Novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intracellular adhesion molecule-1 interaction. 2. Mechanism of inhibition and structure-based improvement of pharmaceutical properties

Article abstract of DOI:10.1021/jm000503f

The interaction between leukocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1) has been implicated in inflammatory and immune diseases. Recently, a novel series of p-arylthio cinnamides has been described as potent

Full text of DOI:10.1021/jm000503f

1202
J . Med. Chem. 2001, 44, 1202-1210
Novel p-Ar ylth io Cin n a m id es a s An ta gon ists of Leu k ocyte F u n ction -Associa ted
An tigen -1/In tr a cellu la r Ad h esion Molecu le-1 In ter a ction . 2. Mech a n ism of
In h ibition a n d Str u ctu r e-Ba sed Im p r ovem en t of P h a r m a ceu tica l P r op er ties
Gang Liu,*,† J effrey R. Huth,*,‡ Edward T. Olejniczak,‡ Renaldo Mendoza,‡ Peter DeVries,† Sandra Leitza,†
Edward B. Reilly,† Gregory F. Okasinski,† Stephen W. Fesik,‡ and Thomas W. von Geldern†
Metabolic Disease Research and Research NMR, Pharmaceutical Products Division, Abbott Laboratories,
Abbott Park, Illinois 60064-6098
Received November 27, 2000
The interaction between leukocyte function-associated antigen-1 (LFA-1) and intracellular
adhesion molecule-1 (ICAM-1) has been implicated in inflammatory and immune diseases.
Recently, a novel series of p-arylthio cinnamides has been described as potent antagonists of
the LFA-1/ICAM-1 interaction. These compounds were found to bind to the I domain of LFA-1
using two-dimensional NMR spectroscopy of 15N-labeled LFA-1 I domain. On the basis of NOE
studies between compound 1 and the I domain of LFA-1, a model of the complex was constructed.
This model revealed that compound 1 does not directly inhibit ICAM-1 binding by interacting
with the metal ion dependent adhesion site (MIDAS). Instead, it binds to the previously proposed
I domain allosteric site (IDAS) of LFA-1 and likely modulates the activation of LFA-1 through
its interaction with this regulatory site. A fragment-based NMR screening strategy was applied
to identify small, more water-soluble ligands that bind to a specific region of the IDAS. When
incorporated into the parent cinnamide template, the resulting analogues exhibited increased
aqueous solubility and improved pharmacokinetic profiles in rats, demonstrating the power of
this NMR-based screening approach for rapidly modifying high-affinity ligands.
In tr od u ction
proposal that one of the observed conformations is a
mimetic of the “activated” ligand bound form while the
other represents the unligated state of the protein.
Differences in the conformation of CD11a I domain were
also observed in crystallography studies.9,10 The pro-
posal that these conformations reflect a regulatory site
was supported by a recent structural study of an I
domain of R2â1/collagen peptide complex in which the
ligated I domain is in the proposed “activated” form.11
In the “activated” ligand bound state, the C-terminal
helix of the I domain drops downward by 10 Å, whereas
in the unligated state, this helix moves away from the
â-sheet core of the protein to expose a hydrophobic cleft.
On the basis of a series of site-directed mutagenesis
studies of amino acid residues in this cleft, we proposed
that this site (termed the I domain allosteric site or
IDAS) could act as a regulatory site for the activation
process.12
Leukocyte function-associated antigen-1 (LFA-1) (RLâ2,
CD11a/CD18) is a heterodimeric transmembrane gly-
coprotein expressed on all leukocytes.1,2 As a cell-surface
adhesion receptor, LFA-1 supports inflammatory and
specific T-cell immune responses through mediating cell
adhesion, leukocyte transmigration, and augmentation
of T-cell receptor signaling.3 These functions are achieved
through an interaction with its counter-receptorssthe
intracellular adhesion molecules (ICAM)-1, -2, and -3
located on endothelial and antigen-presenting cells.4,5
Therapeutic prevention of LFA-1 binding to ICAMs has
potential in the treatment of inflammatory diseases6
and graft rejection after transplantation.7
Similar to other integrins, LFA-1 consists of a large
R-subunit (CD11a) and a smaller â-subunit (CD18). The
most extracellular portion of the R-subunit of LFA-1
contains a magnesium cation-binding domain, called
metal ion dependent adhesion site (MIDAS), which is
involved in the direct interaction with ICAMs.8 Near
the amino terminus of the R-subunit is a 200 amino acid
insert termed the I domain that is required for binding
to ICAMs.3 In the crystal structures of CD11b I do-
mains, the I domain exhibited two different conforma-
tions depending on the crystal contacts.8 This led to the
Recently, a novel series of p-arylthio cinnamide-based
antagonists of the LFA-1/ICAM-1 interaction has been
reported by our laboratories, represented by 1 (Chart
1).13 This series of compounds was identified by screen-
ing for the inhibition of the LFA-1/ICAM-1 interaction
using full length proteins. In this report, we present the
results of NMR studies that defined the binding site of
these compounds. We also describe a rapid synthetic
approach for investigating the A-ring SAR of the p-
arylthio cinnamide antagonists and show how a frag-
ment-based NMR screening approach facilitated the
discovery of antagonists with improved pharmaceutical
properties.
* Address correspondence to Gang Liu at D-47R, AP-10, Abbott
Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6098.
Tel: (847) 935-1224, Fax: (847) 938-1674, E-mail: gang.liu@abbott.com.
Address correspondence regarding NMR spectroscopy to J effrey R.
Huth at D-47G, AP10, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064-6098. Tel: (847) 938-0959, Fax: (847) 938-
2478, E-mail: jeff.huth@abbott.com.
† Metabolic Disease Research.
‡ Research NMR.
10.1021/jm000503f CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/21/2001

Novel p-Arylthio Cinnamides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1203
Ch a r t 1. Representative Examples of the Diaryl
Sulfides as LFA-1/ICAM-1 Antagonists
converted to the cinnamide 6. The masked benzenethiol
was released by treatment of 6 with potassium tert-
butoxide to give potassium thiolate 7 for the Ullmann
coupling. Two types of coupling conditions have been
tried with 5-iodoindole, both of which yielded sufficient
amount of the desired product 8 for SAR studies, albeit
in low yields.14,15 The resulting indole analogue 8 could
be further functionalized to give compounds 9 and 10
through Mannich reaction or simple alkylation, respec-
tively.
The potassium thiolate 7 can also be used for nucleo-
philic substitution to give the nitro-containing com-
pound 11 (Scheme 2). Reduction of the nitro group and
cyclization of the resulting dianilino compound provided
the benzimidazolone analogue 12.
