β-Lactam derivatives as enzyme inhibitors: 1-Peptidyl derivatives of 4-phenylazetidin-2-one as inhibitors of elastase and papain

Article abstract of DOI:10.1002/1521-4184(20008)333:8<243::AID-ARDP243>3.0.CO;2-O

N-Peptidyl substituted azetidin-2-ones were synthesized and evaluated as inhibitors of the serine protease elastase, and the cysteine protease papain. All compounds were synthesized from 4-phenylazetidin-2-one, either from the racemate or from the pure enantiomers. The (S)-enantiomer was prepared by enantioselective synthesis from (S)-β-phenyl-β-alanine, while the (R)-enantiomer was obtained by enzymatic resolution with α-chymotrypsin. N-Alkylation with bromoacetates introduced a spacer group which, after hydrolysis to the free acid, was acylated with amino acid esters or di- or tripeptide esters. The enzymatic assays proved some derivatives to be effective inhibitors of PPE and/or papain. N-BOC protected amino acid derivatives without a spacer group inhibited PPE reversibly, while derivatives with spacer-group showed either weak or no inhibitory properties. On the other hand, papain was inactivated irreversibly by ethyl (RS)-2-oxo-4-phenylazetidin-1-acetate. The highest inhibitory activity against papain was found for the diastereomers of N-(2-oxo-4-phenylazetidin-1-acetyl)-L-alanyl-L-valine benzyl ester, a compound with a spacer group.

Full text of DOI:10.1002/1521-4184(20008)333:8<243::AID-ARDP243>3.0.CO;2-O

Full Papers
β-Lactam Derivatives as Enzyme Inhibitors: 1-Peptidyl Derivatives of
4-Phenylazetidin-2-one as Inhibitors of Elastase and Papain
a)1)
b)
a)
Karin Achilles , Tanja Schirmeister , and Hans-Hartwig Otto *
a)
Lehrstuhl Pharmazeutische/Medizinische Chemie, Institut für Pharmazie, Ernst-Moritz-Arndt-Universität Greifswald, Friedrich-Ludwig-Jahn-Str. 17,
17487 Greifswald, Germany
b) Department of Pharmaceutical Chemistry, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 9, 79104 Freiburg i. Br., Germany
Key Words: 1,4-Disubstituted azetidin-2-ones; serine protease inhibitors; porcine pancreatic elastase; cysteine protease
inhibitors; papain
The development of protease inhibitors is based on the
catalytic mechanisms and structures of the active sites of the
proteases. All inhibitors developed up to now bear a chemi-
cally reactive group (“electrophilic trap”), which allows for
the nucleophilic attack of the active site amino acids of the
proteases. In addition, most inhibitors possess a peptide moi-
ety responsible for recognition by the enzyme. Among the
classes of cysteine protease inhibitors that appear most prom-
Summary
N-Peptidyl substituted azetidin-2-ones were synthesized and
evaluated as inhibitors of the serine protease elastase, and the
cysteine protease papain. All compounds were synthesized from
4-phenylazetidin-2-one, either from the racemate or from the pure
enantiomers. The (S)-enantiomer was prepared by enantioselective
synthesis from (S)-β-phenyl-β-alanine, while the (R)-enantiomer
was obtained by enzymatic resolution with α-chymotrypsin. N-Al-
kylation with bromoacetates introduced a spacer group which,
after hydrolysis to the free acid, was acylated with amino acid
esters or di- or tripeptide esters. The enzymatic assays proved some
derivatives to be effective inhibitors of PPE and/or papain. N-BOC
protected amino acid derivatives without a spacer group inhibited
PPE reversibly, while derivatives with spacer group showed either
weak or no inhibitory properties. On the other hand, papain was
inactivated irreversibly by ethyl (RS)-2-oxo-4-phenylazetidin-1-
acetate. The highest inhibitory activity against papain was found
for the diastereomers of N-(2-oxo-4-phenylazetidin-1-acetyl)-L-
alanyl-L-valine benzyl ester, a compound with a spacer group.
[14]
[15]
ising are epoxides
and aziridines . Effective inhibitors
[16]
of elastase include peptide aldehydes , trifluoromethyl ke-
[17]
[18]
tones , as well as bicyclic
and monocyclic β-lactam
[19,20]
derivatives
. In continuation of our interest in β-lactams
[21]
as enzyme inhibitors we reasoned that modification of our
structures would even lead to potent or more potent inhibitors
against proteases.
In this paper, we describe the syntheses of a number of
aminoacyl and peptidyl substituted azetidin-2-ones and their
activities as inhibitors of elastase and papain. In our concept
we use the β-lactam ring as the electrophilic trap, and choose
amino acids L-alanine, L-valine, and L-proline for the recog-
nition part as they are reported to be effective in other inhibi-
[16, 17]
tors
. Porcine pancreatic elastase (PPE) is similar to
HLE. It is easily available, and therefore it is used in our
experiments.
Introduction
Results and Discussion
Proteases have been found to be involved in the pathogene-
sis of a large number of diseases. In particular, the serine
protease elastase (HLE) is claimed to play a key role in the
Racemic 4-phenylazetidin-2-one (1) was prepared in a two
step synthesis from styrene and chlorosulfonylisocyanate
[1]
pathogenesis of lung diseases such as emphysema
,
[22]
according to Graf . The (S)-enantiomer (S)-1 was obtained
[2]
[3]
[4]
ARDS , fibrosis , but also in arthritis and arteriosclero-
sis . Cysteine proteases , including calpains and most
cathepsins, play a major role in processes with abnormal
protein degradation, e.g. musculardystrophy or myocardial
infarcts . Increased levels of these proteases are also found
in four steps from 3-amino-3-phenylpropionic acid. Esterifi-
cation with methanol/SOCl gave the methyl ester HCl,
[5]
[6]
2
[7]
[8]
[9]
[10]
[11]
in cancer , Alzheimer’s disease , and cataracts . It has
been shown that inhibition of these proteases can have thera-
[12]
peutic effects on some of these diseases . All thiol depend-
[13]
ent cathepsins belong to the papain gene family
.
Therefore, papain from the latex of the carica papaya fruit
represents a simple and inexpensive model for the evaluation
of cysteine protease inhibitors.
———
1) Part of the Ph. D. Thesis of K. A., University of Freiburg, 1997.
Scheme 1
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0365-6233/00/0808/0243 $17.50 +.50/0

244
Achilles, Schirmeister, and Otto
1
which was separated by kinetic resolution with (R,R)-tartaric
The enantiomeric excess usually can be determined by H
NMR shift experiments using optical active shift reagents like
TFMC-praseodymium(III) orTFEC-europium(III) . How-
acid via the diastereomeric salts according to Wassermann
[27]
[23]
and Berger
.
ever, this method failed, even when we used different con-
centrations of 3 and the reagents. Alternatively, we used
HPLC with a Whelck-O1 column, showing that the product
of enzymatic hydrolysis of 2b consists of a 92:8 mixture of
the enantiomers. The absolute configuration was established
The formation of the β-lactam ring was directly achieved
from methyl (S)-3-amino-3-phenylpropionate with tert-
butylmagnesium chloride after N-silylation with chloro-
trimethylsilane in the presence of triethylamine and
4-dimethylaminopyridine.
[28]
by CD spectroscopy. In accordance with the literature , we
A very mild method for the N-alkylation of azetidin-2-ones
[24]
postulate for the compound with a positive Cotton effect at λ
= 220 nm (∆ε = +4.9), a negative effect at λ = 232 nm, and a
positive effect at λ = 241 nm the (S)-configuration, (S)-2b.
Vice versa, the CD spectrum of the acid (R)-3 shows negative
Cotton effects at λ = 217 nm (∆ε = –10.7), and λ = 259 nm,
and a positive effect at λ = 235 nm (∆ε = +6.6). Furthermore,
is the use of phase-transfer catalysis (ptc) . In agreement
with this method described by Reuschling and Pietsch, N-al-
kylation of 1 and of (S)-1 with alkyl bromoacetates was
performed via solid/liquid phase transfer catalysis
[25]
(Scheme 2)
yielding the corresponding compounds 2.
S-2b remaining from the enzymatic hydrolysis gave an
25
equivalent CD spectrum and nearly identical [α]
values as
D
S-2b synthesized from S-1.
Hydrolysis of (S)-2b with NaOH or with PLE led to the
(S)-enantiomer (S)-3 with 51% or 59% yield. The catalytic
hydrogenolysis of 2c seemed to be not an appropriate route
to 3. Using an excess of hydrogen, the main product of this
reaction was not 3, but the ring opened N-(3-phenyl-
[29]
propionyl)glycine . Reaction with equivalent amounts of
H showed preferred hydrogenolysis of the ester (83%), but
2
the cyclic hetero benzyl bond was still attacked (17%).
The structures of compounds 2 and 3 were characterized by
their spectroscopic data. The IR spectra show a strong car-
Scheme 2
–1
bonyl absorption between 1740 and 1765 cm establishing
Hydrolysis of 2a with trifluoroacetic acid gave the racemic
β-lactam carboxylic acid 3 (83%). Alkaline hydrolysis of 2b
with refluxing 1 M NaOH gave the β-lactam carboxylic acid
3 and a ring opened by-product 3A. This side reaction was
avoided by reduced temperature (60 °C) during hydrolysis.
the intact β-lactam ring. Furthermore, the ring is charac-
1
terized by the ABX system of ring protons found in the H
NMR spectra with three signal groups around δ = 2.9, 3.45
and 4.85 ppm, and J = 2.5, J = 15.0 and J = 5.2–5.5
AX
AB
BX
Hz. The methylene group at nitrogen is detected by an AB
system at δ = 3.3–3.5 and 4.25–4.40 ppm, J ≈ 17.5 Hz.
The enzymatic hydrolysis of 2b with α-chymotrypsin under
pH-stat conditions
AB
[26]
Due to the diminished reactivity of the nitrogen in 1 the
acylation of the β-lactam ring was achieved only by deproto-
nation with BuLi followed by the reaction with N-protected
yielded the (R)-enantiomer (R)-3 with
84% enantiomeric excess. An enzyme screening including
porcine liver esterase (PLE), cholesterol esterase (ChE), por-
cine pancreatic lipase (PPL), lipase from Penicillium roque-
forti (PrL), proteinase K (PrK), and α-chymotrypsin (CTR)
selected α-chymotrypsin as the most appropriate enzyme for
the enzymatic resolution of 2b (Table 1).
[30]
amino acid N-carboxyanhydrides (NCA) . In accordance
[31]
with former results , under these conditions only acylation
of the nitrogen occured. No C-3 acylated products were
detected. The yields varied between 18 and 46%. From 1 and
L-alanine we obtained 4. Compound 5 was prepared from
(S)-1 and L-alanine, 6 was obtained from 1 and L-valine, and
7 from L-valine and (S)-1. The reaction failed when TEA was
used for the deprotonation. Furthermore, experiments using
anhydrides or chloroformates for activation of the amino acid
were unsuccessful. During the syntheses no racemization was
Table 1. Enzyme screening for 2b.
——————————————————————————————
Enzyme (amount*)
Turnover (%)
after 3 d
Opt. rotation
acid / ester
——————————————————————————————
1
observed, as could be demonstrated by H NMR-spectros-
without
0
100
19
0
0 / 0
copy.
PLE (200 µl)
ChE (50 mg)
PPL (500 mg)
PrL (50 mg)
PrK (50 mg)
CTR (64 mg)
0 / 0
n.d.** / –7°
0 / 0
0
0 / 0
39
~50
56° / –91°
80° / –113°
——————————————————————————————
* Amount used for 150 mg 2b. ** n.d. = not determined
Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)

Inhibitors of Elastase and Papain
245
Table 3. Peptidyl β-lactams (free acids).
