N-Acyl-l-phenylalanine derivatives as potent VLA-4 antagonists that mimic a cyclic peptide conformation

Article abstract of DOI:10.1016/S0960-894X(01)00711-9

A series of N-benzylpyroglutamyl-l-phenylalanine derivatives bearing carbamoyl substituents in the 3- or 4-positions was prepared and assayed for inhibition of the interaction between VCAM and VLA-4. Potent inhibition was observed in a number of analogues with substitution in the 4-position favored over the 3-position. A crystal structure of the key intermediate 25 indicates that it accesses a low energy conformation which closely matches key pharmacophores of a structurally characterized cyclic peptide.

Full text of DOI:10.1016/S0960-894X(01)00711-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 137–140
N-Acyl-L-phenylalanine Derivatives as Potent VLA-4 Antagonists
that Mimic a Cyclic Peptide Conformation
Li Chen,* Jefferson Tilley, Richard V. Trilles, Weiya Yun, David Fry, Charles Cook,
Karen Rowan, Virginia Schwinge and Robert Campbell
Roche Research Center, Hoffmann-La Roche Inc, Nutley, NJ 07110, USA
Received 30 August 2001; accepted 9 October 2001
Abstract—A series of N-benzylpyroglutamyl-l-phenylalanine derivatives bearingcarbamoyl substituents in the 3- or 4-positions
was prepared and assayed for inhibition of the interaction between VCAM and VLA-4. Potent inhibition was observed in a number
of analogues with substitution in the 4-position favored over the 3-position. A crystal structure of the key intermediate 25 indicates
that it accesses a low energy conformation which closely matches key pharmacophores of a structurally characterized cyclic peptide.
# 2002 Elsevier Science Ltd. All rights reserved.
The very late antigen 4 (VLA-4, a4b1) is an integrin
receptor expressed on many lymphocytes and mediates
cell adhesion, infiltration and the co-stimulation
response, when it interacts with its counter ligands, such
as VCAM-1 and fibronectin. VLA-4 antagonists that
inhibit the interaction of VCAM-1/VLA-4 are being
intensively sought for the treatment of inflammatory
diseases such as asthma (Fig. 1).1À3
minus, the proline ringand elements of the disulfide
bridge were key pharmacophores. In addition, the
potency is dependent on the selection of an appropriate
N-terminal reside, such as a tyrosine residue.
NMR studies of cyclic peptides related to 14,5 suggested
a consistent picture of a relatively planar ringstructure
of the cyclic peptide core with the two ringamide bonds
perpendicular to the ringand pointingin opposite
directions from one another to minimize the overall
dipole interaction. The precise orientation of the critical
distal N-terminal group is ill defined. In the course of
this work, we obtained a crystal structure of the N-
(benzylpyroglutamyl)Phe derivative 25 that represents a
low energy gauche (À) conformation with the two ben-
zene rings in close proximity. Overlaying of the X-ray
Studies on cyclic peptide based VLA-4 antagonists,4,5
such as 1 (IC50 1.8 nM in an ELISA assay), suggested
that a tyrosine residue at the N-terminus was beneficial
to the activity of this class. We have previously descri-
bed our efforts to scan a variety of N-(benzylpyro-
glutamyl)phenylalanines for their ability to inhibit the
VCAM–VLA-4 interaction.6,7 The most potent com-
pounds to emerge from this work were the 4-acylamino-
phenylalanine derivatives typified by 2 and 3 with IC50s
of 0.37 and 6.5 nM, respectively. As a result of this work,
we proposed that members of N-Acyl-Phe series may
mimic the cyclic peptide inhibitors interaction with VLA-
4. In the present paper, we provide additional support for
this hypothesis and extend our SAR studies to the cor-
responding3- and 4-aminocarbonyl-phenylalanines 4.
