Parallel synthesis of isatin-based serine protease inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00523-0

The synthesis of N-functionalised isatins using parallel, solution synthesis is described. Functionalised polymers were employed as stoichiometric and catalytic reagents as well as purification media in the exercise, and the derivatives were screened against a panel of serine proteases; high percentage inhibition was observed in several cases. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00523-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2501±2504
Parallel Synthesis of Isatin-Based Serine Protease Inhibitors
Stephen J. Shuttleworth,* Daniel Nasturica, Christian Gervais, M. Arshad Siddiqui,
Robert F. Rando and Nola Lee
BioChem Pharma Inc., 275 Armand-Frappier Boulevard, Laval, Quebec, H7V 4A7, Canada
Received 19 July 2000; accepted 30 August 2000
AbstractÐThe synthesis of N-functionalised isatins using parallel, solution synthesis is described. Functionalised polymers were
employed as stoichiometric and catalytic reagents as well as puri®cation media in the exercise, and the derivatives were screened
against a panel of serine proteases; high percentage inhibition was observed in several cases. # 2000 Elsevier Science Ltd. All rights
reserved.
ꢀ
As part of ongoing investigations into targeted libraries
based on privileged structural motifs, we turned our
attention to isatin. Over recent years, isatin derivatives
have been studied as putative acyl traps for serine and
cysteine proteases. In 1995, Iyer and co-workers repor-
ted that simple N-BOC and N-CBZ-isatin analogues
acted as reversible, slow-binding inhibitors of serine
proteases, and exhibited selectivity for a-chymotrypsin
over porcine pancreatic elastase.1 Webber and co-
workers at Agouron subsequently outlined a compre-
hensive and successful study of isatin-derived inhibitors
reaction mixtures were agitated at 45 C for 16 h, after
which the crude products were treated with a functiona-
4
lised amine resin to remove any unreacted electrophile.
2
of HRV 3C protease. In view of the evident potential
of isatin to serve as a biologically-relevant motif for
serine and cysteine proteases, we set out to make a series
of arrays that could serve as probes for these families of
enzyme targets. We chose to prepare a variety of ana-
logues, ranging from simple N-functionalised isatins to
more complex derivatives, and in all cases employed a
solution chemistry approach that was amenable to the
rapid, parallel synthesis and puri®cation of at least 10
milligram quantities of material.
Scheme 1. Preparation of N-alkylated isatin analogues.
Supported BEMP has been used successfully as a strong
base by other researchers in similar contexts.5 During
our method development studies, we observed complete
consumption of starting isatin using BEMP as the base,
and isolated yields of desired N-alkylated products were
high, typically exceeding 90% following polymeric
scavenging of excess electrophile. When we examined
the method using parallel synthesis, we obtained similar
encouraging results: complete conversion of isatins was
observed, and the desired products were isolated in
similarly high yields, generally with good levels of che-
,6
Scheme 1 outlines the method used for preparing of N-
alkylated isatins 1±10. For the initial deprotonation
step, we judged that use of a polymer-supported strong
base would be more suitable to high throughput parallel
synthesis than sodium hydride; accordingly, we treated
a series of 5- and 5,7-functionalised isatins with 2-tert-
butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-
1
mical purity as determined by H NMR and HPLC-MS
analysis.7 Random examples of the members of this
,8
array are summarised in Table 1.
3
diazaphosphorine on polystyrene (BEMP, 2.3 mmol/g
loading, 1.05 equiv) in acetonitrile, and quenched the
We then extended this exercise to the preparation of an
additional series of isatin analogues based loosely on the
neutrophil elastase inhibitors developed by Zeneca in
resulting anion with a-bromoketone (1.05 equiv). The
9
the mid-1990s. As for the approach outlined above, we
chose to prepare the analogues in solution using
*
0
Corresponding author. E-mail: sjshuttleworth@earthlink.net
960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00523-0

2
502
S. J. Shuttleworth et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2501±2504
supported reagents. 1,4-Piperazine was selected as the
core heterocycle in this study owing to its ubiquitous use
material was rapidly puri®ed on a cartridge pre-packed
with normal-phase silica using a 20-channel vacuum
manifold, eluting with ethyl acetate:hexanes (3:1).
11
10
12
in the area of protease inhibitors. The piperazine
moiety was attached to the isatin sca€old via a simple
acetyl linker. Reactive electrophilesÐexcesses of which
could be removed by polymeric scavengingÐwere
chosen to allow for the incorporation of a diverse range
of nitrogen capping groups, which encompassed polar
and lipophilic motifs. Scheme 2 outlines the synthetic
approach adopted, and results obtained in the exercise
are listed in Table 2.