——————————————————————————————
No. AA1 AA2 AA3 Educt Yield * Optical
mg/% rotation
——————————————————————————————
The peptides needed for the reactions with 3 were synthe-
sized by coupling the respective C-protected amino acids or
peptides with DCC/N-hydroxysuccinimide. Sterically hin-
dered peptides could be obtained by coupling with diphenyl
phosphorazidate (DPPA) (0 °C, DMF). The coupling of 3,
(S)-3, and (R)-3 with peptides was achieved with DPPA
or N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide HCl
(EDCl), alternatively to give the compounds 8–34 (Table 2).
To obtain the unprotected analogs 35–42, either the phenyl
35
36
37
38
39
40
41
42
Ala(OH)
Ala(OH)
Val
–
–
8
9
115/46% –22.9
.
–
–
39/47%
–37.8
–30.7
Ala(OH)
Ala(OH)
Ala(OH)
Pro
–
23 75/98%
esters (8, 9) were hydrolyzed (pH 10) with hydrogen perox-
Val
–
24 129/98% –12.5
[32]
ide
to yield 35 and 36, or the trichloroethyl-protecting
in the derivatives 23–25 and 32–34 was deprotected
Val
–
25 73/97%
32 78/99%
–7.1
[33]
group
Val
Ala(OH)
Ala(OH)
Ala(OH)
–61.8
with zinc in glacial acetic acid to give 37–42 in quantitative
[34]
yields
.
Val
Pro
33 139/98% –90.4
34 91/97% –30.5
Val
Pro
——————————————————————————————
* All compounds are obtained as viscous materials.
Enzyme Kinetics
In order to evaluate whether the inhibition is reversible or
irreversible, the time-dependent decrease of enzyme activity
was monitored by the increase of UV absorption at 405 nm
due to the release of p-nitroaniline from the respective sub-
α
strates: Ac-Ala-Ala-Ala-pNA for elastase, and N -benzoy-
larginine-p-nitroanilide-HCl (L-BAPA) for papain.
Table 2. Peptidyl β-lactams prepared from 3.
————————————————————————————————————————————
No.
AA1
AA2
AA3
Educt
Yield *
mg/%
Optical
rotation
————————————————————————————————————————————
8
Ala(OPh)
Ala(OPh)
Ala(OBn)
Phe(OMe)
Phe(OMe)
Phe(OMe)
Val(OBn)
Ala
–
–
3
98/19%
–34.7
–111.4
–35.4
–17.3
–33.3
–20.3
–22.2
–45.3
–114.6
–28.6
–110.9
–17.0
–52.1
–111.4
–7.4
9
–
–
(S)-3
3
100/19%
229/51%
250/36%
270/50%
200/56%
213/44%
114/22%
248/39%
322/29%
456/41%
275/48%
453/40%
264/39%
130/23%
302/62%
230/47%
155/42%
170/28%
181/30%
85/14%
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
–
–
–
–
3
–
–
(S)-3
(R)-3
3
–
–
–
–
Ala(OBn)
Ala(OBn)
Val(OBn)
Val(OBn)
Val(OBn)
Ala(OBn)
Ala(OBn)
Ala(OBn)
Ala(OTCE)
Ala(OTCE)
Ala(OTCE)
Pro(OBn)
Pro(OBn)
Pro(OBn)
Pro
–
3
Ala
–
(S)-3
3
Ala
–
Ala
–
(S)-3
(R)-3
3
Ala
–
Val
–
Val
–
(S)-3
(R)-3
3
Val
–
Val
–
–45.2
–101.1
–42.3
–77.4
–128.9
–45.9
–65.2
–150.7
–55.4
–76.9
–134.4
–31.8
Val
–
(S)-3
(R)-3
3
Val
–
Val
–
Val
–
(S)-3
(R)-3
3
Val
–
Val
Ala(OBn)
Ala(OBn)
Ala(OBn)
Ala(OTCE)
Ala(OTCE)
Ala(OTCE)
126/18%
165/24%
165/24%
300/41%
325/44%
152/43%
Val
Pro
(S)-3
(R)-3
3
Val
Pro
Val
Pro
Val
Pro
(S)-3
(R)-3
Val
Pro
————————————————————————————————————————————
* The yields refer to the last step of the synthesis.
Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)

246
In cases where no time-dependent inhibition was observed,
Achilles, Schirmeister, and Otto
The mixture of diastereomers 17 and the diastereomers 18
the inhibitor constants K were calculated from Dixon plots. and 19 were the most potent inhibitors of papain. The dia-
I
Lineweaver-Burke and Eadie-Hofstee plots indicated that the stereomeric compounds 18 and 19 did not show a stereodif-
mode of inhibition of PPE is competitive for the substrate.
ferentiating profile. Furthermore, the N-alkylated compound
Alternatively, inactivation rates for time-dependent inhibi- 2b, the N-acylated compounds 4, and the β-lactam peptides
tion were determined by dilution assays according to Kitz and 11 and 14 were weak inhibitors of papain. All compounds
[35]
[36]
Wilson or continuously as described by Tian und Tsou
.
were irreversible inhibitors.
K values characterizing the inhibition of PPE and inactiva-
I
The different mechanisms of inhibition for PPE and papain
tion rates characterizing the inhibition of papain are presented may be explained by the higher nucleophilic properties of the
in Table 4.
thiolate residue of the catalytic active cysteine residue com-
pared to the hydroxy group of the active site serine residue.
Therefore, a nucleophilic attack with formation of a covalent
bond to the carbonyl carbon is facilitated. Furthermore, the
suitability of 2b as a substrate for the enzymatic hydrolysis
with a serine protease also indicates that the phenyl-substi-
tuted β-lactam ring-system can act only as a reversible inhibi-
tor of elastase. Thus, the inhibitors either compete with the
substrate for the active site or bind at a site near the active
site, which also hinders the interaction of the substrate with
the catalytic site.
Discussion
The highest inhibitory activity against PPE was found for
the N-acylated compounds 4, 5, and 6. This is in good accord-
ance with the results from Doherty et al. who found high
inhibition constants for N-acylated β-lactams bearing a leav-
[37]
ing group in position 4 . Compounds 10, 11, 14, 15, 17, 18,
20, 26, and 29 showed no or only weak inhibitory activity
against PPE. Compared to the C-protected compound 20, the
deprotected compound 37 was a less efficient inhibitor. Com-
pounds 2b and 3 did not inhibit PPE. This does not seem to
be surprising since 2b serves as a substrate for the serine
protease α-chymotrypsin, and the product of this enzymatic
hydrolysis was (R)-3. Interestingly, the pure (S,R)-diastereo-
mer 5 was less effective than the diastereomeric mixture 4.
This might suggest that the (R,S)-diastereomer of 5, which
was not available, is the better inhibitor than the (S,R)-dia-
stereomer 5. All compounds inhibited PPE in a competitive,
reversible manner.
Acknowledgment
We wish to thank Degussa AG, for generous support with amino acids, E.
Merck AG, Darmstadt, for the HPLC experiments and the support with
chromatography materials, and the “Fonds der Chemischen Industrie” for
financial support. T. S. gratefully acknowledges the continuous support from
the “Deutsche Forschungsgemeinschaft”.
Table 4. Results of the enzymatic tests.
——————————————————————————————
Experimental
No.
Elastase
KI [mM] —————————————————
Papain k2nd [M–1min–1
]
General: Mp: not corrected; Linström apparatus. IR spectra (cm–1): Per-
kin-Elmer IR 841; in KBr, if not noted otherwise. NMR spectra: Varian
U-300 (300 MHz) for 1H; Varian U-300 (75.4 MHz) for 13C; δ in ppm;
1H-values and 13C-values from spectra in CDCl3, if not noted otherwise.
Standard ref. for all spectra in CDCl3 or D4-MeOH was the signal of the
undeuterated solvent obtained in D2O. UV spectra: Pharmacia Ultrospec III.
Polarimeter: Perkin-Elmer 241. Mass Spectra: Finnigan MAT 312. pH-stat:
Metrohm 702 SM Titrino. Elementary analyses: Pharmazeutisches Institut or
Chemisches Laboratorium der Universität Freiburg. All compounds gave
satisfactory elemental analyses or were proven by high resolution MS. Rf
values from cc (silica gel 60 Merck 7734). Tlc (Merck Alufolien, silica gel
60 F254, No. 5549).
Continuous
Assay
Dilution
Assay
——————————————————————————————
2a
2b
(S)-2b
3
–
–
–
–
8
57
n.i.
–
n.i.
–
n.i.
–
9
7
(S)-3
(R)-3
4
–
–
–
–
–
–
19
24
–
0.76
2.08
1.58
n.i.
n.i.
1.6
n.i.
1.4
n.i.
–
Tetrahydrofuran (THF) was stored with KOH, then refluxed with Na and
benzophenone, and distilled prior to use. Other solvents were dried/ purified
according to literature procedures. Unless noted otherwise, reactions were
conducted under an atmosphere of dry nitrogen.
5
6
–
10
11
14
15
17
18
19
20
26
29
37
40
n.i.
11
–
PPE was purchased fromServa(Nr. 20929), α-chymotrypsin, and L-BAPA
from Merck Darmstadt, proteinase K from Sigma, cholesterol esterase from
Boehringer Mannheim, and papain from carica papya, PLE, PPL, and lipase
from penicillium roqueforti from Fluka. All enzymes were used without
further purification. All substrates were purchased from Bachem.
Abbreviations: L-BAPA = Nα-Benzoylarginine-p-nitroanilide HCl; BuLi
= n-Butyllithium, 1.6 M solution in n-hexane; EtOAc = Ethyl acetate; TEA
= Triethylamine; CTMS = Chlorotrimethylsilane; DMAP = 4-Dimethylami-
nopyridine; TMEDA = Tetramethylethylenediamine; DPPA = Diphenyl
phosphorazidate; EDCl = N-(3-Dimethylaminopropyl)-N′-ethylcarbodi-
imide HCl; DCC = Dicyclohexylcarbodiimide; ar = aromatic.
–
78
–
n.i.
773
563
761
n.i.
n.i.
n.i.
n.i.
n.i.
–
–
–
–
2.4
n.i.
n.i.
2.1
n.i.
–
–
–
–
–
(RS)-4-Phenylazetidin-2-one (1)
——————————————————————————————
–: not determined; n.i.: no inhibition
Overall yield: 60%.– Mp 105 °C (ref.[22] 107 °C).
Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)

Inhibitors of Elastase and Papain
247
IR (film): ν = 3440 (OH), 2928 (CH), 1737 (C=O).– 1H NMR: δ = 2.94,
3.48, 4.88 (ABX, JAX = 2.3 Hz, JBX = 5.2 Hz, JAB = 15.0 Hz, 3H, 3-H, 3-H′,
4-H), 3.47, 4.27 (AB, J = 18.3 Hz, 2H, CH2), 7.35 (m, 5H, ar-H), 9.07 [s(br),
1H, CO2H].– MS (70 eV): m/z (%) = 205 (2.3) [M+], 177 (19), 160 (2.9)
[M+-CO2H], 146 (1.5) [M+-CH2CO2H], 104 (100).– C11H11NO3 (205.21).–
HRMS: Calcd. 205.0738; found 205.0739.
