Diamine containing VLA-4 antagonists

Article abstract of DOI:10.1016/S0968-0896(01)00132-8

Design and synthesis of a library as potential VLA-4 antagonists has been accomplished, based around a proposed pharmacophoric model. Compounds possessing submicromolar potency were identified and structure-activity relationships were seen across the library. Further derivatisation produced compounds with IC₅₀'s < 10 nmol for inhibiting the VLA-4 mediated binding of fibronectin to RAMOS cells, providing an ideal starting point for a lead optimisation Programme.

Full text of DOI:10.1016/S0968-0896(01)00132-8

Bioorganic & Medicinal Chemistry 9 (2001) 2195–2202
Diamine Containing VLA-4 Antagonists
Peter C. Astles,y Neil V. Harris{ and Andrew D. Morley*
Aventis Pharma Ltd, Dagenham Research Centre, Rainham Road South, Dagenham, Essex RM10 7XS, UK
Received 9 March 2001;accepted 20 April 2001
Abstract—Design and synthesis of a library as potential VLA-4 antagonists has been accomplished, based around a proposed
pharmacophoric model. Compounds possessing submicromolar potency were identified and structure–activity relationships were
seen across the library. Further derivatisation produced compounds with IC50’s <10 nmol for inhibiting the VLA-4 mediated
binding of fibronectin to RAMOS cells, providing an ideal starting point for a lead optimisation Programme. # 2001 Elsevier
Science Ltd. All rights reserved.
Introduction
reported data, it was rationalised that an aromatic ring
present in the terminal amide cap and the b-carboxylate
of the aspartic acid (or related residue) were essential for
biological activity. De novo design was undertaken
based around these moieties, with a view to constructing
non-peptidic libraries which incorporated these phar-
macophores.
The cell adhesion molecule VLA-4 (a4b1) is a member of
the integrin super family of heterodimeric cell surface
receptors and is found on all haemopoetic cells with the
exception of neutrophils. Its primary ligands are the
endothelial cell surface protein, vascular cell adhesion
molecule-1 (VCAM-1) and the alternatively spliced CS-1
isoform of fibronectin, which is localised in the extra-
cellular matrix. Recently, it has been demonstrated that
besides mediating cell trafficking, VLA-4 also regulates
the activation and survival of a number of cell types.
Monoclonal antibodies directed against VCAM-1 and
the a4 subunit have been shown to be useful modulators
in animal models of chronic inflammatory diseases,
including asthma and rheumatoid arthritis.1À4 More
recently, small peptide antagonists have been shown to
be efficacious in preclinical models of asthma.5À7 Sub-
sequently, the identification of non peptide antagonists
has been reported in the literature.8,9 This has led to
the belief that non-peptidic VLA-4 antagonists could be
identified and used to treat diseases of this type.
Chemistry
The generic library 1 employing the inputs depicted in
Table 1 was ideal for preparation on solid phase,
employing the terminal acid pharmacophore as a point
of attachment to the resin. By synthesising compounds
this way, it was found that the construction, isolation
and purification processes could be greatly accelerated
and simplified.
The proposed synthetic sequence for the preparation of
1 was to alkylate resin bound bromoacetate, 2 and
acrylate, 3, with a series of diverse diamines (4–14) to
furnish the core diamine-acid templates. Acylation of
the resulting primary or secondary amine of these tem-
plates with a series of aromatic carboxylic acids caps
(15–28) would provide the immobilised library members
which could be cleaved from the resin using standard
procedures.
At the commencement of this research a small number
of peptidic antagonists had already been described in
the literature.10À12 Most interesting was a series of LDV
peptidomimetics reported by Biogen.13,14 From the
A representative synthetic example for the preparation
of the library is depicted in Scheme 1, using 4-(amino-
methyl)piperidine, 6, as the diamine component. Esters
*Corresponding author at present address: AstraZeneca, Mereside,
Alderley Park, Macclesfield, Cheshire SK10 4TG, UK. Tel.: +44-
1625-512782;fax: +44-0-1625-516667;e-mail: andy.morley@
astrazeneca.com
yPresent address: Eli Lilly and Co., Lilly Research Centre Ltd, Erl
Wood Manor, Windlesham, Surrey GU20 6PH, UK.