Resu lts a n d Discu ssion
NMR Scr een in g. Because of the central importance
of the I domain for LFA-1 function, we reasoned that
the small molecule antagonists identified by high-
throughput screening may exert their functions by
binding to the I domain. To test this hypothesis, two-
dimensional 15N-heteronuclear single-quantum correla-
tion (15N/1H HSQC) spectra16 of uniformly 15N-labeled
LFA-1 I domain12 were recorded in the presence and
absence of the original diaryl sulfide lead 2 (Chart 1).
Ch em istr y
To expedite the exploration of different types of
heterocycles for replacing the i-propylphenyl of 1, a
versatile synthetic sequence involving Ullmann coupling
was devised (Scheme 1). Aromatic nucleophilic substitu-
tion of 3-chloro-4-fluorobenzaldehyde (4) with methyl
3-mercaptopropionate yielded the aldehyde 5, which was
Sch em e 1a
a
Reagents and conditions: (a) K2CO3, HSCH2CH2CO2Me, DMF, 70 °C, 95%; (b) malonic acid, cat. piperidine, pyr., 110 °C, 98%; (c) i.
(COCl)2, CH2Cl2, cat. DMF, rt; ii. N-acetylpiperazine, NEt3, cat. DMAP, CH2Cl2, 84% two steps; (d) t-BuOK, THF, -78 °C-rt; (e) Cu
powder, CuI, K2CO3, DMF, 90 °C, 25% or Pd(PPh3)4, n-BuOH, 100 °C, 22%; (f) CH2dN(CH3)2I, dioxane/H2O, rt, 59%; (g) KOH, DMSO,
R-X, rt, 63%.
Sch em e 2a
a
Reagents and conditions: (a) 4,5-Dichloro-2-nitroaniline, K2CO3, DMF, 70 °C, 93%; (b) SnCl2, EtOH, reflux, 44%; (c) CDI, reflux,
THF, 32%.

1204 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Liu et al.
F igu r e 2. Average model of the CD11a I domain/compound
1 complex. Side chains of the IDAS that contact 1 are shown
in green with nitrogens in blue and oxygens in red. Side chains
of the MIDAS that chelate magnesium are shown in yellow
with oxygens in red. Compound 1 is drawn in magenta with
nitrogens in blue, oxygens in red, and the sulfur in gold. This
figure was created using the program Ribbons (Carson, M. J .
J . Mol. Graph. 1987, 5, 103-106).
F igu r e 1. Backbone atom superposition of 10 models of
compound 1 bound to the CD11a I domain. The protein
backbone is shown in black, compound 1 in red, and protein
side chains that create the binding pocket in blue.
Numerous chemical shift changes were observed upon
the addition of 2, indicating that the diaryl sulfide binds
to the I domain of LFA-1. NMR titration studies showed
that compound 2 is in intermediate exchange which
corresponds to a dissociation constant (Kd) less than 20
µM (Kd values for tight binders cannot be determined
by NMR). This is consistent with the measured IC50 of
2.3 µM in the LFA-1/ICAM-1 binding assay using the
entire extracellular domain of LFA-1. Compound 1 was
found to be in slow exchange when titrated with the I
domain, indicating a tighter binding than that of
compound 2, which was reflected as higher potency of
compound 1. That compound 1 also shifts similar
resonances as compound 2 indicates both compounds
bind to a similar region of the LFA-1 I domain. Thus,
the interaction of the p-arylthio cinnamides with the I
domain of LFA-1 can account for the inhibition of the
full length LFA-1 protein without invoking binding to
another site on LFA-1.
The location of the binding site within the I domain
for this series of compounds was identified by NMR.
Backbone and side chain assignments of the I domain
and the I domain/1 complex were obtained using stan-
dard heteronuclear multidimensional NMR experiments
(Experimental Section). Nineteen NOEs between 1 and
the I domain were obtained and assigned to residues
within the IDAS of the I domain. Structure calculations
using these NOEs and the X-ray coordinates for the free
I domain12 unambiguously determined the binding site
and orientation of compound 1 within the IDAS (Figure
1). The model reveals that compound 1 binds to a
hydrophobic pocket formed by strands one, three, and
four of the core â-sheet and R-helices one and seven
(Figure 2). Ring A of compound 1 interacts with a
hydrophobic surface involving Ile259 and Ile235. The
isopropyl substitution on ring A points away from the I
domain pocket toward the hydrophobic portions of
Lys287 and Lys305 on the surface of the I domain. The
sulfur of compound 1 is buried within the hydrophobic
Ch a r t 2. Fragments Identified from NMR Screening in
the Presence of Compound 3
pocket near Val157 and Leu302. Ring B is also buried
in the hydrophobic pocket and shows NOE contacts to
both residues Leu161 and Leu132. The ethenylcarbonyl
group and attached N-acetylpiperazine point toward the
N- and C-termini of the I domain, as indicated by the
NOEs observed between the acetyl methyl group and
Ile255. The absence of any NOEs to amino acid residues
within the MIDAS proved that no direct interaction with
the MIDAS by compound 1 was observed.
The binding of the p-arylthio cinnamide 1 to the IDAS
of LFA-1 suggests an indirect mechanism of inhibition
of the LFA-1/ICAM-1 interaction. Rather than directly
inhibiting ICAM-1 binding by blocking the MIDAS of
LFA-1, this series of compounds appears to act by
disrupting a regulatory site that encompasses a deep
hydrophobic pocket in the I domain of LFA-1. Lovastatin
(30) (Chart 3) has recently been found to be an antago-
nist of the LFA-1/ICAM-1 interaction,17 and its binding
site has been determined using both NMR and X-ray
crystallography. A superposition of our model with the
X-ray structure of lovastatin complex shows that 30 and
1 bind in a similar manner (Figure 3). The RMSD of
the protein backbones between the lovastatin complex
and the complex 1 is 0.6 Å, and the position of the

Novel p-Arylthio Cinnamides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1205
Ch a r t 3. Structure of Lovastatin, a Reported LFA-1/
ICAM-1 Interaction Antagonist
thiadiazole (15), and benzodioxane (16) as shown in
Chart 2. NMR structural studies indicate that these
molecules bind to the A-ring site. For instance, NMR
experiments on a ternary complex of compound 3 and
16 bound to the I domain revealed NOEs between
compound 16 and residue Ile259 of the IDAS. This
indicates that compound 16 binds to the pocket occupied
by the A-ring of the parent compound 1. NOEs between
compound 3 and methyl groups of the IDAS were also
observed, indicating that 3 and 16 bind to the IDAS in
the same region as the parent compound 1.