(S)-4-Phenylazetidin-2-one ((S)-1)
To a solution of methyl (S)-3-amino-3-phenylpropionate (6.0 g, 33 mmol)
in diethyl ether (150 ml) at 0 °C were added TEA (4.6 ml, 33 mmol), a
catalytic amount of DMAP (≈ 50 mg) and CTMS (4.2 ml, 33 mmol). After
stirring for 3 h, the mixture was maintained at room temp. for 12 h. The
precipitate was filtered off, diethyl ether (600 ml) was added, and the
solution was cooled to –5 °C. tert-Butylmagnesium chloride (18.2 ml,
37 mmol, as 2 M solution in diethyl ether) was added dropwise. The mixture
was stirred vigorously for 30 min at –5 °C, and then 12 h at room temp. With
cooling, the solution was acidified with HCl-satd. diethyl ether. The precipi-
tated salt was dissolved in water (100 ml), the phases were separated and the
aqueous layer was extracted with diethyl ether (100 ml). The combined
organic layers were dried (Na2SO4) and concentrated to dryness. The residue
was dissolved in methanol (50 ml), warmed to 50 °C and the solvent was
removed in vacuo. The product was crystallized with cyclohexane. Recrys-
tallization from cyclohexane/chloroform (1:1). Yield: 1.5 g (31%). Light
yellow crystals.– Mp 108 °C (ref.[22] 107 °C).– [α]D25 = –130.2° (c = 1.12,
EtOH).
(S)-2-(2-Oxo-4-phenylazetidin-1-yl)acetic Acid ((S)-3)
From (S)-2b (2.0 g, 8.6 mmol) as described for 3 (method b). Yield: 0.9 g
(51%). Colourless liquid.– [α]D25 = –98.2° (c = 0.97, CHCl3).
(R)-2-(2-Oxo-4-phenylazetidin-1-yl)acetic Acid ((R)-3)
Phosphate buffer (350 ml, pH 8, 0.05 M), and α-chymotrypsin (350 mg)
were added to a solution of 2b (3.5 g, 15 mmol) in acetone (35 ml). As soon
as the pH started decreasing, the solution was adjusted to the initial pH with
0.1 M NaOH. When 0.4 eq. of sodium hydroxide was used, the reaction was
stopped by adding EtOAc (200 ml). The phases were separated, and the
aqueous layer was extracted with EtOAc (3 x 200 ml). The combined organic
layers were dried (Na2SO4) and evaporated (phase I). The aqueous phase was
adjusted to pH 2 with 2 M HCl, and extracted with EtOAc (3 x 200 ml). The
combined organic layers were dried and evaporated (phase II). Phase I: Yield:
1.0 g (57%) (S)-2b. Colourless liquid. Phase II: Yield: 1.1 g (65%) (R)-3.
Colourless liquid.– [α]D25 = +80.3° (c = 1.09, CHCl3).
General Procedure for the N-Alkylation of 1
The appropriate bromoacetate (10 mmol), and powdered KOH (0.62 g,
11 mmol) were added to a solution of 1 or (S)-1 (1.47 g, 10 mmol), and
tetrabutylammonium bromide (0.36 g, 1 mmol) in THF (50 ml). The mix-
ture was stirred for 8–10 h (tlc control), filtered, and the filtrate was concen-
trated in vacuo. The residue was purified by cc with EtOAc or recrystallized
from ethanol/water (1:1).
(RS)-3-Aza-4-phenylhexane-1,6-diacid (3A)
By-product from the synthesis of 3, method b. Yield: ca. 10%.– Mp.
201 °C.– IR: ν = 3426 (OH, NH), 2929 (CH), 1716, 1656 (C=O).– 1H NMR
([D4]MeOH): 3.07, 3.23, 4.77 (ABX, JAX = 7.8 Hz, JBX = 6.7 Hz, JAB = 16.8
Hz, 3H, 3-H, 3-H′, 4-H), 3.61, 3.75 (AB, J = 16.8 Hz, 2H, CH2), 7.29 (m,
5H, ar-H).– MS (70eV): m/z (%) = 223 (2), 177 (57) [M+-CH2CO2H], 151
(44) [M+-NH-CH2-CO2H]. HREIMS: Calcd. 223.0842; found 223.0845.–
Anal. (C11H13NO4).
tert-Butyl (RS)-2-(2-Oxo-4-phenylazetidin-1-yl) Acetate (2a)
Yield: 2.51 g (96%). Colourless crystals.– Mp 64 °C.– IR: ν = 3006, 2977,
2931 (CH), 1753 (C=O).– 1H NMR: δ = 1.44 [s, 9H, C(CH3)3], 2.89, 3.45,
4.88 (ABX, JAX = 2.5 Hz, JBX = 5.5 Hz, JAB = 15.0 Hz, 3H, 3-H, 3-H′, 4-H),
3.33, 4.27 (AB, J = 17.7 Hz, 2H, CH2), 7.31 (m, 5H, ar-H).– Anal.
(C15H19NO3).
General Procedure for the N-Acylation of 1, (S)-1
Ethyl (RS)-2-(2-Oxo-4-phenylazetidin-1-yl)acetate (2b)
A solution of 1 or (S)-1 (6.8 mmol) in THF (50 ml) was cooled to –78 °C,
BuLi (4.7 ml, 7.5 mmol) was added, and the mixture was stirred for 10 min.
A solution of the appropriate BOC-amino-acid-NCA (6.8 mmol) in THF
(10 ml) was added, and the reaction was completed by stirring at –78 °C for
1 h. The mixture was warmed to room temp., and subsequently quenched
with a satd. solution of NH4Cl (100 ml). The layers were separated, and the
aqueous layer was extracted with EtOAc (2 × 50 ml). The combined organic
layers were dried (Na2SO4) and concentrated in vacuo. The product was
purified by cc (silica gel/EtOAc), if not otherwise noted.
Yield: After cc 1.65 g (71%). Colourless liquid [24].– Rf = 0.59.
Ethyl (S)-2-(2-Oxo-4-phenylazetidin-1-yl)acetate ((S)-2b)
Yield: After cc 1.51 g (65%). Colourless liquid.– Rf = 0.59.– [α]D25 = –
117.3° (0.99, CHCl3).– IR (film): ν = 2983 (CH), 1765 (C=O).– 1H NMR:
δ = 1.25 (t, J = 7.0 Hz, 3H, CH3), 2.92, 3.48, 4.88 (ABX, JAX = 2.4 Hz,
JBX = 5.2 Hz, JAB = 15.0 Hz, 3H, 3-H, 3-H′, 4-H), 3.44, 4.35 (AB,
J = 17.4 Hz, 2H, CH2), 4.16 (m, 2H, CH2CH3), 7.37 (m, 5H, ar-H).– Anal.
(C13H15NO3).
(S,R)-1-[(S)-N-(tert-Butoxycarbonyl)alanyl]-4-phenylazetidin-2-one (4)
From 1 and BOC-ala-NCA. Yield: 1.00 g (46%). Colourless solid.– Mp
140 °C.– Rf = 0.64.– αD25 = –68.9° (c = 0.92, EtOH).– 1H NMR: δ = 1.40
[2s, 9H, C(CH3)3], 1.42 (2AB, 3H, CH3), 2.98, 3.53, 5.08 (2ABX, 3H, 3-H,
3-H′, 4-H), 4.92 [m, 1H, α-H(ala)], 5.08 (m, 1H, N-H), 7.31 (m, 5H, ar-H).–
C17H22N2O4 (318.38).
Benzyl (RS)-2-(2-Oxo-4-phenylazetidin-1-yl)acetate (2c)
Yield: After cc 2.48 g (84%). Colourless liquid. – Rf = 0.58.– IR (film): ν
= 3033, 2958 (CH), 1759 (C=O).– 1H NMR: δ = 2.87, 3.42, 4.84 (ABX, JAX
= 2.6 Hz, JBX = 5.4 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H), 3.42, 4.40 (AB,
J = 18.1 Hz, 2H, CH2), 5.13 [AB, J = 12.2 Hz, 2H, CH2(benzyl)], 7.30 (m,
10H, ar-H).– Anal. (C18H17NO3).
(S)-1-[(S)-N-(tert-Butoxycarbonyl)alanyl]-4-phenylazetidin-2-one (5)
From (S)-1 and BOC-ala-NCA. Yield: 0.48 g (22%). Light yellow solid.–
(RS)-2-(2-Oxo-4-phenylazetidin-1-yl)acetic Acid (3)
25
Mp 137 °C.– Rf = 0.64.– [α]D = –147.7° (c = 0.89, EtOH).– IR:
ν = 3411 (NH), 1777, 1705 (C=O).– 1H NMR: δ = 1.44 [d, 3H, CH3; s, 9H,
C(CH3)3], 2.98, 3.53, 5.07 (ABX, JAX = 3.4 Hz, JBX = 6.6 Hz,
Method a: Trifluoroacetic acid (5 ml) was added at 0 °C to a solution of
2a (4.0 g, 15.3 mmol) in CH2Cl2 (5 ml). After stirring for 1 h at 0 °C, the
mixture was hydrolyzed with 0.1 M NaOH. The layers were separated, the
aqueous layer was adjusted to pH 2 with 2 M HCl and extracted with CH2Cl2
(2 × 25 ml). The combined organic layers were dried (Na2SO4) and evapo-
rated. Yield: 2.6 g (83%). Colourless liquid.
Method b: A solution of 2b (2.0 g, 8.6 mmol) in 1 M NaOH solution (20
ml) was warmed to 60 °C for 20 min. After cooling, the mixture was
extracted with EtOAc, and the organic phase was discharged. The aqueous
layer was adjusted to pH 2 with 2 M HCl and extracted with EtOAc (3 ×
50 ml). The combined organic layers were dried (Na2SO4) and evaporated.
Yield: 1.5 g (87%). Colourless liquid.
JAB = 16.5 Hz, 3H, 3-H, 3-H′, 4-H), 4.93 [m, 1H, α-H(ala)], 5.10 (m, 1H,
NH), 7.31 (m, 5H, ar-H).– Anal. (C17H22N2O4).
(S,R)-1-[(S)-N-(tert-Butoxycarbonyl)valyl]-4-phenylazetidin-2-one (6)
From 1 and BOC-val-NCA. Yield: 0.42 g (18%). Colourless solid.– Mp
1
40 °C.– Rf = 0.52.– αD25 = -34.8° (c = 0.99, EtOH).– H NMR: δ = 0.81,
1.07 (2d, J = 6.8 Hz, 6H, 2 CH3), 1.43 [s, 9H, C(CH3)3], 2.25 [m, 1H,
β-H(val)], 3.00. 3.53, 5.05 (ABX, JAX = 3.7 Hz, JBX = 6.7 Hz,
JAB = 16.6 Hz, 3H, 3-H, 3-H′, 4-H), 4.85 [m, 1H, α-H(val)], 5.0 (m, 1H,
Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)

248
Achilles, Schirmeister, and Otto
N-H), 7.30 (m, 5H, ar-H).– MS (70 eV): m/z (%) = 346 (6) [M+], 290 (12)
[M+-tBu], 132 (64), 116 (97).– C19H26N2O4 (346.42).– HRMS: Calcd.
346.1889; found 346.1893.
(C-3), 47.87, 48.00 [C-α (ala)], 55.00, 55.06 (C-4), 66.76, 66.85 [CH2(ben-
zyl)], 126.29, 126.35, 127.77, 127.85, 128.09, 128.15, 128.30, 128.35,
128.44, 128.76, 128.79, 135.09, 135.12, 137.02, 137.16 (ar-C), 166.67,
166.78, 168.02, 168.08, 172.11 (C=O).– MS (70 eV): m/z (%) = 366 (8.0)
[M+], 338 (46), 275 (98), 189 (26), 118 (76), 91 (100).– C21H22N204
(366.42).– HRMS: Calcd. 366.1579; found 366.1579. Ratio of diastereomers
(S)-1-[(S)-N-(tert-Butoxycarbonyl)valyl]-4-phenylazetidin-2-one (7)
(
13C NMR) 1:1.
