Discovery of potent antagonists of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. 3. Amide (C-ring) structure - Activity relationship and improvement of overall properties of arylthio cinnamides

Article abstract of DOI:10.1021/jm010059w

The interaction of LFA-1 and ICAM-1 plays an important role in the cell adhesion process. On the basis of previously reported SAR and structural information on the binding of our p-arylthiocinnamide series to LFA-1, we have identified the cyclic amide (C-

Full text of DOI:10.1021/jm010059w

J . Med. Chem. 2001, 44, 2913-2920
2913
Discover y of P oten t An ta gon ists of Leu k ocyte F u n ction -Associa ted An tigen -1/
In ter cellu la r Ad h esion Molecu le-1 In ter a ction . 3. Am id e (C-Rin g)
Str u ctu r e-Activity Rela tion sh ip a n d Im p r ovem en t of Over a ll P r op er ties of
Ar ylth io Cin n a m id es
Zhonghua Pei,*,† Zhili Xin,† Gang Liu,† Yihong Li,† Edward B. Reilly,‡ Nathan L. Lubbers,§ J effery R. Huth,
J ames T. Link,† Thomas W. von Geldern,† Bryan F. Cox,§ Sandra Leitza,‡ Yi Gao,| Kennan C. Marsh,|
Peter DeVries,‡ and Greg F. Okasinski†
Departments of Metabolic Disease Research, Integrative Pharmacology, Advanced Technology, and Drug Analysis,
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064
Received February 5, 2001
The interaction of LFA-1 and ICAM-1 plays an important role in the cell adhesion process. On
the basis of previously reported SAR and structural information on the binding of our
p-arylthiocinnamide series to LFA-1, we have identified the cyclic amide (C-ring) as a site for
modification. Improvement in potency and, more importantly, in the physical properties and
pharmacokinetic profiles of the leading compounds resulted from this modification. One of the
best compounds (11f) is also shown to reduce myocardial infarct size in rat.
In tr od u ction
of LFA-1,9 we have proposed a mechanism of inhibition
for the p-arylthio cinnamides. They lock the I domain
in an inactivated state by hindering the conformational
change of the C-terminal R helix that accompanies
LFA-1 activation. The nuclear Overhauser effect (NOE)
based model of how compound 9a binds to the I domain
showed that the A- and B-rings are packed between I235
and L302, thereby inhibiting the packing of the C-
terminal R helix with the core â sheet.
However, one of the limitations of these early com-
pounds in our series for potential clinical use is that
they generally exhibit very poor water solubility (e.g.,
the solubility of 9a is <1 µg/mL at pH 7.4 in 50 mM
phosphate buffer) and poor pharmacokinetic profiles
(e.g., compound 9a was not orally bioavailable in rats,
F ) 0%). A modest improvement in solubility was
achieved by replacing the isopropylphenyl A-ring with
a more hydrophilic benzodioxane substituent.8 In this
report, we disclose our progress on the modification of
the C-ring of the molecule aimed at dramatically
improving water solubility and the pharmacokinetic
profile while maintaining or improving potency.
The process of cell adhesion plays an important role
in many fundamental biological processes, including
development, differentiation, and immune response.
Dysfunction of such a process is implicated in pathologi-
cal conditions such as chronic inflammation, cancer
metastasis, and viral infections.1 Several families of
molecules serve specialized adhesive functions at dif-
ferent stages of the multistep process of cell adhesion.2,3
Of particular interest is the interaction between leuko-
cyte function-associated antigen-1 (LFA-1, CD11a/CD18,
RLâ2) and intercellular adhesion molecule-1 (ICAM-1,
CD54), which is a major adhesive force for leukocyte
adhesion and extravasation from the bloodstream into
tissue sites. Monoclonal antibodies (MAbs) have been
used to demonstrate the critical role of the LFA-1/
ICAM-1 interaction in models of inflammation or im-
mune reaction,4 and some have been reported as po-
tential therapeutic agents.5 Reports on the more
challenging task of designing small molecules to block
this protein/protein interaction are beginning to emerge.6
We have reported the discovery of novel p-arylthio-
cinnamides as antagonists of LFA-1/ICAM-1 interac-
tion.7 The critical structural features of this series
include (1) the arylthio group (A-ring), (2) the properly
substituted cinnamyl moiety (B-ring), and (3) the cyclic
amides (C-ring). We have shown by NMR studies that
these compounds bind to a proposed I domain allosteric
site (IDAS) of LFA-1.8 Considering the results of several
biochemical studies that reveal the activation process
Ch em istr y
The synthesis is similar to what was described in a
previous article of this series.7 In brief, nucleophilic
substitution between properly substituted thiols 1 and
aldehyde halides 2 gave the sulfide aldehydes 3 (Scheme
1). The cinnamic acids 4 were obtained by either a
Horner-Emmons-Wittig reaction or condensation-
decarboxylation between aldehydes 3 and malonic acid.
The acids were then converted to the amides 5 through
either the corresponding acid chlorides or direct coupling
with the addition of an activating reagent such as
TBTU.10 The substituted piperazine intermediates 6a
and 6b were prepared from the fully protected inter-
mediate 7b. Compound 7b was synthesized from the
acid 7a , which itself was prepared from 2-piperazine-
carboxylic acid according to literature protocols.11
* To whom correspondence should be addressed. Address: D-47R,
AP-10, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL
60064-6098. Phone: (847) 935-6254. Fax: (847) 938-1674. E-mail:
zhonghua.pei@abbott.com.
† Department of Metabolic Disease Research.
‡ Current address: Biologics Development, Hospital Products Divi-
sion, Abbott Laboratories.
§ Department of Integrative Pharmacology.
Department of Advanced Technology.
| Department of Drug Analysis.
10.1021/jm010059w CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/02/2001

2914 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Pei et al.