With A-ring replacement fragments identified, a
strategy was needed for the final assembly of the linked
molecules. From the previous A-ring SAR study for
increasing aqueous solubility with heterocycles, it was
determined that when the sulfide is linked to the
heteroatom-containing rings, the resulting compounds
exhibited dramatically decreased potency.19 This infor-
mation led to the strategy of linking to the benzo portion
of the A-ring replacement fragments. Indole fragment
13 was used to explore the effect of different positions
for the sulfide bond. Linking at the 5-position of the
indole yielded compound 8 which possessed slightly
improved potency compared to 1 (Table 1). The hydrogen-
bonding character of the indole does not appear to be
important because the N-methylated indole yielded an
equipotent compound 10a . Linking at the 6-position of
the indole resulted in compound 17 which displayed
greater than 7-fold decrease in potency versus the
5-position analogue 8. N-Methylindole linked at the
7-position (18) is over 3-fold less potent than the 5-N-
methylated analogue 10a , while ethylation further
decreased the potency of the resulting compound 19. So,
the 5-position of indole appears to be the preferred
position for forming the sulfide bond. Accordingly,
methylenedioxybenzene, a bioisoster of benzothiadiaz-
ole,20 was linked at the 5-position to provide 20 which
possessed a 3-fold loss of potency compared with 1. In
addition, the benzodioxane was linked at the 6-position
to yield 21 (A-292949) with comparable potency as 1.
The NOE evidence suggests that the aromatic portion
of the benzodioxane packs against the side chain of I259.
This orientation would allow the dioxane to point toward
the solvent where the interactions with water and the
side chains of L287 and L305 are possible.
F igu r e 3. Superposition of compound 1 and lovastatin in the
IDAS of LFA-1. Compound 1 is drawn in magenta with
nitrogens in blue, oxygens in red, and the sulfur in gold.
Lovastatin 30 (lactone form) is drawn in green with oxygens
in red.
C-terminal helix is in the conformation of the “unli-
gated” I domain for both complexes. Secondly, rings B
and C of 1 occupy the same space in the IDAS as does
30. These results show that blocking LFA-1 activation
through this IDAS site can be achieved with different
molecular scaffolds by locking the I domain in a low-
avidity conformation.
F r a gm en t -Ba sed NMR Scr een in g To Op t im ize
Com p ou n d 1. It has recently been shown that high-
affinity ligands can be designed by using information
derived from the NMR-based screening of fragments.18
The method involves the fragmentation of an existing
lead molecule, the identification of suitable replace-
ments for the fragments, and incorporation of the newly
identified fragments into the original scaffold. In the
case of the p-arylthio cinnamide, the presence of hydro-
philic residues L287 and L305 in the A-ring binding
pocket offered opportunities to improve the potency and
physical properties of 1 through identification of more
hydrophilic replacements of the i-propylphenyl moiety.
Toward this end, compound 3 (Chart 1) was synthe-
sized as a fragment of 1 to block the binding sites for
the B- and C-rings on the I domain. Subsequently, 2500
molecules with a molecular weight less than 150 Da
were tested for binding to the I domain in the presence
of saturating amounts of 3. Several classes of hetero-
cycles were identified which bind to the A-ring site with
Kd values in the submillimolar to millimolar range,
including indole (13), N-methylindole (14), 2,1,3-benzo-
It is interesting to note that, as fragments, indole and
benzodioxane showed a 30-fold difference in binding
affinity as estimated from NMR titration in the presence
of 3, yet the potencies of the corresponding linked
compounds 8 and 21 differ by only 2-fold. It is not
unexpected that variation exists before and after frag-
ments being linked to the core structure. Introduction
of a linker atom between two ligands may alter the
binding orientation of the A- and B-rings relative to the
unlinked fragments. Such a small change in binding
orientation could account for the narrowing of 30-fold
affinity difference to near equipotency for 8 and 21.21
A cell-based adhesion assay, which measures the
ability of the antagonists to block the adherence of J Y-8
cells expressing LFA-1 to immobilized ICAM-1, was
used to confirm the functional activity in vitro. Among
the more potent compounds listed in Table 1, indole
compounds 8 and 10a gave IC50 values of 15 nM and

1206 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Liu et al.
Ta ble 2. LFA-1/ICAM-1 Binding Inhibition from A-Ring
Ta ble 1. LFA-1/ICAM-1 Binding Inhibition from A-Ring
Modification of the p-Arylthio Cinnamides
Modification (Substituted Indoles and Benzodioxanes) of the
p-Arylthio Cinnamides
a
IC50 calculated using a mean of at least two measurements
(all duplicates) for six concentrations from 6.4 × 10-9 to 2 × 10-5
unless otherwise noted.
30 nM, respectively, while the benzodioxane analogue
21 exhibited an IC50 of 80 nM in this assay.
Attention was then turned to attaching more hydro-
philic groups to the indole and benzodioxane portions
of the molecules (Table 2). The N,N-dimethylamino-
methyl-substituted indole analogue 9 exhibited over a
20-fold loss in potency compared to 8. This loss in
potency was partially recovered with N,N-dimethylamino-
methyl-substituted indole analogue 22. Attachment of
a negatively charged carboxy group (23) to the methyl
group of 10a resulted in an over 15-fold loss of potency.
Furthermore, the addition of a neutral methoxymethyl
group (24) to the methyl group of 10a led to 3-fold loss
of potency.
For the benzodioxane-based analogue, addition of an
ethoxy group to the 5-position of the benzodioxane of
21 resulted in 25 with over 2-fold decrease of potency
(Table 2). Attaching a hydroxymethyl group to the 2/3-
position of the benzodioxane yielded compound 26 which
a
IC50 calculated using a mean of at least two measurements
(all duplicates) for six concentrations from 6.4 × 10-9 to 2 × 10-5
b
unless otherwise noted. 1:1 mixture of 2- and 3-position regioi-
somers.

Novel p-Arylthio Cinnamides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1207
Ta ble 3. Pharmacokinetic Profiles of Representative Compounds in Ratsa,b
solubility
(µg/mL)
Vâ
(l/kg)
i.v. t1/2
(h)
AUCi.v.
(µg‚h/mL)
oral Cmax/Tmax
(µg/mL; h)
oral t1/2
(h)
AUCoral
(µg‚h/mL)
F
(%)
compd
1
0.9 ( 0.1
0.8 ( 0.1
4.1 ( 0.5
2.0
(1.1-3.0)
0.4
(0.3-0.9)
i.v. dosing not possible
1.9
(1.8-2.1)
0
NDc
0
0
8
0.6/6.0
(0.4-0.8)/(4.0-8.0)
0.19/0.5
4.7
(4.1-5.5)
1.1
5.8
(4.2-8.8)
0.28
NAc
21
3.0
(0.9-6.0)
0.55
(0.30-1.79)
2.2
(2.1-2.3)
12.4
(8.7-15.8)
(0.17-0.20)/(0.25-1.0)
(0.99-1.14)
(0.19-0.35)
a
b
A total of 5 mg/kg dosed for both intravenous injection and oral gavage. Harmonic mean from three rats each listed with the range
of the data in parentheses. c ND, not determined. NA, not applicable.
under nitrogen atmosphere unless specifically noted. Flash
chromatography was performed using silica gel (230-400
mesh) from E. M. Science. Proton NMR spectra were recorded
on a General Electric QE300 instrument with Me4Si as an
internal standard and are reported as shift (multiplicity,
coupling constants, proton counts). Mass spectral analyses
were accomplished using desorption chemical ionization (DCI),
atmospheric pressure chemical ionization (APCI), and elec-
trospray ionization (ESI), as specified for individual com-
pounds. 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 × 30 mm i.d.,
S-5 µM, 120 Å, and a flow rate of 25 mL/min; λ ) 214, 245
nm; mobile phase A, 0.05 M NH4OAc or 0.1% TFA in H2O,
and mobile phase B, CH3CN; linear gradient 20-100% of B
in 20 min. The purified fractions were evaporated to dryness
on a Savant SpeedVac.
