6-Acylamino-2-[(alkylsulfonyl)oxy]-1H-isoindole-1,3-dione mechanism- based inhibitors of human leukocyte elastase

Article abstract of DOI:10.1016/S0960-894X(99)00588-0

A study of various 2-[(alkylsulfonyl)oxy]-6-substituted-1H-isoindole- 1,3-diones' inhibition of chymotrypsin compared to inhibition of HLE reveals that acylamino substitution in the 6-position increases selectivity and potency of these inhibitors for HLE.

Full text of DOI:10.1016/S0960-894X(99)00588-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 27±30
6-Acylamino-2-[(alkylsulfonyl)oxy]-1H-isoindole-1,3-dione
Mechanism-Based Inhibitors of Human Leukocyte Elastase
John E. Kerrigan, a,b,* Matthew C. Walters, a Kerri J. Forrester, a Jason B. Crowder a
and Lisa J. Christopher a,y
aDepartment of Chemistry, University of South Alabama, Mobile, AL 36688, USA
bDepartment of Pharmaceutical Chemistry, College of Pharmacy, Rutgers University, 160 Frelinghuysen Road, Piscataway,
NJ 08854, USA
Received 25 August 1999; accepted 8 October 1999
AbstractÐA study of various 2-[(alkylsulfonyl)oxy]-6-substituted-1H-isoindole-1,3-diones' inhibition of chymotrypsin compared to
inhibition of HLE reveals that acylamino substitution in the 6-position increases selectivity and potency of these inhibitors for HLE.
The best HLE inhibitor in this series was 6-(methylglutaryl)amino-2-[(ethylsulfonyl)oxy]-1H-isoindole-1,3-dione with a kobs/[I]=
220,000 M 1 s 1. # 1999 Elsevier Science Ltd. All rights reserved.
Chronic obstructive pulmonary disease (COPD) is a
growing concern as the nation's population steadily
ages. In the United States alone, more than 15 million
Americans su€ered from COPD in 1994.1 The 1994 ®g-
ure is a substantial increase from the 1986 ®gure of 10
million.2 Pulmonary emphysema and chronic bronchitis
are two major components of COPD. Human leukocyte
elastase (HLE; EC 3.4.21.37), a digestive serine protease
found in polymorphonuclear leukocytes, has been
me's active site. A wide range of mechanism-based
inhibitors have been developed to inhibit serine pro-
teases: 3,4-dichloroisocoumarin,10 3-alkoxy-7-amino-4-
chloroisocoumarins,11,12 haloenol lactones,13 N-((alkyl-
sulfonyl)oxy)succinimides,14,15 saccharin derivatives,16
1,2,5-thiadiazolidin-3-one-1,1-dioxides,17,18 isoxazo-
lines,19 sulfonyloxyketones20 and N-((alkylsulfonyl)-
oxy)phthalimides.21
implicated as a causative agent of emphysema.3,4
A
The N-((alkylsulfonyl)oxy)phthalimides are an attrac-
tive structural target for further development. Neumann
and Gutschow found that simple N-((alkylsulfonyl)-
oxy)phthalimides are very e€ective inhibitors of chy-
motrypsin (ChT) and HLE in vitro.21 While Neumann's
compounds are indeed very good inhibitors of HLE,
they are not selective for HLE.
delicate balance between HLE and a-1-antitrypsin, a
natural inhibitor of HLE, can be upset by a long history
of cigarette smoking.5,6 Cigarette smoking reduces the
association rate constant of a-1-antitrypsin for HLE in
the lower respiratory tract.7
A wide variety of di€erent types of inhibitors of HLE
are known.8 Mechanism-based inhibitors have the
advantage of being, chemically, fairly inactive until they
reach their target enzyme.9 These inhibitors take
advantage of binding speci®city to accomplish their
goal. The enzyme recognizes the inhibitor as a substrate.
The enzyme cleaves the inhibitor, releasing a reactive
functionality. The reactive functional group covalently
bonds to the enzyme, and e€ectively blocks the enzy-
The N-((alkylsulfonyl)oxy)succinimide inhibitors, upon
acylation of the catalytic serine residue, release a sul-
phone amide anion group (Fig. 1) that undergoes a
Lossen rearrangement to release a latent isocyanate A.22
The released isocyanate reacts with the catalytic histi-
dine residue forming an imidazole-N-carboxamide B,
irreversibly inactivating the enzyme. Experimental evi-
dence in support of the formation of the imidazole-N-
carboxamide B in the inhibition of a-chymotrypsin by
N-((methylsulfonyl)oxy)succinimides has been demon-
strated by 13C NMR studies.23 The reactivation studies
of chymotrypsin inactivated by N-((alkylsulfonyl)oxy)-
phthalimides provides additional support for this
*Corresponding author. Tel.: +1-732-445-5763; fax: +1-732-445-
6312.
yPresent address: Bristol-Myers Squibb Co., PO Box 191, New
Brunswick, NJ 08903-0191, USA.