Sch em e 1a
a
Reagents and conditions: (a) Cs2CO3, MeCN, heat; (b) EtO2CCH2P(O)(OEt)2, NaH, THF then NaOH; (c) CH2(COOH)2, pyridine, cat.
piperidine, 110 °C; (d) (COCl)2, cat. DMF, then R3R3, NH, 6, DCM; (e) TBTU, R3R3, NH, i-Pr2NEt, DMF.
Ta ble 1. Biological Activites of Compound 8a
Ta ble 2. Biological Activities of Compound 9a
a
IC50 calculated using a mean of at least two measurements
(all duplicates) for six concentrations from 3.2 × 10-8 to 10-4 unless
otherwise noted. The ranges are shown in parentheses.
Resu lts a n d Discu ssion
At the early stage of our SAR investigation using 2,4-
dichlorophenyl as the A-ring,7 it was found that the
C-ring tolerates different groups (Table 1); 1-(3-amino-
propyl)-2-pyrrolidinyl (8a ), morpholinyl (8c), and sub-
stituted piperazinyl amides (8b and 8d ) are all moder-
ately potent LFA antagonists. This clearly suggested to
us that there is room for C-ring modification and that
this position could be used to identify an additional
binding pocket and/or to fine-tune physical properties.
On the observation that the N-methyl analogue (8b)
is about 8-fold less potent than the corresponding N-acyl
analogue (8d ), the effect of the N substituent of the
piperazine ring using the more potent 2-isopropylphenyl
as the A-ring and 2-nitrophenyl as the B-ring was
a
IC50 calculated using a mean of at least two measurements
(all duplicates) for six concentrations from 3.2 × 10-8 to 10-4 unless
otherwise noted. The ranges are shown in parentheses.
explored. Compound 9b (Table 2) with the N-acyl group
removed shows much better water solubility (7 µg/mL
at pH 7.4). Unfortunately, it is 10-fold less potent than
compound 9a , confirming the importance of the carbonyl
group. N-Methyl substitution in the C-ring (9c) im-
proved the potency by 3.5-fold compared to unsubsti-
tuted analogue 9b. A 4-pyridine carbonyl group (9d )
maintained most of the potency. The corresponding
piperidine analogues with a para ketone or a para ketal

Amide SAR and Cinnamides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2915
Ta ble 4. Biological Activities of Compound 11a
Ta ble 3. Biological Activities of Compound 10a
a
IC50 calculated using a mean of at least two measurements
(all duplicates) for six concentrations from 3.2 × 10-8 to 10-4 unless
otherwise noted. The ranges are shown in parentheses.
ring; the potency of the resulting analogue (10f) is
reduced when compared to that of 9a . However, when
the free carboxylic acid is moved to the 2-position of the
piperazine ring, the compound (10g) has maintained
most of the potency (IC50 ) 0.130 µM) but now the water
solubility has dramatically increased to >3000 µg/mL
at pH 7.4. Another analogue with a free carboxylic acid
group attached to a five-membered ring (10h ) has
comparable potency but now has only one carbonyl
group in it.
The other challenge presented by this series of
analogues is the pharmacokinetic (PK) profile as pointed
out earlier. For example, compound 9a was not detect-
able in plasma at all when administered orally in rat
(Table 5, dosage of 5 mg/kg for all testings). Similar
results were obtained with 10c, although it has im-
proved water solubility. After the discovery that a free
carboxylic group on the C-ring provides much improved
water solubility with little decrease in potency, numer-
ous analogues with free carboxylic acid attached were
made (data not shown). We were pleased to find that
C-rings bearing a free carboxylic group simultaneously
improved the PK properties significantly in addition to
increasing water solubility. Among them, the iso-
nipecotic acid and nipecotic acid-derived analogues (11a
and 11b, Table 4) started to show promising overall
properties; both compounds show both excellent poten-
cies (IC50 ) 0.030-0.031 µM) and water solubility
(>3000 µg/mL at pH 7.4), and at the same time the PK
profile is significantly improved (Table 5). The PK
profile of 11a following a 5 mg/kg oral dosage looks very
a
IC50 calculated using a mean of at least two measurements
(all duplicates) for six concentrations from 3.2 × 10-8 to 10-4 unless
otherwise noted. The ranges are shown in parentheses.
group (9e and 9f) show similar potencies. A urea-
substituted piperazine analogue (9g) indicated a trend
of increasing (1.8-fold) potency for the first time.
Since a carbonyl group attached to the C-ring is
beneficial to the potency, the effect of the position of the
carbonyl on the piperazine ring was investigated. A
methyl ester attached to the 2-position (10a ) leads to a
marginal (1.3-fold) increase of potency compared to
unsubstituted analogue 9b (Table 3). When the methyl
ester is moved to the 3-position (10b), the IC50 was
improved to 0.060 µM. When the ester was reduced to
the alcohol, the resulting analogue 10c showed signifi-
cantly improved solubility (5 µg/mL at pH 7.4), but
again, the potency suffered. An N,N-dimethylamide at
the 3-position is also well tolerated (10d ). When the
nitrogen at the piperazine is methylated, the resulting
tertiary amine 10e is more potent than secondary amine
analogues (10a -10d ).
promising: area under curve AUC0-8h ) 1.3 µg h mL-1
;
mean residence time MRT ) 3.5 h; terminal half-life
t1/2 ) 1.0 h; oral bioavailability F ) 21%. The PK profile
The next interesting observation came when a free
carboxylic acid was put at the 3-positon of the piperazine
for 11b after oral dosing is AUC0-8h ) 0.47 µg h mL-1
,

2916 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Pei et al.