This allowed for 11 to be isolated on a gram scale as a
white solid in 90% yield.13 Compound 11 was then
treated with isatin anions, generated as outlined above
using polymeric BEMP in acetonitrile, and, after 16 h at
ꢀ
45 C, the solutions were cooled to room temperature
and treated with functionalised amine resin to remove
any unreacted starting material. The resulting isatin
analogues, 12±17, were isolated in purities exceeding
9
0%, again following rapid puri®cation through pre-
Initially, mono-BOC-protected piperazine (1.0 weight)
in dichloromethane at 0 C was treated with chloracetyl
packed silica cartridges.11 These analogues were
prepared on a 100 mg scale, and were analysed by
ꢀ
chloride (1.05 equiv) and polymer-supported DMAP
1
14
400 MHz H NMR and LCMS. Consumption of
starting material was monitored in all cases, and
isolated yields of all analogues exceeded 80%. Libera-
tion of the corresponding free bases, 18±23, was
accomplished in quantitative yield using tri¯uoroacetic
acid (50%) in dichloromethane at room temperature.
Subsequent capping with isocyanates, and with a-bro-
moketones in the presence of polymer-supported
Hunigs base (3.0 mmol/g loading, 2.0 equiv),15 followed
by scavenging of excess electrophile with amine resin
furnished the desired products. All N-capping experiments
were conducted using stock solutions of starting
materials in acetonitrile at elevated temperature, using a
48-well Flex-Chem block.16 Consumption of electro-
philes was monitored by TLC, and the products 24±35
were subsequently isolated by ®ltration. The presence of
the ®nal products, with purities greater than 70%, was
con®rmed by LCMS.5
(
10 mol%, 2.0 mmol/g loading). The reaction mixture
was warmed to room temperature over 2 h, after which
the resin was removed by ®ltration. The resulting crude
Table 1. Data for examples of N-alkyl isatins prepared using parallel
synthesis
Compound
R1
R2
R3
R4
Isolated purity7,8
1
2
3
4
5
6
7
8
9
CH
H
H
3
H
H
H
H
H
H
H
H
H
H
CH
H
H
3
m-OCH -Ph
3
NH
p-Cl-Ph
tert-Bu
p-Br-Ph
>90
>90
>90
>90
>90
>90
>90
>90
40
2
H
CH
H
3
CH
H
3
CF
3
O
2-Naphthyl
H
CH
H
p-CH
CH
p-CH
p-F-Ph
3
-Ph
3
CH
H
3
3
NO
H
2
3
-Ph
1
0
H
63
Scheme 2. Synthesis of piperazine-functionalised isatin analogues using polymer-supported reagents.

S. J. Shuttleworth et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2501±2504
Table 2. Representative piperazine-functionalised isatin analogues
2503
We then evaluated the biological activity of the com-
pounds against a panel of mammalian serine proteases,
including human chymotrypsin, human leukocyte elas-
tase and human plasmin.17 Biological data for selected
compounds is listed in Table 3.18 As can be seen, good
activity was observed in a number of cases, though
selectivity between each enzyme target was limited.
Compound R1
R2
R3
Theoretical
mass
Mass
determined
(M+1)
2
4
NO
2
2
H
509.48
510.0
In summary we have outlined the convenient, parallel-
solution synthesis of isatin analogues using functionalised
polymers; several hundred analogues were prepared in
this exercise. The supported reagents were employed as
stoichiometric reagents, catalysts, and puri®cation
media. Activity of these compounds in serine protease
assays was observed.
2
2
2
2
2
3
3
3
3
3
3
5
6
7
8
9
0
1
2
3
4
5
NO
H
H
582.64
526.48
501.43
476.92
462.51
426.51
450.45
421.46
343.39
357.41
509.95
583.0
527.0
502.0
477.2
463.0
427.0
450.9
422.0
344.0
358.2
510.2
CF
3
3
O
O
CF
H
Cl
H
Acknowledgements
We would like to thank Mrs Andrea Petschner for her
assistance in this work, and Dr. Richard Storer and Dr.
Terry Bowlin for their comments.
CH
CH
H
3
CH
CH
H
3
3
3
References and Notes
H
H
1. Iyer, R. A.; Hanna, P. E. Bioorg. Med. Chem. Lett. 1995, 5,
8
2
9.
. Webber, S. E.; Tikhe, J.; Worland, S. T.; Fuhrman, S. A.;
H
H
Hendrickson, T. F.; Matthews, D. A.; Love, R. A.; Patick, A.
K.; Meador, A. W.; Ferre, R. A.; Brown, E. L.; DeLisle, D.
M.; Ford, S. E.; Binford, S. L. J. Med. Chem. 1996, 39, 5072.