Detection of Liga n d Bin d in g Usin g NMR. The I domain
of LFA-1 was isotopically labeled and purified as previously
described.12 NMR samples contained 0.4 µM uniformly 15N-
labeled LFA-1 I domain, 0.6 mM MgCl2, 100 mM sodium
phosphate (pH 7.2) in H2O/D2O (9:1). Ligand binding was
detected at 30 °C by acquiring sensitivity-enhanced [15N]HSQC
spectra16 on a Bruker DRX500 spectrometer in the presence
and absence of added compound. Dissociation constants were
estimated by monitoring chemical shift changes as a function
of added ligand.22
exhibits a potency improvement over the parent com-
pound 21. In contrast, attachment of a carboxy group
to the benzodioxane of 21 led to a 30-fold decrease of
potency with the resulting analogue 27. N,N-Dimeth-
ylcarboxamide attachment at the 2/3-position of the
benzodioxane gave compound 28 which had a much
reduced potency compared with compound 21. Benz-
imidazolone-based compound 12 exhibited submicro-
molar potency, as did the quinoline-derived compound
29.
The solubility and pharmacokinetic profiles of repre-
sentative compounds were measured, and the results
are shown in Table 3. The indole replacement of
i-propylphenyl (8) did not improve the aqueous solubility
over that of 1, while the benzodioxane replacement (21)
yielded at least 4-fold increase of aqueous solubility.
Although the solubility improvement observed with the
incorporation of heteroatoms did not appear to be
dramatic, this modification did render an improvement
in the rat pharmacokinetic profile of the resulting
analogues over 1. Compound 1 could not be detected
following 5 mg/kg oral dosing in rats. In contrast,
compound 8 (unable to dose i.v. due to poor solubility)
was detected after oral dosing and yielded an oral half-
life of 4.7 h. The oral AUC is 5.8 µg‚h/mL after 5 mg/kg
oral gavage in rats. The solubility improvement ob-
tained for 21 enabled i.v. administration and yielded
an oral bioavailability of ∼13%.
NOE -Ba sed Mod el of LF A-1 I Dom a in /Com p ou n d 1
Com p lex. NMR experiments were performed at 30 °C on
Bruker DRX spectrometers at 500, 600, and 800 MHz.
Backbone and side chain assignments of the I domain in the
absence of ligand were obtained as previously reported.12
Assignments for the complex with compound 1 were obtained
using similar experiments. Amides not able to be assigned with
backbone triple resonance experiments were assigned using
15N-edited NOE experiments. Carbon and proton assignments
for methyl group were obtained from 13C-edited NOE experi-
ments using 15N, 13C, 2H-labeled protein with protons incor-
porated at the γ-methyls of Val, δ-methyls of Leu, and â-, γ1-,
γ2-, and δ1-positions of Ile.23 NOEs between the ligand and
protein were obtained from isotope-edited/filtered 3D experi-
ments.24 Ligand assignments were obtained from two-dimen-
sional double-filtered NOESY and TOCSY experiments.25
A model of the complex was obtained by manually position-
ing compound 1 near the C-terminus of the I domain followed
by energy minimization with the XPLOR.26 In the calculations,
the backbone atoms for residues 130-303 were fixed. The side
chain atoms were allowed to move to accommodate the ligand.
A total of 19 intermolecular and two intraligand distance
restraints were used with a square well potential and placed
into two loose distance categories, 1.8-5.0 and 1.8-6.0 Å. An
additional angstrom was added to the upper limit for NOEs
involving methyl groups of valine or leucine that were not
stereoassigned. Two constraints were included to reflect the
lack of an observed NOE. These constrains kept the closest
approach for these pairs of protons at 4 Å.
Con clu sion s
Using NMR, the binding site of compound 1 was
established as the proposed IDAS of the I domain of
LFA-1. The NMR studies suggest a possible mechanism
of inhibition of the LFA-1/ICAM-1 interaction by our
novel series of p-arylthio cinnamides that involves the
disruption of a hydrophobic regulatory site of the I
domain of LFA-1. The application of fragment-based
NMR screening in combination with an Ullmann reac-
tion-based divergent synthetic sequence significantly
decreased the time and effort involved in modifying this
series of high-affinity ligands. A-ring modification of
compound 1 resulted in high-affinity antagonists of the
LFA-1/ICAM-1 interaction, such as 21, with increased
aqueous solubility and improved pharmacokinetic pro-
files in rats. In addition, heterocycle-based A-ring
replacements provided a diverse set of fragments that
can be combined with the optimized B- and C-rings of
the p-arylthio cinnamides, which will be reported in the
following papers.
Exp er im en ta l Section
F r a gm en t-Ba sed NMR Scr een in g. 15N-LFA-1 I domain
(0.3 mM) in 0.45 mM MgCl2 and 100 mM sodium phosphate
(pH 7.0) in H2O/D2O (9:1), in the presence of 0.3 mM compound
3, was analyzed with and without mixtures of five compounds
Gen er a l. Unless otherwise specified, all solvents and
reagents were obtained from commercial suppliers and used
without further purification. All reactions were performed

1208 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Liu et al.
at 5 mM each. Compounds were added from 1 M stocks in
deuterated DMSO. Ligand binding was detected by acquiring
[15N]HSQC spectra. To deconvolute mixtures that induced
chemical shift changes of the I domain, binding of each of the
five compounds was evaluated individually. Binding affinities
for compounds that bind in the presence of compound 3 were
determined by NMR as described previously.22 Confirmation
that these molecules bind to the pocket occupied by the A-ring
of compound 1 was based on observed NOEs between com-
pound 16 and the protein for a ternary complex composed of
the I domain, compound 3, and compound 16.
