Inhibitors of human neutrophil elastase based on a highly functionalized N-amino-4-imidazolidinone scaffold

Article abstract of DOI:10.1016/j.ejmech.2010.06.028

A series of compounds based on the N-amino-4-imidazolidinone scaffold was synthesized and screened against human neutrophil elastase (HNE). These studies lead to the identification of a selective, low micromolar reversible competitive inhibitor of HNE. A series of derivatives based on the N-amino-4- imidazolidinone scaffold (I) was synthesized and shown to inhibit human neutrophil elastase.

Full text of DOI:10.1016/j.ejmech.2010.06.028

European Journal of Medicinal Chemistry 45 (2010) 4280e4287
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibitors of human neutrophil elastase based on a highly functionalized
N-amino-4-imidazolidinone scaffold
*
Guijia He, Dengfeng Dou, Liuqing Wei, Kevin R. Alliston, William C. Groutas
Department of Chemistry, Wichita State University, 206 McKinley Hall, 1845 Fairmount, Wichita, Kansas 67260, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of compounds based on the N-amino-4-imidazolidinone scaffold was synthesized and screened
against human neutrophil elastase (HNE). These studies lead to the identification of a selective, low
micromolar reversible competitive inhibitor of HNE.
Received 9 February 2010
Received in revised form
17 May 2010
Accepted 18 June 2010
Available online 30 June 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Protease inhibitors
Neutrophil elastase
1. Introduction
a scaffold that docks to the active site in a predictable fashion and
orients appended recognition elements toward the S0 subsites.
Chronic obstructive pulmonary disease (COPD) is a major health
problem that affects more than 16 million people in the U.S. [1]. The
disorder is associated with airflow limitation that is not easily
reversible [2,3]. Currently, there are no drugs on the market for the
specific treatment of COPD.
Although the pathogenesis of COPD is poorly understood, the
disorder is characterized by increased numbers of alveolar macro-
phages, neutrophils and cytotoxic T lymphocytes, increased elas-
tolysis by an array of proteases [4], oxidative stress [5], apoptosis
[6], and the release of multiple inflammatory mediators,
including cytokines and chemokines [7e10].
Thus, based on previous studies involving the use of functionalized
4-imidazolidinone derivatives as reversible competitive inhibitors
of HNE [14], we reasoned that inhibitors based on the N-amino-4-
imidazolidinone scaffold (I) (Fig. 1) may exhibit high selectivity
toward HNE over Pr 3 (and vice versa) through exploitation of
differences in the S0 subsites of the two enzymes. We describe
herein the results of exploratory studies related to the selective
inhibition of HNE by derivatives based on scaffold (I) (Fig. 1).
2. Chemistry
Several lines of evidence suggest that macrophage- and
neutrophil-derived oxidative and proteolytic mediators released at
sites of inflammation play an important role in the pathophysiology
of COPD [11]. Most prominent among these mediators are human
neutrophil elastase (HNE), proteinase 3 (Pr 3), and MMP-12.
Elucidation of the biochemical and molecular mechanisms related
to the pathophysiology of COPD may lead to the emergence of
effective therapeutics [11].
The desired compounds 7e13, 14e18 and 19e26 were synthe-
sized as shown in Scheme 1. Cbz-DL-phenylalanine (or Cbz-DL-
leucine) was coupled to t-butyl carbazate using isobutyl
chloroformate in the presence of N-methylmorpholine. Removal of
the protective group followed by sodium triacetoxyborohydride-
mediated reductive amination [15] yielded intermediate 3 which
was cyclized with aqueous formaldehyde. The formation of the
4-imidazolidinone ring was also accompanied by N-hydroxy-
methylation of the product to afford compound 4. Stirring
compound 4 with aqueous sodium hydroxide resulted in clean
removal of the hydroxymethyl moiety to yield the desired inter-
mediate 5. Initial attempts at removing the Boc group using tri-
fluoroacetic acid or dry HCl lead to intractable mixtures, however,
microwave irradiation of 5 in the presence of silica gel [16] yielded
the desired N-amino-4-imidazolidinone which could be function-
alized via reaction with an array of structurally-diverse isocyanates,
0
0
HNE and Pr 3 show significant diffe0rences in their S2 and S1 eS3
0
subsites [12]. For instance, the S1 eS3 pockets in Pr 3 are smaller
and more polar than those of HNE because of the presence of Asp61
and Arg143 [13]. These differences can be exploited by utilizing
* Corresponding author. Tel.: þ1 316 978 7374; fax: þ1 316 978 3431.
E-mail address: bill.groutas@wichita.edu (W.C. Groutas).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.06.028

G. He et al. / European Journal of Medicinal Chemistry 45 (2010) 4280e4287
4281
intoreversiblecompetitiveinhibitor(III)(Fig. 2)[14];(c)groupsR4 and
R5 in (I) are oriented toward the S0 subsites; (d) HNE is a basic, 218
amino acid single polypeptide glycoprotein whose primary structure
shows considerable homology (54%) [13] with Pr 3. Furthermore, the
two enzymes have extended binding sites and show a preference for
hydrophobic substrates/inhibitors. However, although HNE and Pr 3
exhibit high sequence identity, their S0 subsites show significant
differences which can be exploited [13,19]. As mentioned earlier, the
S1
O
P1
R4
R5
Sn'
N
N
N
R2
R3
S2
(I)
0
S1 eS30 pockets in Pr 3 are smaller and more polar than those of HNE
Fig. 1. General structure of inhibitor (I).
because of the presence of Asp61 and Arg143 while the presence of
Val190, Phe192, Ala213, Val216, Phe228 and the disulfide bridge
Cys191eCys220 makes the active site of HNE hemispherical and
hydrophobic.
Based on the aforementioned considerations, it was envisaged
that derivatives based on scaffold (I) might make possible the
attainment of optimal potency and selectivity toward HNE (or Pr 3)
through favorable binding interactions with the S0 subsites of the
two enzymes. Thus, initial exploratory studies focused on gener-
ating a series of focused libraries by varying diversity sites R4 and
R5. All compounds were derived from (DL) Phe (P1 ¼ benzyl) and
(DL) Leu (P1 ¼ isobutyl) and listed in Table 1. The inhibitory activity
against HNE and Pr 3 is summarized in Fig. 3. Compound 24 was
carboxylic acids, acid anhydrides, sulfonyl chlorides or N-protected
amino acids. The resulting products could be readily debenzylated
using catalytic hydrogenation.