Ta ble 5. Water Solubility and Pharmacokinetic Profiles of
Ta ble 6. Summary of Inhibition of the J Y8 Cell Adhesion
Selected Compoundsa
Processa
a
a
iv dosing
po dosing
MRT
compd
IC50 (µM)
compd
IC50 (µM)
CLp
compd (L h-1 kg-1
Vâ
t1/2
(h)
Cmax
F
(%)
9d
9e
10b
10e
11a
0.020
0.024
11b
11c
11e
11f
0.09 (0.040-0.14)
)
(L kg-1
)
(µg mL-1
)
(h)
0.002
0.15
0.14
0.098 (0.02-0.20)
0.11 (0.030-0.190)
0.165 (0.10-0.17)
9a b
<1
5
>3300
>3300
>3300
>3300
2.7
3.2
0.4
1.9
1.15
0.4
3.9
2.3
5.7
0
0
NA
NA
3.5
11.3
6.7
0
0
10cb
11a b
11bb
11eb
11fb
11fc
0.791
2.097
0.12
0.29
0.34
0.50
0.45
2.8
1.5
3.7
21
19
39
29
55
a
IC50 calculated using a mean of at least measurements (all
duplicates) for six concentrations from 6.4 × 10-9 to 2 × 10-5
unless otherwise noted. The assay was run with 1% fetal bovine
serum. The ranges are shown in parentheses. For structures, see
previous tables.
5.0
5.7
a
All were dosed at 5 mg/kg. MRT is mean residence time. NA
b
means not applicable. In rat. c In dog.
not pack in the hydrophobic pocket of the I domain
(Figure 1). The N-acetylmethyl group of this ring binds
near I255, as determined from NOE measurements.
This positions the ring near the N- and C-termini of the
I domain that link it to rest of the LFA-1 molecule. No
structural information exists to explain how the I
domain interacts with the other domains of LFA-1. As
a result, it is unknown whether the C-ring makes
important contacts with residues outside the I domain.
However, the solvent-exposed position of the C-ring in
the NMR model is consistent with the SAR of the
p-arylthiocinnamide series in that a variety of polar
groups at three different positions were tolerated on this
ring without a loss in potency.
Some of the active compounds were also tested in our
secondary cellular assaysthe J Y-8 cell adhesion assay,
where the inhibition of the adhesion of J Y-8 cells with
ICAM-1 expressed on the cell surface to immobilized
LFA-1 was measured. The IC50 values of some selected
compounds are shown in Table 6. Generally those
compounds are quite active in this cellular assay and
the IC50 values are consistent with those obtained in
the binding assay (e.g., they appear to be approximately
MRT ) 11.3 h, t1/2 ) 0.68 h, F ) 19%. The analogue
with a tetrazole group (11c), a common carboxylic acid
isostere, is both potent and water-soluble (IC50 ) 0.020
µM, water solubility of >3000 µg/mL at pH 7.4), but the
PK does not improve (F ) 0%). The analogue with a
sulfonic acid group (11d ) maintained most of the
potency. Combined with the A-ring and B-ring improve-
ments,8 compounds 11e and 11f were prepared and they
displayed excellent overall properties: for both 11e and
11f, IC50 ) 0.025-0.030 µM, water solubility is >3000
µg/mL at pH 7.4. The PK profile of 11e is defined by
the following: for iv dosing, AUC is much larger
(AUC0-8h ) 43.3 µg h mL-1) and the clearance is now
very low (CLp ) 0.12 L h-1 kg-1), t1/2 ) 3.9 h; for oral
dosing, AUC0-8h ) 16.8 µg h mL-1, MRT ) 6.7 h,
estimated terminal half-life t1/2 ) 12.2 h, F ) 39%. The
PK profile of 11f is similar: for oral dosing in rat,
AUC0-8h ) 5.1 µg h mL-1, MRT ) 5.0 h, estimated
terminal t1/2 ) 12.6 h, F ) 28%; for oral dosing in dog,
AUC0-8h ) 9.15 µg h mL-1, t1/2 ) 5.4 h, F ) 55%.
From the NOE-based model of the binding of com-
pound 9a to LFA, it can be seen that the C-ring does
F igu r e 1. NMR model of 9a bound to the LFa-1 I domain. The surface of the LFA-1 ligand binding site shows that one-half of
the compound is buried in the pocket. Atoms of the ligand with <30% solvent-accessible surface area are shown in green, and
accessible atoms are shown in gray. The oxygen atoms of the ligand are in red. Positions 2 and 3 of the C-ring are labeled to show
the location of chemical modifications of this series, which are described in this paper.

Amide SAR and Cinnamides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2917
Ta ble 7. Hempdynamic Dataa
ischemia (min)
Pre-I
reperfusion (min)
n
15
5
60
120
180
control
MAP
HR
11
126 ( 5
109 ( 9
106 ( 7
93 ( 5
87 ( 7
384 ( 5
333 ( 25
84 ( 6
378 ( 7
320 ( 22
417 ( 11
525 ( 22
386 ( 18
427 ( 45
379 ( 11
300 ( 26
374 ( 15
348 ( 25
RPP
3 mg/kg
MAP
HR
4
118 ( 5
393 ( 14*
464 ( 35
107 ( 6
391 ( 15
418 ( 37
98 ( 10
379 ( 18
375 ( 53
99 ( 10
382 ( 16
381 ( 50
85 ( 11
371 ( 13*
318 ( 53
95 ( 2
376 ( 12
351 ( 11
RPP
50 mg/kg
MAP
HR
5
128 ( 6
419 ( 7
540 ( 34
105 ( 11
400 ( 13
427 ( 57
105 ( 8
382 ( 6
402 ( 34
118 ( 5*
396 ( 9
467 ( 24*
96 ( 4
397 ( 5
381 ( 19
80 ( 6
399 ( 5
320 ( 27
RPP
a
n ) number of rats per group; Pre-I ) immediately prior to occlusion; MAP ) mean arterial pressure (mmHg); HR ) heart rate
(beats per min); RPP ) rate pressure product [(mmHg‚beats/min)/100]. Values are mean ( SEM. *p < 0.05 vs corresponding time point
in controls.
