N-Cycloalkanoyl-L-phenylalanine derivatives as VCAM/VLA-4 antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00386-4

A systematic structure-activity relationship investigation of the lead compound 1 resulted the identification of several N-[(substituted alkyl)cycloalkanoyl]-4-[((2,6-dichlorophenyl)carbonyl)amino]-L-phenylalanine derivatives as potent VCAM/VLA-4 antagonists. The data are consistent with a model of these compounds in which these alkanoylphenylalanines reside in a compact gauche (-) bioactive conformation.

Full text of DOI:10.1016/S0960-894X(02)00386-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2475–2478
N-Cycloalkanoyl-L-Phenylalanine Derivatives as VCAM/VLA-4
Antagonists
Achyutharao Sidduri, Jefferson W. Tilley,* Kenneth Hull, Jian Ping Lou, Gerry Kaplan,
Allen Sheffron, Li Chen, Robert Campbell, Robert Guthrie, Tai-Nan Huang,
Nicholas Huby, Karen Rowan, Virginia Schwinge and Louis M. Renzetti
Roche Research Center, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ07110, USA
Received 19 February 2002; accepted 22 April 2002
Abstract—A systematic structure–activity relationship investigation of the lead compound 1 resulted the identification of several
N-[(substituted alkyl)cycloalkanoyl]-4-[((2,6-dichlorophenyl)carbonyl)amino]-l-phenylalanine derivatives as potent VCAM/VLA-4
antagonists. The data are consistent with a model of these compounds in which these alkanoylphenylalanines reside in a compact
gauche (À) bioactive conformation. # 2002 Elsevier Science Ltd. All rights reserved.
The integrin receptor VLA-4 (a4b1) is expressed on a
variety of leucocytes including B-cells, T-cells, basophils
and eosinophils and is involved in the recruitment, acti-
vation and survival of these cell types.1 Data supporting
a role for VLA-4 in a number of inflammatory diseases
including asthma, rheumatoid arthritis, inflammatory
bowel diseases, multiple sclerosis, atherosclerosis, and
diabetes have emerged and is summarized in recent
reviews.2 This putative role of VLA-4 in disease has
prompted an intense search for effective inhibitors of
the VCAM–VLA-4 interaction.
include the five-membered ring and the carboxylic acid
with the phenylalanine aromatic ring of 1 occupying the
same region of space as the disulfide moiety of 2.4,5
We now report a detailed investigation of the structure–
activity relationship of cycloalkanoyl derivatives related
to 1 as well as the X-ray crystal structure of the methyl
ester of 16 that is fully consistent with our bioactive
conformational hypothesis.
The new analogues were generally prepared from the
l-phenylalanine derivative 36 in two straight forward
steps as shown in Scheme 1. The desired 1-(substituted
alkyl)cycloalkanecarboxylic acids were generally pre-
pared by alkylation of the anion derived from methyl
cycloalkanecarboxylic acid esters as depicted in Scheme 2.
The p- or m-(1-methyltetrazol-5-yl)benzyl chlorides
were prepared in three steps from the corresponding
We have previously reported identification of the potent
a-branched cycloalkanoyl phenylalanine derivative 1,
which was designed on the premise that it mimicked
essential pharmacophoric features of a NMRderived
three dimensional model of the cyclic peptide 2.3 Key
common elements of the two classes of inhibitors
*Corresponding author. Tel.: +1-973-235-4660; fax: +1-973-235-
7122; e-mail: jefferson.tilley@roche.com
Scheme 1. (a) 1-(Substituted alkyl)cycloalkanecarboxylic acid, HBTU,
DIPEA, DMF, rt, 15–48 h; (b) 1.0 N NaOH, EtOH, 50–55 ꢀC, 15 h.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00386-4