3-[4-[3-(4-Acetyl-piperazin-1-yl)-3-oxo-propenyl]-2-chloro-
p h en ylsu lfa n yl]-p r op ion ic Acid Met h yl E st er (6). To a
stirred solution of 3-chloro-4-fluorobenzaldehyde (3.3 g, 20.8
mmol) in 25 mL of anhydrous DMF with potassium carbonate
(4.3 g, 31.1 mmol) was added methyl-3-mercaptopropionate
(2.3 mL, 20.8 mmol) via syringe dropwise. The mixture was
then heated at 60 °C for 2 h. The reaction mixture was then
cooled to room temperature and partitioned between ether and
water. The aqueous layer was extracted with ether once, and
the combined organic layer was washed with water and brine,
dried over Na2SO4, and condensed in vacuo to give 5.1 g (19.7
mmol, 95%) of the desired aldehyde 5 as a colorless oil.
ature and poured into water. The aqueous mixture was
extracted with EtOAc (2 × 25 mL). The combined organic layer
was then washed with water and brine, dried over Na2SO4,
filtered, and concentrated. The crude product was purified
using a Gilson preparative HPLC to give compound 8 (115 mg,
0.58 mmol, 25%) as an amorphous off-white solid. 1H NMR
(DMSO-d6, 300 MHz) δ 2.03 (s, 3H), 3.40-3.78 (m, 8H), 6.51
(d, J ) 8.4 Hz, 1H), 6.53 (s, 1H), 7.23 (dd, J ) 2.1, 8.4 Hz,
1H), 7.27 (d, J ) 15.6 Hz, 1H), 7.39 (d, J ) 15.6 Hz, 1H), 7.41
(dd, J ) 1.8, 8.4 Hz, 1H), 7.49 (t, J ) 2.7 Hz, 1H), 7.56 (d, J )
8.4 Hz, 1H), 7.85 (d, J ) 1.8 Hz, 1H), 7.99 (d, J ) 1.8 Hz, 1H);
MS (APCI+) (M + NH4)+ at m/z 440, 442. Anal. (C23H22
ClN3O2S‚0.53CH2Cl2) C, H, N.
-
3-(Dim eth yla m in om eth yl)in d ol-5-yl 2-Ch lor o-4-(E-((4-
acetylpiper azin -1-yl)car bon yl)eth en yl)ph en yl Su lfide (9).
To a stirred solution of Eschenmoser’s salt (15 mg, 0.081 mmol)
in 1 mL of dioxan/H2O (3:1) at ambient temperature was added
compound 8 (30 mg, 0.068 mmol). The mixture was then
stirred at room temperature overnight. Solvent was then
removed on
a rotavap under reduced pressure, and the
resulting crude product was purified on a Gilson preparative
HPLC to give compound 9 as a light brown solid (20 mg, 0.040
1
mmol, 59%). H NMR (CDCl3, 300 MHz) δ 2.15 (s, 3H), 2.54
(s, 6H), 3.47-3.85 (m, 8H), 4.05 (s, 2H), 6.56 (d, J ) 8.7 Hz,
1H), 6.77 (d, J ) 15.6 Hz, 1H), 7.09 (d, J ) 8.7 Hz, 1H), 7.36
(dd, J ) 1.5, 8.7 Hz, 1H), 7.50 (d, J ) 8.7 Hz, 1H), 7.52 (s,
2H), 7.56 (d, J ) 15.6 Hz, 1H), 7.88 (s, 1H), 9.27 (s, 1H); MS
(ESI+) (M + H)+ at m/z 497, 499. Anal. (C26H29ClN4O2S‚
1.24TFA) C, H, N.
1-Meth ylin d ol-5-yl 2-Ch lor o-4-(E-((4-a cetylp ip er a zin -
1-yl)ca r bon yl)eth en yl)p h en yl Su lfid e (10a ): white solid;
1H NMR (DMSO-d6, 300 MHz) δ 2.04 (s, 3H), 3.40-3.80 (m,
8H), 3.86 (s, 3H), 6.49 (d, J ) 8.4 Hz, 1H), 6.52 (d, J ) 3.0 Hz,
1H), 7.27 (d, J ) 15.6 Hz, 1H), 7.31 (dd, J ) 2.4, 8.4 Hz, 1H),
7.39 (d, J ) 15.6 Hz, 1H), 7.41 (dd, J ) 1.8, 8.4 Hz, 1H), 7.48
(d, J ) 3.0 Hz, 1H), 7.63 (d, J ) 8.4 Hz, 1H), 7.85 (d, J ) 1.8
Hz, 1H), 7.99 (brs, 1H); MS (APCI+) (M + H)+ at m/z 454, 456.
Anal. (C24H24ClN3O2S‚0.11H2O) C, H, N.
6-Ch lor oben zim idazol-2-on -5-yl 2-Ch lor o-4-(E-((4-acetyl-
p ip er a zin -1-yl)ca r bon yl)eth en yl)p h en yl Su lfid e (12). The
potassium thiolate 7 (150 mg, 0.37 mmol) in 3.0 mL of DMF
was heated with K2CO3 (76 mg, 0.55 mmol) and 4,5-dichloro-
2-nitroaniline (76 mg, 0.37 mmol) at 70 °C for 3 h. Reaction
was then quenched with water after cooling to room temper-
ature. The yellow precipitates were collected through filtration,
washed with water, and dried in a vacuum oven to give the
nitro compound 11 (170 mg, 0.34 mmol, 93%).
To a stirred solution of nitrobenzene (170 mg, 0.34 mmol)
in 2 mL of EtOH was added anhydrous SnCl2 (325 mg, 1.72
mmol). The mixture was then refluxed for 2 h. The reaction
was allowed to cool to ambient temperature, quenched with
saturated NaHCO3, and extracted with EtOAc (2 × 20 mL).
The combined organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was then
purified on a Gilson preparative HPLC to give the dianilino
compound as a light yellow solid (70 mg, 44%).
A mixture of the aldehyde 5 (1.0 g, 3.9 mmol), malonic acid
(0.88 g, 8.5 mmol), and piperidine (57 µL, 0.58 mmol) in 4 mL
of anhydrous pyridine was heated at 110 °C for 2 h during
which the gas evolution ceased. After pyridine was removed
under vacuum, water and 3 N aqueous HCl were then added
with stirring. The desired cinnamic acid was collected through
filtration, washed with cold water, and dried in a vacuum oven
overnight to give 1.15 g (4.8 mmol, 98%) of the cinnamic acid
as a white solid.
A suspension of the acid (220 mg, 0.73 mmol) in 5 mL of
CH2Cl2 was stirred with (COCl)2 (71 µL, 0.81 mmol) and one
drop of DMF for 90 min. The solvent was then removed under
vacuum, and the residual (COCl)2 was removed with benzene
(2 × 2 mL) in vacuo. A separate flask was charged with
N-acetylpiperazine (113 mg, 0.88 mmol), NEt3 (257 µL, 1.83
mmol), and DMAP (4.4 mg, 0.032 mmol) in 10 mL of CH2Cl2.
The acid chloride in 5 mL of CH2Cl2 was then dropped in
slowly. After 30 min, the reaction mixture was poured into 3
N aqueous HCl and extracted with EtOAc. The organic layer
was washed with brine, dried with Na2SO4, and condensed
under reduced pressure. The crude product was purified on a
silica gel flash column (5-10% MeOH in EtOAc as eluent) to
give 250 mg (0.61 mmol, 84%) of cinnamide 6 as a white solid.
