1,4-Diazepane-2,5-diones as novel inhibitors of LFA-1

Article abstract of DOI:10.1016/j.bmcl.2004.11.072

1,4-Diazepane-2,5-diones (2) are found to be a new class of potent LFA-1 inhibitors. The synthesis, structure, and biological evaluation of these 1,4-diazepine-2,5-diones and related derivatives are described.

Full text of DOI:10.1016/j.bmcl.2004.11.072

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1217–1220
1,4-Diazepane-2,5-diones as novel inhibitors of LFA-1
Sompong Wattanasin,a,* Joerg Kallen,b Stewart Myers,a Qin Guo,a Michael Sabio,a
Claus Ehrhardt,b Rainer Albert,b Ulrich Hommel,b Gisbert Weckbecker,b
Karl Welzenbachb and Gabriele Weitz-Schmidtb
aNovartis Institutes for BioMedical Research, Cambridge, MA 02139, USA
bNovartis Institutes for BioMedical Research, Basel 4002, Switzerland
Received 27 October 2004; revised 19 November 2004; accepted 29 November 2004
Available online 23 December 2004
Abstract—1,4-Diazepane-2,5-diones (2) are found to be a new class of potent LFA-1 inhibitors. The synthesis, structure, and bio-
logical evaluation of these 1,4-diazepine-2,5-diones and related derivatives are described.
Ó 2004 Elsevier Ltd. All rights reserved.
Lymphocyte function-associated antigen-1 (LFA-1,
CD11a/CD18, aLb2) is a heterodimeric adhesion recep-
tor belonging to the b2 integrin family. LFA-1 is ex-
pressed on all leukocytes. Interactions between LFA-1
and intercellular adhesion molecules, such as ICAM-1,
play central roles in mediating immune and inflamma-
tory responses and are essential in the pathogenesis of
many diseases.1 Together with other adhesion receptors,
LFA-1 mediates leukocyte migration to sites of inflam-
mation and antigen exposure.2–4 LFA-1 is involved in
both firm adhesion and locomotion of leukocytes.5
Moreover, during an immune response, LFA-1 binding
to ICAM-1 can enhance T-cell-receptor-dependent acti-
vation and proliferation of T-cells.6 Blockade of LFA-1
interactions with their ligands is an attractive therapeu-
tic target. The intervention of integrin LFA-1 by mono-
clonal antibodies (mAbs) has shown efficacy in animal
models of inflammation and autoimmune disease, for
example, arthritis,7 ischemia/reperfusion injury,8 and
transplant rejection.9 Clinical studies suggest that anti-
LFA-1 therapy is beneficial in bone marrow and solid
organ transplantation.10,11 Recent data show that a
humanized monoclonal antibody of the CD11a subunit
of LFA-1 inhibits the activation of T cells and is effica-
cious in patients suffering from moderate to severe pla-
que psoriasis12a and in allergic asthma.12b Because of
the importance of this integrin and its implication in
various disease processes, efforts toward the design
and synthesis of orally bioavailable small molecule
inhibitors have intensified in recent years.1,13 In addi-
tion, understanding the mechanism of LFA-1 binding
to ligands has become fundamental in developing thera-
peutic agents against this integrin.1d
In a previous communication,14 we disclosed the identi-
fication of 1, as a potent LFA-1 antagonist, from a
structure-based combinatorial library. This compound
exhibited nanomolar binding activity in an LFA-1/
ICAM-1 ELISA-type binding assay.15 As part of an
effort to optimize 1 and identify novel LFA-1 antagonists,
we sought to explore the structure–activity relationship
(SAR) of related templates (2 and 3). The efforts led to
the discovery of a new class of potent LFA-1 antago-
nists whose central core is comprised of 1,4-diazepane-
2,5-diones (2). Although we introduced a second sp2
carbon atom in 2 and 3 and shortened the ring in 3, a
molecular modeling analysis suggested that the side
chains in 2 and 3 can adopt an arrangement similar to
that in 1. Herein, we describe the synthesis and initial
SAR studies of this novel series.
N
O
O
O
O
N
N
R1
O
N
N
Keywords: Integrin; LFA-1; Diazepanediones; I-domain; L-site
binding.
O
H2N
N
H
( )n
*
Corresponding author. Tel.: +1 617 871 7645; fax: +1 617 871
7042; e-mail: sompong.wattanasin@pharma.novartis.com
2
3
(n=2)
(n=1)
1
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.072

