Synthesis and evaluation of δ-lactams (piperazones) as elastase inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00995-2

A series of monocyclic δ-lactams (piperazones) was prepared and analysed as inhibitors of porcine pancreatic elastase and human neutrophil elastase.

Full text of DOI:10.1016/S0960-894X(02)00995-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 387–389
Synthesis and Evaluation of ꢀ-Lactams (Piperazones) as
Elastase Inhibitors
Jurgen Seibel,a David Brown,c Augustin Amour,c Simon J. Macdonald,b
Neil J. Oldhama and Christopher J. Schofielda,*
aThe Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, UK
bMedicinal Chemistry 1, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
cSystems Research, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
Received 1 August 2002; revised 28 October 2002; accepted 6 November 2002
Abstract—A series of monocyclic d-lactams (piperazones) was prepared and analysed as inhibitors of porcine pancreatic elastase
and human neutrophil elastase.
# 2002 Elsevier Science Ltd. All rights reserved.
Serine proteases and related enzymes are a diverse and
ancient family of hydrolytic enzymes that share com-
mon active site characteristics including a nucleophilic
serine residue and an ‘oxy-anion’ hole.1 Included in the
best characterized members of the family are the pan-
creatic serine proteases, trypsin, and chymotrypsin.
Elastases are relatively non-selective enzymes belonging
to the chymoptrypsin sub-family. They have been
implicated in the development of various human dis-
eases including emphysema, cystic fibrosis, and rheu-
matoid arthritis.2 Elastases and other serine proteases,
including those involved in blood clotting and infection,
are current targets for medicinal chemistry.
b-Lactam inhibitors of serine proteases react to form
relatively stable acyl–enzyme complexes (cf. that
involved in catalysis) via ring-opening of the lactam by
the nucleophilic side chain of Ser-195.4ꢀ7 In one case the
stability of the acyl–enzyme complex is related to rota-
tion of the carbonyl group of the ester link out of the
oxy-anion hole.6
5,5-trans-Fused g-lactams are potent inhibitors of
human neutrophil elastase (HNE); IC50 values of
<0.05 mM being observed in some cases.8,9 Certain
monocyclic g-lactams (3) have also been shown to inhi-
bit elastase, but are relatively weak inhibitors compared
to their b-lactam analogues. In one g-lactam series, this
is due, in part at least, to reversible formation of the
g-lactam from the acyl–enzyme and in another series
also due to the hydrolytic lability of the acyl–enzyme in
which the ester carbonyl was located in the oxyanion
hole. Hydrolytically stable acyl–enzyme complexes are
thus only formed when the hydrolytic water molecule
itself is displaced or another aspect of the catalytic
machinery is perturbed.10,11
Many different types of serine protease inhibitor have
been developed. Amongst those forming a covalent bond
with the target enzyme, extensive studies have been carried
out with acylating agents, including studies on the inhibi-
tion of elastases by both monocyclic b-lactams (1) and
bicyclic b-lactams (2).3 Both a sufficient rate of formation
and a threshold stability of the complex are required for
useful inhibition via an acyl–enzyme complex.
Imming et al. have reported an interesting NMR study
on the rates of hydrolysis of a series of lactams.12
g-Butyrolactam was found to be hydrolyzed con-
siderably slower than b-propiolactam, but it was noted
that ‘d-valerolactam and b-propiolactam had the same
reactivity’. Penicillin antibiotics were more reactive than
both by approximately a factor of 103. Medium-sized
lactams were the least susceptible lactams to hydrolysis.
*Corresponding author. Fax: +44-1865-275674; e-mail:
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00995-2

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J. Seibel et al. / Bioorg. Med. Chem. Lett. 13 (2003) 387–389
This study suggested that d-lactams have potential for
use as antibacterials and serine protease inhibitors.
piperaz-2-ones via variation of the starting materials
and/or the N-substituents.