3. Biochemical studies
Enzyme assays and inhibition studies using neutrophil elastase
and proteinase 3 were conducted as previously described [17]. The
inhibition constant (KI) of compound 24 was determined using
a Dixon plot [18].
4. Results and discussion
found to be a highly selective, low micromolar (KI 7.34 mM)
reversible competitive inhibitor of HNE, as evidenced by the
construction of a Dixon plot [18] (Fig. 4). Compound 24 was devoid
of any inhibitory activity toward Pr 3 while the rest of the
compounds showed minimal inhibition (0e20%) toward both
enzymes (Fig. 3). Inhibition of HNE and Pr 3 appears to be insen-
sitive to variations in the structural diversity of R4 and R5, sug-
gesting that R4 and R5 are oriented toward the S0 subsites in a non-
optimal manner. The results also suggest that it may be possible to
design non-covalent inhibitors of HNE and Pr 3 that exhibit opti-
mized potency and selectivity through the utilization of appro-
priate recognition elements. These inhibitors are unlike most
4.1. Inhibitor design rationale
The design of inhibitor (I) was based on the following consider-
ations: (a) recent studies in our laboratory have demonstrated that
inhibitor(II)(Fig. 2) isa highlyefficientmechanism-based irreversible
inhibitor of serine proteases that docks to the active site of HNE with
the R1 and R2 groups occupying the S1 and S2 subsites [13]; (b)
replacement of the sulfonyl group with a methylene group lowers the
reactivity of the amide carbonyl in the resulting 4-imidazolidinone
ring making possible the transformation of irreversible inhibitor (II)
O
O
P1
(DL)
COOH
NHCbz
P1
P1
b
c
a
NHNHBoc
NHNHBoc
NHCbz
1
NH2
(P1 = benzyl)
2
O
O
N
O
P1
P1
P1
N
NHNHBoc
d
NBoc
N
NH
N
e
NHBoc
f
R3
OH
R3
4 (R3 = H)
5 (R3 = H)
3
O
O
P1
P1
O
R4
P1
N
N
N
NH2
R4
N
R5
N
N
b
g
N
R5
HN
R3
R3
R3
14-18 (R3 = H)
7-13 (R3 = H)
6 (R3 = H)
Scheme 1. General synthesis of inhibitor I. Reagents and conditions: a) IBCF/NMM THF then NH2NHBoc; b) H2/10%PdeC/CH3OH; c) RCHO/NaBH(OAc)3/HOAc; d) HCHO or R3CHO;
e) NaOH/aq dioxane; f) silica gel/microwave; g) RSO2Cl/base or RN]C]O or (RCO)2O or RCOOH/EDCI/DMF or protected amino acid/EDCI/DMF.

4282
G. He et al. / European Journal of Medicinal Chemistry 45 (2010) 4280e4287
O
O
O
P1
P1
P1
R4
R5
N N
N
N
N
N
N
S
L
NHR3
R2
R2
R2
O
R3
O
O
(I)
(II)
(III)
(Mechanism-based
irreversible in hibitors)
(reversible competitive
in hibitors)
(seletive reversible
competitive in hibitors)
Fig. 2. Design rationale of reversible competitive inhibitors of human neutrophil elastase and proteinase 3.
reported inhibitors of mammalian and viral serine proteases which
incorporate in their structures a warhead that leads to covalent
inhibition or, in the case of transition state inhibitors, the formation
of a reversible covalent adduct [20].
In conclusion, we have demonstrated that the multiple points of
diversityafforded byscaffold (I) render it well-suited to the design of
non-covalent inhibitors of serine proteases with similar active sites.
reaction mixture was stirred at room temperature under hydrogen
(low pressure) for 3 h. The catalyst was filtered off, rinsed with
methanol and the solvent was evaporated, leaving a crude product
which was purified by recrystallization (ethyl acetate/hexanes) to
yield the product as a white solid (26.53 g, 82% yield), mp
115.0e116.0 ꢁC 1H NMR (CDCl3):
d 1.48 (s, 9H), 2.68e2.76 (dd, 1H,
J ¼ 15, 9 Hz), 3.28e3.36 (dd, 1H, J ¼ 15, 6 Hz), 3.70e3.75 (dd, 1H,
J ¼ 9, 6 Hz), 6.45 (s, 1H), 7.20e7.36 (m, 5H).
5. Experimental section
5.2.3. (RS)-tert-Butyl 2-(2-(benzylamino)-3-phenylpropanoyl)
hydrazinecarboxylate 3
5.1. General methods
A solution of compound 2 (23.40 g; 83.8 mmol) and benzal-
dehyde (10.68 g; 100.6 mmol) in dry 1, 2-dichloroethane (150 mL)
was treated with glacial acetic acid (7.05 g; 117.3 mmol), followed
by sodium triacetoxyborohydride (25.56 g; 117.3 mmol). The
reaction mixture was stirred for 4 h at room temperature and then
neutralized via the dropwise addition of cold 10% aqueous NaOH
solution to pH 9e10 while stirring. The organic phase was sepa-
rated and the aqueous phase was extracted with ethyl acetate
(3 ꢀ 150 mL), the combined organic extracts were dried over
anhydrous Na2SO4 and the solvent removed. The crude product
was purified by flash chromatography (silica gel/hexane/ethyl
acetate) to yield a pure product as a colorless oil (14.04 g, 45%
The 1H spectra were recorded on a Varian XL-300 or XL-400 NMR
spectrometer. A HewlettePackard diode array UV/Vis spectropho-
tometer was used in the in vitro evaluation of the inhibitors. Human
neutrophil elastase, proteinase 3 and Boc-Ala-Ala-Nva thiobenzyl
ester were purchased from Elastin Products Company, Owensville,
MO. Methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide and 5,50-dithio-
bis (2-nitrobenzoic acid) were purchased from Sigma Chemicals, St.