F igu r e 3. Graph showing area at risk as a function of the
F igu r e 2. Graph showing infarct size in control rats and rats
left ventricle in control rats and rats receiving 11f at 3 and
receiving 11f at 3 and 50 mg/kg after 25 min of ischemia and
50 mg/kg after 25 min of ischemia and 3 h of reperfusion. Bars
3 h of reperfusion. Bars represent mean ( SEM. The asterisk
represent mean ( SEM.
(/) indicates P < 0.05 vs control.
of 11f resulted in an IS/AAR of 53.6 ( 12.9%. Rats
treated with 50 mg/kg of 11f had an IS/AAR of 47.2 (
5.8% (p < 0.05 vs control). The area at risk as a
percentage of the total left ventricle (AAR/LV) is shown
in Figure 3 and showed no significant difference be-
tween the different treatment groups, indicating that
similar areas of the left ventricular tissue were jeopar-
dized by occlusion of the LCA in each group. The rate-
pressure product was significantly affected by 50 mg/
kg of 11f at one time point at the end of the infusion,
indicating that 11f had no sustained effect on myocar-
dial oxygen demand (Table 7). These results demon-
strate the efficacy of 11f in reducing myocardial infarct
size and highlight the importance of the LFA-1/ICAM-1
interaction in the pathogenesis of myocardial ischemia
and reperfusion.
5-fold less potent than in the binding assay). However,
an exception does exist (e.g., compound 11c appears to
be more potent in the functional assay (2 nM) than in
the biochemical assay (20 nM). It is believed that the
apparent discrepancy is mainly due to the differences
in protein binding properties among those molecules,
which is being addressed aggressively.
With the in vitro potency established, it was decided
to test the efficacy of our best compound in vivo. It is
known that neutrophils can play an important role in
producing the myocardial necrosis that follows ischemia
and reperfusion. After ischemia/reperfusion, neutrophils
adhere to the endothelium and then migrate into the
myocardium. This process is dependent on specific
integrins such as Mac-1 (CD11b/CD18) and intracellular
adhesion molecules (ICAM-1). Inhibition of neutrophil/
endothelial cell interaction with monoclonal antibodies
against Mac-1 protects against injury after myocardial
ischemia and reperfusion.12 In addition, monoclonal
antibodies against the CD11a epitope of LFA-1 (CD11a/
CD18) have been shown to be effective in reducing
myocardial infarct size.13 One study has demonstrated
that canine neutrophils utilize the integrin LFA-1 to
adhere to ICAM-1 on cardiac myocytes.14 Here, we
describe the cardioprotective effect of a small molecule
antagonist (11f) of the LFA-1/ICA-1 interaction in a rat
model of ischemia and reperfusion.
Con clu sion s
In conclusion, on the basis of the previous SAR and
structural information from NMR studies, we were able
to identify the C-ring in our p-arylthiocinnamide series
as a site to simultaneously improve both the potency
and physical properties. By combination of these obser-
vations with our previous results on A-ring and B-ring
optimization, potent LFA-1/ICAM-1 antagonists with
desirable physical and pharmacokinetic properties were
prepared. Of particular note is compound 11f, with IC50
) 25 nM in the binding assay, greater than 3 mg/mL
water solubility, and F ) 55% (5 mg/kg, po in dog). It is
also shown to reduce myocardial infarct size in rat.
The administration of 11f (see Experimental Section
for details) resulted in a dose-dependent reduction in
myocardial infarct size (Figure 2). The control group had
an IS/AAR of 68.0 ( 2.5%. Administration of 3 mg/kg

2918 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Pei et al.
Hz), 4.01 (br s, 4H), 3.44 (quintet, 1H, J ) 6.8 Hz), 2.56 (br m,
4H), 1.18 (d, 6H, J ) 7.1 Hz). MS (ESI): m/z 425, 457. Anal.
(C23H24N2O4S) C, H, N.
(2-Isopr opylph en yl)[2-n itr o-4-(E-((3-aza-6,9-diooxaspir o-
[5.4]d eca n -1-yl)ca r bon yl)eth en yl)p h en yl] su lfid e (9f). 1H
NMR (CDCl3, 300 MHz): δ 8.44 (s, 1H), 7.50-7.62 (m, 4H),
7.41 (d, 1H, J ) 8.0 Hz), 7.30 (m, 1H), 6.96 (br d, 1H, J ) 15.6
Hz), 6.69 (d, 1H, J ) 9.4 Hz), 4.00 (s, 4H), 3.75 (br m, 4H),
3.44 (m, 1H), 1.75 (br s, 4H), 1.18 (d, 6H, J ) 7.0 Hz). MS
(ESI): m/z 439, 937. Anal. (C25H28N2O5S) C, H, N.
Exp er im en ta l Section
LFA-1/ICAM-1 biochemical assay and J Y-8/ICAM-1 adhe-
sion assay and solubility testing procedure were performed as
previously described.7
Gen er a l. All solvents and reagents were obtained from
commercial suppliers and used without further purification.
All reactions were performed under nitrogen atmosphere
unless otherwise noted. Flash chromatography was performed
using silica gel (230-400 mesh) from E. M. Science. Mass
spectral analyses were accomplished using one of the three
ionization methods: desorption chemical ionization (DCI),
atmospheric pressure chemical ionization (APCI), and elec-
trospray ionization (ESI), as specified for individual com-
pounds. Proton NMR spectra were recorded on a General
Electric QE300 instrument with tetramethylsilane (TMS) as
an internal standard and are reported as shift (multiplicity,
coupling constants, proton counts). Elemental analyses were
performed by Robertson Microlit Laboratories, Madison, NJ ,
and are consistent with theoretical values to within 0.4%
unless otherwise indicated. Preparative HPLC was performed
on an automated Gilson HPLC system, using an YMC C-18
column, 75 mm × 30 mm i.d., S-5 µM, 120 Å at a flow rate of
25 mL/min. UV absorption detection at λ ) 214 and 245 nm
was used. The mobile phase consists of (A) 0.05 M NH4OAc or
0.1% TFA in H2O and (B) CH3CN; linear gradient is 20-100%
of B in 20 min. The purified fractions were evaporated to
dryness on a Savant SpeedVac.