1H NMR (CDCl3, 300 MHz) δ 2.16 (s, 3H), 2.72 (t, J ) 7.5 Hz,
2H), 3.25 (t, J ) 7.5 Hz, 2H), 3.54 (t, J ) 4.95 Hz, 2H), 3.60-
3.83 (brm, 6H), 6.83 (d, J ) 15.6 Hz, 1H), 7.28 (d, J ) 8.7 Hz,
1H), 7.37 (dd, J ) 2.1, 8.7 Hz, 1H), 7.56 (d, J ) 2.1 Hz, 1H),
7.61 (d, J ) 15.6 Hz, 1H); MS (APCI+) (M + NH4)+ at m/z 428,
430.
P ot a ssiu m -4-[3-(4-a ce t yl-p ip e r a zin -1-yl)-3-oxo-p r o-
p en yl]-2-ch lor o-ben zen eth iola te (7). To a stirred solution
of the compound 6 (105 mg, 0.26 mmol) in 2 mL of THF at 0
°C was added t-BuOK solution (1.0 M, 281 µL, 0.29 mmol).
Light orange precipitates appeared immediately. After comple-
tion of the addition, the reaction mixture was stirred at room
temperature for 1 h before the solvent was removed on a
rotavap under reduced pressure. The residue was dried under
high vacuum to give the potassium thiolate 7 as a light yellow
A mixture of dianiline (35 mg, 0.075 mmol) and CDI (13
mg, 0.075 mmol) in THF was stirred at ambient temperature
for 1 day. Solvent was then removed under reduced pressure,
and the crude product was purified on a Gilson preparative
1
HPLC to give compound 12 (12 mg, 32%) as a white solid. H
1
solid. H NMR (DMSO-d6, 300 MHz) δ 2.02 (s, 3H), 3.46 (brs,
NMR (DMSO-d6, 300 MHz) δ 2.04 (s, 3H), 3.40-3.80 (m, 8H),
6.63 (d, J ) 8.4 Hz, 1H), 7.11 (d, J ) 2.4 Hz, 1H), 7.12 (s, 1H),
7.23 (s, 1H), 7.32 (d, J ) 15.6 Hz, 1H), 7.43 (d, J ) 15.6 Hz,
1H), 7.50 (d, J ) 8.4 Hz, 1H), 8.03 (brs, 1H); MS (APCI+) (M
- CO + H)+ at m/z 465, 467. Anal. (C22H20Cl2N4O3S‚0.16H2O)
C, H, N.
6H), 3.54-3.80 (m, 2H), 6.88 (d, J ) 14.9 Hz, 1H), 6.96 (d, J
) 8.7 Hz, 1H), 7.26 (dd, J ) 2.1, 8.7 Hz, 1H), 7.33 (d, J ) 2.1
Hz, 1H), 7.46 (d, J ) 14.9 Hz, 1H); MS (ESI+) (M + H)+ at m/z
324, 326.
5-In d olyl 2-Ch lor o-4-(E-((4-a cetylp ip er a zin -1-yl)ca r bo-
n yl)eth en yl)p h en yl Su lfid e (8). Meth od A of Su lfid e
F or m a tion . To a stirred solution of 5-iodoindole (255 mg, 1.05
mmol) in 5 mL of anhydrous DMF were added the potassium
thiolate 7 (457 mg, 1.26 mmol), followed by K2CO3 (174 mg,
1.26 mmol) and cuprous iodide (20 mg, 0.11 mmol). The
resulting mixture was then heated at 120 °C overnight. The
reaction mixture was then allowed to cool to ambient temper-
In d ol-6-yl 2-Ch lor o-4-(E-((4-a cetylp ip er a zin -1-yl)ca r -
1
bon yl)eth en yl)p h en yl Su lfid e (17): white solid; H NMR
(DMSO-d6, 300 MHz) δ 2.03 (s, 3H), 3.40-3.77 (m, 8H), 6.52-
6.55 (m, 1H), 6.60 (d, J ) 8.4 Hz, 1H), 7.13 (dd, J ) 1.8, 8.4
Hz, 1H), 7.27 (d, J ) 15.6 Hz, 1H), 7.40 (d, J ) 15.6 Hz, 1H),
7.43 (dd, J ) 1.8, 8.4 Hz, 1H), 7.51 (t, J ) 3.0 Hz, 1H), 7.64
(m, 1H), 7.70 (d, J ) 8.4 Hz, 1H), 7.99 (d, J ) 1.8 Hz, 1H); MS

Novel p-Arylthio Cinnamides
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1209
(APCI+) (M + H)+ at m/z 440, 442. Anal. (C23H22ClN3O2S‚
1H), 7.25 (d, J ) 15.6 Hz, 1H), 7.38 (d, J ) 15.6 Hz, 1H), 7.40
(d, J ) 3.0 Hz, 1H), 7.47 (d, J ) 8.4 Hz, 1H), 7.80 (d, J ) 2.1
Hz, 1H), 7.97 (s, 1H); MS (ESI+) (M - H)+ at m/z 496, 498.
Anal. (C25H24ClN3O4S‚0.18H2O) C, H, N.
0.04TFA) C, H, N.
1-Meth ylin d ol-7-yl 2-Ch lor o-4-(E-((4-a cetylp ip er a zin -
1-yl)ca r bon yl)eth en yl)p h en yl Su lfid e (18): light brown
solid; 1H NMR (CDCl3, 300 MHz) δ 2.14 (s, 3H), 3.47-3.56
(m, 2H), 3.56-3.83 (m, 6H), 3.96 (s, 3H), 6.42 (d, J ) 8.4 Hz,
1H), 6.55 (d, J ) 3.6 Hz, 1H), 6.76 (d, J ) 15.6 Hz, 1H), 6.99
(d, J ) 3.6 Hz, 1H), 7.09 (dd, J ) 2.1, 8.4 Hz, 1H), 7.15 (t, J )
7.65 Hz, 1H), 7.42 (dd, J ) 0.9, 7.5 Hz, 1H), 7.53 (d, J ) 1.8
Hz, 1H), 7.55 (dd, J ) 15.6 Hz, 1H), 7.77 (dd, J ) 0.9, 7.5 Hz,
1-(2-Meth oxyeth yl)in dol-5-yl 2-Ch lor o-4-(E-((4-acetylpip-
er a zin -1-yl)ca r bon yl)eth en yl)p h en yl Su lfid e (24): white
1
solid; H NMR (CDCl3, 300 MHz) δ 2.14 (s, 2H), 3.35 (s, 3H),
3.46-3.56 (m, 2H), 3.56-3.80 (m, 6H), 3.75 (t, J ) 5.6 Hz, 2H),
4.33 (t, J ) 5.6 Hz, 2H), 6.54 (d, J ) 3.3 Hz, 1H), 6.61 (d, J )
8.7 Hz, 1H), 6.75 (d, J ) 15.3 Hz, 1H), 7.09 (dd, J ) 2.1, 11.7
Hz, 1H), 7.26 (overlapping d, 1H), 7.36 (dd, J ) 2.1, 8.7 Hz,
1H), 7.44 (d, J ) 8.7 Hz, 1H), 7.51 (d, J ) 2.1 Hz, 1H), 7.56 (d,
J ) 15.3 Hz, 1H), 7.88 (d, J ) 1.5 Hz, 1H); MS (ESI+) (M +
H)+ at m/z 498, 500. Anal. (C26H28ClN3O3S‚0.12 H2O) C, H, N.