0960-894X/00/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00588-0

28
J. E. Kerrigan et al. / Bioorg. Med. Chem. Lett. 10 (2000) 27±30
Figure 1. Mechanism of inhibition.
mechanism.21 It was found that only a maximum of
25% activity was restored for the mesyloxy (R0=Me)
and 18% activity restored for the dansyloxy (R0=
Dansyl).21 Neumann and Gutschow have suggested a
similar mechanism for their N-((alkylsulfonyl)oxy)-
phthalimides (R=H).21
For the preparation of compound 5, tosyl-l-phenyl-
alanine acid chloride was prepared from tosyl-l-phenyl-
alanine in re¯uxing SOCl2. Compound 12 (R=ethyl)
was prepared from 3 via the same procedure used to
prepare compound 4 (R=methyl). Compounds 13±15
were prepared from 12 as shown in Scheme 1 below.
Powers et al. observed that hydrophobic side-chains
enhanced potency and selectivity of their mechanism-
based isocoumarin inhibitors for HLE.11 Based on
structural similarity between the isocoumarin and
phthalimide ring systems (Fig. 2), we surmised that 6-
Discussion
Most of the compounds in this study exhibited good
selectivity for HLE over chymotrypsin (see Table 1).
The notable exception was the phenylalanine mimic 6,
which was only a 2-fold better inhibitor of HLE over
chymotrypsin and was the least potent. The result with
compound 6 indicates that the (tosyl)amino group in the
Tos-l-Phe derivative 5 may play an important role in
binding to the enzyme. Inclusion of a trans double bond
(R0=trans-PhCHˆCHCONH) in derivative 7 moder-
ately increased inhibitor selectivity and potency for
HLE relative to compound 6. The (R0=methylglutaryl/
R=CH3CH2) derivative 15 was the most potent and
acylamino-substituted-1H-isoindole-1,3-diones
might
bind to HLE in a similar fashion and bene®t from
hydrophobic substituents in the 6-position.
Synthesis
Compound 4 was synthesized via our previously pub-
lished procedure.24 Compounds 5±11 were synthesized
from 4 via reaction with the appropriate acid chloride.25
Figure 2. Isocoumarins and sulfonyloxy phthalimides.
Scheme 1. Synthesis of acylaminosulfonyloxy phthalimides.25

J. E. Kerrigan et al. / Bioorg. Med. Chem. Lett. 10 (2000) 27±30
29
Table 1. Inhibition of chymotrypsin and human leukocyte elastase by 6-acylamino-2-[(alkylsulfonyl)oxy]-1H-isoindole-1,3-dionesa
1
1
kobs/[I] (M
s )
Compound
R0
R
Chymotrypsin
HLE
kobs/[I] Ratio HLE/ChT
4
5
6
7
8
9
10
11
12
13
14
15
NH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
12,000
11,000
4,800
1,600
3,600
3,300
5,400
6,600
13,000
5,300
9,600
14,000
110,000
160,000
9,700
9.2
15
2.0
13
6.9
7.6
5.6
8.6
4.6
11
Tos-L-PheCONH
PhCH2CH2CONH
trans-PhCH=CHCONH
PhCONH
(1-Naphthyl)CONH
CH3OCOCONH
CH3OCO(CH2)3CONH
NH2
20,000
25,000
25,000
30,000
57,000
60,000
56,000
25,000
220,000
CH3OCOCONH
CH3OCO(CH2)2CONH
CH3OCO(CH2)3CONH
2.6
16
aConditions: 0.1 M HEPES, 0.5 M NaCl, pH 7.5 at 25 ꢀC and <6% DMSO. Substrate used for chymotrypsin was Suc-Ala-Ala-Pro-Phe-pNA
([S]=80 mM; [E]=4 nM). Substrate used for HLE was MeO-Suc-Ala-Ala-Pro-Val-pNA ([S]=100 mM; [E]=30 nM). All values were calculated from
the initial part of the progress curve via the method of Tian and Tsou (Tian, W.; Tsou, C. Biochemistry 1982, 21, 1028). All data was collected in
triplicate with an average relative standard deviation of ‹20%. All log plots had R2ꢁ0.99.
