N-Benzoyl amino acids as ICAM/LFA-1 inhibitors. Part 2: Structure-activity relationship of the benzoyl moiety

Article abstract of DOI:10.1016/j.bmcl.2004.02.046

o-Bromobenzoyl L-tryptophan 1 inhibits the association of LFA-1 with ICAM-1 with an IC₅₀ of 1.7μM. Evaluation of the structure-activity relationship of the benzoyl moiety shows that 2,6-di-substitutions greatly enhance potency of this class of inhibitors. Electronegative substitutions that favor a 90°angle between the benzoyl ring and the amide bond yield the most potent compounds. There is a strong correlation between the potency of the compounds and the difference between the ab initio energy at 90°and the global minima energy for given compounds. Combining the favored benzoyl substitutions with L-histidine and L-asparagine resulted in a 15-fold increase in potency over compound 1.

Full text of DOI:10.1016/j.bmcl.2004.02.046

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2055–2059
N-Benzoyl amino acids as ICAM/LFA-1 inhibitors. Part 2:
Structure–activity relationship of the benzoyl moiety
Daniel J. Burdick,a,* James C. Marsters, Jr.,a Ignacio Aliagas-Martin,a Mark Stanley,a
Maureen Beresini,b Kevin Clark,b Robert S. McDowella, and Thomas R. Gadeka
aDepartment of Medicinal Chemistry, Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080, USA
bBioanalytical Research and Development, Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080, USA
Received 24 December 2003; revised 12 February 2004; accepted 12 February 2004
Abstract—o-Bromobenzoyl -tryptophan 1 inhibits the association of LFA-1 with ICAM-1 with an IC50 of 1.7 lM. Evaluation of
L
the structure–activity relationship of the benzoyl moiety shows that 2,6-di-substitutions greatly enhance potency of this class of
inhibitors. Electronegative substitutions that favor a 90° angle between the benzoyl ring and the amide bond yield the most potent
compounds. There is a strong correlation between the potency of the compounds and the difference between the ab initio energy at
90° and the global minima energy for given compounds. Combining the favored benzoyl substitutions with
-asparagine resulted in a 15-fold increase in potency over compound 1.
L-histidine and
L
Ó 2004 Elsevier Ltd. All rights reserved.
The interaction of leukocyte function-associated anti-
gen-1 (LFA-1, aLb2, CD11a/CD18) with intercellular
adhesion molecule 1 (ICAM-1) has been implicated
in numerous autoimmune diseases such as psoriasis,
asthma, rheumatoid arthritis, and graft rejection.1–3 In
the past several years, there have been reports of several
monoclonal antibodies and small-molecule antagonists
to this interaction.4–7 We have previously described the
structure–activity relationship (SAR) of the amino acid
portion of compound 1, and in this communication we
will turn our attention to the SARof the benzoyl moiety 8
(Fig. 1).
NH
O
OH
N
H
O
Br
1
Figure 1.
benzoic acids. All compounds were evaluated in an
ICAM-1/LFA-1 enzyme linked immunosorbant assay
(ELISA).4 Potency is calculated as the concentration of
inhibitor needed to inhibit 50% of the ICAM-1/LFA-1
complex formation (IC50). Reported IC50 values are the
arithmetic average of at least two assays.
We initially generated a set of compounds to evaluate
the optimal distance of the benzoyl ring from the amino
acid. Extending the ring out from the amide bond one to
five methylene units diminished potency. Therefore, we
decided to concentrate on improving the inhibitors
through modifications of the benzoyl ring itself. Four
classes of acids were coupled to L-tryptophan: mono-
substituted benzoic acids; monosubstituted heterocyclic
aromatic acids; saturated cyclic acids, and di-substituted
Compounds 1–58 were synthesized using standard
Fmoc solid phase synthetic techniques (Scheme 1) on
commercially available Na-Fmoc-Nin-tert-butyloxycar-
bonyl-L-tryptophan 4-hydroxymethylphenoxy resin
(Wang resin).9;10 Following removal of the Fmoc group
with 20% piperidine in DMF and rinsing of the resin,
the amine was acylated with the appropriate commer-
cially available acid using HBTU, HOBT, and DIPEA
in DMF. After drying the resin to a constant weight, it
was then treated with a solution of TFA containing
2.5% water and 2.5% anisole. Excess TFA was removed
Keywords: LFA-1; ICAM-1; Inhibitor.
* Corresponding author. Tel.: +1-650-225-1368; fax: +1-650-225-2061;
e-mail: burdick.dan@gene.com
Current address: Sunesis Pharmaceuticals, 341 Oyster Point Boule-
vard, South San Francisco, CA 94080, USA.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.046