1218
S. Wattanasin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1217–1220
O
O
O
Compounds 2, 3, and related derivatives were evaluated
H
N
H
N
O
O
NH2.HCl
N
H
HO
N
H
for their activity in the LFA-1/ICAM-1 binding as-
say.15b The results are outlined in Table 1. Initially, we
investigated the SAR of 2 around the R1 moiety, be-
cause this moiety has been shown in 1 to be vital for
binding potency.14 The phenyl derivative (2a) is a weak
LFA-1 inhibitor. Substitution of the benzene ring in the
4-bromophenyl (2b) derivative provides no improve-
ment in potency. The bulky 3-benzyloxyphenyl deriva-
tive (2c) is less active. Polar substitution in the 3-
hydroxyphenyl (2d), 4-hydroxy-3-methoxyphenyl (2e),
and 4-dimethylaminophenyl (2f) derivatives, however,
shows a two or threefold binding improvement. The 4-
pyridyl derivative (2g) is totally inactive. As observed
earlier in 1 for the effect of a 3-quinolyl group, the 3-
quinolyl derivative (2h) shows a remarkable enhancing
ability in inhibiting ICAM-1 and LFA-1 interactions
(IC50 = 69 nM). It is as active as the previous lead struc-
ture (1). The 6-quinolyl derivative (2i) is twofold less ac-
tive. Compound 2j, which is a longer chain analogue of
2h, is about 30-fold less active. The 4-pyridylethylene
analogue (2k), however, is also an excellent inhibitor
of LFA-1 (IC50 = 24 nM). The corresponding amide
analogue (2l) is more than 100-fold less active. Data of
the piperazine-2,4-dione series (3a–c) indicated that they
are only weak inhibitors of LFA-1. Interestingly, while
the corresponding 4-bromophenyl derivatives (2b and
3a) are comparable in potency, a 187-fold loss in po-
tency was observed with the 6-quinolyl derivative (3b)
in the six-membered ring series.
O
O
a, b
c
3
4
5
O
R1
O
O
d
NH
NH
O
O
N
R1
O
O
O
N
N
OH
O
N
H
N
O
N
H
N
H
O
g
O
b,e, f
2
7
6
Scheme 1. Regents and conditions: (a) DCCI (2 equiv), HOBt
(2 equiv), methylamine (2 equiv), NMM (1 equiv), DMF, rt, 12 h; (b)
HCL (gas), CH2Cl2, rt, 2 h; (c) methyl acrylate (excess), DIPEA
(2 equiv), MeOH, rt, 14 h; (d) N-t-Boc-leucine (1 equiv), HATU
(5 equiv), DIPEA(5 equiv), DMF,
0 °C–rt, 48 h; (e) R1–CHO,
Na(OAc)3BH (1.5 equiv), HOAc (0.5 equiv), THF, rt, 1 h; (f) LiOH
(2.5 equiv), THF, water, rt, 1 h; (g) HATU (1.2 equiv), DIPEA
(1.2 equiv) DMF, 0 °C–rt, 3 h.
1,4-Diazepane-2,5-diones were synthesized as outlined
in Scheme 1. The synthesis starts with the conversion
of Boc-amino acid 3 into the amino intermediate 4, after
removal of the Boc protective group. 1,4-Addition of 4
to methyl acrylate leads to 5. Coupling of 5 with N-t-
Boc protected leucine and coupling conditions, such
as PyBroP/NMM/DMAP/DMF/rt,16a DIPC/DMF/rt,
CIP/HOBt/DIEA/THF/rt,16b and DCC/HOBt/NMM/
rt,16c gave only trace amounts or low yields of the de-
sired coupling product 6. Good yields (75–90%) of the
coupling reaction, however, could be obtained if excess
amounts of HATU were used. Deprotection of 6, reduc-
tive amination, and ester hydrolysis afforded the amino
acid intermediate 7. Cyclization of 7 led to the desired
1,4-diazepane-2,5-diones (2).17 The overall yield of this
unoptimized sequence is in the range of 10–19%.
Three other stereoisomers of 2h also have been synthe-
sized according to Scheme 1 and were found to be much
less active (Table 2), suggesting that the S,S configura-
tion of the chiral centers in this series of compounds is
optimal for binding potency.
The synthesis of piperazine-2,4-diones (3) is shown in
Scheme 2. Alkylation or reductive amination of L-leu-
cine t-butyl ester hydrochloride gave amino acid inter-
mediate 8 (30–40%). Coupling of 8 with chloroacetic
acid (90%) and reaction of the resulting chloro com-
pound 9 with 4 gave compound 10 (35–40%). Ester
deprotection of 10 followed by cyclization afforded
(15–20%) the piperazine-2,5-diones (3).18
Compound 2h is a potent and selective inhibitor of
LFA-1 with no inhibition of binding to the closely re-
lated integrin Mac-1 (CD11b/CD18), with an IC50 value
greater than 100 lM (cell-free ICAM-1/Mac-1 binding
assay).19 It is also inactive (IC50 > 100 lM) in a VLA-4
Table 1. In vitro cell-free LFA-1/ICAM-1 binding assay15b
Compd
R1
IC50 (nM)a
1
—
Phenyl
70
2850
2950
6600
1300
950
2a
2b
2c
2d
2e
2f
2g
2h
2i
4-Bromophenyl
3-Benzyloxyphenyl
3-Hydroxyphenyl
4-Hydroxy-3-methoxyphenyl
4-Dimethylaminophenyl
4-Pyridyl
O
R1
c
a or b
O
O
R1
N
NH2.HCl
N
H
O
O
O
O
O
8
9
Cl
800
d
HO
O
O
33,000
69
130
R1
O
R1
O
N
N
R1
O
N
O
H
N
H
N
3-Quinolinyl
6-Quinolinyl
N
N
H
O
O
N
H
O
N
H
f
e
2j
3-Quinolinylmethyl
4-Pyridyl–CH@CH–
4-Pyridyl–NHCO–
4-Bromophenyl
6-Quinolinyl
890
24
3
11
10
2k
2l
3700
2950
24,400
13,300
Scheme 2. Regents and conditions: (a) R1–CHO, Na(OAc)3BH
(1.5 equiv), HOAc (0.5 equiv), THF, rt, 1 h; (b) R1–CH2Br, DIEA,
DMF, rt, 16 h; (c) chloroacetic acid, DIPCl (2 equiv), DMF; (d) 4,
DIEA, DMF, 40 °C, 16 h; (e) TFA, CH2Cl2, rt, 3 h; (f) HATU
(1.2 equiv), DIPEA(1.2 equiv) DMF, 0 °C–rt, 4 h.
3a
3b
3c
4-(3-Pyridyl)phenyl
a Each value is a mean of two experiments.

