
medulla, where it has been proposed to play a special-
ized role in volume regulation.6 NHE-5 is present in
several nonepithelial tissues, including brain, spleen,
and skeletal muscle, and its role is unknown. NHE-6 is
ubiquitous and is believed to be most abundant in
mitochondria.
F igu r e 1.
underlies calcium overload and contractile dysfunction
observed during ischemia and reperfusion. NHE-1
inhibition limits ischemic damage by indirectly inhibit-
ing calcium overload.8 A major advantage of an NHE-1
inhibitor is that NHE-1 is believed to be inactive in the
normal myocardium, and thus, any effects due to
inhibition of NHE-1 should be specific for the ischemic
region.
Cariporide (HOE-642) (Figure 1) was the first selec-
tive NHE-1 inhibitor discovered, and it is currently in
phase II/III clinical trials as a potential treatment for
myocardial infarction (MI) and ischemic damage due to
angioplasty and reperfusion following thrombolysis.9
Another NHE-1 inhibitor, EMD-96-875 (eniporide) is
also reported to be in phase II clinical trials.10
Our objective was to identify a potent and selective
NHE-1 inhibitor that is suitable for once or twice daily
dosing. Since most known NHE inhibitors are acyl
guanidines, we synthesized a series of acyl guanidine
analogues from diverse carboxylic acids using high-
throughput solution phase chemistry. From this effort,
several novel acyl guanidines were identified as potent
Intracellular pH changes have been implicated in a
variety of pathophysiological conditions including hy-
pertension and myocardial ischemia. NHE-1 is the
predominant isoform in myocardial cells, and it is
believed to be responsible for exchanging intracellular
protons generated by anaerobic metabolism for extra-
cellular sodium ions during ischemia.7 As such, it is
implicated in the increase in intracellular sodium that
* Corresponding author. Tel: 609-252-6955. Fax: 609-252-6804.
E-mail: saleem.ahmad@bms.com.
† Department of Medicinal Chemistry.
Solid State Chemistry Department.
§ Metabolism and Pharmacokinetics Department.
‡ Department of Metabolic and Cardiovascular Drug Discovery.
10.1021/jm010100v CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/30/2001

Arylcyclopropanecarboxyl Guanidines as NHE-1 Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3303
Sch em e 1. Synthesis of Arylcyclopropanecarboxyl
Guanidines (3a -k and 4) via Palladium Catalyzed
Cyclopropanation of Cinnamic Acids or Estersa
Sch em e 2. Synthesis of
gem-Dimethylcyclopropanecarboxyl Guanidines from
Ethyl or tert-Butyl Cinnamates via Cyclopropanation
with Isopropylidenetriphenyl Phosphoranea
a
Reagents: (a) Pd(OAc)2/CH2N2/ether/THF; (b) guanidine/DMF.
inhibitors of NHE-1, including analogues containing an
arylcyclopropane moiety. Herein we report the design,
synthesis, and NHE-1 inhibitory activity of a novel
series of arylcyclopropanecarboxyl guanidines including
compound 1 (BMS-284640, NHE-1 IC50 ) 9 nM), which
in our hands is ca. 380-fold more potent than cariporide
and orally bioavailable in rats and dogs.11
Ch em istr y
The arylcyclopropanecarboxyl guanidines described
herein may be divided into an unsubstituted cyclopro-
pane series and a gem-dimethylcyclopropane series.
Synthesis of the first series of analogues 3a -3k and 4
was carried out via the sequence of steps outlined in
Scheme 1. Most of the cinnamic acids or esters used in
the preparation of this series are commercially avail-
able. Synthesis of the unsaturated acid required for the
preparation of 4 has been described previously by Catt
et al.12
a
Reagents: (a) isopropyltriphenylphosphonium iodide/n-BuLi/
THF; (b) TFA-CH2Cl2 1:1 (used only for the tert-butyl esters); (c)
CDI/DMF then guanidine; (d) dimethyl malonate/piperidine/acetic
acid; (e) 2-nitropropane/potassium tert-butoxide/DMSO; (f) sodium
cyanide/DMSO; (g) chiral resolution (see Experimental Section).
The various cyclopropanecarboxyl esters 2 were pre-
pared by treatment of a THF solution of the respective
trans-cinnamic acids (or esters) with an ether solution
of diazomethane in the presence of palladium acetate
and used as such without further purification.13 The
various esters 2 could be converted to the corresponding
acyl guanidines by treatment with excess guanidine
(free base) in DMF for 12 h. In each case, the products
isolated after workup were subjected to reversed-phase
preparative HPLC to isolate pure acyl guanidines as
TFA salts. It is noteworthy that the various cyclopro-
panecarboxyl guanidines described herein exhibited
limited stability when they were exposed as free bases
to methanol or ethanol yielding the corresponding
cyclopropyl esters. However, these compounds could be
isolated in pure and stable form as acid salts.
Synthesis of the gem-dimethyl series of analogues
7a -7i involved cyclopropanation of ethyl or tert-butyl
cinnamates (5a -5i). While some of the cinnamic acids
or esters used to prepare this series of analogues could
be obtained from commercial sources, most of them were
prepared from the corresponding aldehydes via Horner-
Emmons or Wittig reactions.14 Generally a THF or DMF
solution of tert-butyl diethylphosphonoacetate was treated
at low temperature with sodium bistrimethylsilylamide
followed by treatment with a solution of the correspond-
ing aldehyde. Alternatively, stirring the aldehydes with
(carbethoxymethylene)triphenylphosphorane or (carb-
tert-butoxymethylene)triphenyl-phosphorane in reflux-
ing dichloromethane also afforded the corresponding
cinnamates.
The cyclopropanation typically involved treating a
THF solution of the cinnamate at -78 °C to room
temperature with isopropylidenetriphenylphosphorane
(generated by treating a THF solution of isopropyl-
triphenylphosphonium iodide with n-BuLi).15 In most
cases the crude cyclopropyl tert-butyl esters thus pre-
pared were converted to the corresponding acids by
treatment with excess trifluoroacetic acid in methylene
chloride at room temperature (Scheme 2). The yields for
the cyclopropanation step ranged from 20 to 95%. In
general, the presence of an ortho substituent on the aryl
group resulted in reduced yields most likely due to steric
hindrance. Nevertheless, the crude products were sub-
jected to the usual acid/base extractive workup to afford
acids of sufficient purity. Treatment of a solution of
crude carboxylic acids in DMF with stoichiometric
quantities of 1,1′-carbonyldiimidazole (CDI) for 1 h at
room temperature followed by treatment with guanidine
(free base) or guanidine carbonate afforded the various
gem-dimethylcyclopropanecarboxyl guanidines. Most of
the final compounds in this series were purified by
reversed phase preparative HPLC and isolated as TFA
salts.
The benzodihydrofuranyl analogue 1 was initially
prepared in a manner similar to that described above
for synthesis of the acyl guanidines 7a -7i. However,
this approach was problematic due to poor yields for the
cyclopropanation step. An alternate procedure was
therefore developed which involved condensation of
dimethyl malonate with the known aldehyde 812 to

3304 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Ahmad et al.
Ta ble 1. Sodium Hydrogen Exchange-1 (NHE-1) Activity of
the Various Arylcyclopropanecarboxyl Guanidinesa
Sch em e 3. Synthesis of Various
3-Alkyl-4-fluorobenzaldehydesa
compd no.
R1
R2
IC50 (µM)b
3.5
0.98
0.28
2.9
1.6
0.54
1.6
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
H
H
H
H
H
H
H
H
H
2-Cl
3-Cl
4-Cl
4-F
a
3-OMe
3-NO2
4-NO2
2,5-diCl
3-Br, 4-F
4-iPr
Reagents: (a) methylboronic acid/CsF/Pd(Ph3P)4/DME/argon;
(b) ethyl or isopropyl magnesium chloride/ZnCl2PdCl2(dppf)/CuI/
THF; (c) LAH/THF; (d) Swern oxidation.
>30
c
0.04 ( 0.0015
0.09 ( 0.0565
>30
3j
3k
4
0.12 ( 0.0268c
0.069
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
3-Cl
0.014 ( 0.0047c
0.012 ( 0.0056
0.006 ( 0.0004c
0.005 ( 0.0014c
0.003 ( 0.0014
0.032
3-OMe
3,5-diCl
2,5-diCl
3-Br, 4-F
3-Me, 4-F
3-Et, 4-F
3-iPr, 4-F
F igu r e 2.
afford the corresponding unsaturated diester and sub-
sequent cyclopropanation with 2-nitropropane in the
presence of potassium tert-butoxide to afford 9. The
diester 9 was converted to the acid 10 by treatment with
sodium cyanide in dimethyl sulfoxide.