5-Eth oxyben zod ioxa n -6-yl 2-Ch lor o-4-(E-((4-a cetylp ip -
er a zin -1-yl)ca r bon yl)eth en yl)p h en yl Su lfid e (25): white
1H); MS (APCI+) (M + H)+ at m/z 454, 456. Anal. (C24H24
ClN3O2S) C, H, N.
-
1-Eth ylin d ol-7-yl 2-Ch lor o-4-(E-((4-a cetylp ip er a zin -1-
yl)ca r bon yl)eth en yl)p h en yl Su lfid e (19). Meth od B for
Su lfid e F or m a tion . A stirred solution of thiolate 7 (65 mg,
0.18 mmol) in 1 mL of anhydrous DMSO was charged with
7-bromo-1-ethylindole (44 mg, 0.20 mmol), Pd (PPh3)4 (10.4 mg,
0.090 mmol), and t-BuONa (18 mg, 0.18 mmol). The mixture
was heated in a sealed tube at 110 °C for 2 h. The reaction
mixture was then cooled to room temperature and quenched
with 3 mL of water. The crude product was extracted out with
EtOAc (2 × 10 mL). The combined organic layer was then
washed with water and brine, dried over Na2SO4, filtered, and
concentrated. The desired product was isolated from a Gilson
preparative HPLC as an off-white solid (19 mg, 0.041 mmol,
1
solid; H NMR (CDCl3, 300 MHz) δ 1.28 (t, J ) 7.2 Hz, 3H),
2.14 (s, 3H), 3.54 (brs, 2H), 3.60-3.88 (m, 6H), 4.06 (q, J )
7.2 Hz, 2H), 4.33 (s, 4H), 6.70 (d, J ) 8.4 Hz, 1H), 6.73 (d, J
) 8.4 Hz, 1H), 6.78 (d, J ) 15.6 Hz, 1H0, 6.98 (d, J ) 8.4 Hz,
1H), 7.17 (dd, J ) 1.8, 8.4 Hz, 1H), 7.50 (d, J ) 1.8 Hz, 1H),
7.57 (d, J ) 15.6 Hz, 1H); MS (APCI+) (M+H)+ at m/z 503,
505. Anal. (C25H27ClN2O5S‚0.11H2O) C, H, N.
2-(H yd r oxym et h yl)-b en zod ioxa n -6-yl 2-Ch lor o-4-(E-
((4-a cet ylp ip er a zin -1-yl)ca r b on yl)et h en yl)p h en yl Su l-
fid e (26): light yellow oil; 1H NMR (CDCl3, 300 MHz, mixture
of 1:1 regioisomers) δ 2.15 (s, 3H), 3.46-3.83 (m, 8H), 3.83-
4.01 (m, 2H), 4.10-4.42 (m, 4H), 6.75 (d, J ) 8.4 Hz, 1H), 6.79
(d, J ) 15.9 Hz, 1H), [6.95 (d), 6.98 (d), J ) 4.8 Hz, 1H in
total], [7.04 (t), 7.07 (t), J ) 1.5 Hz, 1H in total], [7.10 (d),
7.11 (d), J ) 2.4 Hz, 1H in total], 7.19 (d, J ) 8.4 Hz, 1H),
7.53 (s, 1H), 7.58 (d, J ) 15.6 Hz, 1H); MS (APCI+) (M + H)+
at m/z 489. Anal. (C24H25ClN2O5S‚0.23H2O) C, H, N.
2-Car boxy-ben zodioxan -6-yl 2-Ch lor o-4-(E-((4-acetylpip-
er a zin -1-yl)ca r bon yl)eth en yl)p h en yl Su lfid e (27): light
brown solid; 1H NMR (DMSO-d6, 300 MHz, 1:1 mixture of
regioisomers) δ 2.04 (s, 3H), 3.47 (brs, 6H), 3.55-3.77 (m, 2H),
[5.01 (d), 5.03 (d), J ) 5.1 Hz, 2H in total], [5.39 (brs), 5.43
(brs), 1H in total], [6.61 (d), 6.69 (d), J ) 8.4 Hz, 1H in total],
6.73-6.95 (m, 2H), 6.95-7.23 (m, 2H), 7.24-7.60 (m, 2H),
7.92-8.01 (m, 1H); MS (ESI+) (M + Na)+ at m/z 503, 505. Anal.
(C24H23ClN2O6S‚0.15H2O) C, H, N.
2-(Dim eth ylam in ocar bon yl)-ben zodioxan -6-yl 2-Ch lor o-
4-(E-((4-acetylpiper azin -1-yl)car bon yl)eth en yl)ph en yl Su l-
fid e (28): white solid; 1H NMR (CDCl3, 300 MHz, 1:1 mixture
of regioisomers) δ 1.93 (s, 3H), 2.15 (s, 6H), 3.53 (brs, 2H),
3.59-3.90 (brm, 8H), 4.86-5.01 (m, 1H), 6.74-6.81 (m, 1H),
6.80 (d, J ) 15.3 Hz, 1H), 6.93 (d, J ) 8.7 Hz, 1H), 7.02 (d, J
) 1.8 Hz, 1H), 7.13 (dd, J ) 1.8, 8.4 Hz, 1H), 7.16-7.25 (m,
1H), 7.54 (s, 1H), 7.58 (d, J ) 15.6 Hz, 1H); MS (ESI+) (M +
Na)+ at m/z 552, 554. Anal. (C26H28ClN3O5S‚0.35H2O) C, H,
N.
1
22%). H NMR (CDCl3, 300 MHz) δ 1.30 (t, J ) 7.05 Hz, 3H),
2.14 (s, 3H), 3.52 (brs, 2H), 3.58-3.84 (m, 6H), 4.42 (q, J )
7.05 Hz, 2H), 6.42 (d, J ) 8.4 Hz, 1H), 6.59 (d, J ) 3.0 Hz,
1H), 6.76 (d, J ) 15.6 Hz, 1H), 7.08 (d, J ) 8.4 Hz, 1H), 7.10
(d, J ) 3.0 Hz, 1H), 7.16 (t, J ) 7.65 Hz, 1H), 7.42 (dd, J )
0.9, 7.5 Hz, 1H),7.53 (d, J ) 1.8 Hz, 1H), 7.54 (d, J ) 15.6 Hz,
1H), 7.78 (dd, J ) 0.9, 7.5 Hz, 1H); MS (APCI+) (M + H)+ at
m/z 468, 470. Anal. (C25H26ClN3O2S) C, H, N.