S. Wattanasin et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1217–1220
1219
Table 2. In vitro cell-free LFA-1/ICAM-1 binding assay of stereo-
isomers of 2h
Leu298, Leu302, Ile306, and the aliphatic moiety of
Lys287. The Leu side chain of the ligand binds in a
hydrophobic subsite formed mainly by the side chains
of Phe153, Val157, Ile235, Tyr257, Ile259, and Ile306.
The quinolyl moiety binds to a pocket that is formed
mainly by the side chains of Val130, Leu132, Tyr166,
Thr231, Val233, and Ile255. There are two direct hydro-
gen bonds between 2h and the I-domain, specifically
from the amide-nitrogen atom to the Lys305 carbonyl
group and from the quinolyl nitrogen atom to Tyr166-
OH. In addition, there is a system of water-mediated
weak hydrogen bonds between the amide oxygen atom
and both Glu284 and Tyr257. There is also a weak
water-mediated hydrogen bond between the oxygen
atom at position 2 of the diazepane ring and the car-
bonyl group of Ile306. An analysis of these interactions
provides a better understanding of the SAR and offers a
basis for further optimization of this class of inhibitors.
Compound
Chiral configurationsa
IC50 (nM)b
2h
2m
2n
2o
S,S
S,R
R,S
R,R
69
510
5700
4200
a Configurations of the naphthylalanine side chain and the leucine side
chain are listed, respectively, for each isomer.
b Each value is a mean of two experiments.
In summary, we have discovered 1,4-diazepane-2,5-
diones as a new class of inhibitors of LFA-1. Com-
pounds 2h and 2k are potent inhibitors of LFA-1 and
ICAM-1 interactions. The X-ray crystal structure
reveals the L-site as the binding site of 2h with the
I-domain of LFA-1. Further optimization of this novel
class of compounds is in progress and will be reported
in due course.
Figure 1. X-ray crystallographic representation of the complex of
compound 2h with the I-domain of LFA-1. The continuous and dotted
black lines indicate, respectively, direct and water-mediated intermo-
lecular hydrogen-bond interactions.
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