In some instances the aldehydes used to prepare the
respective cinnamates also had to be prepared (Scheme
3). Aldehyde 12 was synthesized from the corresponding
bromo aldehyde 11 as described by Wright et al.16 The
3-ethyl- and 3-isopropylbenzaldehydes 14 and 15, re-
spectively, were prepared from the corresponding 3-
bromobenzoate 13 via treatment with ethyl or isopropyl
Grignard reagent by using the procedure of Weichert
et al.17 followed by LAH reduction of the resulting
benzoates and subsequent Swern oxidation.
Most of the compounds were initially prepared and
evaluated as racemates. However, several key com-
pounds including the racemic form of 1 were subse-
quently resolved via chromatography on a chiral pre-
parative HPLC column, and each enantiomer was
independently characterized and evaluated in relevant
biological assays. It is noteworthy that in all cases most
of the NHE-1 inhibitory activity could be attributed to
a single enantiomer.18 This article enlists physical data
only for the enantiomers that exhibited enhanced
potencies in the primary screen. Enantiomerically pure
forms of 1 and compound 7h were also prepared by
resolving the corresponding racemic acids via chroma-
tography using a chiral column prior to coupling with
guanidine. The absolute stereochemistry of 1 was
determined to be (R,R) by a single-crystal X-ray analysis
of the (R)-phenylglycinol amide 16 (Figure 2) derived
from the acid (+)-10.
0.005 ( 0.0003c
0.017
1
0.009 ( 0.0013c
3.4 ( 1.19
HOE-642 (cariporide)
EMD-96-875 (eniporide)
0.38 ( 0.14
a
Sodium hydrogen exchange activity is determined in AP1 cells
b
expressing human NHE-1. The IC50 values are means ( SEM
of 3-6 experiments; single values indicate the results of one
experiment. c IC50 values are reported only for the more active (+)-
enantiomers.
an imposed acidosis.20 Using this protocol, IC50 values
for cariporide and eniporide were measured as 3.4 and
0.38 µM, respectively.
The starting acyl guanidine 3a showed NHE-1 inhibi-
tory potency (IC50 ) 3.5 µM) similar to cariporide (Table
1). Introduction of a chloro group at the 2- or 3-position
of the phenyl group resulted in a modest 3- or 12-fold
increase in potency (compounds 3b and 3c, respectively),
perhaps indicating a role of hydrophobic interactions at
the receptor and/or increased affinity resulting from
conformational preference. While incorporation of the
4-chloro group (compound 3d ) did not affect the IC50
value of the parent compound 3a , introduction of a
smaller fluoro group (compound 3e) at this site en-
hanced the NHE-1 inhibitory activity by approximately
2-fold. The 3-methoxy analogue 3f also showed a ca.
6-fold increase in binding affinity. While the 3-nitro
compound 3g displayed ca. 2-fold enhancement in
potency, the 4-nitro analogue 3h was essentially devoid
of NHE-1 inhibitory activity. The 2,5-dichloro and
3-bromo-4-fluoro analogues 3i and 3j, with IC50 values
of 40 and 90 nM respectively, are 87- and 39-fold more
potent than the unsubstituted compound 3a . The 4-iso-
propyl analogue 3k did not significantly inhibit NHE-1
at 30 µM concentration. The lack of inhibitory activity
of the 4-nitro and 4-isopropyl analogues, 3h and 3k
respectively, may be attributed to steric restrictions at
the binding site. Constraining the 3-methoxy analogue
3f to afford the more rigid benzodihydrofuran-derived
analogue 4 resulted in further 4-fold enhancement in
potency.
Resu lts a n d Discu ssion
NHE inhibitors were screened in an AP1 cell line19
(devoid of NHE activity) expressing the human NHE
isoforms-1, -2, -3, and -5. The IC50 values were deter-
mined by measuring the ability of compounds to inhibit
50% of the sodium dependent recovery of pH following

Arylcyclopropanecarboxyl Guanidines as NHE-1 Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3305
Ta ble 2. Specificity of 1, Cariporide, and Eniporide for NHE-1
Ta ble 3. Pharmacokinetic Parameters and Oral Bioavailability
Isoform vs NHE-2, -3, and -5 Isoformsa,b
of Selected Arylcyclopropanecarboxyl Guanidines in Ratsa
NHE-1
IC50 (µM)
NHE-2
NHE-3
NHE-5
compd
no.
Vssb
(L/kg)
clearance
(mL/min/kg)
t1/2
(h)
% oral
bioavailability
compd
IC50 (µM) IC50 (µM) IC50 (µM)
1
0.009 ( 0.0013 1.8 ( 0.44
>30
>30
>30
3.36 ( 0.14
>30
3i
7e
4
21 ( 7
23 ( 3
6 ( 1
143 ( 4
44 ( 12
120 ( 4
74 ( 9
3.4 ( 1.7
8 ( 2
13 ( 0
47 ( 21
49 ( 9
63 ( 24
cariporide
eniporide
3.4 ( 1.19
62 ( 7.00
17 ( 2.43
0.38 ( 0.14
>30
1.3 ( 0.3
3 ( 2
1
8.6 ( 2.9
a
Sodium hydrogen exchange activity is determined in AP1 cells
b
a
expressing human NHE-1-5. The IC50 values are means ( SEM
of 2-6 experiments.
Plasma concentrations were determined after a single (10
µmol/kg, n ) 3) intra-arterial and oral dose to rats; the vales are
b
means ( SEM of the three experiments. Volume of distribution
at steady state.
Efforts were also made to explore substituent effects
at the cyclopropyl ring. Table 1 outlines a series of gem-
dimethylcyclopropyl analogues 1 and 7a -7i. The phenyl
analogue 7a was ca. 50-fold more potent than the lead
compound 3a . The substantial increase in potency of 7a
induced by the gem-dimethyl group was also exhibited
by a number of additional analogues (Table 1). This may
be attributed to further hydrophobic interactions achieved
by the inhibitor at the receptor site. Alternatively, the
conformational rigidity imposed by introducing steric
bulk at the cyclopropane ring may hinder rotation of
aryl and acyl guanidine groups relative to the cyclopro-
pane ring, thus optimizing interactions at respective
binding sites. Combination of this “gem-dimethyl effect”
with groups capitalizing on interactions at the phenyl
ring resulted in the discovery of some of the most potent
NHE-1 inhibitors known to date. For instance, the
3-bromo-4-fluoro-gem-dimethyl analogue 7f (IC50 ) 3
nM) is ca. 1100-fold more potent than the lead com-
pound 3a and cariporide. Attempts were made to
investigate steric requirements of hydrophobic groups
at the 3-position of the phenyl ring as well as to find a
replacement for the 3-bromo group of 7f. The 3-alkyl-
4-fluoro analogues 7g-7i were therefore prepared and
evaluated for NHE-1 potency. The 3-ethyl-4-fluoro
analogue 7h with an IC50 value of 5 nM is 3- and 6-fold
more potent than the corresponding isopropyl and
methyl analogues, respectively. Introduction of the gem-
dimethyl group to 4 resulted in 13-fold enhanced
potency yielding 1 (IC50 ) 9 nM).
cariporide is only 26-fold selective for NHE-1 vs the
cardiac sodium current (IC50 ) 88 µM).
P h a r m a cok in etic P r op er ties
Several analogues reported here were evaluated for
pharmacokinetic (PK) properties in rats. Various com-
pounds were dosed orally or intra-arterially at 10 µmol/
kg (n ) 3), and plasma samples were analyzed by LC/
MS/MS. Table 3 outlines oral bioavailability and various
PK parameters of selected NHE-1 inhibitors. In cases
where both the gem-dimethyl and the des-dimethyl
analogues were tested, the dimethyl analogues exhibited
longer plasma elimination half-life (t1/2) and improved
oral bioavailability. For example, after a single intra-
arterial and oral dose to rats, the t1/2 and oral bioavail-
ability values for the 2,5-dichlorophenyl analogue 3i and
the corresponding dimethyl analogue 7e were 3.4 h and
13% vs 8 h and 47%. Similarly, 1 with 63% oral
bioavailability and a 3 h t1/2 showed superior PK profile
over the corresponding analogue without the gem-
dimethyl group (compound 4).