Here, we report the synthesis of a series of monocyclic
d-lactams/piperaz-2-ones and its evaluation for elastase
inhibition. The choice of target took into account pre-
vious structure–activity work on b- and g-lactams with
respect to the group a- to the lactam carbonyl and the
N-substituent. The choice of piperaz-2-ones as templates
reflected their potential ease of synthesis, including a
desire to minimize the number of chiral centres, and the
possibility of modification in a ‘combinatorial’ manner.
Positive ion electrospray mass spectrometry (ESI-MS)
studies on the interaction of 15b, 19, 20 and 21 with
PPE gave rise to mass shifts corresponding to the
formation of both 1:1 and 1:2 complexes (Table 1). In
each case the E*I2 complexes were observed at lower
levels (ca. 10% relative intensity of E+EI+EI2). For
15b, further MS analyses were carried out. No peak was
observed for the anticipated hydrolysis product 22.
Further incubation of 22 with PPE in H2O18 followed
by ESI-MS analysis did not result in incorporation of
18O label into 22. This result contrasts with that
obtained with certain monocyclic-g-lactam inhibitors 3
under analogous conditions, where clear evidence for
acylation of Ser-195 was obtained.6
The first explored route involved lactam formation as
the final or penultimate step. Alkylation of a tert-butyl
ester derivative of isoleucine 5 with (2-chloro-ethyl)car-
bamic acid tert-butyl ester 4 followed by double-depro-
tection of amine
6 with CF3CO2H yielded the
cyclization precursor 7. Lactamization could be achieved
with N,N0-dicyclohexylcarbodiimide (DCC) and pyridine
in moderate yields to give the d-lactam 8. Sulfamoylation
of both amide and amine nitrogens was achieved by
reaction 2.1 equiv BuLi in THF at 0 ꢁC followed by TsCl
(2.2 equiv) to provide 9. Although this approach could be
improved by differential N-protection, it was limited since
lactam formation could not be achieved (in sufficiently
high yield if at all) when electron withdrawing groups
(e.g., para-toluenesulphonyl) were present on the primary
amine of the cyclisation precursor (Scheme 1).
The lactam derivatives (15), (19), (20) and (21) were
tested for inhibition of both HNE and PPE by standard
kinetic analyses.6,13 No inhibition of either was
observed for either enzyme for 8ab, 9, 14ab, 15a, or
18a,b. In some cases, for example, 9, lack of solubi-
lity was problematic and may explain the lack of
observed inhibition for these and other compounds.
Weak inhibition of PPE, but not HNE, was observed
in the case of 16b (IC50 ca. 2000 mM), 19 (IC50 ca.
293 mM), and 20 (IC50 923 mM).14 Each of these three
compounds displayed apparent slow binding kinetics.
Compound 15b was a more potent inhibitor, but was
still slow binding with IC50 values of 28 and 62 mM
versus HNE and PPE, respectively. In the latter case
a Ki value of 154 mM was obtained. The apparent
difference in activity between 15a (inactive) and 15b
(active) which differ only in the substituent a- to the lactam
carbonyl, being 1-methylpropyl and isobutyl in the latter is
interesting and supports the argument that in order to
achieve inhibition correct fit is as important as reactivity of
the amide moiety. In this regard it is also notable that the
most active compound (15b) has an N-benzyl substituent
on its lactam nitrogen and is thus chemically less reactive
than the targets with N-sulphonyl groups most of which
were inactive.
A second approach was based on the Mitsunobu cycli-
zation reaction and is exemplified in Scheme 2. This route
proved to be straightforward and flexible. Coupling of
N-t-Boc amino acids 11 with N-benzylethanolamine 10
mediated by 1,10-carbonyldiimidazole (CDI) gave 12.
Subsequent removal of the Boc group followed by
cyclization via the Mitsunobu protocol gave the
piperazin-2-ones 14 in 66–68% overall yield from 11.
Different N-substituents could be introduced via Boc
protection and removal of the benzyl group by Birch
reduction (Schemes 3 and 4). The amides were reacted
with LHMDS (lithium hexamethyldisilazide) or nBuLi,
followed by reaction with TsCl or MsCl to form targets
19, 20, 21.