Louis, MO. Melting points were determined on a Mel-Temp apparatus
and are uncorrected. Reagents and solvents were purchased from
various chemical suppliers (Aldrich, Acros Organics, TCI American,
and Bachem). Silica gel (230e450 mesh) used for flash chromatog-
raphy was purchased from Sorbent Technologies (Atlanta, GA). Thin
layer chromatography was performed using Analtech silica gel plates
and the TLC plates were visualized using iodine and/or UV light. All
compounds synthesized as diastereomeric mixtures were screened
against human neutrophil elastase and proteinase 3 as such without
separating the individual diastereomers.
yield). 1H NMR (CDCl3):
d 1.45 (s, 9H), 2.74e2.82 (m, 1H),
3.19e3.24 (dd, 1H, J ¼ 12, 6 Hz), 3.46e3.54 (dd, 1H, J ¼ 9, 6 Hz),
3.54e3.58 (d, 1H, J ¼ 12 Hz), 3.78e3.52 (d, 1H, J ¼ 12 Hz), 6.82 (s,
1H), 7.08e7.32 (m, 10H).
5.2.4. (RS)-tert-Butyl 3,4-dibenzyl-5-oxoimidazolidin-1-yl
(hydroxymethyl)carbamate 4
5.2. Representative syntheses
A mixture of compound 3 (11.20 g; 30.31 mmol) and 37%
aqueous formaldehyde (50 mL) was refluxed for 4 h. The reaction
mixture was cooled to room temperature, water (50 mL) was
added, and the solution was extracted with CH2Cl2 (3 ꢀ 70 mL).
The organic phase was dried over anhydrous Na2SO4 and the
solvent was removed in vacuo. The crude product was purified by
flash chromatography (silica gel/hexane/ethyl acetate) to yield
a pure product as a colorless oil (6.48 g; 52% yield). 1H NMR
5.2.1. (RS)-tert-Butyl 2-(2-(benzyloxycarbonylamino)-3-
phenylpropanoyl)hydrazine-carboxyl-ate 1
AsolutionofZ-DL-Phe-OH(71.84g, 240mmol)indryTHF(200mL)
cooled in an ice-bath was treated with isobutyl chloroformate
(32.78 g; 240 mmol), followed with N-methylmorpholine (24.28 g;
240 mmol). The resulting mixture was stirred for 5 min, and t-butyl
carbazate (31.60 g; 240 mmol) was added. The ice-bath was removed
and the reaction mixture was stirred for 7 h at room temperature. The
solvent was removed on the rotary evaporator and the residue was
taken up in methylene chloride (200 mL). The resulting solution was
washed sequentially with 5% aqueous HCl (70 mL), 5% aqueous
NaHCO3 (3 ꢀ 70 mL) and brine (3 ꢀ 70 mL). The organic phase was
dried overanhydrous Na2SO4 andthesolventwasremovedtoyieldthe
(CDCl3):
d 1.42 (s, 9H), 2.98e3.02 (m, 1H), 3.08e3.15 (m, 1H),
3.45e3.60 (m, 2H), 3.80e3.85 (s, 1H), 4.05 (s, 2H), 4.70e4.82 (m,
2H), 7.05e7.50 (m, 10H).
5.2.5. (RS)-tert-Butyl 3,4-dibenzyl-5-oxoimidazolidin-1-
ylcarbamate 5
Compound 4 (12.47 g; 30.31 mmol) in 1, 4-dioxane (50 mL) was
treated with a solution of KOH (29.68 g; 530 mmol) in H2O (53 mL)
and the reaction mixture was stirred at room temperature for 1 h.
The solvent was removed on a rotary evaporator and the resulting
residue was extracted with ethyl acetate (3 ꢀ 70 mL). The organic
phase was dried over anhydrous Na2SO4 and the solvent was
removed. The crude product was purified by flash chromatography
(silica gel/hexane/ethyl acetate) to yield a pure product as a color-
1
product (97.25 g; 98% yield) as a white solid, mp 120.0e121.5 ꢁC H
NMR (CDCl3): d 1.45(s, 9H), 3.00e3.20(m, 2H), 4.41e4.52(m,1H), 5.05
(s, 2H), 5.22e5.30(s, 1H), 6.38(s, 1H), 7.08e7.38(m, 10H).
5.2.2. (RS)-tert-Butyl 2-(2-amino-3-phenylpropanoyl)
hydrazinecarboxylate 2
A solution of compound 1 (47.79 g; 116 mmol) in methanol
(250 mL) was treated with 10% palladium on carbon (7.89 g). The
less oil (7.51 g; 65% yield). 1H NMR (CDCl3):
d 1.42 (s, 9H), 2.95e3.08

G. He et al. / European Journal of Medicinal Chemistry 45 (2010) 4280e4287
Table 1 (continued )
4283
Table 1
N-Amino-4-imidazolidinone derivatives (I)a
Compound P1
R2
R4
COOH
R5
H
O
O
P1
R4
R5
N
N
18
benzyl
H
N
R2
R3
O
.
O
NH
19
20
21
isobutyl benzyl
isobutyl benzyl
isobutyl benzyl
H
H
H
Compound P1
R2
R4
R5
H
O
O
O
O
NH
7
8
benzyl
benzyl
benzyl
O
NH
O
O
OH
benzyl
benzyl
H
H
NH
O
O
OH
O
9
benzyl
O
22
23
isobutyl benzyl
isobutyl benzyl
H
H
O
O
O
COOH
10b
benzyl
benzyl
benzyl
benzyl
H
H
N
O
O
F
O
11b
N
H
N
O
24b
isobutyl benzyl
H
O
O
O
O
12b
benzyl
benzyl
benzyl
benzyl
H
H
N
25
26
isobutyl benzyl
isobutyl benzyl
eSO2eCH3
SO2
eSO2eCH3
SO2
O
COOH
13
a
R3 ¼ H for all.
b
Compounds were screened as diastereomeric mixtures.
O
14
15
benzyl
benzyl
H
H
H
H
NH
O
(m, 1H), 3.12e3.22 (dd, 1H, J ¼ 12, 6 Hz), 3.44e3.50 (d, 1H,
J ¼ 12 Hz), 3.54e3.58 (s, 1H), 3.78e3.86 (d, 1H J ¼ 12 Hz), 3.96 (s,
1H), 4.02e4.12 (m, 1H), 6.30e6.42 (s, 1H), 7.10e7.22 (m, 10H).