2056
D. J. Burdick et al. / Bioorg. Med. Chem. Lett. 14(2004) 2055–2059
combination of steric and electronic effects. For exam-
ple, the medium sized hydroxy (8) and methoxy substi-
tutions are approximately 300- and 100-fold,
respectively, less potent than the bromo substitution (1)
and they are approximately 4- and 2-fold, respectively,
less potent than the un-substituted benzoyl ring (2) it-
self. The alkoxy groups stabilize the co-planarity of the
ring and amide bond with a large electronic resonance
effect, as well as, internal hydrogen bonding with the
amide bond NH hydrogen. The methyl substitution (6)
has a larger steric effect and a smaller electronic reso-
nance effect than the alkoxy groups. This could help
force the amide bond to twist out of the plane of the
benzoyl ring. The methyl inhibitor (6) is 2.3 times less
potent than the bromo analogue (1) and 32 times more
potent than the un-substituted analogue (2).
NBoc
NH
OH
a,b, c
O
O
FmocHN
N
H
R
O
O
Scheme 1. Reagents and conditions: (a) 20% piperidine/DMF, rt; (b)
appropriate acid (RCOO2H), HBTU, HOBT, DIPEA, DMF, 2 h, rt;
(c) triisopropylsilane, water, TFA, 1 h, rt.
in vacuo and the cleaved molecule was extracted away
from the resin with a 10% solution of acetic acid in
water. The crude product was purified by reverse phase
HPLC, lyophilized, and its molecular weight verified by
electro spray mass spectroscopy. Compounds 59–67
where synthesized in a similar manner with the excep-
tion that Na-Fmoc-
Fmoc-Nim-tert-butyloxycarbonyl-
were used in place of Na-Fmoc-Nin-tert-butyloxycar-
bonyl- -tryptophan Wang resin where appropriate.
Halogen substitutions (1, 3–5) constitute the best overall
groups of monosubstituted benzoyl inhibitors. With the
exception of the fluoro analogue (3), the halogens all
have a similar steric and electronic resonance effect, and
in general their potency increases as they become smaller
and more electronegative.11 The chloro analogue (4) is
slightly more potent than the bromo analogue (1), but it
is six times more potent than the iodo compound (5).
The fluoro analogue (3) is the anomaly. Its ability to
hydrogen bond with the amide NH can favor a co-pla-
nar relationship between the amide bond and the phenyl
ring and seems to overcome any steric effect.12
L
-asparagine Wang resin and Na-
L
-histidine Wang resin
L
ELISA assay results for the monosubstituted benzoyl
analogues are shown in Table 1. Two observations can
be made from this data. First, compounds with halogen
(1, 3–5) or methyl (6) substitutions at the ortho position
are more potent than the same substitution at either the
meta or para position. For example, N-ortho-chloro-
benzoyl-
potent than N-meta-chlorobenzoyl-
and more than 100 times more potent than N-para-
chlorobenzoyl- -tryptophan (18). In general, potency
L
-tryptophan (4) is almost 200 times more
L
-tryptophan (11)
Figure 2 shows a plot of the strain energy at 90°
ðDEstrainÞ of model N-methyl ortho-monosubstituted-
benzamides (H, Me, F, Cl, Br, OMe) versus the log of
the IC50 value for the analogous N-ortho-substituted
L
for regional isomers of a given substituent rank
ortho > para > meta.
benzoyl L-tryptophan inhibitors (1–6, 8) for which there
was quantitative data.13 DEstrain values estimate the en-
ergy needed to rotate the substituted amide from its
global minima to a preferred angle of 90° in the bound
state. With the exception of the fluoro-substituted ana-
logue, there is a strong linear correlation found between
the calculated strain energies and the experimental
binding energies suggesting that DDG is directly corre-
lated to DEstrain. The calculated DEstrain value for the
The second observation has to do with the potency of
the ortho substituted analogues. Substitutions that help
force the amide bond out of the plane of the benzoyl
ring lead to better inhibitors than those that enforce
co-planarity. This influence on potency appears to be a
Table 1. ELISA assay results for monosubstituted N-benzoyl-L-tryp-
tophan analogues
Strain at 90° vs. log IC50
NH
2.5
O
Ome
OH
N
H
2.0
R
H
O
1.5
R
ortho
meta
para
Compd IC50
(lM)
Compd IC50
(lM)
Compd IC50
(lM)
1.0
0.5
F
Me
H
2
3
4
1
5
6
7
8
9
127
7.4
Cl
Br
F
Cl
10
11
12
13
14
148
295
17
18
19
20
21
22
23
24
136
169
61
0.0
1.5
1
2
3
4
5
6
Br
I
1.72
8.9
>100
>100
295
Strain (Kcal/mol)
92
Me
Ph
OMe
OH
4
129
>100
178
>100
Figure 2. Plot of DEstrain versus log IC50 for monosubstituted ortho-
benzoyl -tryptophan inhibitors. There is a strong correlation between
>100
225
>500
L
15
16
438
>100
DEstrain and potency of the inhibitors. The fluoro analogue does not
follow the trend due to enhanced hydrogen bond capability.