Con clu sion
This report describes the design and synthesis of
various novel arylcyclopropanecarboxyl guanidines as
potent and selective inhibitors of NHE-1. Systematic
structure-activity relationships were used to optimize
the inhibition by various cyclopropyl acyl guanidines of
NHE-1 by studying the effect of substituents at the aryl
ring. In general, introduction of chloro, methoxy, and
lower alkyl groups at the meta position and a fluoro
group at the para position result in significant enhance-
ment in potency. It was also realized that incorporation
of a gem-dimethyl group at the cyclopropane ring
further improves the NHE-1 inhibitory activity. In
addition, the gem-dimethyl series of analogues display
improved pharmacokinetic properties when compared
to the corresponding analogues without the dimethyl
group.
Evaluation of cariporide, a compound under investi-
gation in human clinical trials, reveals that this com-
pound exhibits only modest potency against the human
NHE-1 (IC50 ) 3.4 µM) with modest 18-fold selectivity
over NHE-2. It has also been shown that compound 1
shows 380- and 40-fold improved potency for NHE-1
over cariporide and eniporide, respectively, as well as
improved NHE-1/NHE-2 selectivity. In addition, 1 has
a good oral bioavailability (63%) and modest plasma
half-life (3 h) in rats. Furthermore, in dogs, the oral
bioavailability of 1 is 34% with a 4.4 h half-life. On the
basis of the improved selectivity and superior pharma-
cokinetic profile, compound 1 was selected for further
Selectivity
The selectivity of several acyl guanidines discussed
herein for NHE-1 was investigated in cell lines express-
ing NHE-2, NHE-3, and NHE-5. Table 2 outlines the
various IC50 values for 1, cariporide, and eniporide.
Compound 1 exhibits improved selectivity for NHE-1.
It is ca. 200-fold selective for NHE-1 over NHE-2, nearly
an order of magnitude more selective than cariporide,
and 4-fold more selective than eniporide. Chronic inhi-
bition of NHE-2 activity may lead to gastric problems,
since the NHE-2 knockout transgenic mouse shows
decreased acid secretion linked to reduced viability of
gastric parietal cells.3 Loss of parietal cells in man would
lead to inhibition of vitamin B12 uptake due to loss of
intrinsic factor. None of the compounds show significant
inhibition of NHE-3 at concentrations up to 30 µM.
Selectivity of 1 against NHE-5 is intermediate to NHE-2
and NHE-3.
Standard electrophysiological techniques were used
to measure the effects of various NHE inhibitors on
cardiac sodium channel currents.21 Both compound 1
and eniporide had little effect on sodium current at 100
µM (30% and 15% inhibition, respectively). However,

3306 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Ahmad et al.
(()-tr a n s-N-(Am in oim in om eth yl)-2-(2-ch lor op h en yl)-
cyclop r op a n eca r boxa m id e (3b): obtained as a white solid
(TFA salt, 96% yield); 1H NMR (500 MHz, CDCl3) δ 12.29 (brs,
1H), 8.85 (brs, 2H), 7.95 (brs, 2H), 7.38-7.36 (m, 1H), 7.22-
7.20 (m, 2H), 7.10-7.08 (m, 1H), 2.85-2.82 (m, 1H), 2.01-
1.99 (m, 1H), 1.79-1.77 (m, 1H), 1.53-1.52 (m, 1H); 13C NMR
(125 MHz, CD3OD) δ 174.78, 155.49, 136.68, 135.41, 129.20,
128.47, 127.61, 127.17, 26.23, 24.97, 15.70; HRMS (ESI) calcd
for C11H12ClN3O (M + H)+ 238.0748, found 238.0738; Anal.
HPLC tR ) 2.7 min.
development. Efforts are underway to evaluate 1 in
animal models, and the results of which will be pub-
lished in due course.
Exp er im en ta l Section
Reagents and solvents were purchased from Aldrich Chemi-
cal Co. and used without further purification. Silica gel 60
(Merck) was used for flash chromatography, and silica gel 60
F254 (Merck) was used for thin-layer chromatography (TLC).
The mobile systems are indicated in the procedures. TLC spots
were examined under UV light at 254 nm. Analytical HPLC
analyses were performed under the following conditions: YMC
S5 ODS 4.6 × 50 mm column/linear gradient system of H2O-
MeOH-H3PO4 90:10:0.2 to 10:90:0.2 over 4 min at a flow rate
of 4 mL/min with a 1 min hold time at 10:90:0.2 H2O-MeOH-
H3PO4. All final compounds were purified by preparative
HPLC on a YMC ODS A 30 × 250 mm column using a water-
MeOH-TFA 90:10:0.1 to 10:90:0.1 linear gradient over 30 min
at a flow rate of 25 mL/min. Unless otherwise noted, all
reactions were monitored by LC/MS (Shimadzu-Micromass
system/YMC S5 ODS 4.6 × 50 mm column/water-MeOH-
TFA 90:10:0.1 to 10:90:0.1 linear gradient over 4 min followed
by a 1 min hold at 10:90:0.1 of water-MeOH-TFA at a flow
rate of 4 mL/min. NMR data were obtained using a J EOL CPF-
270, Delta Eclipse 270, 400, or 500 spectrometer. Several
compounds were resolved by chromatography on a chiral
column (CHIRALPAK AD 50 × 500 mm column manufactured
by Chiral Technologies, Inc., Exton, PA) to obtain each
enantiomer in pure form. Optical rotations were determined
by using a Perkin-Elmer 241 polarimeter. All final compounds
were of g98% purity by the LC/MS and analytical HPLC
systems and were characterized by NMR and high-resolution
mass spectral (HRMS) analyses.
(()-tr a n s-N-(Am in oim in om eth yl)-2-(3-ch lor op h en yl)-
cyclop r op a n eca r boxa m id e (3c): obtained as a gummy solid
(TFA salt, 26% yield); 1H NMR (500 MHz, CDCl3) δ 12.14 (brs,
1H), 8.57 (brs, 2H), 8.16 (brs, 2H), 7.22-7.21 (m, 2H), 7.12 (s,
1H), 6.99-6.98 (m, 1H), 2.63-2.62 (m, 1H), 2.07-2.04 (m, 1H),
1.76-1.74 (m, 1H), 1.48-1.47 (m, 1H); 13C NMR (125 MHz,
CDCl3) δ 174.85, 155.96, 140.65, 134.55, 129.90, 127.24, 126.82,
124.44, 28.05, 25.98, 18.58; HRMS (ESI) calcd for C11H12ClN3O
(M + H)+ 238.0748, found 238.0742; Anal. HPLC tR ) 2.9 min.
(()-tr a n s-N-(Am in oim in om eth yl)-2-(4-ch lor op h en yl)-
cyclop r op a n eca r boxa m id e (3d ): obtained as a gummy solid
(TFA salt, 38% yield); 1H NMR (500 MHz, CDCl3) δ 12.08 (brs,
1H), 8.54 (brs, 2H), 8.16 (brs, 2H), 7.26 (d, 2H, J ) 8.2 Hz),
7.04 (d, 2H, J ) 8.8 Hz), 2.63-2.61 (m, 1H), 2.04-2.02 (m,
1H), 1.75-1.72 (m, 1H), 1.45-1.43 (m, 1H); 13C NMR (125
MHz, CDCl3) δ 174.91, 155.90, 137.10, 132.83, 128.74, 127.73,
27.97, 25.98, 18.66; HRMS (ESI) calcd for C11H12ClN3O (M +
H)+ 238.0748, found 238.0748; Anal. HPLC tR ) 2.9 min.
(()-tr a n s-N-(Am in oim in om et h yl)-2-(4-flu or op h en yl)-
cyclop r op a n eca r boxa m id e (3e): obtained as a white solid
(TFA salt, 47% yield); 1H NMR (500 MHz, CDCl3) δ 12.19 (brs,
1H), 8.63 (brs, 2H), 8.09 (brs, 2H), 7.10-7.08 (m, 2H), 6.98 (t,
2H, J ) 8.2 Hz, 8.8 Hz), 2.64-2.63 (m, 1H), 2.03-2.01 (m,
1H), 1.68-1.66 (m, 1H), 1.44-1.43 (m, 1H); 13C NMR (125
MHz, CDCl3) δ 175.13, 161.25, 156.70, 135.00, 128.10, 128.06,
115.50, 115.40, 28.03, 25.92, 18.62; HRMS (ESI) calcd for
P r oced u r e A. Gen er a l P r oced u r e for P r ep a r in g Ar yl-
cyclop r op a n eca r boxyl Gu a n id in es 3a -k fr om Cin n a m ic
Acid s or Ester s.13 To a solution of the cinnamic acid or ester
(0.5 mmol) in THF (10 mL) were added Pd(OAc)2 (5 mg) and
a cold (0 °C) ethereal solution of diazomethane (6 mL, freshly
prepared from 1-methyl-3-nitro-1-nitrosoguanidine (MNNG, 74
mg, 4.5 mmol)) alternately in small portions with continuous
stirring during 10-15 min (the addition of diazomethane was
done in small portions just following each addition of the
palladium catalyst). It is noteworthy that the analytical HPLC
retention times and the TLC Rf values for the cinnamates and
the corresponding cyclopropyl esters are almost indistinguish-
able. Furthermore, we could not observe the molecular ions
(M + H)+ for the cyclopropyl esters in LC/MS. Therefore, the
C
11H12FN3O (M + H)+ 222.1043, found 222.1046; Anal. HPLC
tR ) 2.5 min.