OH
5.2.6. (RS)-3-Amino-1,5-dibenzylimidazolidin-4-one 6
O
Compound 5 (10.53 g; 27.60 mmol) was dissolved in CH2Cl2
(300 mL) and silica gel (Sorbent Technologies, 60 Å, 32e63 mm)
O
(140 g) was added. The solvent was removed on a rotary evaporator
and the dry powdered solid was irradiated in a microwave oven in
an open crystallizing dish for 10 min at 860 W. The solid was
thoroughly washed with methanol and the solvent was removed.
The crude product was purified by flash chromatography (silica gel/
hexane/ethyl acetate) to yield a pure product as a colorless oil
O
16b
17b
benzyl
benzyl
H
H
H
H
N
O
O
F
(2.33 g; 30% yield). 1H NMR (CDCl3):
d 2.85e3.02 (m, 1H), 3.09e3.17
N
(dd, 1H, J ¼ 12, 6 Hz), 3.38e3.46 (d, 1H, J ¼ 12 Hz), 3.46e3.56
(m, 1H), 3.72e3.84 (m, 2H), 3.95e4.04 (d, 1H, J ¼ 12 Hz), 7.08e7.18
(m, 10H).
(continued on next page)

4284
G. He et al. / European Journal of Medicinal Chemistry 45 (2010) 4280e4287
3.44e3.54 (d, 1H, J ¼ 12 Hz), 3.51e3.63 (m, 1H), 3.80e4.00 (m, 3H),
4.64e4.78 (m, 1H), 6.64e6.76 (s, 1H), 7.00e7.44 (m, 15H); 13C NMR
(CDCl3): 173.05, 156.87, 139.07, 137.68, 137.11, 130.15, 128.99,
d
128.85, 128.72, 128.65, 128.39, 127.82, 126.79, 126.49, 69.72, 65.33,
58.87, 41.54, 37.01, 36.22.
5.2.9. (RS)-4-(3,4-Dibenzyl-5-oxoimidazolidin-1-ylamino)-4-
oxobutanoic acid 9
To compound 6 (0.56 g; 2 mmol) in dry CH2Cl2 (10 mL) was
added succinic anhydride (0.20 g; 2 mmol) and the reaction
mixture was stirred overnight at room temperature. The solvent
was removed on a rotary evaporator and the crude product was
washed with 20 mL diethyl ether to give a pure product as a white
solid (0.54 g; 71% yield), mp 131.5e133.0 ꢁC 1H NMR (CDCl3):
d
2.39e2.59 (m, 2H), 2.64e2.74 (m, 2H), 2.91e3.19 (m, 2H),
3.49e3.58 (d, 1H, J ¼ 12 Hz), 3.56e3.64 (m, 1H), 3.73e3.82 (d, 1H,
J ¼ 12 Hz), 4.04e4.23 (m, 2H), 7.12e7.36 (m,10H), 8.33e8.46 (s,1H);
13C NMR (CD3OD):
d 174.69, 172.41, 171.46, 137.59, 137.03, 129.67,
128.57, 128.36, 128.17, 127.44, 126.43, 68.61, 65.32, 58.95, 36.85,
28.63, 28.15.
Fig. 3. Inhibition of human neutrophil elastase and proteinase 3 by compounds 7e26.
Values were expressed as percent inhibition using an inhibitor/enzyme ratio of 500,
and an incubation time of 30 min for human neutrophil elastase and no incubation for
proteinase 3.
5.2.10. (RS)-1-Benzyl-N-[(RS)-3,4-dibenzyl-5-oxoimidazolidin-1-
yl]-5-oxopyrrolidine-3-carbox-amide 10
A
solution of 1-benzyl-5-oxopyrrolidine-3-carboxylic acid
5.2.7. (RS)-Ethyl 2-(3-(3,4-dibenzyl-5-oxoimidazolidin-1-yl)
ureido)acetate 7
(0.44 g; 2 mmol) dissolved in 20 mL DMF was treated with 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.46 g;
2.4 mmol) and the solution was stirred for 20 min. Compound 6
(0.56 g; 2 mmol) was added and the reaction mixture was stirred at
room temperature overnight. The solvent was removed in vacuo
and the residue was dissolved in CH2Cl2 (70 mL) and then was
washed sequentially with 5% aqueous HCl (3 ꢀ 20 mL), 5% aqueous
NaHCO3 (3 ꢀ 20 mL) and brine (3 ꢀ 20 mL). The organic phase was
dried over anhydrous Na2SO4 and the solvent was removed. The
crude product was purified by flash chromatography (silica gel/
hexane/ethyl acetate) to yield a pure product as a colorless oil
Compound 6 (0.56 g; 2 mmol) in dry CH2Cl2 (3 mL) was cooled
to 0 ꢁC and then treated with triethylamine (0.33 mL, 2.4 mmol)
and ethyl isocyanatoacetate (0.26 g; 2 mmol). The reaction mixture
was stirred overnight at room temperature. The mixture was
treated with ethyl acetate (40 mL) and then washed successively
with 5% aqueous HCl (10 mL), 5% aqueous NaHCO3 (10 mL) and
brine (10 mL). The organic phase was dried over anhydrous Na2SO4
and the solvent was removed. The crude product was purified by
flash chromatography (silica gel/hexane/ethyl acetate) to yield
a pure product as a colorless oil (0.15 g; 18%). 1H NMR (CDCl3):
(0.28 g; 29% yield). 1H NMR (CDCl3):
d 2.50e2.80 (m, 2H), 2.86e2.98
d
1.20e1.28 (t, 3H, J ¼ 6 Hz), 2.92e3.15 (m, 2H), 3.48e3.55 (d, 1H,
J ¼ 9 Hz), 3.55e3.61 (m, 1H), 3.75e3.92 (m, 3H), 4.00e4.19 (m, 4H),
5.80 (s, 1H), 7.15e7.28 (m, 10H); 13C NMR (CDCl3):
173.37, 170.86,
(m, 1H), 3.00e3.17 (m, 2H), 3.28e3.49 (m, 3H), 3.48e3.56 (m, 1H),
3.71e3.86 (m, 1H), 3.93e4.02 (t, 1H, J ¼ 6 Hz), 4.02e4.11 (m, 1H),
4.30e4.42 (t, 2H, J ¼ 6 Hz), 7.05e7.40 (m, 15H), 9.16e9.22 (d, 1H,
d
156.88, 137.83,137.22,130.06,129.78, 128.85, 128.64,128.55,128.52,
128.41, 127.71, 126.68, 69.65, 65.44, 61.51, 59.03, 41.94, 37.11, 14.27.