(2-Isop r op ylp h en yl)[2-n itr o-4-(E-((4-m eth yla m in oca r -
b on ylp ip er a zin -1-yl)ca r b on yl)et h en yl)p h en yl] Su lfid e
1
(9g). Yellow solid. H NMR (DMSO-d6, 300 MHz): δ 1.14 (d,
J ) 6.8 Hz, 6H), 2.58 (d, J ) 4.4 Hz, 3H), 3.30-3.40 (m, 1H),
3.28-3.70 (br m, 8H), 6.52 (q, J ) 4.4 Hz, 1H), 6.64 (d, J )
8.5 Hz, 1H), 7.32-7.62 (m, 6H), 7.90 (dd, J ) 8.5, 1.8 Hz, 1H),
8.64 (d, J ) 1.8 Hz, 1H). MS (APCI): (M + NH4)+ at m/z 486.
Anal. (C24H28N4S1O4‚0.36EtOAc) C, H, N.
(2-Isop r op ylp h en yl)[2-n it r o-4-(E-((2-ca r b om et h oxy-
p ip er a zin -1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e (10a ).
Yellow solid. 1H NMR (DMSO-d6, 300 MHz): δ 1.14 (d, J )
6.6 Hz, 6H), 2.52-2.91 (br m, 5H), 3.30-3.40 (m, 1H), 3.68,
3.69 (s, s, 3H), 4.10-4.25 (br m, 1H), 5.00-5.21 (br m, 1H),
6.60-6.65 (m, 1H), 7.29-7.62 (m, 6H), 7.85-7.95 (m, 1H),
8.64-8.68 (m, 1H). MS (APCI): (M + H)+ at m/z 470. Anal.
(C24H27N3O5S‚0.46EtOAc) C, H, N.
(2-Isop r op ylp h en yl)[2-n it r o-4-(E-((3-ca r b om et h oxy-
p ip er a zin -1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e (10b). A
mixture of (2-isopropylphenyl)[2-nitro-4-E-(carboxyethenyl)-
phenyl] sulfide, the amine (1.0 equiv), 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (1.0 equiv),
and diisopropylethylamine (2.0 equiv) in DMF was stirred at
ambient temperature for 4 h. Ethyl acetate was added, and
the mixture was washed sequentially with 1 N HCl, sodium
bicarbonate, and brine. The resultant yellow solid was treated
with 1:1 TFA/dichloromethane at ambient temperature to give
Compounds 8a -8d and 9a were reported before.
Gen er a l P r oced u r e for Ma k in g th e Am id es. The pro-
cedure used for preparing 9b is typical. A mixture of 3-[4-(2-
isopropylphenylsulfanyl)-3-nitrophenyl]acrylic acid (1.84 g,
5.37 mmol), BOC-piperazine (1.0 g, 5.37 mmol), 2-(1H-benzo-
triazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate
(TBTU) (1.72 g, 5.37 mmol), and diisopropylethylamine (1.87
mL, 10.74 mmol) in 20 mL of DMF was stirred at ambient
temperature overnight. Ethyl acetate was added, and the
mixture was washed sequentially with 1 N HCl, saturated
NaHCO3, and brine. The crude product was purified by flash
chromatography to give tert-butyl 4-{3-[4-2-isopropyl-phenyl-
sulfanyl)-3-nitrophenyl]acryloyl}piperazine-1-carboxylate as a
yellow solid (2.2 g, yield 80%). The above tert-butyl ester (2.1
g, 4.11 mmol) was treated with 1:1 of TFA/CH2Cl2 at room
temperature for 2 h. The reaction mixture was concentrated,
and the residue was basified by saturated NaHCO3 solution
and extracted with ethyl acetate. The organic phase was
washed with brine, dried, and concentrated to give (2-isopro-
pylphenyl)[2-nitro-4-(E-((4-piperazin-1-yl)carbonyl)ethenyl)-
phenyl] sulfide (9b) as a yellow solid (1.6 g, yield 95%).
(2-Isop r op ylp h en yl)[2-n it r o-4-(E-((4-p ip er a zin -1-yl)-
ca r bon yl)eth en yl)p h en yl] Su lfid e (9b). Yellow solid. 1H
NMR (DMSO-d6, 300 MHz): δ 1.14 (d, J ) 7.1 Hz, 6H), 2.82
(br s, 4H), 3.29-3.39 (m, 1H), 3.52-3.72 (br m, 4H), 6.64 (d, J
) 8.5 Hz, 1H), 7.32-7.62 (m, 6H), 7.88 (dd, J ) 8.8 Hz, 1.8
Hz, 1H), 8.63 (d, J ) 1.7 Hz, 1H). MS (APCI): (M + H)+ at
m/z 412. Anal. (C22H25N3S1O3‚1.64H2O) C, H, N.
1
the title compound as a yellow solid. H NMR (DMSO-d6, 300
MHz): δ 1.15 (d, J ) 6.6 Hz, 6H), 2.52-3.16 (br m, 4H), 3.25-
3.47 (m, 1H), 3.60-3.65 (br d, 3H), 3.60, 3.66 (br s, br s, 3H),
6.61-6.67 (br m, 1H), 7.30-7.62 (m, 6H), 7.88-7.93 (br m,
1H), 8.58-8.65 (br m, 1H). MS (APCI): (M + H)+ at m/z 470.