3. Available from Fluka.
CH
3
H
4
. Aminomethyl polystyrene resin (1.0 equiv, 1.2 mmol/g
loading) from Novabiochem, or preferably a pre-packed car-
tridge containing an amine sorbent [bonded silica (aminopro-
pyl functionality) of 0.6 meq/g capacity] from IST
Technologies Ltd were both used e€ectively. Isolation of pro-
ducts in parallel was accomplished using a 20 channel vacuum
manifold available from IST Technologies Ltd.
Cl
H
5
Lett. 1998, 8, 1089.
6. Habermann, J.; Ley, S. V.; Scott, J. S. J. Chem. Soc., Per-
kin Trans. 1 1999, 1253.
. Xu, W.; Mohan, R.; Morrissey, M. M. Bioorg. Med. Chem.
1
7,18
Table 3. Percentage inhibition @ 20 mg/mL
Compound
Human
chymotrypsin
Human
leukocyte
elastase
Human
plasmin
7
4
. Purities were judged by HPLC-MS at 215 nm and by
1
00 MHz H NMR. In several cases, trituration with methyl-
tert-butyl ether greatly enhanced the levels of purity obtained,
and allowed for the products to be isolated as ®ne powders.
We judged the lower purities observed in entries 9 and 10 of
Table 1 to be unique cases, perhaps resulting from a degree of
experimental error, and not to be representative of the success
of the approach as a whole.
2
3
4
6
7
5
89
85
97
94
96
83
87
79
77
82
60
74
95
3
56
51
64
45
90
95
94
79
26
74
97
79
NA
29
87
55
86
84
89
77
82
77
57
80
66
61
NA
1
2
2
2
2
2
2
3
0
4
5
6
7
8
9
5
8
. Data: 1: d
s), 7.20 (1H, d), 6.90 (1H, s), 6.50 (1H, s), 5.10 (2H, s), 3.90
3H, s), 2.10 (3H, s), 2.05 (3H, s). M+1 found 324;
requires 323.35. 2: d (400 MHz, DMSO-d ) 7.80
H 6
(400 MHz, DMSO-d ) 7.40 (2H, m), 7.35 (1H,
(
C
19
H17NO
4
H
6
(1H, s), 7.70 (1H, t), 7.60 (1H, d), 7.30 (1H, s), 7.20 (1H, t),
7.00 (1H, d), 4.20 (2H, s). M+1 found 205; C H N O
3
10
8
2
requires 204,19. 3: d
H
6
(400 MHz, DMSO-d ) 8.10 (2H, d), 7.65
Chymostatin
0.5 mg/mL)
BOC-Ala-Ala-Ala-NH-Bz
100 mM)
Antiplasmin
0.5 mg/mL)
(
2H, d), 7.60 (2H, m), 7.20 (2H, m), 5.40 (2H, s). M+1 found
(
3
00; C H ClNO requires 299.72. 4: d (400 MHz, DMSO-
16 10 3 H
NA
NA
72
NA
92
d6) 7.70 (1H, d), 7.55 (1H, t), 7.20 (1H, t), 6.60 (1H, d), 4.80
1H, s), 1.30 (9H, s). M+1 found 246; C14 requires
45.28. 5: d (400 MHz, DMSO-d ) 7.80 (4H, 2d), 7.60 (1H,
d), 7.30 (1H, s), 7.00 (1H, d), 5.59 (1H, q), 2.20 (3H, s), 1.45
(
(
H15NO
3
a
2
NA
2
H
6
(

2
504
S. J. Shuttleworth et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2501±2504
(
(
2H, d). M+1 found 373; C18
400 MHz, DMSO-d ) 9.00 (1H, s), 8.20±7.90 (4H, m), 7.80±
H
14BrNO
3
requires 372.22. 6: d
H
requires 418.41. 15: d
(1H, d), 7.00 (1H, br d), 4.50 (2H, s), 3.80±3.40 (8H, m), 1.50
(9H, s). M+1 found 452; C20 requires 457.41. 16:
(400 MHz, CDCl ) 7.40 (1H, s), 7.05 (1H, s), 6.95 (1H, d),
H 3
(400 MHz, CDCl ) 7.60 (1H, s), 7.10
6
7
.60 (3H, m), 7.40 (1H, d), 7.00 (1H, d), 5.70 (2H, s). M+1
found 400; C21 NO requires 399.33. 7: d (400 MHz,
CDCl ) 8.00 (2H, d), 7.75 (1H, d), 7.60 (1H, t), 7.30 (2H, d),
22 3 3 6
H F N O
H
12
F
3
4
H
d
H
3
5.10 (2H, s), 3.80±3.40 (8H, m), 2.20 (3H, s), 1.50 (9H, s).