J ¼ 15 Hz); 13C NMR (CDCl3):
d 172.75, 172.47, 171.77, 137.74, 137.09,
135.73, 129.79, 128.91, 128.75, 128.62, 128.43, 128.09, 127.89, 127.71,
126.69, 68.73, 65.29, 59.09, 49.08, 46.63, 37.09, 34.88, 34.56, 34.39.
5.2.8. (RS)-1-(3,4-Dibenzyl-5-oxoimidazolidin-1-yl)-3-
phenethylurea 8
Prepared from compound 6 and 2-phenethyl isocyanate using
a procedure similar to that used in the synthesis of compound 7 to
give a pure product as a colorless oil (0.61 g; 72% yield). 1H NMR
5.2.11. (RS)-N-[(RS)-3,4-Dibenzyl-5-oxoimidazolidin-1-yl]-1-
(4-fluorobenzyl)-5-oxopyrrolidine-3-carboxamide 11
Prepared from compound 6 and 1-(4-fluorobenzyl)-5-oxo-
pyrrolidine-3-carboxylic acid using a procedure similar to that used
in the synthesis of compound 10 to give a pure product as a color-
(CDCl3): d 2.56e2.79 (m, 2H), 2.91e3.12 (m, 2H), 3.14e3.44 (m, 2H),
less oil (0.34 g; 34% yield). 1H NMR (CDCl3):
d 2.54e2.83 (m, 2H),
2.91e3.18 (m, 3H), 3.30e3.51 (m, 3H), 3.51e3.62 (m, 1H), 3.78e3.90
(t, 1H, J ¼ 12 Hz), 3.97e4.12 (m, 2H), 4.27e4.51 (m, 2H), 6.94e7.08
(m, 2H), 7.12e7.40 (m, 12H), 7.66e7.80 (d, 1H, J ¼ 24 Hz); 13C NMR
[S] = 3.5x10
[S] = 7.0x10
M
M
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
(CDCl3):
d 172.65, 172.61, 172.49, 172.43, 171.74, 164.04, 160.78,
137.68, 137.07, 137.04, 131.63, 131.59, 129.93, 129.82, 128.75, 128.66,
128.44, 127.78, 126.73, 115.96, 115.67, 68.74, 65.25, 58.99, 48.88,
45.93, 37.02, 34.83, 34.54.
5.2.12. (RS)-N-[(RS)-3,4-Dibenzyl-5-oxoimidazolidin-1-yl]-1-
(furan-2-ylmethyl)-5-oxopyrrolidine-3-carboxamide 12
Prepared from compound 6 and 1-(furan-2-ylmethyl)-5-oxo-
pyrrolidine-3-carboxylic acid using a procedure similar to that used
in the synthesis of compound 10 to give a pure product as a color-
-2.E-05 -1.E-05 0.E+00
1.E-05
2.E-05
[I]
3.E-05
4.E-05
5.E-05
6.E-05
less oil (0.51 g; 40% yield). 1H NMR (CDCl3):
d 2.50e2.75 (m, 2H),
2.90e3.01 (m, 1H), 3.01e3.17 (m, 2H), 3.37e3.63 (m, 4H), 3.76e3.90
(m, 1H), 4.00e4.15 (m, 2H), 4.28e4.53 (m, 2H), 6.20e6.30 (m, 2H),
Fig. 4. Dixon plot for the interaction of compound 24 with human neutrophil elastase.

G. He et al. / European Journal of Medicinal Chemistry 45 (2010) 4280e4287
4285
7.07e7.42 (m, 11H), 8.67e8.82 (d, 1H, J ¼ 27 Hz); 13C NMR (CDCl3):
5.2.18. (RS)-2-(4-Benzyl-5-oxoimidazolidin-1-ylcarbamoyl)benzoic
acid 18
d
172.63, 172.54, 171.78, 149.32, 142.80, 137.74, 137.11, 129.81, 128.78,
128.63, 128.45, 127.73, 126.71, 110.59, 108.90, 68.77, 65.33, 59.13,
49.16, 39.35, 37.09, 34.92, 34.51.
Prepared from compound 13 using a similar procedure as that of
compound 14 to give a pure product as a light yellow solid (0.32 g;
81% yield), mp 123.5e125.0 ꢁC 1H NMR (DMSO):
d 2.66e2.80 (m,
5.2.13. (RS)-2-(3,4-Dibenzyl-5-oxoimidazolidin-1-ylcarbamoyl)
benzoic acid 13
1H), 2.92e3.06 (m, 1H), 3.15 (s, 1H), 3.51e3.63 (m, 1H), 4.00e4.16
(m, 1H), 4.22e4.34 (m, 1H), 7.12e7.40 (m, 5H), 7.42e7.74 (m, 4H),
Prepared from compound 6 and phthalic anhydride using
a similar procedure as that used in the synthesis of compound 9 to
give a pure product as a white solid (0.47 g; 47% yield), mp
7.07 (s, 1H),10.43 (s, 1H); 13C NMR (CD3OD):
d 173.01,164.66,164.16,
135.51, 134.50, 131.82, 131.36, 130.63, 130.19, 129.50, 128.51, 124.23,
65.24, 59.54, 37.15.
The compounds derived from Z-DL-Leu-OH (compounds
19e26) were synthesized using procedures as those used in the
synthesis of compounds 1e18.