From (S)-1 and BOC-val-NCA. Yield: 0.14 g (6%). Colourless solid.– Mp
38 °C.– Rf = 0.52.– [α]D25 = –129.7° (c = 0.78, EtOH).– IR: ν = 3377 (NH),
2971, 2933 (CH), 1793, 1707 (C=O).– 1H NMR: δ = 0.81, 1.07 (2d,
J = 6.8 Hz, 6H, 2 CH3), 1.43 [s, 9H, C(CH3)3], 2.25 [m, 1H, β-H(val)], 3.00.
3.53, 5.05 (ABX, JAX = 3.7 Hz, JBX = 6.7 Hz, JAB = 16.6 Hz, 3H, 3-H, 3-H′,
4-H), 4.85 [m, 1H, α-H(val)], 5.0 (m, 1H, N-H), 7.30 (m, 5H, ar-H).– MS
(70 eV): m/z (%) = 346 (6) [M+], 290 (12) [M+-tBu], 132 (64), 116 (97).–
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-phenylalanine Methyl Es-
ter (11)
From 3 (400 mg, 1.9 mmol), and L-phenylalanine methyl ester HCl
(463 mg, 2.0 mmol). Yield: 250 mg (36%). Colourless liquid.– Rf = 0.52.–
αD25 = –17.3° (c = 0.97, EtOH).
C
19H26N2O4 (346.42).– HRMS: Calcd. 346.1889; found 346.1893.
General Procedure for the Coupling of 3, S-3 or R-3 with Amino Acid or
Peptide Esters
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-phenylalanine Methyl Ester
Method a: DPPA (1.1 eq) and TEA (2.1 eq) were added at 0 °C to a
solution of 3, (S)-3 or (R)-3 (1 eq) and the N-deprotected peptide or amino
acid ester (1.04 eq) in DMF (8 ml). The mixture was stirred at 0 °C for 10 h.
EtOAc was added, and the mixture was washed with water (3× 50 ml), then
with a satd. NaHCO3 solution (50 ml), and then with brine (50 ml). The
organic layer was dried (Na2SO4), the solvent was removed in vacuo, and
the residue was purified by cc (silica gel, EtOAc).
Method b: EDCl (1.1 eq) and TEA (1 eq) were added to a solution of 3,
(S)-3, or (R)-3 (1 eq) and the N-deprotected peptide or amino acid ester (1 eq)
in DMF (20 ml). The mixture was stirred for 2 h at room temp., EtOAc was
added, and the organic phase was separated, washed with water (50 ml), 1 M
HCl (50 ml), water (50 ml), a satd. NaHCO3 solution (50 ml), and finally
with water (50 ml). The organic layer was dried (Na2SO4), and the solvent
was removed in vacuo. The residue was purified by cc (silica gel/EtOAc).
Method a was used, if not noted otherwise.
(12)
From (S)-3 (300 mg, 1.46 mmol), and L-phenylalanine methyl ester HCl
(328 mg, 1.52 mmol). Yield: 270 mg (50%). Colourless liquid.– Rf = 0.42.–
[α]D25 = –33.3° (c = 0.70, EtOH).– IR (film): ν = 3299 (NH), 1749, 1687
(C=O).– 1H NMR: δ = 2.90, 3.43, 4.68 (ABX, JAX = 2.4 Hz, JBX = 5.1 Hz,
JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H), 3.04, 3.12, 4.81 [ABX, JAX = 6.4 Hz,
JBX = 5.6 Hz, JAB = 13.9 Hz, 3H, β-H(phe), α-H(phe)], 3.37, 4.12 (AB,
J = 16.9 Hz, 2H, CH2), 3.74 (s, 3H, OCH3), 6.46 (d, J = 8.1 Hz, 1H, NH),
7.25 (mc, 10H, ar-H).– 13C NMR: δ = 37.27 [β-C(phe)], 44.44 (CH2), 47.08
(C-3), 52.41 (OCH3), 53.19 [α-C(phe)], 55.24 (C-4), 126.50, 127.22, 128.64,
128.80, 129.12, 129.19, 135.56, 137.10 (ar-C), 166.84, 168.09, 171.58
(C=O).– MS (70 eV): m/z (%) = 366 (39) [M+], 205 (75), 175 (55), 162 (96),
149 (67) 131 (70), 118 (100).– C21H22N2O4 (366.42).– HRMS: Calcd.
366.1580; found 366.1580.
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanine Phenyl Ester (8)
{2-[(R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-phenylalanine Methyl Ester
(13)
Method a: From 3 (300 mg, 1.46 mmol), and L-alanine phenyl ester HCl
(307 mg, 1.52 mmol). Yield: 98 mg (19%).
Method b: From 3 (450 mg, 2.2 mmol), and L-alanine phenyl ester HCl
(442 mg, 2.2 mmol). Yield: 93 mg (12%). Colourless liquid.– Rf = 0.63.–
From (R)-3 (200 mg, 0.97 mmol), and L-phenylalanine methyl ester HCl
(218 mg, 1.01 mmol). Yield: 200 mg (56%). Colourless liquid.– Rf = 0.42.–
[α]D25 = –20.3° (c = 1.53, EtOH).– IR (film): ν = 3304 (NH), 1742, 1680
(C=O).– 1H NMR: δ = 2.87, 3.36, 4.49 (ABX, JAX = 2.6 Hz, JBX = 5.2 Hz,
JAB = 14.7 Hz, 3H, 3-H, 3-H′, 4-H), 3.04, 3.18, 4.80 [ABX, JAX = 5.8 Hz,
JBX = 5.8 Hz, JAB = 14.0 Hz, 3H, β-H(phe), α-H(phe)], 3.36, 4.08 (AB,
J = 16.9 Hz, 2H, CH2), 3.70 (s, 3H, OCH3), 6.40 (d(br), 1H, N-H), 7.25 (m,
10H, ar-H).– 13C NMR: δ = 37.60 [β-C(phe)], 44.41 (CH2), 47.06 (C-3),
52.40 (OCH3), 53.10 [α-C(phe)], 55.24 (C-4), 126.47, 127.23, 128.65,
128.75, 129.07, 129.24, 135.73, 137.10 (ar-C), 166.89, 168.06, 171.49
(C=O).– MS (70 eV): m/z (%) = 366 (1.9) [M+], 162 (44), 131 (32), 118 (99),
91 (100).– C21H22N2O4 (366.42).– HRMS: Calcd. 366.1580; found
366.1583.
1
αD25 = –34.7° (c = 1.13, EtOH).– H NMR: δ = 1.48, 1.53 (d, J = 7.3 Hz,
3H, CH3), 2.89 (2.91), 3.46, 4.78 (ABX, JAX = 2.7 Hz, JAB = 15.0 Hz, 3H,
3-H, 3-H′, 4-H), 3.46, 4.15 (AB, 2H, CH2), 4.70 [m, 1H, α-H(ala)], 6.74 [m,
1H, N-H(ala)], 7.17 (m, 10H, ar-H).– C20H20N204 (352.39). Ratio of dias-
tereomers (13C NMR) 1:1.
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanine Phenyl Ester (9)
From (S)-3 (300 mg, 1.46 mmol), and L-alanine phenyl ester HCl (307 mg,
1.52 mmol). Yield: 100 mg (19%). Hard, glasslike liquid.– Rf = 0.63.–
αD25 = 111.4° (c = 0.90, EtOH).– IR (film): ν = 3291 (NH), 3067, 2936 (CH),
1746, 1674 (C=O).– 1H NMR: δ = 1.53 (d, J = 7.6 Hz, 3H, CH3), 2.98, 3.49,
4.77 (ABX, JAX = 2.6 Hz, JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H),
3.49, 4.20 (AB, J = 16.6 Hz, 2H, CH2), 4.77 [dq, J = 7.3 Hz, 1H, α-H(ala)],
6.74 (d, J = 7.3 Hz, 1H, N-H), 7.26 (m, 10H, ar-H).– 13C NMR: δ = 17.76
[CH3(ala)], 44.41 (CH2), 46.85 (C-3), 48.21 [α-C(ala)], 55.32 (C-4), 121.15,
126.12, 126.53, 128.73, 128.85, 129.04, 129.44, 137.04, 150.32 (ar-C),
167.02, 168.42, 171.31 (C=O).– MS (CI): m/z (%) = 353 (100) [M++1].– MS
(70 eV): m/z (%) = 259 (74), 231 (50) [M+-COOPh], 189 (100) [M+-
ala(OPh)], 160 (36) [M+-CH2COala(OPh)], 146 (41), 127 (76), 118 (52), 104
(34), 91 (48).– C20H20N204 (352.39).
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valine Benzyl Ester (14)
From 3 (250 mg, 1.22 mmol), and L-valine benzyl ester tosylate (509 mg,
1.27 mmol). Yield: 213 mg (44%). Colourless liquid.– Rf = 0.42.– αD25 = –
22.2° (c = 1.22, EtOH).– IR (film): ν = 3302 (NH), 1742, 1683 (C=O).–
1H NMR: δ = 0.88 [m, 6H, CH3(val)], 2.19 [m, 1H, β-H(val)], 2.94, 3.45,
4.71, 4.78 (2 ABX, JAX = 2.4 Hz, JBX = 5.4 Hz, 3H, 3-H, 3-H′, 4-H), 3.43,
3.53, 4.12, 4.20 (2 AB, J = 16.6 Hz, 2H, CH2), 4.55 [m, 1H, α-H(val)], 5.18
[m, 2H, CH2(benzyl)], 6.65 (m, 1H, N-H), 7.35 (m, 10H, ar-H).– 13C NMR:
δ = 17.62, 17.67, 18.92 [CH3(val)], 31.03, 31.07 [β-C(val)], 44.28, 44.50
(CH2), 46.89, 46.92 (C-3), 55.24, 55.40(C-4), 57.08, 57.33 [α-C(val)], 66.97,
67.07 [CH2(benzyl)], 126.49, 128.28, 128.36, 128.39, 128.46, 128.54,
128.59, 128.64, 128.71, 129.02, 129.06, 129.16, 135.26, 135.28, 137.29,
137.37 (ar-C), 167.42, 168.23, 168.34, 171.39, 171.44 (C=O).– MS (70 eV):
m/z (%) = 394 (16) [M+], 366 (40), 303 (83), 259 (41), 216 (74), 118 (92),
104 (75), 91 (100).– C23H26N204 (394.47).– HRMS: Calcd. 394.1893; found
394.1892. Ratio of diastereomers (13C NMR) 1:1.
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanine Benzyl Ester (10)
From 3 (250 mg, 1.22 mmol), and L-alanine benzyl ester tosylate (445 mg,
1.26 mmol). Yield: 229 mg (51%). Colourless liquid.– Rf = 0.52.– αD25 = –
35.4° (c = 1.21, EtOH).– IR (film): ν = 3306 (NH), 1745, 1675 (C=O).–
1H NMR: δ = 1.33, 1.40 (2d, J = 7.2 Hz, 3H, CH3), 2.90, 2.95, 3.45, 4.70,
4.75 (2 ABX, 3H, 3-H, 3-H′, 4-H), 3.45, 4.10 (AB, 2H, CH2), 4.55 [m, 1H,
α-H(ala)], 5.16 [2AB, 2H, CH2(benzyl)], 6.60 (m, 1H, N-H), 7.31 (m, 10H,
ar-H).– 13C NMR: δ = 17.59 [2 CH3(ala)], 43.65, 43.90 (CH2), 46.61, 46.67
Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)

Inhibitors of Elastase and Papain
249
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanyl-L-alanine Benzyl
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-alanine Benzyl Es-
Ester (15)
ter (20)
From 3 (250 mg, 1.22 mmol), and L-alanyl-L-alanine benzyl ester HCl
(361 mg, 1.26 mmol). Yield: 114 mg (22%). Colourless liquid.– Rf = 0.29.–
αD25 = –45.3° (c = 1.30, EtOH). Ratio of diastereomers (13C NMR) 1:1.