{Present address: Argenta Discovery Ltd, 8/9 Spire Green Centre, Flex
Meadow, Harlow, Essex CM195TR, UK.
2
and 3 were prepared on Wang (p-benzyloxy-
benzylalcohol) resin using standard symmetrical anhy-
dride conditions.15 Initial attempts to achieve high
regioselectivity for the alkylation with 6 (preferential
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00132-8

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P. C. Astles et al. / Bioorg. Med. Chem. 9 (2001) 2195–2202
reaction of secondary amine with 2 or 3) was not suc-
cessful. Therefore, protection of the primary amino
group of 6 and related non-symmetrical diamines was
deemed necessary. Finding a suitable group that could
be introduced easily and which was also compatible
with solid phase chemistry was not trivial. The solution
was to protect the primary amine as an imine.16 Treat-
ment of 6 with one equivalent of 3,4-dimethoxy-
benzaldehyde in toluene with azeotropic removal of water
afforded the crude imine 29 which showed >95% purity
by NMR and HPLC without the need for purification.
Reaction of 5–10 equiv of 29 with resins 2 or 3 was
achieved in a DMSO/diisopropylethylamine (DIPEA)
mixture for 24 h. After washing of the resin, cleaved
samples could be analysed by HPLC using evaporative
light scattering (ELS) detection. Loadings were deter-
mined by NMR analysis employing p-tolylsulphone as
an internal standard. Cleavage of a known quantity of
resin was undertaken in 50% deuterated TFA in CDCl3
for 0.5 h. The resulting spectrum were of good quality,
allowing accurate integration measurement to be
achieved, with purity generally >95% (typical loading
values being in the region of 0.4–0.6 mmol/g).
The imine protecting group of 30 was easily removed by
treating the resin with a known volume of a mixture of
MeCN/H2O/TFA (80:20:2). By employing HPLC, the
progression of the reaction could be monitored, mea-
suring the concentration of the liberated 3,4-dimethoxy-
benzaldehyde. Reactions were generally complete within
an hour.
Table 1. Components of diamine library
Acylation of the resulting amines, 31, with caps 15–28
was achieved using o-(7-azabenzotriazol-1-yl)1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU)
mediated coupling17 to afford the resin bound products,
32. These could be cleaved from the resin using a 1:1
TFA/CH2Cl2 mixture for 1 h.
No. Diamine (RHN-X-NHR0)
No.
Terminal cap
Using the protocol depicted in Scheme 1, a library of
approximately 200 compounds was synthesised. Pro-
ducts were analysed for purity by reverse phase LC/MS
(>85% in all cases) and the presence of the requisite
mass ion confirmed by electrospray mass spectrometry.
4
15
5
6
7
16
17
Subsequent work focused on using 33 as a pivotal
intermediate for the synthesis of additional VLA-4
inhibitors. It is available from commercial 4-(amino-
methyl)piperidine as outlined in Scheme 2. Selective
protection of the secondary amine was achieved follow-
ing the method of Prugh16 to afford 34. Acylation, using
standard conditions, gave 35 and removal of the BOC
protecting group, with trifluoroacetic acid, provided 33
in quantitative yield.
18 (R¼H)
19 (R¼Me)
8
20 (R¼H)
21 (R¼Me)
The solution synthesis of 36 is depicted in Scheme 3.
Reaction of ethyl crotonate and 33 proceeds smoothly
at room temperature and saponification of the resulting
ester provided 36 in acceptable yield.