(()-tr a n s-N-(Am in oim in om eth yl)-2-(3-m eth oxyph en yl)-
cyclop r op a n eca r boxa m id e (3f): obtained as a white solid
(TFA salt, 98% yield); 1H NMR (500 MHz, CDCl3) δ 12.04 (brs,
1H), 8.55 (brs, 2H), 8.15 (brs, 2H), 7.20 (t, 1H, 7.7 Hz, 8.2 Hz),
6.77 (d, 1H, J ) 8.2 Hz), 6.69 (d, 1H, J ) 7.7 Hz), 6.64 (s, 1H),
3.78 (s, 3H), 2.64-2.63 (m, 1H), 2.07-2.05 (m, 1H), 1.74-1.71
(m, 1H), 1.48-1.45 (m, 1H); 13C NMR (125 MHz, CDCl3) δ
174.50, 159.82, 156.20, 140.21, 129.65, 118.57, 112.61, 111.94,
55.16, 28.69, 26.02, 18.89; HRMS (ESI) calcd for C12H15N3O2
(M + H)+ 234.1243, found 234.1241; Anal. HPLC tR ) 2.6 min.
(()-tr a n s-N-(Am in oim in om et h yl)-2-(3-n it r op h en yl)-
cyclop r op a n eca r boxa m id e (3g): obtained as an off-white
solid (TFA salt, 68% yield); 1H NMR (500 MHz, CD3OD) δ 8.10
(d, 1H, J ) 8.7 Hz), 8.08 (s, 1H), 7.63 (d, 1H, J ) 7.7 Hz), 7.56
(t, 1H, J ) 7.7 Hz, 8.3 Hz), 2.80-2.77 (m, 1H), 2.17-2.15 (m,
1H), 1.82-1.80 (m, 1H), 1.67-1.65 (m, 1H); 13C NMR (125
MHz, CD3OD) δ 178.00, 156.0, 149.0, 142.00, 133.40, 130.49,
122.36, 121.73, 27.81, 27.24, 17.88; HRMS (ESI) calcd for
1
progress of these reactions was monitored by H NMR. Upon
completion of the reaction, a few drops of glacial acetic acid
were added to quench excess diazomethane. The reaction
mixture was filtered through Celite, and the filtrate was
concentrated to give a thick dark brown residue. The crude
cyclopropyl esters thus prepared were dissolved in 0.5 mL of
DMF and treated with a solution of guanidine (148 mg, 2.5
mmol) in DMF (1 mL). The reaction mixtures were stirred for
12 h at room temperature, diluted with water, and extracted
with EtOAc. The organic layer was dried over magnesium
sulfate and concentrated, and the residue was subjected to
reversed-phase preparative HPLC (ODS column/H2O-MeOH-
TFA, 90:10:0.1 to 10:90:0.1 gradient) to afford the correspond-
ing acyl guanidines as TFA salts. The following compounds
(3a -k ) were prepared by the general procedure A described
above.
(()-tr a n s-N-(Am in oim in om et h yl)-2-p h en ylcyclop r o-
p a n eca r boxa m id e (3a ): obtained as a white solid (TFA salt,
14% yield, two steps); 1H NMR (500 MHz, CDCl3) δ 12.03 (brs,
1H), 8.58 (brs, 2H), 8.06 (brs, 2H), 7.22 (t, 2H, J ) 7.2 Hz, 7.8
Hz), 7.17 (t, 1H, J ) 7.8 Hz, 7.2 Hz), 7.05 (d, 2H, 9.1 Hz), 2.60-
2.59 (m, 1H), 2.00-1.99 (m, 1H), 1.69-1.66 (m, 1H), 1.43-
1.41 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 177.00, 156.00,
138.00, 128.64, 127.20, 126.36, 28.90, 26.08, 19.10; HRMS
(ESI) calcd for C11H13N3O (M + H)+ 204.1138, found 204.1139;
Anal. HPLC tR ) 2.4 min.
C
11H12N4O3 (M + H)+ 249.0988, found 249.0982; Anal. HPLC
tR ) 2.3 min.
(()-tr a n s-N-(Am in oim in om et h yl)-2-(4-n it r op h en yl)-
cyclop r op a n eca r boxa m id e (3h ): obtained as a white solid
1
(TFA salt, 31% yield). H NMR (500 MHz, CD3OD) δ 8.17 (d,
2H, J ) 8.8 Hz), 7.42 (d, 2H, J ) 8.8 Hz), 2.77-2.74 (m, 1H),
2.24-2.21 (m, 1H), 1.83-1.79 (m, 1H), 1.68-1.64 (m, 1H); 13
C
NMR (125 MHz, CD3OD) δ 175.38, 158.50, 149.00, 148.00,
128.65, 125.05, 28.78, 28.48, 19.20; HRMS (ESI) calcd for
C
11H12N4O3 (M + H)+ 249.0988, found 249.0989; Anal. HPLC
tR ) 2.3 min.
(+)-t r a n s-N -(Am in o im in o m e t h y l)-2-(2,5-d ic h lo r o -
p h en yl)cyclop r op a n eca r boxa m id e (3i): The title com-
pound was prepared in racemic form (57% yield, white solid)
from the corresponding cinnamate 5e as described in procedure
A except that the crude product was purified by flash chro-
matography (silica gel/ethyl acetate-MeOH-triethylamine 80:
20:0.3). The racemic product was submitted to preparative

Arylcyclopropanecarboxyl Guanidines as NHE-1 Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3307
chromatography on a chiral column (CHIRALPAK AD/92:8:
0.3 hexanes-EtOH-triethylamine) to afford 3i as the faster
moving enantiomer (white solid, free base); 1H NMR (500 MHz,
CDCl3) δ 7.26 (d, J ) 8.2 Hz, 1H, Ar), 7.09 (dd, 1H, J ) 8.2
Hz, 2.2 Hz, Ar), 6.98 (d, 1H, J ) 2.2 Hz, Ar), 2.71-2.67 (m,
1H), 1.88-1.84 (m, 1H), 1.62-1.58 (m, 1H), 1.25-1.20 (m, 1H);
13C NMR (125 MHz, CDCl3) δ 184, 161.04, 140.57, 133.66,
132.52, 130.24, 127.35, 127.09, 38.98, 24.12, 16.38; [R]D +220°
aqueous layer was extracted with EtOAc three times. The
combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. Purification of the crude
residue by silica gel flash chromatography (9:1 hexanes-ethyl
1
acetate) provided 5d as a white solid (5.19 g, 77% yield); H
NMR (270 MHz, CDCl3) δ 7.51 (d, 1H, J ) 17.8 Hz), 7.31 (s,
1H), 7.29-7.23 (m, 2H), 6.29 (d, 1H, J ) 17.8 Hz), 1.48 (s,
9H).
(c ) 0.4, MeOH); MS (ESI) 274 (M + H)+; Anal. HPLC tR
)
3-Eth yl-4-flu or oben za ld eh yd e18 (14): Ethylmagnesium
chloride (2.0 M in ether, 17.1 mL, 34.24 mmol) was added
slowly to anhydrous zinc chloride (flame dried, 4.67 g, 34.24
mmol) in 50 mL THF under argon. The resulting white slurry
was stirred at 50 °C for 3 h. In a separate flask a solution of
3-bromo-4-fluorobenzoic acid methyl ester (5.00 g, 22.83 mmol)
in 50 mL of THF was sequentially treated with PdCl2(dppf)23
(0.835 g,1.14 mmol) and copper(I) iodide (0.261 g, 1.37 mmol)
under argon. The alkyl zinc slurry that had been stirring at
50 °C for 3 h was added slowly to the ester mixture at room
temperature. The resulting dark brown slurry was stirred in
the dark at room temperature for 48 h. The reaction mixture
was concentrated in vacuo to give a dark brown residue, which
was taken up in EtOAc and washed sequentially with 1 M
HCl, saturated NaHCO3, and brine. The organic layer was
dried over MgSO4 and concentrated in vacuo to obtain crude
methyl 3-ethyl-4-fluorobenzoate as dark brown oil (4.8 g).