From 3 (500 mg, 2.44 mmol), and L-valyl-L-alanine benzyl ester HCl
(767 mg, 1.22 mmol), method b. Yield: 453 mg (40%). Colourless liquid.–
Rf = 0.43.– αD25 = –52.1° (c = 0.58, EtOH).– Ratio of diastereomers (13
C
NMR) 1:1.– C26H31N3O5 (465.55).
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanyl-L-alanine Benzyl
Ester (16)
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-alanine Benzyl Ester
(21)
From (S)-3 (300 mg, 1.46 mmol), and L-alanyl-L-alanine benzyl ester HCl
(436 mg, 1.52 mmol). Yield: 248 mg (39%). Colourless solid.– Mp 108 °C.–
Rf = 0.29.– [α]D25 = 114.6° (c = 0.96, EtOH).– IR: ν = 3305 (NH), 1741,
1656 (C=O).– 1H NMR: δ = 1.30, 1.43 (2d, each J = 6.8 Hz, 3H, CH3), 2.97,
3.51, 4.78 (ABX, JAX = 2.4 Hz, JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′,
4-H), 3.46, 4.12 (AB, J = 16.9 Hz, 2H, CH2), 4.42, 4.59 [2dq, each
J = 7.3 Hz, 1H, α-H(ala)], 5.18 [AB, J = 12.2 Hz, 2H, CH2(benzyl)], 6.47
(d, J = 7.1 Hz, 1H, N-H), 6.69 (d, J = 7.3 Hz, 1H, N-H), 7.38 (m, 10H,
ar-H).– 13C NMR: δ = 17.88, 18.30 [CH3(ala)], 44.24 (CH2), 46.99 (C-3),
48.23, 48.73 [α-C(ala)], 55.38 (C-4), 67.11 [CH2(benzyl)], 126.55, 128.08,
128.39, 128.56, 128.74, 129.07, 135.28, 137.26 (ar-C), 166.96, 168.33,
171.57, 172.39 (C=O).– MS (70 eV): m/z (%) = 437 (3.7) [M+], 259 (31),
189 (40), 91 (100).– C24H27N3O5 (437.50).– HRMS: Calcd. 437.1950; found
437.1949.
From (S)-3 (300 mg (1.46 mmol), and L-valyl-L-alanine benzyl ester HCl
(503 mg, 1.60 mmol), method b. Yield: 264 mg (39%). Colourless solid.–
25
Mp 133 °C.– Rf = 0.40.– [α]D = –111.4° (c = 0.90, EtOH).– IR: ν = 3293
1
(NH), 3067, 2963 (CH), 1748, 1642 (C=O).– H NMR: δ = 0.86, 0.91 [2d,
each J = 6.6 Hz, 3H, CH3(val)], 1.43 [d, J = 7.3 Hz, 3H, CH3(ala)], 2.06 [m,
1H, β-H(val)], 2.95, 3.51, 4.76 (ABX, JAX = 2.4 Hz, JBX = 5.4 Hz,
AB = 15.0 Hz, 3H, 3-H, 3-H′, 4-H), 3.44, 4.15 (AB, J = 16.9 Hz, 2H, CH2),
J
4.22 [dd, J = 6.1 Hz, J = 8.8 Hz, 1H, α-H(val)], 4.60 [dq, J = 7.3 Hz, 1H,
α-H(ala)], 5.15 [AB, J = 12.2 Hz, 2H, CH2(benzyl)], 6.36 [d, J = 7.3 Hz, 1H,
N-H(ala)], 6.67 [d, J = 8.8 Hz, 1H, N-H(val)], 7.37 (m, 10H, ar-H).–
13C NMR: δ = 17.95 [CH3(ala), CH3(val)], 18.99 [CH3(val)], 31.24 [β-
C(val)], 44.47 (CH2), 47.04 (C-3), 48.15 [α-C(ala)], 55.30 (C-4), 58.33
[α-C(val)], 67.13 [CH2(benzyl)], 126.49, 128.13, 128.40, 128.55, 128.74,
129.09, 135.24, 137.28 (ar-C), 167.18, 168.34, 170.35, 172.38 (C=O).– MS
(70 eV): m/z (%) = 465 (3.5) [M+], 258 (75), 217 (79), 156 (73), 118 (81),
94 (100).– C26H31N3O5 (465.55).– HRMS: Calcd. 465.2264; found
465.2264.
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanyl-L-valine Benzyl
Ester (17)
From 3 (500 mg, 2.44 mmol), and L-alanyl-L-valine benzyl ester HCl
(798 mg, 2.53 mmol). Yield: 322 mg (29%). Colourless liquid.– Rf = 0.52.–
αD25 = –28.6° (c = 1.26, EtOH). Ratio of diastereomers (13C NMR) 2 : 1.
{2-[(R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-alanine Benzyl Ester
(22)
From (R)-3 (250 mg, 1.22 mmol), and L-valyl-L-alanine benzyl ester HCl
(384 mg, 1.22 mmol), method b. Yield: 130 mg (23%). Colourless solid.–
Mp 142 °C.– Rf = 0.40.– [α]D25 = –7.4° (c = 1.07, EtOH).– IR: ν = 3295
(NH), 1763, 1643 (C=O).– 1H NMR: δ = 0.89, 0.91 [2d, each J = 6.6 Hz, 3H,
CH3(val)], 1.36 [d, J = 7.3 Hz, 3H, CH3(ala)], 2.05 [m, 1H, β-H(val)], 2.91,
3.44, 4.78 (ABX, JAX = 2.4 Hz, JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′,
4-H), 3.44, 4.16 (AB, J = 16.6 Hz, 2H, CH2), 4.25 [dd, J = 6.6 Hz,
J = 8.5 Hz, 1H, α-H(val)], 4.56 [dq, J = 7.3 Hz, 1H, α-H(ala)], 5.11, 5.18 (d,
AB, J = 12.4 Hz, CH2), 6.67 [d, J = 7.3 Hz, 1H, N-H(ala)], 6.67 [d,
J = 8.5 Hz, 1H, N-H(val)], 7.33 (m, 10H, ar-H).– 13C NMR: δ = 17.99
[CH3(ala)], 18.07 [CH3(val)], 19.00 [CH3(val)], 31.21 [β-C(val)], 44.41
(CH2), 47.10 (C-3), 48.16 [α-C(ala)], 55.52 (C-4), 58.45 [α-C(val)], 67.21
[CH2(benzyl)], 126.51, 128.19, 128.46, 128.60, 128.75, 129.10, 135.20,
137.25 (ar-C), 167.27, 168.18, 170.11, 172.31 (C=O).– MS (70 eV): m/z
(%) = 465 (0.9) [M+], 259 (27), 217 (79), 188 (12), 155 (30), 118 (45), 91
(100).– C26H31N3O5 (465.55).– HRMS: Calcd. 465.2264; found 465.2274.
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanyl-L-valine Benzyl Ester
(18)
From (S)-3 (500 mg, 2.44 mmol), and L-alanyl-L-valine benzyl ester HCl
(798 mg, 2.53 mmol). Yield: 456 mg (41%). Colourless solid.– Mp 95 °C.–
25
Rf = 0.43.– [α]D = 110.9° (c = 0.97, EtOH).– IR: ν = 3309 (NH), 1739,
1655 (C=O), 1534.– 1H NMR: δ = 0.85, 0.89 [2d, each J = 6.9 Hz, 3H,
CH3(val)], 1.28 [d, J = 7.0 Hz, 3H, CH3(ala)], 2.17 [m, 1H, β-H(val)], 2.92,
3.46, 4.79 (ABX, JAX = 2.5 Hz, JBX = 5.3 Hz, JAB = 14.8 Hz, 3H, 3-H, 3-H′,
4-H), 3.40, 4.15 (AB, J = 16.8 Hz, 2H, CH2), 4.44 [dq, J = 7.3 Hz, 1H,
α-H(ala)], 4.52 [dd, J = 4.9 Hz, J = 8.5 Hz, 1H, α-H(val), 5.14 (AB,
J = 12.2 Hz, 2H, CH2), 6.76 [d, J = 8.4 Hz, 1H, N-H(val)], 7.04 [d,
J = 7.3 Hz, 1H, N-H(ala)], 7.33 (m, 10H, ar-H).– 13C NMR: δ = 17.55
[CH3(ala)], 18.08, 18.94 [CH3(val)], 31.12 [β-C(val)], 44.49 (CH2), 47.02
(C-3), 48.90 [α-C(ala)], 55.47 (C-4), 57.27 [α-C(val)], 67.10 [CH2(benzyl)],
126.58, 128.36, 128.47, 128.59, 128.84, 129.14, 129.29, 135.24, 137.19
(ar-C), 167.00, 168.31, 171.45, 171.73 (C=O).– MS (70 eV): m/z (%) = 465
(19) [M+], 330 (50), 258 (54), 191 (75), 127 (75), 118 (78), 94 (98), 73 (100).–
C26H31N3O5 (465.55).– HRMS: Calcd. 465.2264; found 465.2264.
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-alanine
2,2,2-Trichloroethyl Ester (23)
From 3 (250 mg, 1.22 mmol), and L-valyl-L-alanine 2,2,2-trichloroethyl
ester HCl (452 mg, 1.27 mmol). Yield: 302 mg (62%). Colourless solid.– Mp
71 °C.– Rf = 0.63.– αD = –45.2° (c = 1.06, EtOH).– C21H26Cl3N3O5
{2-[(R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanyl-L-valine Benzyl Ester
(19)
25
(508.83).
From (R)-3 (250 mg, 1.22 mmol), and L-alanyl-L-valine benzyl ester HCl
(399 mg, 1.27 mmol). Yield: 275 mg (48%). Colourless liquid.– Rf = 0.43.–
[α]D = –17.0° (c = 0.64, EtOH).– IR: ν = 3306 (NH), 1747, 1658 (C=O).–
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-alanine
2,2,2-Trichloroethyl Ester (24)
25
1H NMR: δ = 0.82, 0.84 [2d, each J = 7.1 Hz, 3H, CH3(val)], 1.28 [d,
J = 6.8 Hz, 3H, CH3(ala)], 2.14 [m, 1H, β-H(val)], 2.85, 3.38, 4.81 (ABX,
JAX = 2.4 Hz, JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H), 3.43, 4.21
(AB, J = 16.9 Hz, 2H, CH2), 4.48 [dd, J = 5.1 Hz, J = 8.2 Hz, 1H, α-H(val)],
4.53 [dq, J = 7.1 Hz, 1H, α-H(ala)], 5.07, 5.17 (AB, J = 12.2 Hz, 2H, CH2),
7.10 [d, J = 8.2 Hz, 1H, N-H(val)], 7.33 [m, 10H, ar-H; 1H, N-H(ala)].–
13C NMR: δ = 17.46 [CH3(ala)], 17.94, 18.76 [CH3(val)], 30.72 [β-C(val)],
43.53 (CH2), 46.87 (C-3), 48.68 [α-C(ala)], 55.17 (C-4), 57.20 [α-C(val)],
66.78 [CH2(benzyl)], 126.35, 128.12, 128.21, 128.36, 128.46, 128.84,
135.14, 137.21 (ar-C), 167.05, 168.10, 171.31, 172.04 (C=O). Mixture of
diastereomers 5 : 1; only the signals of the main product are listed.– MS
(FAB): m/z (%) = 466 (100) [M++1].– C26H31N3O5 (465.55).