In conclusion, we have prepared a series of piperaz-2-
ones/d-lactams, containing a single chiral centre, via
synthetic routes that are amenable to optimization by
Both synthetic strategies, but particularly the latter, are
amenable to the production of large numbers of
Scheme 1. Synthesis of d-lactams. Reagents and conditions: (a) (iPr2)EtN (2 equiv), (NBu4)I, MeCN, reflux, 12 h; (b) CF3CO2H/CH2Cl2 (1:1), rt,
3 h; (c) DCC, pyridine (2 equiv), MeCN, rt, 6 h; (d) BuLi (2.1 equiv), THF, TsCl (2.2 equiv), 0 ꢁC, 3 h.

J. Seibel et al. / Bioorg. Med. Chem. Lett. 13 (2003) 387–389
389
Scheme 2. Synthesis of d-lactams using a Mitsunobu strategy. Reagents and conditions: (a) 11, CDI, THF, rt, 1 h, then 10, THF, 12 h; (b) TFA/
CH2Cl2 (1:2), 0 ꢁC, 90 min; (c) PPh3 (1.3 equiv), DEAD (1.3 equiv), THF, rt, 5 h; (d) NEt3 (1.5 equiv), TsCl (1.3 equiv), CH2Cl2, rt, 12 h; (e) NEt3
(1.1 equiv), MsCl (1.1 equiv), CH2Cl2, rt, 12 h.
of Imming12 that d-lactams be evaluated as inhibitors of
serine-enzymes.
References and Notes
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B. G.; Chabin, R.; Shah, S.; Mumford, R.; Dickinson, T. A.;
Griffin, P. R. Biochemistry 1993, 22, 2031.
Scheme 3. Reagents and conditions: (a) Boc2O (1.2 equiv), CH2Cl2, rt,
12 h; (b) Na, NH3, THF, ꢀ78 ꢁC, 20 min.
6. Wilmouth, R. C.; Westwood, N. J.; Anderson, K.; Brown-
lee, W.; Claridge, T. D. W.; Clifton, I. J.; Pritchard, G. J.;
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J. Med. Chem. 1998, 41, 3919.
Scheme 4. Reagents and conditions: (a) LHMDS (1 equiv), MsCl
(1.2 equiv), THF, ꢀ78 ꢁC, 2.5 h; (b) TFA/CH2Cl2 (1:1), 0 ꢁC, 90 min;
(c) BuLi (1.3 equiv), THF, TsCl (1.5 equiv), 0 ꢁC, 3 h.
Table 1. ESIMS studies on the Interaction of d-Lactams with PPEa
9. Macdonald, S. J. F.; Dowle, M. D.; Harrison, L. A.; Shah,
P.; Johnson, M. R.; Inglis, G. G. A.; Clarke, G. D. E.; Smith,
R. A.; Humphreys, D.; Molloy, C. R.; Amour, A.; Dixon, M.;
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Singh, O. M. P.; Kumar, K. A.; Fleetwood, G.; Hodgson,
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Compd Mass inhibitor PPE
E/I
Relative int. (%)b Diff.c
15b
19
20
21
400.5
410.5
334.4
234.3
25,904 26,303
25,908 26,317
25,901 26,236
25,903 26,137
42
74
64
28
399
409
335
234
aIncubations contained PPE/inhibitor in a 1:5 ratio (except for com-
pound 15b which was a 1:2 ratio). The incubation times for 15b, 19, 20
and 21 were 30, 12, 5 and 10 min.
bRelative intensity refers to the intensity of the adduct peaks normal-
ized to E+EI+EI2.
cMass shift relative to unmodified PPE observed in the same analysis.
solid phase synthesis. Some of the compounds were
shown to be inhibitors of PPE. Whilst inhibition has not
been shown to occur via acylation, and other modes of
inhibition are possible, the results support the proposal
14. Values are means of at least three experiments.