5-Meth ylen eben zod ioxolyl 2-Ch lor o-4-(E-((4-a cetylp ip -
er a zin -1-yl)ca r bon yl)eth en yl)p h en yl Su lfid e (20): white
solid; 1H NMR (CDCl3, 300 MHz) δ 2.14 (s, 3H), 3.48-3.60
(m, 2H), 3.60-3.84 (m, 6H), 6.05 (s, 2H), 6.75 (d, J ) 8.4 Hz,
1H), 6.80 (d, J ) 15.3 Hz, 1H), 6.88 (d, J ) 8.4 Hz, 1H), 6.98
(d, J ) 2.1 Hz, 1H), 7.08 (dd, J ) 2.1, 8.4 Hz, 1H), 7.19 (d, J
) 1.8, 8.4 Hz, 1H), 7.52 (d, J ) 2.1 Hz, 1H), 7.58 (d, J ) 15.6
Hz, 1H); MS (APCI+) (M + NH4)+ at m/z 445, 447. Anal.
(C22H21ClN2O4S‚0.12H2O) C, H, N.
Ben zod ioxa n -6-yl 2-Ch lor o-4-(E-((4-a cetylp ip er a zin -1-
yl)car bon yl)eth en yl)ph en yl Su lfide (21): amorphous white
solid; 1H NMR (CDCl3, 300 MHz) δ 2.14 (s, 3H), 3.44-3.57
(m, 2H), 3.57-3.86 (m, 6H), 4.25-4.35 (m, 4H), 6.75 (d, J )
8.4 Hz, 1H), 6.78 (d, J ) 15.6 Hz, 1H), 6.93 (d, J ) 8.4 Hz,
1H), 7.03 (dd, J ) 2.1, 8.4 Hz, 1H), 7.08 (d, J ) 2.1 Hz, 1H),
7.18 (dd, J ) 2.1, 8.4 Hz, 1H), 7.51 (d, J ) 2.1 Hz, 1H), 7.57
(d, J ) 15.6 Hz, 1H); MS (APCI+) (M + H)+ at m/z 459, 461.
Anal. (C23H23ClN2O4S‚0.05H2O) C, H, N.
1-Eth yl,3-(d im eth yla m in om eth yl)in d ol-7-yl 2-Ch lor o-
4-(E-((4-acetylpiper azin -1-yl)car bon yl)eth en yl)ph en yl Su l-
fid e (22): light-brown solid; 1H NMR (CDCl3, 300 MHz) δ 1.30
(t, J ) 7.05 Hz, 3H), 2.14 (s, 3H), 2.41 (s, 6H), 2.93-3.05 (m,
2H), 3.47-3.55 (m, 2H), 3.55-3.87 (m, 6H), 6.42 (d, J ) 8.4
Hz, 1H), 6.85 (d, J ) 15.6 Hz, 1H), 7.09 (dd, J ) 2.1, 8.4 Hz,
1H), 7.17 (d, J ) 8.4 Hz, 1H), 7.23 (d, J ) 8.4 Hz, 1H), 7.43
(dd, J ) 0.9, 7.8 Hz, 1H), 7.52 (d, J ) 2.1 Hz, 1H), 7.54 (d, J
) 15.6 Hz, 1H), 7.81 (dd, J ) 0.9, 7.8 Hz, 1H); MS (ESI+) (M
+ H)+ at m/z 525, 527. Anal. (C28H33ClN4O2S‚1.09TFA) C, H,
N.
1-(Car boxym eth yl)in dol-5-yl 2-Ch lor o-4-(E-((4-acetylpip-
er a zin -1-yl)ca r bon yl)eth en yl)p h en yl Su lfid e (23). To a
stirred solution of compound 8 (35 mg, 0.080 mmol) in 1 mL
of anhydrous DMSO was added crushed KOH (18 mg, 0.32
mmol). After 45 min, tert-butyl bromoacetate (23.5 mL, 0.16
mmol) was added. The resulting mixture was stirred at
ambient temperature for 10 h. Water was then added, and the
reaction mixture was acidified with 3 N HCl to pH ) 3. Acid
8 (25 mg, 63%) was isolated through filtration and drying in
a vacuum oven as a white solid. 1H NMR (DMSO-d6, 300 MHz)
δ 2.04 (s, 3H), 3.38-3.80 (m, 8H), 4.59 (s, 2H), 6.45 (d, J ) 3.0
Hz, 1H), 6.52 (d, J ) 8.7 Hz, 1H), 7.21 (dd, J ) 2.1, 8.7 Hz,
5-Ch lor o-8-eth oxyqu in olin -7-yl 2-Ch lor o-4-(E-((4-acetyl-
piper azin -1-yl)car bon yl)eth en yl)ph en yl Su lfide (29): white
solid; 1H NMR (DMSO-d6, 300 MHz) δ 1.37 (t, J ) 7.2 Hz,
3H), 2.04 (s, 3H), 3.41-3.82 (m, 8H), 4.46 (q, J ) 7.2 Hz, 2H),
7.29 (s, 1H), 7.37 (d, J ) 8.4 Hz, 1H), 7.42 (d, J ) 15.6 Hz,
1H), 7.51 (d, J ) 15.6 Hz, 1H), 7.68 (dd, J ) 1.8, 8.4 Hz, 1H),
7.74 (dd, J ) 3.9, 8.4 Hz, 1H), 8.15 (s, 1H), 8.55 (dd, J ) 1.8,
8.4 Hz, 1H), 9.05 (dd, J ) 1.8, 3.9 Hz, 1H); MS (APCI+) (M +
H)+ at m/z 530, 532, 534. Anal. (C26H25Cl2N3O3S‚0.28TFA) C,
H, N.
Aqu eou s Solu bility Deter m in a tion . The compound was
suspended in a phosphate buffer (pH 7.4, ion strength adjusted
to 0.15 M with sodium chloride). This suspension was equili-
brated by rotating at 25 rpm for approximately 2 d at 25 °C.
The excess solid was removed by filtration. The resulting
saturated solution was diluted and analyzed for drug concen-
tration by UV spectrophotometry.
For ICAM-1/LFA-1 biochemical interaction assay, ICAM-1/
J Y-8 cell adhesion assay, and pharmacokinetic analysis pro-
tocols, see the preceding paper in the series.13
Ack n ow led gm en t. The authors thank the Abbott
1
Analytical Department for assistance in acquiring H

1210 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8
Liu et al.
mides as Antagonists of Leukocyte Function-associated Antigen-
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