From (S)-3 (250 mg, 1.22 mmol), and L-valyl-L-alanine 2,2,2-trichlo-
roethyl ester HCl (452 mg, 1.27 mmol). Yield: 230 mg (47%). Colourless
solid.– Mp 123 °C.– [α]D = –101.1° (c = 1.05, EtOH).– IR: ν = 3301
25
(NH), 1765, 1650 (C=O).– 1H NMR: δ = 0.89, 0.95 [2d, each J = 6.8 Hz, 3H,
CH3(val)], 1.52 [d, J = 7.3 Hz, 3H, CH3(ala)], 2.10 [m, 1H, β-H(val)], 2.97,
3.52, 4.78 (ABX, JAX = 2.4 Hz, JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′,
4-H), 3.48, 4.16 (AB, J = 16.9 Hz, 2H, CH2), 4.27 [dd, J = 6.3 Hz,
J = 8.8 Hz, 1H, α-H(val)], 4.66, 4.93 (AB, J = 11.9 Hz, 2H, CH2), 4.71 [dq,
J = 7.3 Hz, 1H, α-H(ala)], 6.44 [d, J = 7.3 Hz, 1H, N-H(ala)], 6.69 [d,
J = 8.8 Hz, 1H, N-H(val)], 7.34 (m, 5H, ar-H).– 13C NMR: δ = 17.77
[CH3(ala)], 17.89, 19.03 [CH3(val)], 31.06 [β-C(val)], 44.78 (CH2), 47.01
Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)

250
Achilles, Schirmeister, and Otto
[M+], 287 (29), 259 (71), 217 (79), 155 (52), 91 (100).– HRMS: Calcd.
491.2420, found 491.2430.– C28H33N3O5 (491.59).
(C-3), 48.06 [α-C(ala)], 55.41 (C-4), 58.37 [α-C(val)], 74.20 (CH2), 94.48
(CCl3), 126.47, 128.83, 129.14, 137.14 (ar-C), 167.22, 168.43, 170.45,
171.04 (C=O).– MS (70 eV): m/z (%) = 507 (2) [M+], 287 (17), 259 (82),
217 (100), 188 (47), 155 (73).– C21H26Cl3N3O2 (506.82).
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-prolyl-L-alanine
Benzyl Ester (29)
{2-[(R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-alanine
2,2,2-Trichloroethyl Ester (25)
From 3 (250 mg, 1.22 mmol), and L-valyl-L-prolyl-L-alanine benzyl ester
HCl (523 mg, 1.27 mmol). Yield: 126 mg (18%). Colourless liquid.–
25
αD = –65.2° (c = 0.73, EtOH).– Ratio of diastereomers 4 : 1.–
From (R)-3 (150 mg, 0.73 mmol), and L-valyl-L-alanine 2,2,2-trichloro-
ethyl ester HCl (270 mg, 0.76 mmol). Yield: 155 mg (42%). Colourless
liquid.– [α]D25 = –42.3° (c = 0.55, EtOH).– IR: ν = 3281 (NH), 1758, 1650
(C=O).– 1H NMR: δ = 0.95, 0.97 [2d, each J = 6.8 Hz, 3H, CH3(val)], 1.49
[d, J = 7.3 Hz, 3H, CH3(ala)], 2.12 [m, 1H, β-H(val)], 2.95, 3.49, 4.77 (ABX,
JAX = 2.4 Hz, JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H), 3.49, 4.14
(AB, J = 16.6 Hz, 2H, CH2), 4.24 [dd, J = 6.6 Hz, J = 8.5 Hz, 1H, α-H(val)],
4.64, 4.92 (AB, J = 12.0 Hz, 2H, CH2), 4.67 [dq, J = 7.3 Hz, 1H, α-H(ala)],
6.44 [d, J = 7.3 Hz, 1H, N-H(ala)], 6.72 [d, J = 8.5 Hz, 1H, N-H(val)], 7.35
(m, 5H, ar-H).– 13C NMR: δ = 17.38 [CH3(ala)], 18.12, 18.98 [CH3(val)],
31.12 [β-C(val)], 44.01 (CH2), 47.09 (C-3), 48.00 [α-C(ala)], 55.43 (C-4),
58.27 [α-C(val)], 74.05 (CH2), 94.53 (CCl3), 126.42, 128.63, 129.00, 137.29
(ar-C), 167.49, 168.04, 170.78, 170.97 (C=O). Ratio of diastereomers 3 : 1;
only the signals of the main product are listed.– MS (70 eV): m/z (%) = 507
(3) [M+], 259 (100), 217 (95), 155 (55).– C21H26Cl3N3O2 (506.82).
C
31H38N4O6 (562.67).
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-prolyl-L-alanine
Benzyl Ester (30)
From (S)-3 (250 mg, 1.22 mmol), and L-valyl-L-prolyl-L-alanine benzyl
ester HCl (523 mg, 1.27 mmol). Yield: 165 mg (24%). Colourless solid.– Mp
112 °C.– [α]D = –150.7° (c = 0.89, EtOH).– IR: ν = 3309 (NH), 1742,
25
1675, 1629 (C=O).– 1H NMR: δ = 0.83, 0.95 [2d, each J = 6.8 Hz, 3H,
CH3(val)], 1.38 [d, J = 7.1 Hz, 3H, CH3(ala)], 1.98 [m, 3H, 3-H(pro), 4-
H(pro), 4′-H(pro); m, 1H, β-H(val)], 2.31 [m, 1H, 3-H′(pro)], 2.96, 3.53, 4.79
(ABX, JAX = 2.5 Hz, JBX = 5.4 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H),
3.44, 4.21 (AB, J = 16.8 Hz, 2H, CH2), 3.60, 3.70 [2m, each 1H, 5-H(pro),
5-H′(pro)], 4.56 [m, 1H, α-H(pro); 1H, α-H(val); 1H, α-H(ala)], 5.14, 5.20
[AB, J = 12.5 Hz, 2H, CH2(benzyl)], 6.66 [d, J = 8.8 Hz, 1H, N-H(val)], 7.17
[d, J = 7.3 Hz, 1H, NH(ala)], 7.35 (m, 10H, ar-H).– 13C NMR: δ = 17.58,
19.26 [CH3(val)), 18.05 [CH3(ala)), 25.00 [4′-C(pro)], 27.45 [3′-C(pro)],
31.44 [β-C(val)], 44.14 (CH2), 47.14 (C-3), 47.71 [5′-C(pro)], 48.22 [α-
C(ala)], 55.30 (C-4), 55.55 [α-C(pro)], 59.78 [α-C(val)], 67.04 [CH2 (ben-
zyl)], 126.54, 128.12, 128.37, 128.56, 128.73, 129.09, 135.38, 137.45 (ar-C),
167.10, 168.30, 170.59, 171.36, 172.57 (C=O).– MS (70 eV): m/z (%) = 562
(8.0) [M+], 287 (39), 259 (27), 217 (23).– HRMS: calcd. 562.2791, found
562.2810.– C31H38N4O6 (562.67).
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-proline Benzyl
Ester (26)
From 3 (250 mg, 1.22 mmol), and L-valyl-L-proline benzyl ester HCl
(443 mg, 1.27 mmol). Yield: 170 mg (28%). Colourless liquid.– Ratio of
diastereomers (13C NMR) 3 : 2.– αD25 = –77.4° (c = 0.78, EtOH).
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-proline Benzyl Ester
(27)
{2-[(R)-2- Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-prolyl-L-alanine
Benzyl Ester (31)
From (S)-3 (250 mg, 1.22 mmol), and L-valyl-L-proline benzyl ester HCl
25
(443 mg, 1.27 mmol). Yield: 181 mg (30%). Colourless liquid.– [α]D
=
From (R)-3 (250 mg, 1.22 mmol), and L-valyl-L-prolyl-L-alanine benzyl
ester HCl (523 mg, 1.27 mmol). Yield: 165 mg (24%). Colourless solid.– Mp
125 °C.– [α]D25 = –55.4° (c = 0.52, EtOH).– IR: ν = 3308 (NH), 1741, 1674,
1627 (C=O).– 1H NMR: δ = 0.90, 0.97 [2d, each J = 6.6 Hz, 3H, CH3(val)],
1.37 (d, J = 7.3 Hz, 3H, CH3), 2.06 [m, 3H, 3-H(pro), 4-H(pro), 4-H′(pro);
m, 1H, β-H(val)], 2.27 [m, 1H, 3-H′(pro)], 2.92, 3.48, 4.84 (ABX,
JAX = 2.4 Hz, JBX = 5.1 Hz, JAB = 14.7 Hz, 3H, 3-H, 3-H′, 4-H), 3.39, 4.24
(AB, J = 16.6 Hz, 2H, CH2), 3.64 [m, 2H, 5-H(pro), 5′-H(pro)], 4.48 [dd,
J = 4.4 Hz, J = 8.5 Hz, 1H, α-H(pro)], 4.54 [dq, J = 7.3 Hz, 1H, α-H(ala)],
4.58 [dd, J = 6.1 Hz, J = 8.9 Hz, 1H, α-H(val)], 5.13, 5.19 [AB, J = 12.3 Hz,
2H, CH2(benzyl)], 6.79 [d, J = 8.8 Hz, 1H, N-H(val)], 7.22 [d, J = 7.6 Hz,
1H, N-H(ala)], 7.33 (m, 10H, ar-H).– 13C NMR: δ = 17.49, 19.34
[CH3(val)], 17.65 [CH3(ala)], 25.09 [4′-C(pro)], 28.30 [3′-C(pro)], 31.30
[β-C(val)], 42.78 (CH2), 46.92 (C-3), 47.71 [5′-C(pro)], 48.06 [α-C(ala)],
55.42 [C-4; α-C(pro)], 59.96 [α-C(val)], 66.83 [CH2(benzyl)], 126.53,
128.04, 128.22, 128.46, 128.52, 128.95, 135.49, 137.41 (ar-C), 167.50,
167.80, 170.92, 171.05, 172.53 (C=O).– MS (70 eV): m/z (%) = 562 (0.4)
[M+], 287 (5.7), 259 (6.0), 217 (6.4), 155 (5.9), 118 (9.8), 91 (28).– HRMS:
Calcd. 562.2791, found 562.2805.– C31H38N4O6 (562.67).
–128.9° (c = 1.65, EtOH).– IR (film): ν = 3297 (NH), 1753, 1680, 1629
(C=O).– 1H NMR: δ = 0.81, 0.94 [2d, each J = 6.7 Hz, 3H, CH3(val)], 1.99
[m, 3H, 3-H(pro), 4-H(pro), 4-H′(pro); m, 1H, β-H(val)], 2.23 [m, 1H,
3-H′(pro)], 2.93, 3.51, 4.78 (ABX, JAX = 2.4 Hz, JBX = 5.4 Hz,
JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H), 3.39, 4.21 (AB, J = 16.9 Hz, 2H, CH2),
3.64, 3.75 [2m, each 1H, 5-H(pro), 5-H′(pro)], 4.37 [m, 1H, α-H(pro); 1H,
α-H(val)], 5.11, 5.18 [AB, J = 12.2 Hz, 2H, CH2(benzyl)], 6.69 [d,
J = 9.3 Hz, 1H, N-H(val)], 7.35 (m, 10H, ar-H).–13C NMR: δ = 17.49, 19.15
[CH3(val)], 24.81 [(4′-C(pro)], 28.94 [3′-C(pro)], 31.31 [β-C(val)], 44.06
(CH2), 47.17 [5′-C(pro)], 47.20 (C-3), 55.16 (C-4), 55.47 [α-C(pro)], 58.91
[α-C(val)], 66.83 [CH2(benzyl)], 126.49, 128.12, 128.25, 128.47, 128.65,
129.03, 135.46, 137.39 (ar-C), 166.94, 168.14, 170.27, 171.54 (C=O).– MS
(70 eV): m/z (%) = 491 (1.4) [M+], 259 (16), 217 (23), 91 (85).– HRMS:
Calcd. 491.2420, found 491.2420.– C28H33N3O5 (491.59).