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A. Sidduri et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2475–2478
In an effort to improve on the potency of compounds
related to 1, a series of homologous 1-benzyl cycloalk-
anoyl derivatives was prepared (Table 1). Although, the
unsubstituted benzyl derivative 12a was relatively inef-
fective, addition of a substituent in the 3- or, preferably,
the 4-position lead to a significant improvement in
potency. In contrast to the case of direct analogues of
1,5 comparison of the 4-methoxy analogous 12b and 12h
and the tetrazoles 12i–12k suggest that four- and five-
membered cycloalkyl rings are preferred over the corre-
sponding six-membered rings. Although, we were able
to improve the potency of the benzyl derivatives from
triple digit to the low double digit nanomolar range,
their molecular weight became of concern and we chose
to investigate simpler analogues that might achieve the
same conformational preference.
Scheme 2. (a) LDA, THF, À70 ꢀC, RI, and warm to rt, 15 h; (b) 1.0 N
NaOH, MeOH, THF, reflux, 15 h.
toluic acid as illustrated in Scheme 3. The methyl-
thioalkyl-, methylsulfonylalkyl- (10) and azidoalkyl (11)
cycloalkane carboxylic acids were obtained from a
common
bromoalkyl
cycloalkane
carboxylate
intermediate 9 as shown in Scheme 4.7
In the case of amines, after coupling of azidoalkyl
cycloalkane carboxylic acids, 11 with 3, the azides were
converted to the corresponding amines using 1.0 M tri-
methylphosphine in toluene followed by quenching with
water.7 Subsequent amine acylation followed standard
protocols to give the compounds in Table 3.
This thinking led us to prepare the alkyl and substituted
alkyl derivatives shown in Tables 2 and 3. The data in
Table 2 suggest the following: (a) a variety of functional
groups including methoxy, hydroxy, cyano, methylthio,
and methylsulfonyl are well tolerated, (b) in general,
2-carbon alkyl chain analogues have highest affinity
with the exception of methylsulfonyl analogues where a
four-carbon chain is preferred (29 and 30), (c) compar-
ison of the methoxyethyl analogues 16 and 17 and the
methylsulfones 29 and 30 indicate again that there is
little difference in activity between the cyclobutyl and
cyclopentyl derivatives.
Compounds were assayed for VLA-4 antagonist activity
using a solid-phase, dual antibody ELISA in which
VLA-4 derived from Ramos cells was allowed to com-
pete for bound recombinant human VCAM in the pre-
sence of serial dilutions of test compound. VLA-4
bound to VCAM-1 was detected by a complex of anti-
b1 antibody and HRP-conjugated anti-mouse IgG:
chromogenic substrate (K-Blue).7
They were further evaluated in a cell based assay for
their ability to block the interaction between fluoros-
cently labeled Ramos cells, which express VLA-4, with
VCAM coated microtiter plates. The ELISA assay does
not discriminate well among compounds with
IC50s<1.5 nM, and we rely on the more stringent cell
based assay using Ramos cells to rank order potency.
Most of the aminoalkyl derivatives (Table 3) have
moderate to excellent activity in both assays. Particu-
larly noteworthy are the acetamide (34) and the urea 41.
Table 1. VCAM/VLA-4 binding inhibition of N-[(1-benzyl)cycloalk-
anoyl]-4-[((2,6-dichlorophenyl)carbonyl)amino]-l-phenylalanine deri-
vatives
Scheme 3. (a) NCS, benzoylperoxide, chlorobenzene, 95 ꢀC, 8 h; (b)
SOCl2, toluene, 85–90 ꢀC, 15 h, then MeNH2HCl, DIPEA, CH2Cl2,
0 ꢀC and warm to rt, 1 h; (c) SOCl2, toluene, 85–90 ꢀC, 15 h, then
NaN3, TMSCl, CH3CN, 0 ꢀC to rt, 15 h.
Compd
X
n
ELISA
IC50 (nM)
Ramos
IC50 (nM)
1
—
H
4-OCH3
4-CN
—
2
2
2
2
2
2
2
3
1
2
3
1.7
6.6
0.6
0.5
0.8
1.1
0.4
1.6
2.5
0.42
0.47
1.3
99
340
37
38
95
170
100
350
250
27
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
3,4-(OCH3)2
3-OCH3
3-(1-methyl-tetrazol-5-yl)
2-Cl
4-OCH3
4-(1-Methyl-tetrazol-5-yl)
4-(1-Methyl-tetrazol-5-yl)
4-(1-Methyl-tetrazol-5-yl)
Scheme 4. (a) LDA, THF, À70 ꢀC, 1,o-dibromoalkane and allow to
warm to rt, 15 h; (b) NaSMe, DMF, rt, 15 h; (c) H2O2, AcOH, 70 ꢀC,
15 h; (d) 1.0 N NaOH, MeOH, THF, reflux, 15 h; (e) NaN3, DMF,
50 ꢀC, 15 h.
19
61

A. Sidduri et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2475–2478
2477
Table 2. VCAM/VLA-4 binding inhibition of N-[(1-alkyl)cycloalk-
anoyl]-4-[((2,6-dichlorophenyl)carbonyl)amino]-l-phenylalanine deri-
vatives
Table 3. VCAM/VLA-4 binding inhibition of N-[(1-(aminoalk-
yl))cycloalkanoyl]-4-[((2,6-dichlorophenyl)carbonyl)amino]-l-phenyl-
alanine derivatives
Compd
RELISA IC
50 (nM) Ramos IC50 (nM)
Compd
RELISA IC
50 (nM) Ramos IC50 (nM)
13
1.0
134
31
0.53
47
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1.1
1.5
96
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
1.64
5.1
142
227
8
104
0.26
0.61
0.32
0.50
0.52
0.55
1.1
12
17
29
27
39
64
150
350
62
25
75
528
94
8
0.37
3.6
128
47
0.64
1.15
28
40
635
1034
36
14
0.46
7.8
0.1
11
0.29
0.21
0.8
0.14
1.15
5.8
22
19
914
27
1.8
0.33
3.4
1.1
89
0.15
0.1
2.4
151
6