9
22 (R¼H)
23 (R¼Me)
10
24 (R¼H)
The preparation of 39 is outlined in Scheme 4. The key
Cbz-aziridine derivative, 37, was accessed according to
literature precedent18,19 from commercial ethyl-2,3-
dibromopropionate in modest yields. Ring opening of
37 by 33 was highly regioselective, but slow. Hydrolysis
of the desired regioisomer, 38, provided the target
molecule, 39 in low overall yield.
25 (R¼Me)
11
12
13
26
27
28
Results and Discussion
The target library 1, was chosen with a view to linking
the two proposed VLA-4 pharmacophores (aromatic
ring and carboxylic acid) through a suitable template.
Spatial variation of these moieties was achieved by
employing diverse diamines/linkers as the central
14
Not all combinations synthesised.

P. C. Astles et al. / Bioorg. Med. Chem. 9 (2001) 2195–2202
2197
spacer. The library was tested for its ability to inhibit
VLA-4 mediated binding of H3-RAMOS cells to fibro-
nectin and VCAM-1 at doses of 30, 10 and 3 mmol. A
total of 44 compounds showed >50% inhibition at
30 mmol in the fibronectin assay (inhibitions >100 and
<0% are artefacts of the assay conditions). Data for
selected compounds is shown in Table 2.
yl)propionic acid template. The diphenyl urea is essen-
tial for VLA-4 antagonism in these systems, with the
para ureas always showing superior potency over their
corresponding meta analogues (41 vs 42). Improvements
in activity are seen with the incorporation of the ortho-
methyl substituent in the terminal phenyl ring and more
strikingly, with the introduction of the 3-methoxy moi-
ety into the phenylacetyl portion (40, 41 and 43). The
requirement of a specific distance between the acid and
urea is confirmed by the fact that both 43 and 44 possess
similar potency. This would seem to indicate that the
Clear structure–activity relationships (SAR) can be seen
across the library. The most interesting results generally
being derived from the 3-(4-aminomethyl-piperidin-1-
Scheme 1. Reagents: (a) 3,4-dimethoxybenzaldehyde, PhMe reflux;(b) DIPEA, DMSO;(c) MeCN/H 2O/TFA (80:20:2) 1 h;(d) 15–28, HATU,
DIPEA, DMF;(e) 50% TFA in DCM 1 h.
Scheme 2. Reagents: (a) benzaldehyde, PhMe reflux;(b) di -tert-butylcarbonate;(c) 1 M KHSO 4;(d) 26, HATU, DIPEA, DMF;(e) 25% TFA in
DCM.

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P. C. Astles et al. / Bioorg. Med. Chem. 9 (2001) 2195–2202
Scheme 3. Reagents: (a) ethyl crotonate, DIPEA, MeOH;(b) 1 M NaOH/THF.
Scheme 4. Reagents: (a) ammonia, MeCN;(b) Cbz-succinimide, DIPEA, THF;(c) 33, THF reflux;(d) 1 M NaOH/MeOH.
core template is acting only as a spacer in positioning
the proposed pharmacophores in a suitable orientation,
and not contributing to the mode of binding. However,
varying the positioning of the central amide bond rela-
tive to the diaryl urea dramatically effects potency (cf.
43 with 45), suggesting the amide to be either a key
pharmacophore or exerting a particular conformation
on the molecules which is essential for VLA-4 activity.
Finally, comparisons between 46 and lactam 47,20
highlights either the necessity of the carbonyl group or
the preference for an sp2 nitrogen for good biological
activity with this specific substitution pattern.
increases in biological activity were to be achieved. As
variations in the potency of other series had been iden-
tified by modifying substituents around the carboxylic
acid,20 attention turned to other potential pharmaco-
phores that could be incorporated around this part of
the molecule. Substitution a to the acid had not yet
been investigated and it was decided to introduce an
amino group in this position. If successful, this moi-
ety could also be used as a ‘handle’ to incorporate a
variety of potential pharmacophores. Compound 39
was therefore chosen as an initial target to evaluate this
hypothesis.