132.0e134.0 ꢁC 1H NMR (CDCl3):
d
2.91e3.17 (m, 2H), 3.64e3.74 (m,
2H), 3.74e3.82 (d, 1H, J ¼ 15 Hz), 4.29e4.40 (dd, 1H, J ¼ 18, 6 HZ),
7.04e7.67 (m, 10H), 7.75e8.08 (m, 4H), 9.22(s, 1H); 13C NMR
(CD3OD):
d 172.51, 164.16, 163.66, 137.08, 136.61, 134.89, 134.86,
132.30,130.68,129.48, 129.45, 129.38, 128.82, 128.47, 128.33, 128.18,
128.18, 127.34, 126.35, 123.79, 123.77, 67.96, 64.74, 59.04, 36.65.
5.2.19. (RS)-Ethyl 2-(3-(3-benzyl-4-isobutyl-5-oxoimidazolidin-1-
yl)ureido)acetate 19
White solid (50% yield), mp 114.0e115.5 ꢁC 1H NMR (DMSO):
5.2.14. (RS)-1-(4-Benzyl-5-oxoimidazolidin-1-yl)-3-
phenethylurea 14
d
0.68e0.90 (m, 6H), 1.10e1.24 (t, 3H, J ¼ 9 Hz), 1.42e1.58 (m, 2H),
1.78e1.93 (m, 1H), 3.17e3.26 (m, 1H), 3.57e3.66 (d, 1H, J ¼ 12 Hz),
3.80e3.95 (m, 3H), 3.97e4.14 (m, 3H), 4.22e4.30 (d, 1H, J ¼ 6 Hz),
6.24e6.36 (t, 1H, J ¼ 6 Hz), 7.12e7.36 (m, 5H), 8.05 (s, 1H); 13C NMR
A solution of compound 8 (0.50 g; 1.2 mmol) in dry methanol
(50 mL) was treated with 10% palladium on carbon (0.35 g) under
hydrogen gas and the reaction mixture was stirred for 1 h at room
temperature. The catalyst was filtered off and the solvent was
removed on a rotary evaporator leaving a crude product which was
washed with diethyl ether (2 ꢀ 5 mL) to yield a pure product as
a white solid (0.23 g; 59% yield), mp 130.0e131.0 ꢁC 1H NMR
(CDCl3):
d 175.49, 170.92, 156.76, 137.58, 129.14, 128.74, 127.87,
69.68, 62.75, 61.66, 59.45, 42.10, 39.08, 24.88, 23.17, 22.51, 14.33.
5.2.20. (RS)-1-(3-Benzyl-4-isobutyl-5-oxoimidazolidin-1-yl)-3-
phenethylurea 20
(CDCl3):
d
2.69e2.85(m, 2H), 2.92e3.14 (m, 2H), 3.33e3.43 (m, 2H),
Oil (33% yield). 1H NMR (DMSO):
d 0.75e0.98 (m, 6H), 1.46e1.64
3.70e3.82 (m, 1H), 4.04e4.15 (t, 1H, J ¼ 9 Hz), 4.26e4.37 (dd, 1H,
(m, 2H),1.84e1.98 (m,1H), 2.67e2.82 (t, 2H, J ¼ 9 Hz), 3.19e3.28 (m,
1H), 3.30e3.45 (m, 2H), 3.57e3.67 (d, 1H, J ¼ 12 Hz), 3.90e4.00 (d,
1H, J ¼ 12 Hz), 4.00e4.07 (d, 1H, J ¼ 6 Hz), 4.23e4.51 (d, 1H,
J ¼ 6 Hz), 5.52e5.63 (t, 1H, J ¼ 6 Hz), 7.08e7.42 (m, 10H), 7.72 (s,
J ¼ 12, 6 Hz), 4.98e5.13 (t, 1H, J ¼ 9 Hz), 6.97e7.11 (s, 1H), 7.11e7.38
(m, 10H); 13C NMR (CDCl3):
d 175.59, 156.93, 139.06, 136.95, 129.63,
129.03, 128.88, 128.75, 127.25, 126.64, 64.73, 59.28, 41.56, 36.82,
36.22.
1H); 13C NMR (CDCl3):
d 175.15, 156.82, 139.03, 137.34, 129.08,
128.81, 127.97, 126.67, 69.74, 62.57, 59.22, 41.53, 39.17, 36.23, 24.87,
23.10, 22.63.
5.2.15. (RS)-4-(4-Benzyl-5-oxoimidazolidin-1-ylamino)-4-
oxobutanoic acid 15
Prepared from compound 9 using a similar procedure as that of
compound 14 to give a pure product as a light yellow oil (0.34 g;
5.2.21. (RS)-4-(3-Benzyl-4-isobutyl-5-oxoimidazolidin-1-ylamino)-
4-oxobutanoic acid 21
89% yield). 1H NMR (CDCl3):
d
2.43e2.73 (m, 4H), 3.09e3.26 (m,
1H), 3.30e3.47 (m, 1H), 4.23e4.38 (m, 1H), 4.51e4.69 (m, 2H),
7.16e7.46 (m, 5H); 13C NMR (CD3OD):
174.22, 171.94, 165.82,
Oil (52% yield). 1H NMR (DMSO):
d 0.70e0.82 (m, 3H), 0.82e0.95
(m, 3H), 1.47e1.63 (m, 2H), 1.78e1.95 (m, 1H), 2.40e2.58 (m, 2H),
2.58e2.74 (m, 2H), 3.23e3.33 (m, 1H), 3.64e3.74 (d, 1H, J ¼ 12 Hz),
3.90e3.98 (d, 1H, J ¼ 12 Hz), 4.05e4.13 (d, 1H, J ¼ 6 Hz), 4.28e4.35
(d, 1H, J ¼ 6 Hz), 7.16e7.37 (m, 5H), 9.06 (s, 1H); 13C NMR (CDCl3):
d
133.83, 129.34, 129.03, 128.70, 128.61, 127.33, 57.67, 33.68, 28.45,
28.08, 26.08.
d
173.56, 172.79, 172.29, 137.50, 129.27, 129.19, 128.77, 128.69,
5.2.16. (RS)-1-Benzyl-N-[(RS)-4-benzyl-5-oxoimidazolidin-1-yl]-5-
oxopyrrolidine-3-carboxamide 16
127.96, 67.40, 62.46, 59.89, 38.79, 26.91, 26.77, 24.91, 23.20, 22.21.