Anal. (C24H27N3S1O5) C, H, N.
(2-Isopr opylph en yl)[2-n itr o-4-(E-((3-h ydr oxym eth ylpip-
er a zin -1-yl)-ca r bon yl)eth en yl)p h en yl] Su lfid e (10c). Yel-
1
low solid. H NMR (DMSO-d6, 300 MHz): δ 1.14 (d, J ) 7.0
Hz, 6H), 1.40, 1.41 (s, s, 9H), 2.25-3.08 (m, 6H), 3.29-3.39
(m, 1H), 4.06-4.46 (br m, 2H), 4.68-4.76 (br m, 1H), 6.61-
6.67 (m, 1H), 7.30-7.62 (m, 6H), 7.88-7.93 (br m, 1H), 8.59-
8.66 (br m, 1H). MS (APCI): (M + H)+ at m/z 442. Anal.
(C23H27N3S1O4) C, H, N.
(2-Isopr opylph en yl)[2-n itr o-4-(E-(((3-dim eth ylam in ocar -
bon yl)p ip er a zin -1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e
(10d ). Yellow solid. 1H NMR (DMSO-d6, 300 MHz): δ 1.14 (d,
J ) 6.6 Hz, 6H), 2.50-3.20 (br m, 4H), 2.82 (s, 3H), 3.04 (s,
3H), 3.26-3.49 (m, 1H), 3.52-3.59 (m, 1H), 4.08-4.47 (br m,
2H), 6.63 (d, J ) 8.5 Hz, 1H), 7.31-7.62 (m, 6H), 7.86-7.92
(m, 1H), 8.61 (br m, 1H). MS (APCI): (M + H)+ at m/z 483.
Anal. (C25H30N4S1O4‚0.39EtOAc) C, H, N.
(2-Isop r op ylp h en yl)[2-n itr o-4-(E-((4-m eth ylp ip er a zin -
1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e (9c). Yellow solid.
1H NMR (DMSO-d6, 300 MHz): δ 1.14 (d, J ) 7.0 Hz, 6H),
2.19 (s, 3H), 2.25-2.36 (br m, 4H), 3.30-3.40 (m, 1H), 3.51-
3.72 (br m, 4H), 6.63 (d, J ) 8.5 Hz, 1H), 7.24-7.63 (m, 6H),
7.88-7.92 (dd, J ) 8.8, 1.8 Hz, 1H), 8.64 (d, J ) 1.8 Hz, 1H).
MS (APCI): (M + H)+ at m/z 426. Anal. (C23H27N3S1O3‚0.26
H2O) C, H, N.
(2-Isop r op ylp h en yl)[2-n itr o-4-(E-((3-ca r bom eth oxy-4-
m eth ylp ip er a zin -1-yl)ca r bon yl)eth en yl) p h en yl] Su lfid e
(10e). Yellow solid. 1H NMR (DMSO-d6, 300 MHz): δ 1.14 (d,
J ) 7.0 Hz, 6H); 2.25, 2.26 (s, s, 3H); 2.20-3.98 (br m, 8H);
3.57, 3.63 (s, s, 3H); 6.63 (d, J ) 8.5 Hz, 1H); 7.30-7.63 (m,
6H); 7.91 (dd, J ) 8.5, 1.5 Hz, 1H); 8.60-8.68 (br m, 1H). MS
(APCI): (M + H)+ at m/z 484. Anal. (C25H29N3O5S) C, H, N.
(2-Isopr opylph en yl)[2-n itr o-4-(E-((2-car boxy-4-acylpip-
er a zin -1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e (10f). Yel-
(2-Isop r op ylp h en yl)[2-n it r o-4-(E-(4-(p yr id in e-4-ca r -
bon yl)p ip er a zin -1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e
1
(9d ). Yellow solid. H NMR (DMSO-d6, 300 MHz): δ 1.14 (d,
J ) 6.6 Hz, 6H), 3.30-3.40 (m, 1H), 3.52-3.86 (br m, 8H),
6.61-6.66 (br m, 1H), 7.30-7.62 (m, 8H), 7.83-7.96 (br m,
1H), 8.60-8.71 (m, 3H). MS (APCI): (M + H)+ at m/z 517.
Anal. (C28H28N4S1O4) C, H, N.
1
low solid. H NMR (DMSO-d6, 300 MHz): δ 1.19 (d, J ) 6.6
Hz, 6H), 1.90-2.12 (br m, 2H), 3.35-3.49 (m, 1H), 3.30-3.81
(br m, 2H), 4.41-4.75 (br m, 2H), 5.30-5.41 (br m, 1H), 6.66-
6.72 (m, 1H), 7.25-7.62 (m, 7H), 8.38 (br s, 1H). MS (APCI):
(M + H)+ at m/z 498. Anal. (C25H25N3S1O3) C, H, N.
(2-Isopr opylph en yl)[2-n itr o-4-(E-((4-oxopiper idin -1-yl)-
ca r bon yl)eth en yl)p h en yl] Su lfid e (9e). 1H NMR (CDCl3,
300 MHz): δ 8.45 (s, 1H), 7.50-7.57 (m, 3H), 7.42 (br d, 1H,
J ) 8.1 Hz), 7.30 (m, 1H), 7.02 (br, 1H), 6.72 (d, 1H, J ) 8.4
(2-Isop r op ylp h en yl)[2-n it r o-4-(E-((2-ca r b oxy-4-m et h -
oxycar bon ylpiper azin -1-yl)car bon yl)eth en yl)ph en yl] Su l-

Amide SAR and Cinnamides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2919
fid e (10g). Yellow solid. 1H NMR (DMSO-d6, 300 MHz): δ 1.14
(d, J ) 6.8 Hz, 6H); 2.70-3.95 (br m, 4H); 3.30-3.40 (m, 1H);
3.61, 3.61 (s, s, 3H); 4.16-4.51 (br m, 2H); 5.01-5.28 (br m,
1H); 6.61-6.66 (m, 1H); 7.30-7.63 (m, 6H); 7.83-7.94 (m, 1H);
8.66 (br s, 1H). MS (APCI): (M - H)+ at m/z 512. Anal.