{2-[(R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-proline Benzyl Ester
(28)
From (R)-3 (250 mg, 1.22 mmol), and L-valyl-L-proline benzyl ester HCl
(443 mg, 1.27 mmol). Yield: 85 mg (14%). Colourless liquid.– [α]D25 = –
45.9° (c = 0.85, EtOH).– IR (film): ν = 3252 (NH), 1748, 1632 (C=O).–
1H NMR: δ = 0.88, 0.95 [2d, each J = 6.8 Hz, 3H, CH3(val)], 2.00 [m, 3H,
3-H(pro), 4-H(pro), 4-H′(pro); m, 1H, β-H(val)], 2.18 [m, 1H, 3-H′(pro)],
2.88, 3.46, 4.79 (ABX, JAX = 2.4 Hz, JBX = 5.4 Hz, JAB = 14.7 Hz, 3H, 3-H,
3-H′, 4-H), 3.41, 4.19 (AB, J = 16.6 Hz, 2H, CH2), 3.63, 3.75 [2m, each 1H,
5-H, 5-H′(pro)], 4.53 [m, 1H, α-H(pro); 1H, α-H(val)], 5.09, 5.17 [AB,
J = 12.2 Hz, 2H, CH2(benzyl)], 6.84 [d, J = 8.8 Hz, 1H, N-H(val)], 7.35 (m,
10H, ar-H).– 13C NMR: δ = 17.63, 19.22 [CH3(val)], 24.84 [4′-C(pro)],
28.97 [3′-C(pro)], 31.38 [β-C(val)], 43.77 (CH2), 47.23 [5′-C(pro); C-3],
55.33 (C-4), 55.54 [α-C(pro)], 58.94 [α-C(val)], 66.88 [CH2(benzyl)],
126.50, 128.16, 128.28, 128.50, 128.58, 129.00, 135.49, 137.47 (ar-C),
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-prolyl-L-alanine
2,2,2-Trichloroethyl Ester (32)
From 3 (250 mg, 1.22 mmol), and L-valyl-L-prolyl-L-alanine 2,2,2-trichlo-
roethyl ester HCl (576 mg, 1.27 mmol). Yield: 300 mg (41%). Colourless
liquid.– αD25 = –76.9° (c = 0.93, EtOH).– C26H33Cl3N4O6 (603.93).
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-prolyl-L-alanine
2,2,2-Trichloroethyl Ester (33)
From (S)-3 (250 mg, 1.22 mmol), and L-valyl-L-prolyl-L-alanine 2,2,2-
trichloroethyl ester HCl (576 mg, 1.27 mmol). Yield: 325 mg (44%). Col-
ourless liquid.– [α]D25 = –134.4° (c = 0.85, EtOH).– IR: ν = 3433 (NH),
167.13, 167.94, 170.27, 171.53 (C=O).– Ratio of diastereomers 4 : 1; only 1758, 1686, 1630 (C=O).– 1H NMR: δ = 0.84, 0.95 [2d, each J = 6.8 Hz, 3H,
the signals of the main product are listed.– MS (70 eV): m/z (%) = 491 (11) CH3(val)], 1.47 [d, J = 7.3 Hz, 3H, CH3(ala)], 2.05 [m, 3H, 3-H(pro), 4-
Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)

Inhibitors of Elastase and Papain
251
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-alanine (37)
H(pro), 4-H′(pro); m, 1H, β-H(val)], 2.37 [m, 1H, 3-H′(pro)], 2.96, 3.53, 4.78
(ABX, JAX = 2.4 Hz, JBX = 5.4 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H),
3.44, 4.19 (AB, J = 16.8 Hz, 2H, CH2), 3.60, 3.71 [2m, each 1H, 5-H(pro),
5′-H(pro)], 4.61 [m, 1H, α-H(pro); 1H, α-H(val); 1H, α-H(ala)], 4.64, 4.92
(AB, J = 12.0 Hz, 2H, CH2), 6.63 [d, J = 8.5 Hz, 1H, N-H(val)], 7.23 [d,
J = 7.3 Hz, 1H, N-H(ala)], 7.35 (m, 5H, ar-H).– 13C NMR: δ = 17.67
[CH3(ala), CH3(val)], 19.17 (CH3(val)], 24.93 (4′-C(pro)], 27.50 (3′-C(pro)],
31.35 (β-C(val)], 43.97 (CH2), 47.00 (C-3), 47.74 [5′-C(pro), 48.00 [α-
C(ala)], 55.27 (C-4), 55.59 [α-C(pro)], 59.66 [α-C(val)], 74.14 (CH2), 94.55
(CCl3), 126.47, 128.66, 129.03, 137.46 (ar-C), 167.10, 168.24, 170.85,
171.19, 171.42 (C=O).– MS (70 eV): m/z (%) = 604 (2.3) [M+], 287 (46),
259 (37), 217 (37), 118 (23), 70 (100).– C26H33Cl3N4O6 (603.93).
From 23 (100 mg, 0.20 mmol). Yield: 75 mg (98%). Colourless foam.–
αD25 = 30.7° (c = 0.38, EtOH).– C19H25N3O5 (375.43).
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-alanine (38)
From 24 (180 mg, 0.35 mmol). Yield: 129 mg (98%). Colourless foam.–
[α]D25 = –12.5° (c = 0.40, EtOH).– IR: ν = 3307 (OH), 2967 (CH), 1731,
1653 (C=O).– 1H NMR ([D4]MeOH): δ = 0.86, 0.94 [2d, each J = 6.8 Hz,
3H, CH3(val)], 1.39 [d, J = 7.3 Hz, 3H, CH3(ala)], 2.20 [m, 1H, β-H(val)],
2.88, 3.48, 4.80 (ABX, JAX = 2.4 Hz, JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H,
3-H′, 4-H), 3.50, 4.26 (AB, J = 16.9 Hz, 2H, CH2), 4.21 [d, J = 7.1 Hz, 1H,
α-H(val)], 4.34 [q, J = 7.3 Hz, 1H, α-H(ala)], 7.37 (m, 5H, ar-H).– 13C NMR
([D4]MeOH): δ = 17.99 [CH3(ala)], 18.50, 19.65 [CH3(val)], 32.07 [β-
C(val)], 44.30 (CH2), 47.42 (C-3), 56.45 (C-4), 60.01 [α-C(val)], 127.70,
129.74, 130.14, 138.86 (ar-C), 169.33, 170.61, 172.81, 187.16 (C=O).– MS
(ESI): m/z (%) = 470 (64) [M+2Na+K]3+, 438 (100) [M+1H+Na+K]3+.–
C19H25N3O5 (375.43).
{2-[(R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-prolyl-L-alanine
2,2,2-Trichloroethyl Ester (34)
From (R)-3 (120 mg, 0.58 mmol), and L-valyl-L-prolyl-L-alanine 2,2,2-
trichloroethyl ester HCl (273 mg, 0.61 mmol). Yield: 152 mg (43%). Col-
ourlessliquid.–[α]D25 = –31.8° (c = 0.66, EtOH).– IR: ν = 3417 (NH), 1757,
1685, 1628 (C=O).– 1H NMR: δ = 0.83, 0.94 [2d, each J = 6.8 Hz, 3H,
CH3(val)], 1.38 [d, J = 7.3 Hz, 3H, CH3(ala)], 1.99 [m, 4H, 3-H(pro), 3-
H′(pro), 4-H(pro), 4-H′(pro); m, 1H, β-H(val)], 2.83, 3.41, 5.00 (ABX,
JAX = 2.2 Hz, JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H), 3.13 (AB,
J = 16.6 Hz, 1H, CH2), 3.62, 3.69 [2m, each 1H, 5-H(pro), 5-H′(pro)], 4.53
[m, 1H, α-H(pro); 1H, α-H(val); 1H, α-H(ala); 1H, CH2; 1H, CH2], 4.88
(AB, J = 12.0 Hz, 1H, CH2), 7.29 (m, 5H, ar-H), 7.77 [d, J = 7.1 Hz, 1H,
N-H(ala)], 8.00 [d, J = 8.3 Hz, 1H, N-H(val)].– 13C NMR: δ = 17.19
[CH3(ala)], 17.45, 19.32 [CH3(val)], 25.08 [4′-C(pro)], 28.48 [3′-C(pro)],
31.21 [β-C(val)], 42.53 (CH2), 46.77 (C-3), 47.75 [5′-C(pro), 47.88 [α-
C(ala)], 55.38 (C-4), 55.44 [α-C(pro)], 59.96 [α-C(val)], 74.06 (CH2), 94.60
(CCl3), 126.51, 128.51, 128.92, 137.21 (ar-C), 167.54, 167.87, 170.99,
171.21, 171.28 (C=O).– MS (70 eV): m/z (%) = 604 (2.3) [M+], 455 (2.5),
287 (46), 259 (37), 217 (37), 70 (100).– C26H33Cl3N4O6 (603.93).
{2-[(R)-2- Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-alanine (39)
From 25 (100 mg, 0.20 mmol). Yield: 73 mg (97%). Colourless foam.–
25
[α]D = –7.1° (c = 0.35, EtOH).– IR: ν = 3309 (OH), 2970 (CH), 1752,
1
1643 (C=O).– H NMR ([D4]MeOH): δ = 0.91, 0.96 [2d, each J = 6.8 Hz,
3H, CH3(val)], 1.39 [d, J = 7.3 Hz, 3H, CH3(ala)], 2.05 [m, 1H, β-H(val)],
2.87, 3.46, 4.81 (ABX, JAX = 2.4 Hz, JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H,
3-H′, 4-H), 3.53, 4.22 (AB, J = 16.7 Hz, 2H, CH2), 4.18 [d, J = 6.8 Hz, 1H,
α-H(val)], 4.67 [q, J = 7.3 Hz, 1H, α-H(ala)], 7.36 (m, 5H, ar-H).– 13C NMR
([D4]MeOH): α = 18.11 [CH3(ala)], 18.49, 19.67 [CH3(val)], 32.01 [β-
C(val)], 44.23 (CH2), 47.45 (C-3), 56.62 (C-4), 60.11 [α-C(val)], 127.70,
129.67, 130.08, 138.97(ar-C), 169.35, 169.55, 170.66, 172.89(C=O).–Ratio
of diastereomers 3 : 1; only the signals of the main product are listed.– MS
(ESI): m/z (%) = 470 (100) [M+2Na+K]3+, 438 (77) [M+1H+Na+K]3+.–
C19H25N3O5 (375.43).
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanine (35)
{2-[(S,R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-prolyl-L-alanine
(40)
A solution of H2O2 (30%, 74 µl) was added to a solution of 8 (250 mg,
0.90 mmol) in a mixture of acetone (8 ml) and water (3.3 ml), which was
adjusted with NaOH (1M) to pH = 10.5. The mixture was kept with this pH
for 5 min, then it was diluted with a few ml of water, and twice extracted
with EtOAc. The aqueous layer was adjusted with dil. HCl to pH = 2, and
twice extracted with EtOAc. These combined organic layers were dried
(Na2SO4), and the solvent was removed in vacuo. Yield: 115 mg (46%).