Prepared from compound 10 using a similar procedure as that of
compound 14 to give a pure product as a colorless oil (0.40 g; 65%
5.2.22. (RS)-N-(3-Benzyl-4-isobutyl-5-oxoimidazolidin-1-yl)-3-
phenylpropanamide 22
yield). 1H NMR (CDCl3):
d
2.57e2.84 (m, 2H), 2.85e2.98 (m, 1H),
White solid (60% yield), mp 102.0e103.5 ꢁC 1H NMR (DMSO):
2.98e3.23 (m, 2H), 3.30e3.58 (m, 3H), 3.68e3.78 (m, 1H),
4.20e4.28 (m,1H), 4.34e4.41 (m,1H), 4.44e4.56 (m, 1H), 7.05e7.42
d 0.74e0.97 (m, 6H), 1.47e1.67 (m, 2H), 1.84e1.99 (m, 1H),
2.42e2.58 (t, 2H, J ¼ 9 Hz), 2.84e2.98 (t, 2H, J ¼ 9 Hz), 3.23e3.31 (m,
1H), 3.62e3.70 (d, 1H, J ¼ 12 Hz), 3.89e3.98 (d, 1H, J ¼ 12 Hz),
3.98e4.06 (d,1H, J ¼ 6 Hz), 4.17e4.24 (d,1H, J ¼ 6 Hz), 7.04e7.41 (m,
(m, 10H), 8.68e8.86 (d, 1H, J ¼ 18 Hz); 13C NMR (CDCl3):
d 174.90,
172.89, 171.69, 137.81, 137.02, 129.64, 129.41, 128.94, 128.75, 128.42,
128.07, 127.03, 126.64, 66.72, 59.11, 49.08, 46.68, 40.93, 36.56, 34.81.
10H), 9.17 (s, 1H); 13C NMR (CDCl3):
d 174.34, 171.11, 140.36, 137.72,
129.17, 128.85, 128.76, 128.57, 127.89, 126.69, 68.63, 62.69, 59.67,
39.01, 36.06, 31.32, 24.93, 23.23, 22.44.
5.2.17. (RS)-N-[(RS)-4-Benzyl-5-oxoimidazolidin-1-yl]-1-
(4-fluorobenzyl)-5-oxopyrrolidine-3-carboxamide 17
Prepared from compound 11 using a similar procedure as that of
compound 14 to give a pure product as a colorless oil (0.29 g; 71%
5.2.23. (RS)-2-(3-Benzyl-4-isobutyl-5-oxoimidazolidin-1-
ylcarbamoyl)benzoic acid 23
yield). 1H NMR (CDCl3):
d
2.63e2.82 (m, 2H), 2.90e3.04 (m, 1H),
White solid (53% yield), mp 105.5e107.0 ꢁC 1H NMR (DMSO):
3.04e3.21 (m, 23H), 3.34e3.57 (m, 2H), 3.73e3.81 (m, 1H),
4.21e4.34 (m, 1H), 4.34e4.53 (m, 2H), 6.93e7.07 (t, 2H, J ¼ 9 Hz),
7.10e7.38 (m, 7H), 8.07e8.25 (d, 1H, J ¼ 21 Hz); 13C NMR (CDCl3):
d 0.78e1.07 (m, 6H), 1.60e1.85 (m, 2H), 1.92e2.08 (m, 1H),
3.38e3.47 (m, 1H), 3.86e3.95 (d, 1H, J ¼ 12 Hz), 4.04e4.12 (d, 1H,
J ¼ 12 Hz), 4.17e4.23 (d, 1H, J ¼ 6 Hz), 4.52e4.59 (d, 1H, J ¼ 6 Hz),
d
171.79, 171.73, 164.09, 160.83, 136.92, 131.53, 129.96, 129.85,
7.20e7.48 (m, 5H), 7.71e7.96 (m, 4H); 13C NMR (CDCl3):
d 173.95,
129.68, 129.44, 128.83, 128.48, 127.13, 116.02, 115.74, 63.78, 59.16,
48.93, 46.02, 36.49, 34.86.
164.77, 164.11, 137.82, 136.00, 135.03, 130.10, 130.01, 129.17, 128.66,
128.02,125.94,124.12, 68.09, 62.25, 59.87, 38.91, 24.93, 23.10, 22.45.

4286
G. He et al. / European Journal of Medicinal Chemistry 45 (2010) 4280e4287
5.2.24. (RS)-Benzyl 1-((RS)-3-benzyl-4-isobutyl-5-
oxoimidazolidin-1-ylamino)-1-oxo-3-phenyl-propan-2-
ylcarbamate 24
DMSO were added to a cuvette containing 940
NaCl buffer, pH 7.25. After the solution was well mixed, 20
5,50-dithio-bis(2-nitrobenzioc acid) in DMSO and 10
L of 3.45 mM
solution of proteinase 3 in 0.1 M phosphate/0.25 M NaCl buffer, pH6.50,
were added and the change in absorbance was monitored at 410 nm for
m
L 0.1 M HEPES/0.5 M
mL of 32 mM
m
Oil (48% yield). 1H NMR (DMSO):
d
0.73e0.85 (m, 3H), 0.85e0.98
(m, 3H), 1.47e1.66 (m, 2H), 1.84e1.96 (m, 1H), 2.92e3.17 (m, 2H),
3.20e3.28 (m, 1H), 3.57e3.68 (m, 1H), 3.78e3.86 (m, 1H),
3.86e3.99 (m, 2H), 3.99e4.08 (m, 1H), 4.85e5.06 (m, 2H),
7.05e7.40 (m, 10H), 8.64e8.80 (d, 1H, J ¼ 24 Hz); 13C NMR (CDCl3):
2 min. A control (hydrolysis) containing 940
NaCl, pH 7.25,10 L3.40 M solution of proteinase 3, 20
of 16 mM 5,50-dithio-bis(2-nitrobenzioc acid) in DMSO, and 20
m
L 0.1 M HEPES/0.5 M
LDMSO, 20
L of
m
m
m
mL
m
d
174.01, 170.35,156.25,137.79,136.21,129.81, 129.40,129.21,129.14,
6.49 mM Boc-Ala-Ala-Nva-SBzl in DMSO was also run under the same
condition. Determinations were performed in duplicate or triplicate
and remaining activity was calculated as described for HNE.