Encouraged that novel inhibitors of fibronectin and
VCAM-1 binding to the a4b1 integrin had been identi-
fied from de novo design, the next step was to try and
improve the potency. As limited variations around the
terminal cap and spacer had already been incorporated
into the scaffold, the 4-(aminomethyl)piperidine and
diphenyl urea portions were initially kept constant.
Intermediate 33 was therefore prepared and used to
probe the SAR around the carboxylic acid portion of
the scaffold.
Interestingly, this modification to the general structure
gave a 10-fold increase in potency for the inhibition of
binding of fibronectin to the integrin (Table 3). The Cbz
protecting group was the first amino derivative that had
been incorporated at this position and was unlikely to
be optimal. Thus, 39 provides a useful starting point for
derivatisation in a bid to further optimise potency and
evaluate SAR.
Conclusion
Our previous work, based on the lactam templates, had
shown that increases in biological activity could be
obtained by the introduction of substituents b to the
carboxylic acid.20 Compound 36 was therefore chosen
as a suitable target. Contrary to results experienced in
the lactam series, no increase in activity was observed
compared to the unsubstituted analogue 43 (Table 3).
This again highlights the differences in conformations
and subsequent biological activities between the two
series. An alternative strategy was therefore necessary, if
A de novo library has been designed, based on a puta-
tive pharmacophoric model. This led to the identifica-
tion of novel, non peptidic inhibitors of binding of
fibronectin to the a4b1 integrin, with well defined SAR
being established. Further derivatisation around the
lead template has enabled compounds with low nano-
molar potency to be identified, providing a useful start-
ing point for a lead optimisation process. Additional
work in this area will be reported elsewhere.

P. C. Astles et al. / Bioorg. Med. Chem. 9 (2001) 2195–2202
2199
3
Table 2. Activities of selected library compounds for their inhibition of binding of H-RAMOS cells to Fibronectin and VCAM-1
Inhibition of
VLA-4 binding
to Fibronectin
Inhibition of
VLA-4 binding
to VCAM-1
No.
Structure
30 mM
(%)
IC50
(mM)
30 mM
(%)
IC50
(mM)
Retention
timea
MH+a
Puritya
(%)
40
41
28
9
2.51
2.69
439
453
91%
92%
78
19
7
10
6
>30
42
43
44
45
2.46
2.58
3.92
2.78
453
483
483
483
>99%
>99%
95%
115
124
18
0.1
0.1
88
100
6
1.7
4
95%
46
47
À13
À1
2.49
—
439
—
87%
—
85
4
13
aFor conditions, see Experimental.
Table 3. Activities of selected compounds for their inhibition of binding of RAMOS cells to fibronectin and VCAM-1
Inhibition of VLA-4
binding to Fibronectin
Inhibition of VLA-4
binding to VCAM-1
No.
Structure
IC50 (nM)
100
IC50 (nM)
43
1700
36
39
150
1080
282
7
Experimental
using a Buchi rotary evaporator. Reaction products
were purified, when necessary, by flash chromatography
on silica gel (40–63 mm) eluting with the solvent system
indicated. Yields are not optimised. Melting points were
determined on a Gallenkamp 595 apparatus and are
uncorrected. The structure and purity of all compounds
were confirmed by microanalytical and/or spectro-
Chemical methods
Reagents, starting materials and solvents were pur-
chased from common commercial suppliers and used as
received or purified by standard methods. All organic
solutions were dried over magnesium sulphate and con-
centrated at reduced pressure under aspirator vacuum
1
scopic methods. H NMR spectra were acquired on a

2200
P. C. Astles et al. / Bioorg. Med. Chem. 9 (2001) 2195–2202
Varian VXR-400 or 500 MHz spectrometers;peak
positions are reported in parts per million relative to
internal tetramethylsilane on the d scale. Elemental
analyses were performed by the Physical Chemistry
Department at Aventis. Satisfactory microanalyses
(ꢀ0.4%) were obtained for C, H, N unless otherwise
stated. LC/MS: Samples were run on a Micromass
Platform II mass spectrometer by +ve/Àve ion Elec-
trospray under the following LC conditions.
quantified by counting the plates on a Topcount
Microplate Scintillation counter.