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A. Sidduri et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2475–2478
ranging from phenyl and benzyl to o-substituted alkyl
suggests that these substituents may not be directly
involved in the mediating receptor interaction, but may
play an important role in defining the preferred con-
formation of the ligand through intramolecular interac-
tions. Differences in the solvation of these functional
groups may also play an important role and account for
some of the more subtle potency differences between
individual members of the class. We made similar
observations and conclusions during the course of our
work on N-aroyl phenylalanine derivatives.6 During the
course of this work, we succeed in identifying compact
and practical replacements for the N-benzylpyro-
glutamyl group without loss of potency in our in vitro
assays.
Figure 1. (a) Spacefill representation of the crystal structure of the
methyl ester of 16 (48); (b) overlay of the two most populated con-
formations of cyclic peptide 2 as determined by NMR(black) with 48
(grey).
Increases in the size of alkanoyl group led to a decrease
in potency as evidenced by examples 36–37 and 42–43.
The presence of an aryl group either as part of an amide
(38–39) or urea (44) is detrimental. Among the amides,
acetamido ethyl (34) is preferrable to acetamido butyl
(35) and the presence of a basic nitrogen atom in the
side chain is unfavorable (33, 46, and 47).
References and Notes
1. Elices, M. J. In Cell Adhes. Mol. Matrix Proteins; Mouse,
S. A., Ed.; Springer: Berlin, 1999; p 133.
2. (a) Tilley, J. W.; Sidduri, A. Drugs Future 2001, 26, 985. (b)
Porter, J. R. Idrugs 2000, 3, 788. (c) Adams, S. P.; Lobb, R. R.
Prog. Respir. Res., Basel, Karger 2001, 31, 302. (d) Boer, J.;
Gottschling, D.; Schuster, A.; Semmrich, M.; Holzmann, B.;
Kessler, H. J. Med. Chem. 2001, 44, 2586.
3. Fotouhi, N.; Joshi, P.; Fry, D.; Cook, C.; Tilley, J. W.;
Kaplan, G.; Hanglow, A.; Rowan, K.; Schwinge, V.;
Wolitzky, B. Bioorg. Med. Chem. Lett. 2000, 10, 1171.
4. Chen, L.; Tilley, J. W.; Guthrie, R. W.; Mennona, F.;
Huang, T.-N.; Kaplan, G.; Trilles, R.; Miklowski, D.; Huby,
N.; Schwinge, V.; Wolitzky, B.; Rowan, R. Bioorg. Med.
Chem. Lett. 2000, 10, 729.
5. Chen, L.; Trilles, R. V.; Miklowski, D.; Huang, T.-N.;
Campbell, R.; Rowan, K.; Schwinge, V.; Tilley, J. W. Bioorg.
Med. Chem. Lett. 2001, 11, 137.
6. Sidduri, A.; Tilley, J. W.; Lou, J. P.; Chen, L.; Kaplan, G.;
Mennona, F.; Campbell, R.; Guthrie, R.; Huang, T.-N.;
Rowan, K.; Schwinge, V.; Renzetti, L. M. Bioorg. Med. Chem.
Lett. 2002, 12, 2479.
As previously noted, we believe that the cycloalkanoyl
phenylalanine derivatives related to 1 and 16 are cap-
able of conformationally mimicking the cyclic peptidic
VCAM-VLA-4 inhibitor 2. In support of this notion,
the crystal structure of methyl ester of 16 (48) was
obtained. There are two molecules per unit cell which
differ only by an approximately 180ꢀ rotation of the
cyclopentyl ring about the amide bond. One of these is
shown in Figure 1a.8 As expected, the crystal structure
reveals a low energy, compact, gauche (À) conforma-
tion for the phenylalanine. In one conformation the
cyclopentyl ring occupies the same position as proline
ring of cyclic peptide 2 and the 2-methoxy ethyl side
chain makes a hydrophobic contact with phenylalanine
aromatic ring. An overlay of this conformer with the
NMRderived conformational model of the cyclic
peptide 2 is shown in Figure 1b.
7. Chen, L.; Guthrie, R. W.; Huang, T.-N.; Hull, K. G.; Sid-
duri, A.; Tilley J. W. WO 9910313; Chem. Abstr 1999, 130, 196
952.
8. X-ray: the methyl ester of 16 was crystallized from ethyl
acetate, mp 220–222 ꢀC, crystal size (mm) 0.11Â0.13Â0.18,
space group P22121, R=0.0669, Rw=0.0783. The atomic
coordinates were provided to the CCD with deposition
number CCDC185240.
In summary, within
a series of N-(a-substituted
cycloalkanoyl)phenylalanine derivatives, a variety of
ring sizes and functional groups are tolerated in the side
chain. Furthermore, our finding that a-substituents