128.53,128.34,127.37, 68.23, 67.60, 62.55, 59.55, 59.43, 39.20, 38.31,
24.52, 23.23, 22.67.
5.2.25. (RS)-N-(3-Benzyl-4-isobutyl-5-oxoimidazolidin-1-yl)-N-
(methylsulfonyl)methanesulfon-amide 25
Acknowledgements
White solid (38% yield), mp 88.0e89.5 ꢁC 1H NMR (DMSO):
Support of this work by the Heart and Blood Institute of the
National Institutes of Health (HL 57788) is gratefully acknowledged.
d
0.77e1.03 (m, 6H), 1.48e1.73 (m, 2H), 1.83e2.03 (m, 1H),
3.24e3.35 (m, 1H), 3.36e3.50 (d, 6H, J ¼ 9 Hz), 3.70e3.80 (d, 1H,
J ¼ 18 Hz), 3.88e3.97 (d, 1H, J ¼ 18 Hz), 4.07e4.14 (d, 1H, J ¼ 9 Hz),
4.45e4.53 (d, 1H, J ¼ 9 Hz), 7.23e7.47 (m, 5H); 13C NMR (CDCl3):
References
d
174.67, 137.33, 129.35, 128.90, 128.15, 69.25, 62.08, 59.77, 45.60,
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45.05, 38.03, 24.95, 23.00, 22.26.
5.2.26. (RS)-N-(3-Benzyl-4-isobutyl-5-oxoimidazolidin-1-yl)-4-
methyl-N-tosylbenzenesulfon amide 26
White solid (53% yield), mp 83.0e85.0 ꢁC 1H NMR (DMSO):
d
0.73e0.95 (m, 6H), 1.34e1.71 (m, 2H), 1.82e2.02 (m, 1H), 2.42 (s,
6H), 3.12e3.22 (m, 1H), 3.67e3.77 (d, 1H, J ¼ 12 Hz), 3.84e3.93 (d,
1H, J ¼ 12 Hz), 4.13e4.18 (d, 1H, J ¼ 6 Hz), 4.44e4.56 (d, 1H,
J ¼ 6 Hz), 7.20e7.45 (m, 9H), 7.70e7.92 (m, 4H); 13C NMR (CDCl3):
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(2006) 53e62.
d
174.02, 146.06, 145.99, 137.37, 135.37, 135.27, 129.82, 129.79,
129.42, 129.30, 129.22, 128.78, 127.97, 69.03, 62.18, 59.77, 37.86,
24.78, 23.21, 22.26, 22.03.
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6 (2009)
5.3. Enzyme assay and inhibitory studies
5.3.1. Human neutrophil elastase (HNE)
In a typical inhibition experiment, 10
incubated with 10 L of 3.5 mM solution of inhibitor in DMSO
([inhibitor]/[enzyme] ¼ 500), 970 L 0.1 M HEPES/0.5 M NaCl buffer,
pH 7.25, at 25 ꢁC. After 30 min,10
L of 70 mM methoxysuccinyl-Ala-
mL of 7.0 mM HNE was
m
m
m
[12] Nomenclature used is that of I. Schechter, A. Berger, Biochem. Biophys. Res.
Comm. 27 (1967) 157e162 where S1, S2, S3,.Sn and S10, S20, S30,.Sn0
correspond to the enzyme subsites on either side of the scissile bond. Each
subsite accommodates a corresponding amino acid residue side chain desig-
nated P1, P2, P3, .Pn and P10, P20, P30, .Pn0 of the substrate or (inhibitor). S1
is the primary substrate specificity subsite, and P1eP10 is the scissile bond.
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Biol. 261 (1996) 267e278;
Ala-Pro-Val p-nitroanilide was added and the amount of active
enzyme was determined by monitoring the release of p-nitroaniline
at 410 nm for 2 min. A control (hydrolysis) containing 970
mL 0.1 M
HEPES/0.5 M NaCl buffer, pH 7.25, 10 L of 7.0 M HNE, and 10 mL
m
m
DMSO was run under the same conditions. The remaining activity of
HNE was expressed as %remaining activity ¼ (v/v0) ꢀ 100 and is the
average of duplicate or triplicate determinations.
(c) M. Wysocka, A. Lesner, K. Guzow, L. Mackiewicz, A. Legowska, W. Wiczk,
K. Rolka, Anal. Biochem. 378 (2008) 208e215;
5.3.2. Determination of inhibition constant (KI) for compound 24
against HNE
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J. Reuter, Med. Chem. 49 (2006) 1248e1260;
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M. Brillard-Bourdet, L. Juliano, F.J. Gauthier, Biol. Chem. 277 (2002)
39074e39081;
The KI of inhibitor 24 was determined using a Dixon plot [18].
Briefly, 970
was added in six cuvettes, followed by the sequential addition of 0,
2, 4, 6, 8 and 10 L of 5 mM solution of inhibitor 24 in DMSO and 10,
8, 6, 4, 2, 0 L DMSO for final inhibitor concentrations of 0e50 M.
10 L of 7.0 M HNE and 10 L of 35 mM substrate (methox-
mL 0.1 M HEPES buffer, pH 7.25, containing 0.5 M NaCl,
(g) B. Korkmaz, S. Attucci, T. Moreau, E. Godat, L. Juliano, F. Gauthier, Am. J.
Respir. Cell Mol. Biol. 30 (2004) 801e807.
m
m
m
[14] L. Wei, X. Gan, J. Zhong, K.R. Alliston, W.C. Groutas, Bioorg. Med. Chem. 11
(2003) 5149e5153.
[15] A.F. Abdel-Magid, K.G. Garson, B.D. Harris, C.A. Maryanoff, R.D. Shah, J. Org.
Chem. 61 (1996) 3849e3862.
[16] T. Apelqvist, D. Wensbo, Tetrahedron Lett. 37 (1996) 1471e1472.
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(2009) 3536e3542.
[18] M. Dixon, Biochem. J. 55 (1953) 170e171.
[19] (a) W.C. Groutas, J.R. Hoidal, M.J. Brubaker, M.A. Stanga, R. Venkataraman, B.
H. Gray, N.V. Rao, J. Med. Chem. 33 (1990) 1085e1087;
m
m
m
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