Diamine library
4-(3,4-Dimethoxybenzylideneaminomethyl)piperidine
29 and related compounds were prepared by refluxing
equimolar amounts of diamine and 3,4-dimethoxy-
benzaldehyde in toluene with azetropic removal of
water.16 These compounds were used crude without
purification.
Mobile phase. (A) Water 0.1% formic acid;(B) acetoni-
trile 0.1% formic acid.
A representative example is given below for the synth-
esis of 3-(4-{[3-methoxy-4-(3-(2-methylphenyl)ureido)-
phenyl]-acetylaminomethyl}-piperidin-1-yl)-propionic
acid (43).
Gradient
Time flow (mL/min)
%A
%B
0.00
0.50
4.50
5.00
5.50
2.0
2.0
2.0
2.0
2.0
95
95
5
5
95
5
5
95
95
5
Step 1. A stirred solution of acrylic acid (0.67 g,
9.3 mmol) in dimethylformamide (10 mL) and tetra-
hydrofuran (5 mL) was treated with diisopropyl-carbo-
diimide (0.59 g, 4.67 mmol). After stirring for 5 min, the
solution was treated with N,N-4-(dimethylamino)pyr-
idine (10 mg) and then with Wang resin (1 g, 1 mmol/g,
Advanced ChemTech). The mixture was allowed to
stand at ambient temperature for 18 h. The resin was
drained and then washed three times with dimethylfor-
mamide, then three times with tetrahydrofuran, then
three times with dichloromethane, sucked dry and then
dried under vacuum.
Column Luna 3u C18 (2) 30ꢁ4.6. Detection: diode
array, ELS detection in line ELS, temp 50 ꢂC, Gain 6–
1.8 mL/min, injection volume 10–40 mL dependant on
library. Source temp 150 ꢂC. Approx. 200 mL/min split
to mass spectrometer. Mass spectra were recorded on a
Micromass Platform II mass spectrometer fitted with an
Electrospray source and an HP1100 liquid chromato-
graph;using a mixture of acetonitrile and water (1:1, v/
v) as the mobile phase, a flow rate of 0.3 mL/min, an
injection volume of 20 mL, a run time of 2.0 min, a scan
range of 150–850 Daltons positive/negative, a scan time
of 2.0 s, an ESI voltage of 3.5 Kv, an ESI pressure of
20 N/m2 nitrogen. The ions recorded are positive ions.
Step 2. The resin from Step 1 (0.5 g, 0.92 mmol/g load-
ing) was treated with a mixture of 4-[(3,4-dimethoxy-
benzylidenamino)methyl]piperidine (1.0 g) and DIPEA
(500 mL) in dimethylsulphoxide (10 mL). After standing
at room temperature overnight the resin was drained,
then washed three times with dimethylformamide, then
three times with tetrahydrofuran, then three times with
dichloromethane, sucked dry, and then dried under high
vacuum.
Inhibitory activity in cell adhesion assays
A functional cell adhesion assay was used as the pri-
mary screen to identify and optimise inhibitors of the
interaction between the cell adhesion molecule VLA-4
and its physiological ligands, VCAM-1 and fibronectin.
This assay utilises the VLA-4 positive RAMOS cell line
and measures adhesion to 96-well plates pre-coated with
either VCAM-1 or fibronectin.
Step 3. The resin from Step 2 (50 mg, 0.046 mmol/g
loading) was suspended in a mixture of acetonitrile,
water and trifluoroacetic acid (2.5 mL, 80:20:2, v/v/v) at
room temperature. The mixture was kept at room tem-
perature until HPLC analysis of the supernatant solu-
tion showed no more 3,4-dimethoxybenzaldehyde was
being produced. The resin was then drained, then
washed three times with acetonitrile, then three times
with dimethylformamide, then twice with 5% DIPEA in
dimethylformamide, then three times with dimethylfor-
mamide, then three times with tetrahydrofuran, then
three times with dichloromethane, sucked dry and then
dried under high vacuum.