(C25H27N3O7S) C, H, N.
(2-Isop r op ylp h en yl)[2-t r iflu or om et h yl-4-(E-(((2-ca r -
boxyp yr r ol-3-in -1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e
(10h ). 1H NMR (CDCl3, 300 MHz): δ 7.79 (s, 1H), 7.72 (d, 1H,
J ) 15.5 Hz), 7.49 (d, 1H, J ) 7.4 Hz), 7.36-7.46 (m, 3H),
7.23 (m, 1H), 6.82 (d, 1H, J ) 8.5 Hz), 6.74 (d, 1H, J ) 15.4
Hz), 6.00 (br, 2H), 4.48 (br, 1H), 4.51 (br, 2H), 3.48 (m, 1H),
1.18 (d, 6H, J ) 7.0 Hz). MS (ESI): m/z -460, -492, -921.
Anal. (C24H22 F3N1O3S) C, H, N.
rats were anesthetized with Inactin (120 mg/kg, ip), tra-
cheostomized, and placed on a Harvard rodent respirator.
Catheters were placed in the left femoral artery for the
measurement of arterial pressure and heart rate and in the
vein for administration of compound. The electrocardiogram
(ECG) was recorded with the standard Lead II limb configu-
ration. A left thoracotomy was performed, and the left coronary
artery (LCA) was isolated within a reversible snare occluder.
After a 60 min stabilization period, the LCA was occluded for
25 min and then released for 180 min of reperfusion. Five
minutes prior to reperfusion, rats received 11f at doses of 3
and 50 mg/kg. Fifty percent of the total dose was administered
as an intravenous (iv) bolus, and the remaining 50% was
administered as a 60 min iv infusion. A separate group of rats
received 5% dextrose in water (D5W) and served as the vehicle
control.
(2-Isop r op ylp h en yl)[2-n itr o-4-(E-((4-ca r boxyp ip er id in -
1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e (11a ). Light-yellow
1
solid. H NMR (CDCl3, 300 MHz): δ 1.18 (d, J ) 6.9 Hz, 6H),
In fa r ct Sizin g. After the period of ischemia and reperfu-
sion, the LCA was reoccluded and the heart was rapidly
excised and retrogradely perfused with green fluorescent
microspheres (2.26 µm, Duke Scientific Corp.) to mark the
ischemic area of the myocardium as a nonfluorescent perfusion
defect. This was termed the area at risk of infarction (AAR).
The heart was trimmed of the right ventricle and both atria
and sliced into 1-2 mm thick sections. The slices were
incubated in a triphenyltetrazolium chloride (TTC) solution
for 10 min to stain the viable myocardium brick-red. The heart
slices were fixed in 10% formalin for 24 h. After fixation, the
slices were placed between Plexiglas and both sides of the
entire ventricle and the lumen were traced onto acetate sheets.
The slices were viewed under ultraviolet light (366 nm), and
the fluorescent normal zone (NZ) was delineated within the
slice. Under normal light, the infarct (IS) located within the
AAR was traced onto the acetate. The acetates were placed
under a microscopic video camera (DEI-750, Optronics Engi-
neering), and the resulting images were stored as bitmap files
for analysis. The stored images were displayed on a monitor
using a Windows based image analysis software (Image-Pro
Plus, Media Cybernetics), and the areas of the NZ, AAR, and
IS were traced. The AAR was expressed as a percentage of
the left ventricle, and the IS was expressed as a percentage of
the AAR (IS/AAR).
1.65-1.89 (m, 2H), 1.97-2.14 (m, 2H), 2.59-2.74 (m, 1H),
2.93-3.20 (m, 1H), 3.20-3.42 (m, 1H), 3.44 (septet, J ) 6.9
Hz, 1H), 3.97-4.18 (m, 1H), 4.40-4.65 (m, 1H), 6.70 (d, J )
8.7 Hz, 1H), 6.97 (d, J ) 15.6 Hz, 1H), 7.30 (td, J ) 2.7, 6.9
Hz, 1H), 7.41 (d, J ) 8.7 Hz, 1H), 7.46-7.65 (m, 3H), 7.60 (d,
J ) 15.6 Hz, 1H), 8.43 (s, 1H). MS (ESI+): (M + H)+ at m/z
455. Anal. (C24H26N2O5S‚0.44 H2O) C, H, N.
(2-Isop r op ylp h en yl)[2-n itr o-4-(E-((3-ca r boxyp ip er id in -
1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e (11b). Light-yellow
1
solid. H NMR (DMSO-d6, 300 MHz): δ 1.15 (d, J ) 6.9 Hz,
6H), 1.30-1.50 (m, 1H), 1.50-1.80 (m, 2H), 1.88-2.04 (m, 2H),
2.95-3.17 (m, 1H), 3.94-4.06 (m, 1H), 4.06-4.22 (m, 2H),
4.40-4.52 (m, 1H), 6.63 (d, J ) 8.7 Hz, 1H), 7.33-7.53 (m,
3H), 7.56-7.68 (m, 3H), 7.91 (dd, J ) 1.8, 8.4 Hz, 1H), 8.63
(d, J ) 8.4 Hz, 1H). MS (APCI+): (M + H)+ at m/z 455. Anal.
(C24H26N2O5S) C, H, N.
(2-Isop r op ylp h en yl)[2-n it r o-4-(E-((3-(t et r a zole-5-yl)-
m or p h olin -1-yl)ca r bon yl)eth en yl)p h en yl] Su lfid e (11c).