D. J. Burdick et al. / Bioorg. Med. Chem. Lett. 14(2004) 2055–2059
2057
N-methyl ortho-hydroxybenzamide is 9.75 kcal/mol,
which predicts that the analogous hydroxy compound
(9) would be a very poor inhibitor. This prediction is
borne out in the experimental data; compound 9 has an
IC50 > 500 lM. These calculations support the hypothe-
sis that substitutions that help force the amide bond out
of the plane of the benzoyl ring are better inhibitors than
those that favor co-planarity.
compounds (25–27) show inhibition similar to the
analogous benzoyl compound (2). Compound 28, which
has a bromine meta to the carbonyl, is a poor inhibitor;
while compounds with a chlorine ortho to the carbonyl
(29, 30) are much more potent. The saturated ring
analogues (32–36) do not work as well as the aromatic
compounds. However, the same Ôortho effectÕ can be
seen. Adding a methyl at position-2 (33) increases po-
tency by twofold over cyclohexane ring (32) alone.
Table 2 shows assay data for the aromatic heterocyclic
and saturated analogues. The aromatic heterocyclic
analogues follow the same trend established with the
monosubstituted benzoyl analogues. The three pyridyl
Table 3 shows ELISA assay results for di-substituted N-
benzoyl L-tryptophan analogues. As with the other
series, the position and type of substitution has a great
influence on the compounds potency. For instance, the
3,5-substituted di-fluoro (38) and di-chloro (39) ana-
logues, as well as, the 3,4-di-chloro (40) analogue are
poor inhibitors. However, as with the monobenzoyl
inhibitors, an ortho substitution greatly enhances the
inhibitors potency. The combination of an ortho halo-
gen with a meta substituent (41–47) or a para substituent
(48–50) yield inhibitors that are much more potent than
the corresponding mono meta (10–12, 15) or para ana-
logues (17, 18). As noted previously, if the ortho sub-
stituent is a hydroxy (51, 52) the potency of the analogue
decreases greatly.
Table 2. ELISA results for heterocyclic and saturated L-tryptophan
analogues
NH
O
OH
R
N
H
O
N
Compound
RIC
(lM)
50
The most dramatic increase in potency occurs when
the amide carbonyl is flanked by two ortho substitu-
ents. When both ortho substitutions are the same, the
greatest improvement is seen with the least potent
25
>100
26
27
112
92
N
Table 3. ELISA results for di-substituted N-benzoyl-L-tryptophan
analogues
N
Br
28
29
>200
N
Cl
N
NH
32.6
O
OH
N
R1,R2
H
O
2
30
31
1.2
N
Cl
Compound
2
3
4
5
6
IC50 (lM)
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
F
F
Cl
89.7
200
170.5
21.6
11.5
6.3
Cl
Cl
F
>1000
N
O
Cl
F
F
F
32
33
34
35
61
28
Cl
Cl
Cl
Br
Br
Cl
Cl
Cl
OH
OH
F
Cl
Cl
Br
6.6
17.9
9
OMe
Br
24.3
8.4
Cl
N
380
NO2
NH2
5.2
2
Br
Br
>500
>500
0.46
0.6
>1000
NH
F
Cl
Cl
Me
Cl
F
Me
Me
Br
Cl
1.1
36
37
>1000
>1000
N
H
0.64
0.43
0.25
F
HN
2,6-Di-substitutions greatly enhance potency in this series of inhibitors.