RAMOS cells were seeded at a density of 200,000 cells/
mL in RPMI 1640 culture medium supplemented with
10% FCS and grown for 3 days. Cells were then cen-
trifuged at 800 rpm for 5 min at 20 ꢂC and re-suspended
at a concentration of 500,000 cells/mL in methionine
deficient RPMI 1640 supplemented with 10% FCS and
1 mM l-glutamine. These cells were then radiolabelled
for 18 h with 1 mCi/mL [3H]-methionine and finally
resuspended in Puck’s buffer for assay.
Step 4. The resin from Step 3 (50 mg, nominal
0.046 mmol/g loading) was treated successively at room
temperature with a solution of 3-methoxy-4-[3-(2-
methylphenyl)ureido]phenylacetic acid21 (0.092 mmol)
in dimethylformamide (0.75 mL), then with diisopropyl-
ethylamine (50 mL) and then with a solution of O-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (0.092 mmol) in dimethylformamide
(0.75 mL). The mixture was kept at room temperature
Labelled cells (200,000 per well) were incubated in
FlashplatesTM (96-well plates with scintillant contained
under each well) precoated with fibronectin (30 mg/mL)
or VCAM-1 (10 mg/mL) in the presence of test com-
pound or vehicle (DMSO 1.8% final concentration) at
37 ꢂC for 1 h. Following incubation, adherent cells were

P. C. Astles et al. / Bioorg. Med. Chem. 9 (2001) 2195–2202
2201
for 1–2 h, then the resin drained, and then washed three
1.5H2O) calcd C, 63.2;H, 7.5;N, 12.8;found: C, 63.3;
H, 7.5;N, 12.55.
times with dimethylformamide, three times with tetra-
hydrofuran, three times with dichloromethane, and
dried under high vacuum.
Preparation of 3-[4-({2-[3-Methoxy-4-(3-o-tolyl-ureido)-
phenyl]acetylamino}-methyl)-piperidin-1-yl]butyric acid
(36). A solution of 33 (250 mg, 0.61 mmol) in methanol
(5 mL) was added ethyl crotonate (138 mg, 1.2 mmol)
and triethylamine (1 mL). The mixture was refluxed for
24 h and concentrated under vacuum. The resulting oil
was dissolved in methanol (2 mL) and treated with 1 M
sodium hydroxide (1.8 mL). The mixture was refluxed
for 2 h and concentrated under vacuum. The residue
was taken up in the minimum volume of tetra-
hydrofuran, the pH adjusted to 4 with trifluoroacetic
acid and purified by HPLC [HPLC column Dynamax
60 A C18 column operated under gradient elution con-
ditions with a mixture of acetonitrile and water plus
0.1% trifluoroacetic acid as the mobile phase (0–20 min
0% acetonitrile, ramped up to 100% acetonitrile after
20 min, then maintained at 100% acetonitrile) and UV
detection at 220 nm] to afford 36 as a light tan solid. Mp
Step 5. The resin from step 4 was treated with a mixture
of trifluoroacetic acid and dichloromethane (2 mL, 1:1,
v/v). After 1–2 h at room temperature, the resin was
drained and then washed with a mixture of tri-
fluoroacetic acid and dichloromethane (2 mL). The
combined filtrate and washings were evaporated to give
the 3-(4-{[3-methoxy-4-(3-(2-methylphenyl)ureido)-phe-
nyl]-acetylaminomethyl}-piperidin-1-yl)-propionic acid
(43). MS: MH+ 483. HPLC retention time=2.58 min.
Analytical data for this and other selected members of
the library are shown in Table 2 using standard condi-
tions.