The above crude ester (4.80 g, 26.35 mmol) was dissolved
in THF (100 mL) under argon followed by the addition of LAH
(1.0 M solution in THF, 52.6 mL, 52.6 mmol) dropwise at 0
°C. The reaction was allowed to warm to room temperature
over 1 h, quenched with methanol, and filtered. The filtrate
was diluted with EtOAc, washed sequentially with saturated
NaHCO3 and brine, then dried (MgSO4), and concentrated in
vacuo to obtain 3-ethyl-4-fluorobenzyl alcohol as light brown
oil (3.5 g).
3.2 min.
(()-tr a n s-N-(Am in oim in om et h yl)-2-(3-b r om o-4-flu o-
r op h en yl)cyclop r op a n eca r boxa m id e (3j): obtained as a
white solid (TFA salt, 81% yield); 1H NMR (500 MHz, CDCl3)
δ 12.25 (brs, 1H), 8.64 (brs, 2H), 8.09 (brs, 2H), 7.34 (d, 1H, J
) 5.5 Hz), 7.05-7.04 (m, 1H), 7.03 (s, 1H), 2.64-2.61 (m, 1H),
2.04-2.02 (m, 1H), 1.76-1.73 (m, 1H), 1.46-1.42 (m, 1H);
HRMS (ESI) calcd for C11H11BrFN3O (M + H)+ 300.0149, found
300.0144; Anal. HPLC tR ) 3.0 min.
(()-tr a n s-N-(Am in oim in om eth yl)-2-(4-isopr opylph en yl)-
cyclop r op a n eca r boxa m id e (3k ): obtained as a white solid
(TFA salt, 73% yield); 1H NMR (500 MHz, CDCl3) δ 12.09 (brs,
1H), 8.62 (brs, 2H), 8.17 (brs, 2H), 7.15 (d, 2H, J ) 8.3 Hz),
7.04 (d, 2H, J ) 8.3 Hz), 2.89-2.86(m, 1H), 2.64-2.62 (m, 1H),
2.07-2.03 (m, 1H), 1.72-1.68 (m, 1H), 1.46-1.44 (m, 1H), 1.23
(d, 6H, J ) 7.2 Hz); 13C NMR (125 MHz, CDCl3) δ 175.37,
155.96, 147.79, 135.92, 126.66, 126.34, 33.73, 28.53, 25.98,
23.94, 18.82; HRMS (ESI) calcd for C14H19N3O (M + H)+
246.1607, found 246.1596; Anal. HPLC tR ) 3.3 min.
(+)-tr a n s-N-(Am in oim in om eth yl)-2-(2,3-d ih yd r o-4-ben -
zofu r a n yl)cyclop r op a n eca r boxa m id e (4): 1,1′-Carbonyl-
diimidazole (298 mg, 1.84 mmol) was added to a solution of
2-(2,3-dihydro-4-benzofuranyl)cyclopropanecarboxylic acid12 (300
mg, 1.47 mmol) in THF (3 mL). The reaction mixture was
stirred for 3 h at room temperature followed by the addition
of guanidine (434 mg, 7.35 mmol) in DMF (1 mL). After the
reaction mixture was stirred at room temperature for 1 h, the
solvent was removed in vacuo to afford an off-white residue.
The crude product was purified by silica gel flash chromatog-
raphy as in the case of compound 3i (white solid, 216 mg, 48%)
and submitted to chromatography on a chiral column (CHIRAL-
PAKAD/85:15:0.1 hexanes-ethanol-triethylamine) to afford
4 as the faster moving enantiomer (free base, off-white solid,
mp 149-150 °C); 1H NMR (270 MHz, CD3OD) δ 6.99 (dd, J )
7.62, 8.2 Hz, 1H, Ar), 6.59 (d, J ) 8.2 Hz, 1H, Ar), 6.39 (d, J
) 7.62 Hz, 1H, Ar), 5.96 (b, 4H, NH), 4.51 (t, 2H, J ) 8.8 Hz),
3.56 (d, 2H, J ) 8.8 Hz), 2.25-2.35 (m, 1H), 1.82-1.90 (m,
1H), 1.46-1.50 (m, 1H), 1.20-1.22 (m, 1H); [R]D +136° (c )
0.4, MeOH); 13C NMR (67.5 MHz, CDCl3) δ 185.70, 162.07,
160.29, 138.74, 128.87, 127.11, 116.70, 107.73, 71.79, 29.77,
29.31, 24.81, 17.48; HRMS (ESI) calcd for C13H15N3O2 (M +
H)+ 246.1243, found 246.1244; Anal. HPLC tR ) 2.49 min.
A solution of DMSO (3.56 mL, 49.94 mmol) in 20 mL of
methylene chloride was added dropwise to a stirred solution
of oxalyl chloride (2.18 mL, 24.97 mmol) in methylene chloride
(50 mL) at -78 °C under argon. The reaction mixture was
stirred at -78 °C for 15 min followed by the addition of the
crude alcohol from above (3.5 g, 22.7 mmol) in 15 mL of
methylene chloride. The reaction mixture was stirred at -78
°C for 45 min followed by the addition of triethylamine (10.44
mL, 75 mmol), and the mixture was allowed to come to room
temperature. The mixture was diluted with methylene chlo-
ride, washed sequentially with 10% aqueous sulfuric acid,
saturated sodium bicarbonate solution, and water. The organic
layer was dried over magnesium sulfate and concentrated to
give 3-ethyl-4-fluorobenzaldehyde (14) as a yellow oil (3.3 g,
95% yield); 1H NMR (270 MHz, CDCl3) δ 9.86 (s, 1H), 7.71
(dd, 1H, J ) 2.2, 7.47 Hz), 7.69-7.60 (m, 1H), 7.09 (dd, 1H,
J
) 8.3, 9.2 Hz), 2.66 (q, 2H, J ) 7.9 Hz), 1.20 (t, 3H,
J ) 7.9 H).
tr a n s-3-Ch lor ocin n a m ic Acid , ter t-Bu tyl Ester (5b): To
a solution of 3-chlorocinnamic acid (200 g, 1.095 mol) in 700
mL THF was added di-tert-butyl pyrocarbonate ((Boc)2O, 286.8
g, 1.314 mol) at 0 °C followed by the addition of 4-(dimethyl-
amino)pyridine (26.76 g, 0.219 mol) in small portions. The
reaction mixture was stirred at room temperature for 14 h,
diluted with EtOAc, washed sequentially with water, 2.5%
sulfuric acid, and saturated sodium bicarbonate. The organic
phase was dried over MgSO4 and concentrated to afford a
brown oil which was used as such in the next step without
tr a n s-3-Eth yl-4-flu or ocin n a m ic Acid , ter t-Bu tyl Ester
(5h ): The aldehyde 14 was converted to 5h as described for
5d (brown oil, 65% yield); 1H NMR (CDCl3) δ 7.51 (d, 1H, J )
17.0 Hz), 7.34 (d, 1H, J ) 9.1 Hz), 7.31-7.28 (m, 1H), 6.98
(dd, 1H, J ) 8.6 Hz, 8.4 Hz), 6.27 (d, 1H, J ) 17.0 Hz), 2.66
(q, 2H, J ) 7.7 Hz), 1.51 (s, 9H), 1.22 (t, 3H, J ) 7.7 Hz).
tr a n s-4-F lu or o-3-isop r op ylcin n a m ic Acid , ter t-Bu tyl
Ester (5i): was prepared from 3-bromo-4-fluorobenzoic acid
methyl ester as described for 5h (35% overall yield); 1H NMR
(270 MHz, CDCl3) δ 7.54 (d, 1H, J ) 15 Hz), 7.50-7.30 (m,
2H), 7.06 (t, 1H, J ) 8.2 Hz), 6.29 (d, 1H, J ) 15 Hz), 3.25-
3.15 (m, 1H), 1.57 (s, 9H), 1.27 (d, 6H, J ) 6.4 Hz).