Previous studies6,7 on the SAR of cyclic peptides related
to 1 indicated that the carboxylate group at the C-ter-
*Correspondingauthor. Tel.:+1-973-235-5826; fax:+1-973-235-6085;
e-mail: li.chen@roche.com
Figure 1. N-Acy-Phe and cyclic peptide class of VLA-4 antagonists.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00711-9

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L. Chen, et al. / Bioorg. Med. Chem. Lett. 12 (2002) 137–140
crystal structure of 25 with two most populated families
of NMR structures of the cyclic peptide core suggests
that these two classes of VLA-4 antagonist share key
pharmacophores, includingthe 5-membered pyrrolidine
ringand the C-terminal carboxylic acid, while the aro-
matic ringof 25 occupies the region of the disulfide
bridge of the cyclic peptide (Fig. 2).
an ELISA and Ramos cell based format.11 The results,
shown in Table 1, indicate that 4-tyrosinyl derivative 9
is nearly equal potent to peptide 1 in both the ELISA
and Ramos cell based assays. Comparingthe potencies
of 9 and 3 suggests that it is relative insensitive to the
directionality of the amide bond connectingthe tyrosine
and the phenylalanine. Compounds 10 and 11, derived
from benzyl amine, were less active than compound 9.
Activity of aniline derivative was low and was improved
by incorporation of an acetyl group at the ortho-posi-
tion. Incorporation of acylpiperazines at 4 position gave
low nanomolar analogues, which were 4-fold more
potent than the correspondingsulphonylpiperazine
analogues. The most potent acylpiperazine analogues,
such as compound 17 and 19, are as active as 1 and 9. It
is noteworthy that compound 12 is almost 10-fold less
potent than its reversed amide analogue,7a indicatinga
In this model, the C–H bonds in the 3- and 4-positions
of benzene ringof the phenylalanine of 25 represent
vectors which point toward the region occupied by the
structurally ill defined tyrosine of cyclic peptide 1. With
this understanding, we chose to explore groups in either
position which are capable of mimickingthe function of
the tyrosine of 1.
For the preparation of 4-aminocarbonyl analogues of
phenylalanine, l-tyrosine triflate methyl ester was acy-
lated with N-benzylpyroglutamic acid 6 to give the tri-
flate 7, which was carbonylated with carbon monoxide
at 50 psi in the presence of a palladium catalyst to give
the intermediate acid 8 (Scheme 1). Couplingof 8 with
amines usingHBTU followed by hydrolysis provided
compounds 9–13. Alternatively, reaction of compound
7 with Boc-piperazine under the carbonylation condi-
tions8 followed by removal of the Boc group gave
intermediate piperazine amide 14, which was in turn
acylated by a variety of acids and sulfonyl chlorides.
The acylation products were subsequently hydrolyzed
under basic conditions to give final products 15–21.
The 3-substituted analogues were prepared through an
enantioselective hydrogenation of the dehydro-phenyl-
alanine derivative 23 as shown in Scheme 2. The S-ste-
reochemistry of 24 was assigned based on the literature
precedent9 and was confirmed by X-ray crystallography10
of the alcohol 25. Jones oxidation of 25 followed by cou-
plingwith tyrosine diethyl amide and basic hydrolysis
gave tyrosine analogue 26. Similarly, acylation of inter-
mediate 27 followed by hydrolysis of methyl ester gave 3-
acylpiperazine analogue 28. The sulfonamides 29 and 30
were synthesized by reaction of 27 with the sulfonyl
chlorides followed by hydrolysis of the methyl esters.
Scheme 1. Synthesis of 4-aminocarbonylphenylalanine analogues: (a)
HBTU, DIEPA DMF, rt; (b) PdAc2, dppp, DMSO, CO, Et3N, H2O,
60 ꢀC; (c) NH2R, HBTU, DIEPA, DMF, rt; (d) NaOH, EtOH, H2O,
rt; (e) Pd(OAc)2, dppp, N-Boopiperazine CO, Et3N, 60 ꢀC; (f) 4 N HCl
in cloxane; (g) benzoic acids, HBTU, DIPEA, rt; or benzenesulfonyl
chloride, DCM, DIPEA, rt.