2058
D. J. Burdick et al. / Bioorg. Med. Chem. Lett. 14(2004) 2055–2059
Table 4. Comparison of the various 2,6-di-substituted benzoyl moie-
monosubstituents. For example, the 2,6-di-fluoro com-
pound (53) is 16-fold more potent than the ortho fluoro
compound (3); where as the 2,6-di-chloro compound
(54) is only 2.5-fold more potent than compound 4.
Combinations of different ortho substituents have a
ties in 3N-benzoyl-
L
-amino acid series
L
-Trp (com-
L
-Asn (com-
L-His (com-
pound) IC50
pound) IC50
pound) IC50
O
greater effect. N-2-Chloro-6-methyl-benzoyl
phan (56) shows a moderate increase in potency over N-
2-methyl-benzoyl -tryptophan (6). The 2-bromo-6-flu-
L-trypto-
(4) 1.7
(59) 1.08
(60) 0.75
L
Br
O
oro (57) and 2-chloro-6-fluoro (58) substitutions con-
stitute a 17- and 30-fold, respectively, improvement in
potency over compound 3. Compound 58 is seven times
more potent than our original lead (1).
(55) 1.1
(53) 0.46
(54) 0.6
(58) 0.25
––
(61) 1.77
(63) 0.2
F
O
Figure 3 shows a plot of DEstrain of model N-methyl 2,6-
di-substituted benzamides versus the log of the IC50
value for the analogous inhibitors (53–58). Two obser-
vations can be made from this graph. First, not all 2,6-
di-substitutions result in a DEstrain of 0. The calculated
DEstrain for the di-fluoro analogue (53) is 0.56, which is
about 10-fold lower than the monofluoro analogue. This
result suggests that there is a minimal steric size
requirement for at least one of the ortho substituents to
achieve a DEstrain of 0. Second, compounds with DEstrain
values of 0 (54–58) do not have the same potency.
Activity, once again, appears to be a combination of
steric effect and electronegativity. For example, com-
pound 55 has a DEstrain of 0 due to the large steric con-
tributions of the methyl groups, but its potency is not
that much better than the original lead (1). Compounds
54, 56–58 have at least one substituent that contributes a
large steric component and at least one substituent that
contributes a large electronegative component. Inter-
estingly, as the combination of substituents moves from
less electronegative to more electronegative, the potency
of the inhibitors increases.
(62) 0.23
(64) 0.17
(66) 0.11
F
Cl
F
O
(65) 0.16
(67) 0.13
Cl
O
Cl
However, the enhancement in potency is not additive. In
the case of -tryptophan and -histidine series there is a
L
L
6-fold improvement in potency moving from a 2-bromo
benzoyl ring to a 2-chloro-6-fluoro benzoyl ring, while
this same change in the L-asparagine series results in a
10-fold increase in potency.
Based on these results, we conclude that within the class
of N-benzoyl L-tryptophan antagonists, 2,6-di-substitu-
tions on the benzoyl ring are most favored for inhibition
of the LFA-1/ICAM-1 complex; in particular, N-2-
chloro-6-fluoro benzoyl L-tryptophan shows the highest
potency of the analogues tested. Combining the best
elements of the amino acid SARand the benzoyl SAR
results in two compounds that are 16-fold more potent
than the original screening hit. A subsequent paper will
address finding an additional interaction with the pur-
pose of further increasing potency.
We previously reported the SARof the amino acid
L
-tryptophan antagonists.8
portion of the N-benzoyl
Combining the results of this work with the current
benzoyl SARevaluation, we were able to produce
compounds with further enhanced potency. These
results are displayed in Table 4. The trend found in the
2,6-di-substituted benzoyl L-tryptophan series holds for
-histidine series.
both the
L
-asparagine series and the
L
Strain at 90° vs. log IC50
Acknowledgements
0.2
0.1
Me, Me
The authors would like to thank Martin Struble and his
group for purification of the compounds.
0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
-0.7
Cl, Me
F, Br
Cl, Cl
F,F
References and notes
F, Cl
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-tryptophan analogues. Potency for compounds that have a 90°
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