Mortality: Intra and Intercounty Di€erences; Hensley, M. J.;
Saunders, N. A., Eds.; Marcel Dekker: New York, 1989, pp.
24±30.
3. Jano€, A. Am. Rev. Respir. Dis. 1985, 132, 417.
4. Snider, G. L.; Lucey, E. C.; Stone, P. J. Am. Rev. Respir.
Dis. 1986, 133, 149.
5. Johnson, D.; Travis, J. J. Biol. Chem. 1978, 253, 7142.
6. Beatty, K.; Matteson, N.; Travis, J. Hoppe-Seyler's Z. Phy-
siol. Chem. 1984, 365, 731.
7. Ogushi, F.; Hubbard, R. C.; Vogelmeier, C.; Fells, G. A.;
Crystal, R. G. J. Clin. Invest. 1991, 87, 1060.
most selective inhibitor of HLE in this series of com-
pounds. The tosyl-l-Phe derivative 5 is the next most
selective and potent HLE inhibitor. Most of the other
analogues in this series of compounds were at least
sevenfold more potent inhibitors of HLE relative to
inhibition of chymotrypsin. Changing the R group from
methyl (10 and 11) to ethyl (13 and 15) resulted in a
2- to 4-fold increase in HLE inhibition with the parent
amino compound being the exception. The ethyl analo-
gues 13 and 15 show promise for further development.
8. Bernstein, P. R.; Edwards, P. D.; Williams, J. C. Inhibitors
of Human Leukocyte Elastase; Ellis, G. W., Luscombe, D. K.,
Eds.; Elsevier: Amsterdam, 1994; Vol. 31, pp. 59±120.
9. Powers, J. C.; Harper, J. W. In Proteinase Inhibitors; Barret,
A. J.; Salvensen, G., Eds.; Elsevier: New York, 1986, pp. 131±152.
10. Harper, J. W.; Hemmi, K.; Powers, J. C. Biochemistry
1985, 24, 1831.
In conclusion, we have demonstrated that hydrophobic
substituents in the 6-position enhance potency and
selectivity of mechanism-based sulfonyloxy phthalimide
inhibitors of HLE over chymotrypsin. The aromatic
ring moiety in phenylalanine is well tolerated, as is the
¯exible methyl ester chain of the methylglutaryl group.
11. Kerrigan, J. E.; Oleksyszyn, J.; Kam, C.; Selzler, J.; Pow-
ers, J. C. J. Med. Chem. 1995, 38, 544.
12. Harper, J. W.; Powers, J. C. Biochemistry 1985, 24, 7200.
13. Daniels, S. B.; Cooney, E.; So®a, M. J.; Chakravarty, P.
K.; Katzenellenbogen, J. A. J. Biol. Chem. 1983, 258, 15046.
14. Groutas, W. C.; Brubaker, M. J.; Venkataraman, R.;
Stanga, M. A. Arch. Biochem. Biophys. 1992, 297, 144.
15. Groutas, W. C.; Brubaker, M. J.; Chong, L. S.; Venka-
taraman, R.; Huang, H.; Epp, J. B.; Kuang, R.; Hoidal, J. R.
Bioorg. Med. Chem. 1995, 3, 375.
Acknowledgements
The authors wish to thank the National Institutes of
Health (HL58190-01) for generous support of this work.
High ®eld proton NMR spectra were recorded either
at USA on a Jeol Eclipse 300 MHz NMR or on a
Bruker 400 MHz NMR spectrometer at the UAB
Comprehensive Cancer Center, NMR Core Facility,
Birmingham, AL.
16. Groutas, W. C.; Chong, L. S.; Venkataraman, R.; Epp, J.
B.; Kuang, R.; Houser-Archield, N.; Hoidal, J. R. Bioorg.
Med. Chem. 1995, 3, 187.
17. Groutas, W. C.; Kuang, R.; Ruan, S.; Epp, J. B.; Venka-
taraman, R.; Truong, T. M. Bioorg. Med. Chem. 1998, 6, 661.
18. Kuang, R.; Venkataraman, R.; Ruan, S.; Groutas, W. C.
Bioorg. Med. Chem. Lett. 1998, 8, 539.
References and Notes
1. Peterson, M.; Rahr, R.; Blessing, D.; Ayachi, S. Physician
Assistant 1995, 19, 39.