The N-Acyl-Phe analogues were evaluated for their
ability to inhibit the VCAM–VLA4 interaction in both
Scheme 2. Synthesis of 3-aminocarbonylphenylalanine analogues. (a)
NaH, THF, TBSCI, 53%; (b) MnO2, DCM, 79%; (c) Cbz-trimethyl-
phosphonoglycine, TMG, DCM, 85%; (d) Duphas, MeOH, H2, 98%;
(e) Pd-C, H2, MeOH; (f) N-Benzylpyroglu-OH, HBTU, DIEA, 62%;
(g) HF, MeCN, 62% from 24; (h) Jones reagent, acetone; (i) NH2R,
HBTU, DIEA, DMF, 72% from 25; (j) 1 N NaOH, EtOH/H2O; (k)
N-Boc-piperazine, HBTU, DIPEA, DMF, rt; (l) 4 N HCl in dioxane;
(m) acids, HBTU or sulfonyl chloride, DIPEA.
Figure 2. Overlay of X-ray of 25 (m-hydroxymethyl not shown) in
gold with cyclic peptide core in green and cyan.

L. Chen, et al. / Bioorg. Med. Chem. Lett. 12 (2002) 137–140
139
Table 1. Inhibitory activity of VCAM–VLA-4 interaction in ELISA and Ramos cell-based assay
Compd
R1
R2
ELISA
IC50 (mM)
Ramos
IC50 (mM)
1
2
3
9
0.002
0.200
0.012
2.1
0.00037
0.0065
0.005
H
H
H
H
H
H
H
H
H
H
H
H
0.550
10
11
12
13
15
16
17
18
19
20
21
26
28
29
30
0.061
0.027
0.057
0.004
0.007
0.003
0.004
0.003
0.003
0.011
0.012
0.240
0.120
0.060
0.073
0.940
5.50
0.960
0.320
1.7
0.470
2.2
9.53
24.4
H
H
H
7.8

140
L. Chen, et al. / Bioorg. Med. Chem. Lett. 12 (2002) 137–140
more favorable interaction of 4-aroylaminopheny-
lalanine, such as 2, with the VLA-4 receptor via the
amide carbonyl group. Reports from this laboratory on
a class of imide analogues7b further suggested the
involvement of the amide carbonyl in the receptor
recognition.
burn, B.; Struble, M.; Fitzgerald, G.; Chan, K.; Mullins, S.;
Burnier, J. P.; Fairbrother, W. J.; Clark, K.; Berisini, M.;
Chui, H.; Renz, M.; Jones, S.; Fong, S. J. Med. Chem. 1997,
40, 3359.
5. Fotouhi, N.; Joshi, P.; Fry, D.; Cook, C.; Tilley, J. W.;
Kaplan, G.; Hanglow, A.; Rowan, K.; Schwinge, V.; Wolitzy,
B. Bioorg. Med. Chem. Lett. 2000, 10, 1171.
6. Chen, L.; Tilley, J. W.; Huang, T.-N.; Miklowski, D.;
Trilles, R. V.; Guthrie, R. W.; Luk, K.; Hanglow, A.; Rowan,
K.; Schwinge, V.; Wolitzky, B. Bioorg. Med. Chem. Lett. 2000,
10, 725.
7. (a) 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, K. Bioorg. Med. Chem.
Lett. 2000, 10, 729. (b) Tilley, J.; Kaplan, K.; Rowan, K.;
Schwinge, V.; Wolitzky, B. Bioorg. Med. Chem. Lett. 2001, 11,
1.