M+1 found 388; C H N O requires 387.44. 17: dH
3
7
.15 (1H, t), 6.70 (1H, d), 5.20 (1H, s), 2.45 (3H, s). M+1
found 280; C17 requires 279.30. 8: d (400 MHz,
CDCl ) 7.55 (1H, s), 7.45 (1H, d), 6.60 (1H, d), 5.00 (1H, q),
.30 (3H, s), 2.10 (3H, s), 1.55 (3H, d). M+1 found 231;
requires 231.25. 9: d (400 MHz, CDCl ) 8.50
1H, s), 8.40 (1H, d), 8.00 (2H, d), 8.40 (2H, d), 6.90 (1H, d),
.35 (2H, s), 2.20 (3H, s). M+1 found 325; C17
requires 324.30. 10: dH (400 MHz, CDCl ) 8.10 (2H, d), 7.75
20
25
3
5
H13NO
3
H
3
(400 MHz, CDCl ) 7.35 (1H, s), 7.05 (1H, s), 4.90 (2H, s),
3
3.80±3.40 (8H, m), 2.30 (3H, s), 2.20 (3H, s), 1.50 (9H, s).
M+1 found 402; C H N O requires 401.47.
2
C
(
5
21
27
3
5
13
H13NO
3
H
3
15. Available from Argonaut Technologies Inc.
16. Available from Robbins Scienti®c Inc.
17. Assay protocol: (a) Tris±HCl bu€er (50 mM pH 7.2 @
H
12
N
2
O
5
ꢀ
ꢀ
37 C); (b) glycerol 15%, incubation time 60 min @ 37 C ; (c)
100 mL reaction volume, 96-well plate format; (d) ¯uorescence
based assay; monitoring at excitation/emission wavelengths of
370/460 nm.
3
(
5
1H, d), 7.60 (1H, t), 7.20 (2H, d), 7.15 (1H, t), 6.70 (1H, d),
.20 (2H, s). M+1 found 284; C16
3
H10NFO requires 283.26.
9. Edwards, P. D.; Andisik, D. W.; Strimpler, A. M.; Gomes,
B.; Tuthill, P. A. J. Med. Chem. 1996, 39, 1112 and references
cited therein.
1
1
1
1
18. (a) Human chymotrypsin: Sigma # C 8946 (from human
pancreas) chymotrypsin in Rx: 800 pM; chymotrypsin sub-
strate: Bachem # I-1465 (Suc-Ala-Ala-Pro-Phe-AMC); sub-
0. As determined by MACCS II Drug Data Report analysis.
1. Available from Varian Inc.
2. Available from IST Technologies Ltd.
strate in Rx: 50 mM; K : 33 mM; control inhibitor: Sigma # C
m
7268 (chymostatin). (b) Human elastase: Sigma # E 8140
(from human leukocytes); elastase in Rx: 40 nM; elastase sub-
strate: Bachem # I-1335 (Suc-Ala-Pro-Ala-AMC); substrate in
3. Data 11: d
8H, m), 1.50 (9H, s). M+1 found 263; C11
H
(400 MHz, CDCl
3
) 4.05 (2H, s), 3.80±3.60
2 3
19ClN O
(
requires 262.74.
H
Rx: 100 mM; K : 92 mM; control inhibitor: Calbiochem #
m
1
7
1
1
4. Data: 12: d
.20 (1H, t), 6.95 (1H, br d), 4.60 (2H, s), 3.80±3.40 (8H, m),
.50 (9H, s). M+1 found 373; C19 requires 373.41.
3: dH (400 MHz, CDCl ) 7.70 (1H, s), 7.6 (1H, d), 6.95 (1H,
H
(400 MHz, CDCl
3
) 7.70 (1H, d), 7.6 (1H, t),
324692 (elastase inhibitor). (c) Human plasmin: Enzyme
Research Lab. (from human plasma); plasmin in Rx: 165 pM;
plasmin substrate: Bachem # I-1045 (H-Ala-Phe-Lys-AMC);
23 3 5
H N O
substrate in Rx: 25 mM; K : 19 mM; control inhibitor: Calbio-
3
m
br d), 4.60 (2H, s), 3.80±3.40 (8H, m), 1.50 (9H, s). M+1
found 408; C19 22ClN requires 407.86. 14: d (400 MHz,
CDCl ) 8.70 (1H, s), 8.50 (1H, d), 7.10 (1H, br d), 4.10 (2H, s),
2
chem # 178221 (a -antiplasmin). Isatins: dissolved in DMSO
H
3
O
5
H
@ 10 mg/mL; diluted @ 20 mg/mL in assay. Quenching:
¯uorescence quenching of compounds evaluated on free
AMC in the same bu€er as the enzymatic assays.
3
3
22 4 7
.70±3.40 (8H, m), 1.50 (9H, s). M+1 found 419; C19H N O