2. Higgins, M. W.; Thom, T. In Incidence, Prevalence and
19. Groutas, W. C.; Venkataraman, R.; Chong, L. S.; Yoder,
J. E.; Epp, J. B.; Stanga, M. A.; Kim, E. Bioorg. Med. Chem.
1995, 3, 125.

30
J. E. Kerrigan et al. / Bioorg. Med. Chem. Lett. 10 (2000) 27±30
20. Ohba, T.; Wakayama, J.; Ikeda, E.; Takei, H. Bioorg.
Med. Chem. Lett. 1996, 6, 1127.
21. Neumann, U.; Gutschow, M. J. Biol. Chem. 1994, 269, 21561.
22. Groutas, W. C.; Giri, P. K.; Crowley, J. P.; Castrisos, J.
C.; Brubaker, M. J. Biochem. Biophys. Res. Commun. 1986,
141, 741.
23. Groutas, W. C.; Stanga, M. A.; Brubaker, M. J. J. Am.
Chem. Soc. 1989, 111, 1931.
24. Kerrigan, J. E.; Shirley, J. J. Bioorg. Med. Chem. Lett.
1996, 6, 451.
(DMSO, 300 MHz) d 3.66 (s, 3H), 7.60 (m, 3H), 8.01 (m, 3H),
8.26 (dd, J=8.3, 1.9 Hz, 1H), 8.46 (d, J=1.7 Hz, 1H), 10.94
(s, 1H) ppm; FABHRMS calcd for [C16H12N2O6S+H]+
361.0494, found 361.0560.
6-(1-Naphthoyl)amino-2-[(methylsulfonyl)oxy]-1H-isoindole-1,3-
dione (9). Ethanol (350 mg, 88%); mp 250±252 ꢀC dec; 1H
NMR (DMSO, 300 MHz) d 3.67 (s, 3H), 7.67 (m, 3H), 8.07
(m, 4H), 8.30 (dd, J=8.2, 1.9 Hz, 1H), 8.50 (d, J=1.9 Hz,
1H), 8.66 (s, 1H), 11.10 (s, 1H) ppm; FABHRMS calcd for
[C20H14N2O6S+H]+ 411.0651, found 411.0704.
25. General procedure for the preparation of acylamino analo-
gues (5±11 and 13±15). Triethylamine (0.20 mL, 1.6 mmol) was
added to a stirred solution of compound 4 or 12 (200 mg, 0.8
mmol) and the appropriate acid chloride (1.6 mmol) in THF
(5 mL) at room temperature. The mixture was allowed to stir
overnight at room temperature. The mixture was diluted in
ethyl acetate (15 mL), washed with 10% HCl (8 mL), satu-
rated NaHCO3 (8 mL), brine (8 mL), dried (Na2SO4) and
concentrated. The crude solid obtained was recrystallized in
the solvent indicated.
6-(Tosyl-L-phenylalanyl)amino-2-[(methylsulfonyl)oxy]-1H-iso-
indole-1,3-dione (5). Methanol (220 mg, 53%): mp 243±244 ꢀC
dec; 1H NMR (DMSO, 300 MHz) d 2.19 (s, 3H), 2.81 (dd,
J=13.6, 9.4 Hz, 1H), 2.93 (dd, J=13.5, 9.4 Hz, 1H), 3.65 (s,
3H), 4.14 (q, J=9.4 Hz, 1H), 7.13 (d, J=8.0 Hz, 2H), 7.21 (m,
4H), 7.50 (d, J=8.0 Hz, 2H), 7.63 (d, J=8.8 Hz, 1H), 7.90 (d,
J=9.1 Hz, 2H), 8.33 (s, 1H), 8.45 (d, J=9.3 Hz, 1H), 10.64
(s, 1H) ppm; FABHRMS calcd for [C25H23N3O8S2+H]+
558.1005, found 558.0946.
6-(Oxalyl)amino-2-[(methylsulfonyl)oxy]-1H-isoindole-1,3-dione
(10). Ethanol (213 mg, 80%); mp 232±235 ꢀC; 1H NMR
(DMSO, 400 MHz) d 3.64 (s, 3H), 3.89 (s, 3H), 7.99 (d, J=8.3
Hz, 1H), 8.24 (dd, J=8.3, 1.7 Hz, 1H), 8.37 (d, J=1.6 Hz,
1H), 11.45 (s, 1H) ppm; FABHRMS calcd for [C12H10N2
O8S+H]+ 343.0236, found 343.0291.