1
purification (260 g, 99% yield); H NMR (270 MHz, CDCl3) δ
7.67 (d, 1H, J ) 18.0 Hz), 7.65 (s, 1H), 7.52-7.40 (m, 3H),
6.51 (d, 1H, J ) 18.0 Hz), 1.69 (s, 9H).
tr a n s-3,5-Dich lor ocin n a m ic Acid , ter t-Bu t yl E st er
(5d ): To a solution of tert-butyl diethylphosphonoacetate (6.94
g, 27.50 mmol) in THF (50 mL) at 0 °C was added sodium bis-
(trimethylsilyl)amide (NaHMDS) (1 M in toluene, 27.50 mL,
27.50 mmol) under argon. The resulting solution was warmed
to 25 °C and stirred for 30 min. After cooling the reaction
mixture to 0 °C, a solution of 3,5-dichlorobenzaldehyde (4.38
g, 25.00 mmol) in THF (25 mL) was slowly added. The reaction
mixture was allowed to warm to room temperature, stirred
overnight, and poured onto saturated NH4Cl/EtOAc. The
P r oced u r e B. (()-tr a n s-3-(3,5-Dich lor op h en yl)-2,2-d i-
m eth ylcyclop r op a n eca r boxylic Acid (6d ): To a suspen-
sion of isopropyltriphenylphosphonium iodide (6.48 g, 15
mmol) in THF (45 mL) at -78 °C was added n-BuLi (2.5 M in
hexane, 6.6 mL, 16.5 mmol). The resulting mixture was
warmed to 0 °C and stirred for 30 min. The reaction mixture
was cooled to -78 °C followed by the addition of a solution of
5d (4.11 g, 15 mmol) in THF (30 mL). The reaction mixture
was stirred for 2 h at 0 °C, then slowly warmed to 25 °C, and
stirred overnight. The reaction mixture was poured onto 10%

3308 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Ahmad et al.
H2SO4/EtOAc. The aqueous layer was extracted with EtOAc.
The combined organic layers were sequentially washed with
saturated NaHCO3, brine, dried over Na2SO4, and concen-
trated in vacuo. The crude cyclopropyl tert-butyl ester was
dissolved in 25 mL of trifluoroacetic acid/methylene chloride
(1:1), and the resulting solution was stirred for 1 h at room
temperature. The reaction mixture was concentrated in vacuo.
The residue was partitioned between 10% NaOH and Et2O.
The aqueous layer was washed three times with ether. The
aqueous layer was acidified with dilute sulfuric acid and
extracted with EtOAc. The EtOAc layer was dried over MgSO4
and concentrated in vacuo to afford a white solid (2.32 g, 71%
yield over two steps); H NMR (CDCl3) δ 7.32 (s, 1H), 7.30-
7.27 (m, 2H), 2.77 (d, 1H, J ) 8.9 Hz), 2.20 (d, 1H, J ) 8.9
Hz), 1.48 (s, 3H), 1.05 (s, 3H).
(()-tr a n s-2,2-Dim eth yl-3-p h en ylcyclop r op a n eca r box-
ylic Acid , Eth yl Ester (6a ): prepared by cyclopropanation
of ethyl cinnamate as described in procedure B (gummy solid,
55% yield); 1H NMR (CDCl3) δ 7.58-7.11 (m, 5H), 4.05 (q, 2H,
7.1 Hz), 2.41 (d, 1H, J ) 5.7 Hz), 1.82 (d, 1H, J ) 5.7 Hz),
1.29 (s, 3H), 1.19 (t, 3H, 7.1 Hz), 0.79 (s, 3H).
(()-tr a n s-3-(2,5-Dich lor oph en yl)-2,2-dim eth ylcyclopr o-
p a n eca r boxylic Acid , Eth yl Ester (6e): To a solution of 2,5-
dichlorobenzaldehyde (165 g, 0.94 mol) in dichloromethane
(1300 mL) was added (carbethoxymethylene)triphenyl-
phosphorane (348 g, 1.0 mol) at room temperature. The
resulting solution was stirred under reflux for 4 h, cooled to
room temperature, and concentrated. The residue was dis-
solved in 3:7 ethyl acetate:hexanes (200 mL) and filtered
through a short bed of silica gel. The silica bed was washed
with ethyl acetate-hexanes (3:7). The filtrate was concen-
trated to give 212 g (91%) of the cinnamate 5e as pale yellow
oil. This compound was converted to 6e as described in
procedure B (white solid, 55% yield); 1H NMR (CDCl3) δ 7.58-
7.11 (m, 3H), 4.05 (q, 2 H, 7.1 Hz), 2.41 (d, 1 H, J ) 5.7 Hz),
1.82 (d, 1 H, J ) 5.7 Hz), 1.29 (s, 3H), 1.19 (t, 3H, 7.1 Hz),
0.79 (s, 3H).
with 1 N HCl, and the organic layer was dried (MgSO4) and
concentrated to give 24 g (62% yield) of the corresponding
dimethylcyclopropane diester 9 as yellow oil.
A portion of the dimethylcyclopropane intermediate from
above (20 g, 65.8 mmol) and sodium cyanide (11.6 g, 237 mmol)
in DMSO (125 mL) was heated at 150 °C for 23 h. After cooling,
the mixture was diluted with water and made basic by adding
10% aqueous potassium hydroxide. The mixture was washed
three times with ether, acidified with dilute sulfuric acid, and
extracted with hexanes-ethyl acetate (80:20, needed to filter
through Celite to remove emulsion). The organic layer was
dried (MgSO4) and concentrated to afford a crude brownish
residue, which was treated with activated charcoal in hot
ethanol to afford a light yellow solid. This material (ca. 9:1
trans-cis) was recrystallized from 90:10 acetonitrile-water
to afford 5 g (15% corrected yield from 8) of the racemic form
1
1
of 10 as a white solid; H NMR (270 MHz, CDCl3) δ 7.05 (dd,
1H, J ) 7.9, 7.7 Hz), 6.68 (d, 1H, J ) 7.9 Hz), 6.58 (d, 1H, J
) 7.7 Hz), 4.59 (dd, 2H, J ) 9, 9 Hz), 2.9-3.4 (m, 2H), 2.59 (d,
1H, J ) 5.9 Hz), 1.98 (d, 1H, J ) 5.9 Hz), 1.45 (s, 3H), 0.98 (s,
3H). The racemic acid from above was resolved by preparative
chromatography on a chiral column (CHIRALPAK AD column/
85:15:1.5 hexanes-2-propanol-acetic acid) to give enantio-
merically pure 10 (faster moving) { [R]D +40° (c ) 1, metha-
nol)} as well as the corresponding slower moving (1S,3S)-
enantiomer {[R]D -40° (c ) 1, methanol)}.
(+)-t r a n s-N -(Am in o im in o m e t h y l)-3-(3,5-d ic h lo r o -
p h en yl)-2,2-d im eth ylcyclop r op a n eca r boxa m id e (7d ): To
a solution of the acid 6d (425 mg, 1.60 mmol) in DMF (5 mL)
at room temperature was added CDI (313 mg, 1.90 mmol), and
the resulting solution was stirred for 1 h at room temperature.
Guanidine carbonate (609 mg, 3.20 mmol) was added, and the
reaction mixture was stirred overnight at room temperature.
The reaction mixture was partitioned between water and ethyl
acetate. The aqueous layer was extracted with ethyl acetate,
and the organic layer was washed with brine, then dried
(Na2SO4), and concentrated to afford a yellow gum (373 mg,
77% yield). The above crude racemate was resolved (CHIRAL-
PAK AD column/90:10:0.2 hexanes-EtOH-NEt3) to give the
7d as the faster moving enantiomer; [R]D +22.5° (c ) 1,
isopropyl alcohol). Compound 7d was converted to the corre-
sponding TFA salt by treatment of the free base in methylene
chloride with 1 equiv of TFA; 1H NMR (TFA salt, CDCl3) δ
9.02 (brs, 1H), 7.71 (brs, 2H), 7.37 (s, 1H) 7.30-7.25 (m, 2H),
2.72 (d, 1H, J ) 8.9 Hz), 2.18 (d, 1H, J ) 8.9 Hz), 1.48 (s, 3H),
1.05 (s, 3H); HRMS (ESI) calcd for C13H15Cl2N3O (M + H)+
300.0671, found 300.0659; Anal. HPLC tR ) 3.71 min.