Preparation of 4-({2-[3-methoxy-4-(3-o-tolyl-ureido)-phe-
acid
nyl]acetylamino}-methyl)-piperidine-1-carboxylic
tert-butyl ester (35). A solution of 3-methoxy-4-[3-(2-
methylphenyl)ureido]phenylacetic acid (5 g, 16.12 mmol)
in dimethylformamide (150 mL) was treated with O-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (PerSeptive Biosystems, 6.06 g,
15.9 mmol) and diisopropylethylamine (6.45 mL). 4-
Aminomethylpiperidine-1-carboxylic acid tert-butyl
ester16 (3.4 g, 15.9 mmol) was added and the mixture
stirred at ambient temperature for 3 h. The mixture was
poured into 10% (w/w) sodium carbonate solution
(500 mL) and extracted with tetrahydrofuran (4ꢁ100 mL).
The organic extracts were washed with saturated brine
(250mL) and dried over magnesium sulphate, filtered
and evaporated. The resulting oil was subjected to flash
chromatography on silica gel eluting with dichloro-
methane and methanol (95:5, v/v) to afford 35 (8.0 g,
98%) as a white solid: mp 211–212 ꢂC MS: MH+ 511.
1H NMR (CDCl3) 8.22 (d, J=10 Hz, 1H), 8.02 (s, 1H),
7.71 (m, 2H), 7.19 (m, 2H), 7.04 (m, 1H), 6.79 (m, 2H),
5.85 (m, 1H), 4.04 (m, 2H), 3.83 (s, 3H), 3.51 (s, 2H),
3.06 (m, 2H), 2.62 (m, 2H), 2.24 (s, 3H), 1.59 (m, 3H),
1.04 (m, 2H). Anal. (C28H38N4O5: 0.2CH2Cl2) calcd C,
65.0;H, 7.35;N, 10.8;found: C, 65.3;H, 7.45;N,
10.75.
141–142 ꢂC MS: MH+ 497. H NMR (DMSO) 9.05 (s,
1
1H), 8.59 (s, 1H), 8.43 (s, 1H), 8.05 (m, 1H), 8.00 (d,
J=10 Hz, 1H), 7.76 (m, 1H), 7.14 (m, 2H), 6.94 (m,
2H), 6.77 (m, 1H), 3.85 (s, 3H), 3.6 (m, 2H), 3.38 (m,
4H), 2.86 (m, 4H), 2.22 (s, 3H), 1.77 (m, 2H), 1.62 (m,
2H), 1.36 (m, 2H), 1.23 (d, J=8 Hz, 3H).
Preparation of 2-benzyloxycarbonylamino-3-[4-({2-[3-
methoxy-4-(3-o-tolyl-ureido)-phenyl]acetylamino}-methyl)-
piperidin-1-yl]propionic acid (39). A mixture aziridine-2-
carboxylic acid ethyl ester18 (1.15 g, 10 mmol), N-(ben-
zyloxycarbonyl)succinimide (2.5 g, 10 mmol) and diiso-
propylethylamine (1.95 mL, 10.2 mmol) in acetonitrile
and tetrahydrofuran (100 mL 1:1 v/v) were stirred at
ambient temperature for 4 h. The mixture was con-
centrated under vacuum and ethyl acetate (100 mL)
added. The solution washed with water (3ꢁ100 mL) and
brine (100 mL). The organic layer was dried over mag-
nesium sulphate, filtered and concentrated under
vacuum. The residue was subjected to flash chromato-
graphy on silica gel eluting with ethyl acetate and
cyclohexane (1:6, v/v) to afford 37 (1.4 g) as a pale yel-
low oil. This oil was dissolved in dimethylformamide
(5 mL) and triethylamine (1 mL) and 33 (542 mg,
1.32 mmol) added. The mixture was refluxed for 48 h.