(2-Isopropylphenyl)[2-nitro-4-( E-((3-cyanomorpholin-1-yl)car-
bonyl)ethenyl)phenyl] sulfide (160 mg, 0.336), sodium azide
(56.6 mg, 0.872 mmol), n-Bu3SnCl, and THF were mixed in a
reaction tube, flushed with nitrogen, and heated to reflux
overnight. The mixture was then cooled to ambient tempera-
ture, and 1 N HCl solution was added. The mixture was
extracted with ethyl acetate three times, and the combined
organics were dried over MgSO4. The mixture was filtered
through a short silica gel plug to give 96 mg (56% yield) of the
Sta tistics. All values are expressed as mean ( SEM.
Differences in infarct size, HR, MAP, and RPP (rate-pressure
product) between groups were tested using one-way ANOVA
followed by a post-hoc Dunnett’s test. P < 0.05 was considered
significant.
1
desired material. H NMR (DMSO-d6, 300 MHz): δ 1.14 (d, J
) 6.8 Hz, 6H), 2.96-4.62 (br m, 7H), 4.77 (dd, J ) 10.5, 2.7
Hz, 1H), 6.58-6.67 (m, 1H), 7.32-7.62 (m, 6H), 7.92 (dd, J )
8.8, 2.0 Hz, 1H), 8.62-8.67 (br m, 1H). MS (APCI): (M + H)+
at m/z 481. Anal. (C23H24N6S1O4‚1.2H2O) C, H, N.
P h a r m a cok in etic An a lysis. The pharmacokinetic behav-
ior of compounds was evaluated in male Sprague-Dawley rats
or dogs (for 11f). Briefly, the test compound was prepared as
a 10 mg/mL solution in an ethanol/propylene glycol/D5W (20:
30:50 by volume) vehicle containing 1 mol equiv of sodium
hydroxide. Groups of rats (n ) 4/group) received either a 5
mg/kg intravenous dose administered as a slow bolus in the
jugular vein or a 5 mg/kg oral dose administered by gavage.
Heparinized blood samples (∼0.4 mL/sample) were obtained
from a tail vein of each rat 0.1 (iv only), 0.25, 0.5, 1, 1.5, 2, 3,
4, 6, and 8 h after dosing. The samples were analyzed by
reversed-phase HPLC following liquid/liquid extraction from
the plasma. Initial estimates of the pharmacokinetic param-
eters for NONLIN8416 were obtained with the program
CSTRIP.17 Area under the curve (AUC) values were calculated
by the trapezoidal rule over the time course of the study. The
terminal-phase rate constant (â) was utilized in the extrapola-
tion of the AUC from 8 h to infinity to provide an AUC0-∞ value
and in the calculation of t1/2 values. A comparison of the AUC
following oral dosing with that obtained following an intra-
venous dose provided an estimate of the bioavailability (F).
Mean residence time (MRT) was calculated as the ratio AUMC/
AUC.
(2-Isop r op ylp h en yl)[2-n itr o-4-(E-((4-su lfop ip er id in -1-
yl)ca r b on yl)et h en yl)p h en yl] Su lfid e (11d ). 1H NMR
(DMSO-d6, 300 MHz): δ 8.63 (d, 1H, J ) 1.8 Hz), 7.92 (dd,
1H, J ) 1.8, 8.8 Hz), 7.60 (m, 3H), 7.47 (d, 1H, J ) 14.2 Hz),
7.42 (d, 1H, J ) 14.2 Hz), 6.62 (d, 1H, J ) 8.5 Hz), 4.45 (m,
2H), 4.38 (m, 2H), 3.34 (m, 1H), 3.00 (m, 2H), 2.70 (m, 1H),
2.60 (m,2H), 1.14 (d, 6H, J ) 6.9 Hz). MS (ESI): m/z 491, 981.
Anal. (C23H26 N2O6 S2) C, H, N.
(Be n zod ioxa n -6-yl)[2-t r iflu or om e t h yl-4-(E -((4-ca r -
b oxyp ip er id in -1-yl)ca r b on yl)et h en yl)p h en yl] Su lfid e
(11e). White solid. 1H NMR (DMSO-d6, 300 MHz): δ 1.40 (m,
2H), 1.98 (m, 2H), 2.95 (m, 1H), 3.15 (m, 1H), 3.45 (m, 1H),
4.20 (m, 2H), 4.35 (m, 4H), 7.00 (m, 4H), 7.20 (m, 2H), 7.90
(m, 1H), 8.20 (m, 1H), 12.30 (s, 1H). MS (APCI): m/z 494 (M
+ H)+. Anal. (C24H22F3NO5S‚0.1 H2O) C, H, N.
(Be n zod ioxa n -6-yl)[2-t r iflu or om e t h yl-4-(E -((3-ca r -
b oxyp yr r olid in -1-yl)ca r b on yl)et h en yl)p h en yl] Su lfid e
1
(11f). H NMR (CDCl3, 300 MHz): δ 7.75 (s, 1H), 7.60 (d, 1H,
J ) 15.0 Hz), 7.40 (br, 1H), 7.06 (d, 1H, J ) 2.2 Hz), 6.96-
7.02 (m, 3H), 6.90 (d, 1H, J ) 8.5 Hz), 4.30 (m, 5H), 3.99 (br,
2H), 3.29 (br, 2H), 2.60 (br, 2H), 1.85 (br, 2H). MS (ESI): m/z
-492. Anal. (C24H22F3NO5S‚0.75 H2O) C, H, N.
Ack n ow led gm en t. The authors thank the Abbott
Ra t Mod el of Acu te Myoca r d ia l Isch em ia a n d Rep er -
fu sion . Male Sprague-Dawley rats were subject to coronary
artery ligation by techniques previously described.15 Briefly,
1
Analytical Department for assistance in acquiring H
NMR and mass spectra.

2920 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Pei et al.
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