(()-tr a n s-2,2-Dim et h yl-3-(3-ch lor op h en yl)cyclop r o-
p a n eca r boxylic Acid (6b): prepared as described in proce-
1
dure B (white solid, 65% yield); H NMR (270 MHz, CD3OD)
δ 7.42-7.25 (m, 4H), 2.73 (d, 1H, J ) 8.1 Hz), 2.13 (d, 1H, J
) 8.1 Hz), 1.52 (s, 3H), 1.07 (s, 3H).
(+)-t r a n s-2,2-Dim e t h yl-3-(3-e t h yl-4-flu or op h e n yl)-
cyclop r op a n eca r boxylic Acid (6h ): prepared from 5h by
1
the method described in procedure B (pale oil, 95% yield); H
NMR (270 MHz, CDCl3) δ 10.43 (brs, 1H), 7.07-6.85 (m, 3H),
2.72 (d, 1H, J ) 5.8 Hz), 2.63 (q, 2H, J ) 7.4 Hz), 1.93 (d, 1H,
J ) 5.8 Hz), 1.42 (s, 3H), 1.20 (t, 3H, J ) 7.4), 0.95 (s, 3H).
The racemic acid was separated into the two enantiomers by
preparative chromatography on a chiral column (CHIRALPAK
AD column/97:3:0.3 hexanes-2-propanol-acetic acid) to give
6h (faster moving enantiomer); [R]D +16.9° (c ) 1, methanol).
(()-tr a n s-2,2-Dim eth yl-3-(4-flu or o-3-isop r op ylp h en yl)-
cyclop r op a n eca r boxylic Acid (6i): prepared from 5i by the
(()-t r a n s-N -(Am in o im in o m e t h y l)-2,2-d im e t h y l-3-
p h en ylcyclop r op a n eca r boxa m id e (7a ): prepared from 6a
via treatment with guanidine and subsequent purification as
described in procedure A (white solid, TFA salt, 72% yield);
1H NMR (400 MHz, CDCl3) δ 8.07 (brs, 4H), 7.43-7.01 (m,
5H), 2.47 (d, 1H, J ) 5.7 Hz), 2.19 (d, 1H, J ) 5.7 Hz), 1.21 (s,
3H), 0.82 (s, 3H); HRMS (ESI) calcd for C13H15N3O2 (M + H)+
232.1451, found 232.1460; Anal. HPLC tR ) 3.50 min.
1
method described in procedure B (50% yield); H NMR (270
MHz, CDCl3) δ 7.18-7.10 (brm, 1H), 7.02-6.90 (brm, 2H),
3.24-3.14 (brm, 1H), 2.68 (d, 1H, J ) 2.1 Hz), 1.91 (d, 1H, J
) 2.1 Hz), 1.42 (s, 3H), 1.25 (d, 6H, J ) 6.5 Hz), 0.94 (s, 3H).
(1R,3R)-3-(2,3-Dih yd r o-4-ben zofu r a n yl)-2,2-d im eth yl-
cyclop r op a n eca r boxylic Acid (10): To a solution of 812 (25
g, 169 mmol) and dimethyl malonate (22.3 g, 169 mmol) in
benzene (400 mL) were added piperidine (3 g, 35 mmol) and
acetic acid (10 g, 169 mmol). The resulting solution was stirred
under reflux for 18 h and then concentrated in vacuo. The
residual mixture was taken up in ethyl acetate (200 mL) and
washed sequentially with 1 N HCl and saturated NaHCO3.
The organic layer was dried (MgSO4) and concentrated to give
33 g (73%) of the corresponding arylidenemalonate as yellow
oil.
(+)-tr a n s-N-(Am in oim in om eth yl)-3-(3-ch lor op h en yl)-
2,2-d im eth ylcyclop r op a n eca r boxa m id e (7b): To a solu-
tion of the acid 6b (100 mg, 0.44 mmol) in DMF (2 mL) at 25
°C was added CDI (86 mg, 0.53 mmol). The resulting solution
was stirred for 1 h at room temperature followed by the
addition of guanidine (52 mg, 0.88 mmol). The reaction mixture
was stirred at room temperature for 1 h and partitioned
between water and ethyl acetate. The aqueous layer was
extracted with ethyl acetate, and the combined organic layer
was washed with brine. The ethyl acetate layer was dried
(Na2SO4) and concentrated to provide the racemic form of 7b
as a white solid (90 mg, 75% yield). The crude product was
separated into its two enantiomers (CHIRALPAK AD column/
90:10:0.2 hexanes-EtOH-NEt3) to give the faster moving
enantiomer) {[R]D -27.0° (c ) 1, isopropyl alcohol)} and the
title compound 7b as the slower moving enantiomer {[R]D
+27.1° (c ) 1, isopropyl alcohol)}; 1H NMR (TFA salt, CD3OD)
δ 9.05 (brs, 1H), 7.72 (brs, 2H), 7.51-7.42 (m, 3H), 7.32-7.28
(m, 1H), 3.10 (d, 1H, J ) 8.1 Hz), 2.34 (d, 1H, J ) 8.1 Hz),
To a solution of the crude malonate from above (33 g, 126
mmol) in DMSO (300 mL) was added 2-nitropropane (22.4 g,
252 mmol) and potassium tert-butoxide (28 g, 252 mmol). The
mixture was stirred for 65 h at room temperature, and ethyl
acetate (200 mL) was added. The reaction mixture was washed

Arylcyclopropanecarboxyl Guanidines as NHE-1 Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3309
1.54 (s, 3H), 1.16 (s, 3H); HRMS (ESI) calcd for C13H16ClN3O
(M + H)+ 266.1061, found 266.1052; Anal. HPLC tR ) 3.48
min.
6.92-6.80 (m, 2H), 2.79 (d, 1H, J ) 5.7 Hz), 2.60 (q, 2H, J )
7.4 Hz), 1.99 (d, 1H, J ) 5.7 Hz), 1.36 (s, 3H), 1.18 (t, 3H, J )
7.4 Hz), 0.95 (s, 3H); HRMS (ESI) calcd for C15H20FN3O (M +
H)+ 278.1669, found 278.1659; Anal. HPLC tR ) 3.56 min. A
portion of 7h from above (TFA salt) was dissolved in methylene
chloride and washed with 5% sodium carbonate, and the
methylene chloride layer was treated with 1 equiv of HCl in
ether to give HCl salt of 7h ; [R]D +4.9° (c ) 0.3, MeOH).
(()-tr a n s-N-(Am in oim in om et h yl)-2,2-d im et h yl-3-(3-
m eth oxyp h en yl)cyclop r op a n eca r boxa m id e (7c): A solu-
tion of m-anisaldehyde (1.0 g, 7.34 mmol) and carbethoxy-
methylenetriphenylphosphorane (3.0 g, 8.81 mmol) in di-
chloromethane (50 mL) was refluxed for 3 h. The reaction
mixture was concentrated, and the crude residue was purified
by silica gel chromatography using dichloromethane as an
eluant to give 1.43 g of the cinnamate 5c (95%). Crude 5c was
converted to 6c as described in procedure B. Crude 6c was
converted to 7c via treatment with guanidine as described in
the general procedure A (white solid, TFA salt, 4% overall
yield); 1H NMR (400 MHz, CD3OD) δ 7.24 (t, 1H, J ) 7.89
Hz), 6.81 (m, 3H), 3.80 (s, 3H), 2.86 (d, 1H, J ) 5.79 Hz), 2.16
(d, 1H, J ) 5.76 Hz), 1.39 (s, 3H), 1.01 (s, 3H).
(()-tr a n s-N-(Am in oim in om et h yl)-2,2-d im et h yl-3-(4-
flu or o-3-isop r op ylp h e n yl)cyclop r op a n e ca r b oxa m id e
(7i): prepared from 6i as described for 7b (TFA salt, colorless
1
gum, 15% overall yield); H NMR (CDCl3) δ 11.75 (brs, 1H),
8.60 (brs, 1H), 7.92 (brs, 2H), 6.98-6.87 (m, 3H), 3.21-3.12
(m, 1H), 2.80 (d, 1H, J ) 5.8 Hz), 2.0 (d,1H, J ) 5.8 Hz), 1.35
(s, 3H), 1.21 (d, 6H, J ) 6.9 Hz), 0.94 (s, 3H); HRMS (ESI)
calcd for C16H22FN3O (M + H)+ 292.1826, found 292.1816;
Anal. HPLC tR ) 3.7 min.