This was then concentrated under vacuum and the resi-
due was subjected to flash chromatography on silica gel
eluting with ethyl acetate and methanol (95:5, v/v) to
afford 38 (180 mg) as a pale yellow solid which was dis-
solved in methanol (2 mL) and 1 M sodium hydroxide
(1 mL) added to the mixture. This was heated at 40 ꢂC
for 8 h and concentrated under vacuum. The residue
was taken up in the minimum volume of tetra-
hydrofuran, the pH adjusted to 4 with trifluoroacetic
acid and purified by HPLC, as described above, to
afford 39 (50 mg) as a tan solid: mp 128–129 ꢂC MS:
Preparation of 2-[3-Methoxy-4-(3-o-tolyl-ureido)-phenyl]-
N-piperidine-4-ylmethyl-acetamide (33). 4-({2-[3-Meth-
oxy-4-(3-o-tolyl-ureido)-phenyl]acetylamino}-methyl)-
piperidine-1-carboxylic acid tert-butyl ester (7.9 g,
15.5 mmol) was dissolved in a mixture of dichloro-
methane and trifluoroacetic acid (50 mL, 4:1, v/v) and
stirred at ambient temperature for 1 h. The solution was
concentrated under vacuum. The resulting oil was
rapidly stirred with 4 M sodium hydroxide for 5 min and
the resulting white precipitate filtered off and washed
with water (20ꢁ50 mL) and dried on high vacuum to
afford 33 (5.9 g, 92%) as a white solid: mp 154–160 ꢂC
MS: MH+ 411. 1H NMR (CDCl3) 8.26 (s, 1H), 8.22 (d,
J=10 Hz, 1H), 8.07 (s, 1H), 7.79 (m, 1H), 7.19 (m, 2H),
6.95 (m, 1H), 6.81 (m, 2H), 6.41 (m, 1H), 3.86 (s, 3H),
3.48 (s, 2H), 3.07 (m, 4H), 2.57 (m, 2H), 2.25 (s, 3H),
1.61 (m, 3H), 1.06 (m, 2H). Anal. (C23H30N4O3:
1
MH+ 632. H NMR (DMSO): 8.58 (s, 1H), 8.41 (s,
1H), 8.03 (m, 1H), 8.01 (d, J=10 Hz, 1H), 7.77 (m, 1H),
7.60 (m, 1H), 7.31 (m, 5H), 7.12 (m, 2H), 6.92 (m, 2H),
6.77 (m, 1H), 5.02 (s, 2H), 4.00 (m, 1H), 3.87 (s, 3H),
3.61 (m, 2H), 3.35 (m, 4H), 2.96 (m, 4H), 2.22 (s, 3H),
1.76 (m, 2H), 1.61 (m, 1H), 1.41 (m, 2H).

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P. C. Astles et al. / Bioorg. Med. Chem. 9 (2001) 2195–2202
7. Lin, K.-C.;Zimmerman, C. N.;Castro, A.;Lee, W.-C.;
Acknowledgements
Hammond, C. E.;Ateeq, H. S.;Hsiung, S. H.;Chong, L. T.;
Liao, Y.;Lobb, R. R.;Adams, S. P. J. Med. Chem. 1999, 42,
920.
8. Tilley, J. W.;Kaplan, G.;Rowan, K.;Schwinge, V.;
Wolitzky, B. Bioorg. Med. Chem. Lett. 2001, 11, 1.
9. Archibald, S. C.;Head, J. C.;Gozzard, N.;Howat, D. W.;
Parton, T. A.;Porter, J. R.;Robinson, M. K.;Shock, A.;
Warrellow, G. J.;Abraham, W. M. Bioorg. Med. Chem. Lett.
2000, 10, 997.
The authors would like to thank Mr. B. Wyman, Mr. J.
Shah and Mr. S. Watson for their technical contribu-
tions, Mr. R. Scammell, Miss D. Palmer and Mr. M.
Podmore for assistance with spectroscopy and Mr. C.
Bright and Miss M. Wong for supplying biological data.
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