6-(Methylglutaryl)amino-2-[(methylsulfonyl)oxy]-1H-isoindole-
1,3-dione (11). Ethanol (160 mg, 53%); mp 166±167 ꢀC;
1H NMR (DMSO, 400 MHz) d 1.86 (m, 2H), 2.39 (t,
J=7.3 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 3.60 (s, 3H), 3.63
(3H), 7.91 (m, 2H), 8.26 (d, J=0.8 Hz, 1H), 10.63 (s, 1H) ppm;
FABHRMS calcd for [C15H16N2O8S+H]+ 385.0706, found
385.0715.
6-(Oxalyl)amino-2-[(ethylsulfonyl)oxy]-1H-isoindole-1,3-dione
(13). Ethanol (250 mg, 69%); mp 210±215 ꢀC dec; H NMR
1
(DMSO, 400 MHz) d 1.47 (t, J=7.2 Hz, 3H), 3.79 (q, J=7.2
Hz, 2H), 3.88 (s, 3H), 7.98 (d, J=8.3 Hz, 1H), 8.24 (dd,
J=8.3, 1.8 Hz, 1H), 8.36 (d, J=1.6 Hz, 1H), 11.45 (s, 1H)
ppm; FABHRMS calcd for [C13H12N2O8S+H]+ 357.0393,
found 357.0470.
6-(Methylsuccinyl)amino-2-[(ethylsulfonyl)oxy]-1H-isoindole-
1,3-dione (14). Ethanol (196 mg, 69%); mp 198±200 ꢀC; 1H
NMR (DMSO, 400 MHz) d 1.46 (t, J=6.4 Hz, 3H), 2.64 (t,
J=5.5 Hz, 2H), 2.79 (t, J=5.6 Hz, 2H), 3.60 (s, 3H), 3.77 (q,
J=6.3 Hz, 2H), 7.92 (m, 2H), 8.24 (s, 1H), 10.74 (s, 1H) ppm;
FABHRMS calcd for [C15H16N2O8S+H]+ 385.0706, found
385.0684.
6-(Dihydrocinnamyl)amino-2-[(methylsulfonyl)oxy]-1H-isoindole-
1,3-dione (6). Ethyl acetate (130 mg, 43%); mp 194±196 ꢀC; 1H
NMR (DMSO, 300 MHz) d 2.73 (t, J=7.7 Hz, 2H), 2.94 (t,
J=7.4 Hz, 2H), 3.63 (s, 3H), 7.27 (m, 6H), 7.92 (s, 1H), 8.29
(s, 1H), 10.75 (s, 1H) ppm; FABHRMS calcd for [C18H16
N2O6S+H]+ 389.0807, found 389.0814.
6-(trans-Cinnamyl)amino-2-[(methylsulfonyl)oxy]-1H-isoindole-
1,3-dione (7). Ethyl acetate (60 mg, 30%); mp 243 ꢀC dec; H
1
NMR (DMSO, 300 MHz) d 3.65 (s, 3H), 6.85 (d, J=15.9 Hz,
1H), 7.46 (m, 3H), 7.68 (m, 3H), 7.96 (d, J=8.3 Hz, 1H), 8.04
(dd, J=8.2, 1.7 Hz, 1H), 8.41 (d, J=1.7 Hz, 1H), 10.99 (s, 1H)
ppm; FABHRMS calcd for [C18H14N2O6S+H]+ 387.0651,
found 387.0709.
6-(Methylglutaryl)amino-2-[(ethylsulfonyl)oxy]-1H-isoindole-
1,3-dione (15). Ethanol (26 mg, 9%); mp 114±116 ꢀC; 1H
NMR (DMSO, 400 MHz) d 1.47 (t, J=7.3 Hz, 3H), 1.86 (m,
2H), 2.39 (t, J=7.3 Hz, 2H), 2.46 (t, J=7.4 Hz, 2H), 3.59 (s,
3H), 3.78 (q, J=7.3 Hz, 2H), 7.93 (m, 2H), 8.26 (s, 1H),
10.64 (s, 1H) ppm; FABHRMS calcd for [C16H18N2O8S+H]+
399.0862, found 399.0943.
6-(Benzoyl)amino-2-[(methylsulfonyl)oxy]-1H-isoindole-1,3-dione
(8). Ethanol (70 mg, 20%); mp 200±202 ꢀC dec; 1H NMR