(+)-t r a n s-N -(Am in o im in o m e t h y l)-3-(2,5-d ic h lo r o -
ph en yl)-2,2-dim eth ylcyclopr opan ecar boxam ide (7e): pre-
pared in racemic form from 6e as described for 7b (white solid,
72% yield). The racemic product was subjected to chromatog-
raphy (CHIRALPAK AD column/95:5:0.2 hexanes-2-propanol-
NEt3) giving 7e (faster moving enantiomer); [R]D +22.0° (c )
0.77, isopropyl alcohol); 1H NMR (270 MHz, CDCl3) δ 7.19 (s,
1H), 7.10-7.01 (m, 2H), 2.61 (d, 1H, J ) 5.86 Hz), 1.94 (d,
1H, J ) 5.86 Hz), 1.34 (s, 3H), 0.82 (s, 3H); HRMS (ESI) calcd
for C13H15Cl2N3O (M + H)+ 300.0671, found 300.0661; Anal.
HPLC tR ) 3.567 min.
(()-tr a n s-N-(Am in oim in om et h yl)-3-(3-b r om o-4-flu o-
r op h en yl)-2,2-d im et h ylcyclop r op a n eca r b oxa m id e (7f):
3-Bromo-4-fluorocinnamic acid was converted to the corre-
sponding tert-butyl ester 5f as described for 5b. The crude ester
5f was converted to the acid 6f as described in procedure B.
Compound 6f was coupled with guanidine as in the case
compound 7b to afford 7f after reversed-phase preparative
HPLC purification (TFA salt, white solid, 23% overall yield);
1H NMR (270 MHz, CD3OD) δ 7.52 (brd, 1H, J ) 7.5 Hz), 7.25-
7.20 (m, 1H), 7.18 (t, 1H, J ) 10.5, Hz), 2.84 (d, 1H, J ) 7.5
Hz), 2.17 (d, 1H, J ) 7.5 Hz), 1.37 (s, 3H), 0.98 (s, 3H); HRMS
(ESI) calcd for C13H15BrFN3O2 (M + H)+ 328.0462, found
328.0453; Anal. HPLC tR ) 3.565 min.
(1R ,3R )-t r a n s-N -(Am in o im in o m e t h y l)-3-(2,3-d ih y -
d r ob en zofu r a n -4-yl)-2,2-d im et h ylcyclop r op a n eca r b ox-
a m id e (1): This compound was prepared from the correspond-
ing acid 10 as described for 7b (white solid, HCl salt, 54% yield
from 10); MS m/z 274 (M + H)+; 1H NMR (270 MHz; CDCl3) δ
11.80 (s, 1H); 8.40 (bs, 4H); 7.01 (t, 1H, J ) 7.84); 6.67 (d, 1H,
J ) 7.94); 6.55 (d, 1H, J ) 7.65); 4.58 (t, 2H, J ) 9.2); 3.28-
3.20 (m, 1H); 3.09-3.00 (m, 1H); 2.71 (d, 1H, J ) 5.6); 2.12 (d,
1H, J ) 5.7); 1.4 (s, 3H); 0.99 (s, 3H); 13C NMR (270 MHz;
CDCl3) δ 173.90, 160.30, 156.40, 133.50, 128.50, 127.40, 119.90,
108.60, 71.43, 37.46, 34.43, 33.22, 29.29, 22.21, 20.52; [R]D
+7.3° (c ) 1 CHCl3); Anal. (C15H19N3O2‚HCl) C, H, N.
NHE Assa ys: Stable cell lines expressing four members of
the human Na+/proton exchanger gene family, NHE-1, NHE-
2, NHE-3, and NHE-5, were developed for use as screens. All
clones were expressed in AP1 cells, a CHO (Chinese hamster
ovary) cell derivative that lacks any endogenous NHE activity.
cDNA clones for human NHE-1 and NHE-3 were obtained
using PCR primers based on the published human se-
quences.4,22 A cDNA clone for human NHE-2 was obtained
from Dr. K. Ramaswamy at the University of Illinois at
Chicago. An AP1 cell line expressing the human NHE-5
isoform was obtained from Dr. J . Orlowski (McGill University,
Montreal, Canada).
(()-tr a n s-N-(Am in oim in om et h yl)-2,2-d im et h yl-3-(4-
flu or o-3-m eth ylp h en yl)cyclop r op a n eca r boxa m id e (7g):
To a solution of 3-bromo-4-fluorobenzaldehyde (11) (1.00 g,
4.93 mmol) in ethylene glycol dimethyl ether (18 mL) were
sequentially added cesium fluoride (1.66 g, 10.96 mmol),
methyl boronic acid (0.33 g, 5.48 mmol), and tetrakis(tri-
phenylphosphine)palladium(0) (0.17 g, 0.15 mmol) under
argon. The reaction mixture was refluxed for 17 h, diluted with
EtOAc, and washed with brine. The organic layer was dried
(MgSO4) and concentrated in vacuo, and the crude product was
subjected to flash chromatography (silica gel/hexanes-ethyl
acetate 96:4) to afford 4-fluoro-3-methylbenzaldehyde (12)
(0.523 g) contaminated with ca. 25% of 11.
These cell lines were then used to assay NHE activity.
Monolayers grown in 96-well plates were washed twice in
NaCl-HEPES buffer (100 mM NaCl, 50 mM HEPES pH 7.3,
10 mM glucose, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2) and
incubated at room temperature for 30 min in buffer containing
BCECF-AM (Molecular Probes, #B3051), at a final concentra-
tion of 5 µM. Cells were then washed once with NaCl-HEPES
and acid-loaded by first incubating with NH4Cl-HEPES (20
mM NH4Cl, 80 mM NaCl, 50 mM HEPES pH 7.3, 5 mM KCl,
2 mM CaCl2, 1 mM MgCl2) for 9 min, then by washing twice
with ammonium-free, Na+-free HEPES buffer (100 mM choline
Cl, 50 mM HEPES pH 7.3, 10 mM glucose, 5 mM KCl, 2 mM
CaCl2, 1 mM MgCl2). Drugs were then preincubated with cells
for 7.5 min. NHE activity was initiated by the addition of 450
mM NaCl (final concentration 135 mM). Fluorescence readings
were taken 5 min after the addition of NaCl to each micro-
plate, at 444 nm excitation/538 nm emission and also at 485
nm excitation/538 nm emission. Activity in the absence of NaCl
was subtracted from the activity in the presence of NaCl, and
the ratio of 485 nm/444 nm readings were determined as an
index of pH. The IC50 values reported in Tables 1 and 2 were
determined from an 8-point dose response curve.
This mixture was converted to the corresponding cinnamate
5g as described for 5d . Crude cinnamate 5g was subsequently
transformed to the cyclopropyl acid 6g as described in proce-
dure B. In the final step, the acid 6g was converted to 7g as
described for 7b. The crude product (still contaminated with
ca. 25% of 7f) was purified by preparative HPLC (C18 column/
water-methanol-trifluoroacetic acid 90:10:0.1 to 10:90:0.1
gradient) to afford pure 7g as a colorless gummy solid (TFA
1
salt, 3% overall yield); H NMR (DMSO-d6) δ 8.35-8.15 (m,
4H), 7.16 (d, 1H, J ) 6.8 Hz), 7.09-7.02 (m, 2H), 2.63 (d, 1H,
J ) 6.3 Hz), 2.23-2.17 (m, 4H), 1.28 (s, 3H), 0.89 (s, 3H);
HRMS (ESI) calcd for C14H18FN3O (M + H)+ 264.1513, found
264.1500; Anal. HPLC tR ) 3.4 min.
Ack n ow led gm en t. We greatly appreciate the sup-
port of Bristol-Myers Squibb Department of Discovery
Analytical Sciences.
(+)-tr a n s-N-(Am in oim in om et h yl)-2,2-d im et h yl-3-(3-
eth yl-4-flu or oph en yl)cyclopr opan ecar boxam ide (7h ): Pre-
pared from the acid (+)-6h via the procedure described for 7b.
The crude product was purified by preparative HPLC as
described in general procedure A giving 7h as a colorless gum
(TFA salt, 74% yield); 1H NMR (270 MHz, CDCl3) δ 11.68 (brs,
1H), 8.51 (brs, 1H), 7.98 (brs, 2H), 6.94 (d, 1H, J ) 7.4 Hz),
Su p p or tin g In for m a tion Ava ila ble: Analytical data for
compound 1 and crystallographic data and details of refine-
ment are available for compound 16 and the maleic acid and
mesylate salts of 1. This material is available free of charge
via the Internet at http://pubs.acs.org.

3310 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
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