8. Unpublished results. General procedure for carbonylation
was described as following: To a pressure bottle (250 mL) were
added compound 7 (5.28 g, 10 mmol), Pd(AcO)2 (0.23 g, 0.1
mmol), N-[(dimethylethoxy)-carbonyl]piperazine (7.45 g, 40
mmol), 1,3-bis(diphenyl-phospino)propane (0.42 g, 0.10
mmol) in a mixture of DMSO (50 mL). The bottle was pres-
sured with CO to 40 psi and the pressure released. After four
cycles of pressure/release, the bottle was charged with 40 psi
CO and stirred at 80 ꢀC for 3 h. After coolingto room tem-
perature, the mixture was diluted with 400 mL of ethyl acetate
and washed with water (2Â50 mL), 1N HCl (2Â50 mL),
saturated NaHCO3 (50 mL) and saturated brine (50 mL).
The solvent was then removed and the residue was filtered
through silica gel eluting with ethyl acetate to give 4-
[[4-[(dimethylethoxy)carbonyl]-1-piperazinyl]carbonyl]-N-[5-
oxo-1-(phenylmethyl)-l-prolyl]-l-phenylalanine methyl ester
(3.8 g, 64.5%), HRMS: obs mass, 593.2994. Calcd mass,
593.2975.
In summary, the potent VLA-4 antagonist activity of N-
(benzylpyroglutamyl)phenylalanines together with the
X-ray crystal structure of 25 suggested that this class of
compound can effectively pre-organize into the bioac-
tive conformation presented by the cyclic peptide such
as 1. A remarkable findingfrom this work is that a wide
variety of sub-structure motifs are tolerated on the aro-
matic ringof the N-Acyl-Phe class of VCAM/VLA-4
antagonists. However, the exact nature of interaction
with integrin receptor may vary between chemical clas-
ses. The results further suggested that members of N-
Acyl-Phe class mimic the pharmacophores presented by
the cyclic peptide core structure. Substitution on the
phenylalanine interacts with bindingsites different from
that occupied by the tyrosine of 1. Comparison of pairs
of 3- versus 4-substituted analogues 9 versus 26, 16 ver-
sus 28, 20 versus 29 and 21 versus 30 firmly imply that
substitutions in the 4-position of N-Acyl-Phe is favored
over that in the 3-positions.
References and Notes
1. Elices, M. In Cell Adhesion Molecules and Matrix Proteins:
Role in Health and Diseases; Mousa, S. A., Ed.; Springer:
1998; p 133.
9. Burk, M. J.; Feaster, J. E.; Nugent, W. A.; Harlow, R. L. J.
Am. Chem. Soc. 1993, 115, 10125.
2. (a) Lin, K.-C.; Ateeq, H. S.; Hsiung, S. H.; Chong, L. T.;
Zimmerman, C. N.; Castro, A.; Lee, W.-c.; Hammond, C. E.;
Kalkunte, S.; Chen, L.-L.; Pepinsky, R. B.; Leone, D. R.;
Sprague, A. G.; Abraham, W. M.; Gill, A.; Lobb, R. A.;
Adams, S. P. J. Med. Chem. 1999, 42, 920. (b) Lin, K.-C.;
Castro, A. C. Curr. Opin. Chem. Biol. 1998, 2, 453.
3. Elices, M. Curr. Opin. Antiinflammat. Immunomodulat.
Investigat. Drugs 1999, 1, 14.
10. X-ray report: compound 25 was crystalized from ethyl
acetate, mp 125–125 ꢀC, [a]:+71.2 (c 1, MeOH), crystal size
(mm): 0.15Â0.25Â0.45, space group: P1, Rw: 0.048. The
atomic coordinates are provided to CCD with deposition
number CCDC169413.
11. Chen, L.; Guthrie, R. W.; Huang, T.-N.; Hull, K. G.;
Sidduri, A.; Tilley, J. W. WO 9910312, 1999; WO 9910313,
1999; Chem. Abstr. 1999, 130, 196,952.
4. Jackson, D.; Quan, C.; Artis, D. R.; Rawson, T.; Black-