From 32 (100 mg, 0.17 mmol). Yield: 78 mg (100%). Colourless foam.–
25
αD = –61.8° (c = 0.38, EtOH).– C24H32N4O6 (472.54).
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-prolyl-L-alanine (41)
From 33 (180 mg, 0.30 mmol). Yield: 139 mg (98%). Colourless foam.–
[α]D25 = –90.4° (c = 0.45, EtOH).– IR: ν = 3323 (OH), 2974 (CH), 1740,
1628 (C=O).– H NMR ([D4]MeOH): δ = 0.88, 0.99 [2d, each J = 6.6 Hz,
25
Yellowish viscous liquid.– αD = –22.9° (c = 0.14, EtOH).– 1H NMR
1
([D4]MeOH): δ = 1.34 (d, J = 7.6 Hz, 3H, CH3), 2.88, 3.46, 4.84 (ABX, 3H,
3-H, 3-H′, 4-H), 3.46, 4.22 (2AB, each 2H, CH2), 4.36 [m, 1H, α-H(ala)],
7.36 (m, 5H, ar-H).– C14H16N204 (276.29).
3H, CH3(val)], 1.39 [d, J = 7.3 Hz, 3H, CH3(ala)], 2.07 [m, 4H, 3-H(pro),
3-H′(pro), 4-H(pro), 4-H′(pro); m, 1H, β-H(val)], 2.88, 3.47, 4.79 (ABX,
JAX = 2.4 Hz, JBX = 5.1 Hz, JAB = 14.7 Hz, 3H, 3-H, 3-H′, 4-H), 3.50, 4.21
(AB, J = 16.9 Hz, 2H, CH2), 3.67, 3.85 [2m, each 1H, 5-H(pro), 5-H′(pro)],
4.30 [q, J = 7.3 Hz, 1H, α-H(ala)], 4.43 [m, 2H, α-H(pro), α-H(val)], 7.35
(m, 5H, ar-H).– 13C NMR ([D4]MeOH): δ = 18.31 [CH3(ala)], 18.70, 19.64
[CH3(val)], 25.90 [4′-C(pro)], 30.30 [3′-C(pro)], 31.74 [β-C(val)], 44.08
(CH2), 47.45 (C-3), 56.46 (C-4), 58.00 [α-C(pro)], 61.49 [α-C(val)], 127.71,
129.74, 130.12, 138.89 (ar-C), 169.32, 170.54, 172.28, 173.54, 178.05
(C=O). 5′-C(pro) α-C(ala) are not detectable.– MS (ESI): m/z (%) = 535
(100) [M+1H+Na+K]3+.– C24H32N4O6 (472.54).
{2-[(S)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-alanine (36)
From 9 (110 mg (0.29 mmol), acetone (4 ml), water (1.7 ml), and H2O2
(30%, 26 µl) as described for 35. Yield: 39 mg (47%). Yellowish viscous
liquid.– [α]D25 = –37.8° (c = 0.45, EtOH).– IR: ν = 3404 (OH), 1744, 1665
(C=O).– 1H NMR ([D4]MeOH): δ = 1.34 (d, J = 7.6 Hz, 3H, CH3), 2.88,
3.46, 4.84 (ABX, 3H, 3-H, 3-H′, 4-H), 3.46, 4.22 (2AB, each 2H, CH2), 4.36
[m, 1H, α-H(ala)], 7.36 (m, 5H, ar-H).– 13C NMR ([D4]MeOH): δ = 17.57
[CH3(ala)], 44.11 (CH2), 47.40 (C-3), 56.40 (C-4), 127.73, 129.72, 130.11,
138.82(ar-C), 169.11, 170.52, 175.53(C=O).–MS (CI): m/z (%) = 277 (100)
[M++1].– MS (70 eV): m/z (%) = 259 (48), 248 (41), 231 (37) [M+-COOH],
189 (78) [M+-ala(OH)], 160 (39) [M+-CH2CO-ala(OH)], 146 (68), 132
(100), 118 (71), 104 (49), 91 (60).– C14H16N204 (276.29).
{2-[(R)-2-Oxo-4-phenylazetidin-1-yl]acetyl}-L-valyl-L-prolyl-L-alanine
(42)
From 34 (120 mg, 0.20 mmol). Yield: 91 mg (97%). Colourless foam.–
[α]D = –30.5° (c = 0.30, EtOH).– IR: ν = 3429 (OH), 2975 (CH), 1741,
1632 (C=O).– H NMR ([D4]MeOH): δ = 0.93, 1.00 [2d, each J = 6.8 Hz,
3H, CH3(val)], 1.38 [d, J = 7.1 Hz, 3H, CH3(ala)], 2.07 [m, 5H, 3-H(pro),
3-H′(pro), 4-H(pro), 4-H′(pro), β-H(val)], 2.86, 3.47 (ABX, JAX = 2.4 Hz,
JBX = 5.1 Hz, JAB = 14.9 Hz, 3H, 3-H, 3-H′, 4-H), 3.50, 4.21 (AB,
J = 16.8 Hz, 2H, CH2), 3.67, 3.86 [2m, each 1H, 5-H(pro), 5-H′(pro)], 4.30
[q, J = 7.1 Hz, 1H, α-H(ala)], 4.39 [d, J = 6.8 Hz, 1H, α-H(val)], 4.44 [dd,
25
1
General Procedure for the Cleavage of the 2,2,2-Trichloroethyl Group
The protected compound (100–200 mg) was dissolved in glacial acetic
acid. Zinc powder (400 mg) was added. After stirring for 1 h at room temp.,
EtOAc was added and the suspension filtered. The filtrate was concentrated
in vacuo, and the residue was dried over KOH in vacuo.
Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)

252
Achilles, Schirmeister, and Otto
J = 4.5 Hz, J = 6.1 Hz, 1H, α-H(pro)], 7.38 (m, 5H, ar-H). 4-H not visible.–
13C NMR ([D4]MeOH): α = 18.23 [CH3(ala)], 18.65, 19.66 [CH3(val)],
25.93 [4′-C(pro)], 30.29 [3′-C(pro)], 31.64 [β-C(val)], 44.03 (CH2), 47.49
(C-3), 56.64 (C-4), 58.09 [α-C(pro)], 61.49 [α-C(val)], 127.67, 129.68,
130.08, 138.97 (ar-C), 169.52, 170.58, 172.38, 173.55, 177.08 (C=O). 5′-
C(pro) and α-C(ala) are not detectable.– MS (ESI): m/z (%) = 535 (100)
[M+1H+Na+K]3+.– C24H32N4O6 (472.54).
References
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[4] L. Ekerot, K. Ohlson, Adv. Exp. Med. Biol. 1984, 167, 335–344.
[5] A. Janoff, Ann. Rev. Med. 1985, 36, 207–216.
Enzyme Kinetics
[6] H.-H. Otto, T. Schirmeister, Chem. Rev. 1997, 97, 133–171.
[7] a.) I. Sohar, A. Laszlo, K. Gaal, F. Mechler, Biol. Chem. Hoppe-Seyler
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Elastase
The assays were performed at PPE concentrations of 7.5 µg/ml and sub-
strate concentrations between 0.46–0.52 mM Ac-Ala-Ala-Ala-pNA in 0.1 M
tris-buffer, pH 8.0. Stock solutions of PPE were prepared by dissolving the
enzyme in 1 mM acetic acid. The rate of enzymatic hydrolysis was monitored
at 405 nm continuously for 20 min via the release of p-nitroaniline at 25 °C
[8] R. Bolli, R. Cannon, E. Speir, R. Goldstein, S. Epstein, J. Am. Coll.
Cardiol. 1983, 2, 681–688.
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at 405 nm (ε = 9960 l mol–1 cm–1). The Km-value was calculated to be
0.69 mM. Both substrate and inhibitor were dissolved in DMSO, and all
assays were performed with the same final concentration of DMSO (10–
15%). Enzyme, inhibitor and substrate were mixed and the time-dependent
increase in the UV absorption was monitored over 20 min (final concentra-
tions [I]: 2b: 2.06 mM; 3: 0.46 mM; 4: 0.81 mM; 5: 0.75 mM; 6: 0.63 mM;
10: 1.10 mM; 11: 0.86 mM; 14: 0.69 mM; 15: 0.70 M; 17: 0.82 mM; 18:
0.83 mM; 20: 0.35-0.69 mM; 26: 0.60 mM; 29: 0.54 mM; 37: 0.83 mM; 40:
0.54 mM). Without inhibitor no significant decrease in enzyme activity
occured during the time of the assay (steady-state conditions). For each
inhibitor, two independent experiments were performed, each with at least
three different inhibitor concentrations and two substrate concentrations,
respectively.
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with the program GraFit®[38]
.
Agric. Biol. Chem. 1978, 42, 523–528.
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Papain
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1994, 37, 3090–3099.
The assays were performed at papain concentrations 1.3–3.5 µM and
L-BAPA as substrate at concentrations of 1.37–1.80 mM. Solutions of
papain were prepared freshly by incubating the enzyme (about
100 mg/10 ml = 30–35 µM) in 0.05 M sodium phophate buffer (pH 6.5),
which contained 5 mM EDTA and 5 mM cysteine for 30 min at 25°C. The
Km value was determined as 2.5 mM from five independent experiments. The
substrate was dissolved in 1 ml DMSO and diluted with buffer, the inhibitor
was dissolved in DMSO. All assays were performed with the same final
concentration of DMSO. The rate of enzymatic hydrolysis was monitored by
[18] M. A. Navia, J. P. Springer, T.-Y. Lin, H. R. Williams, R. A. Firestone,
J. M. Pisano, J. B. Doherty, P. E. Finke, K. Hoogsteen, Nature 1987,
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D. Underwood, J. B. Doherty, Biochemistry 1992, 31, 8160–8170.
the release of p-nitroaniline at 25 °C at 405 nm (ε = 9960 l mol–1 cm–1).
Dilution Assay: For the inhibition experiments the enzyme was incubated
with the inhibitor at various inhibitor concentrations (final concentrations [I]:
2a: 2.49–4.97 mM; 2b: 1.88–9.4 mM; (S)-2b: 1.7–9.3 mM; (R)-3: 0.32–
1.93 mM; 4: 0.33–3.28 mM) for varying periods of time (5–40 min, 5–7
values). Following this preincubation with inhibitor, the substrate was added
and the remaining enzyme activity was monitored as described above.
Without inhibitor, no significant decrease in enzyme activity was observed
(steady-state conditions). The experiments were repeated with 3–7 different
inhibitor concentrations. Two independent assays were carried out for each
inhibitor.
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Continuous Assay: Enzyme, inhibitor and substrate were mixed and the
615–618.
time-dependent increase in absorption was monitored at 405 nm over 20 min
(final concentrations [I]: (S)-3: 1.8 mM; 3: 2.8 mM; 10: 1.1 mM; 11:
1.1 mM; 14: 0.70 mM; 15: 0.71 mM; 17: 0.17–0.83 mM; 18: 0.17–0.84 mM;
19: 0.82 mM; 20: 0.70 mM; 26: 0.61 mM; 29: 0.55 mM; 37: 0.84 mM; 40:
0.63 mM). Without inhibitor, no significant decrease in enzyme activity
occured (steady-state conditions). Non-linear or linear regression analysis
with the program GraFit® gave the kinetic constants.
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Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)

Inhibitors of Elastase and Papain
253
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Received: March 30, 2000 [FP473]
Arch. Pharm. Pharm. Med. Chem. 333, 243–253 (2000)