Synthesis and evaluation of novel β-lactam inhibitors of human leukocyte elastase

Article abstract of DOI:10.1139/v01-062

A series of β-lactam derivatives were synthesized and tested to determine the structure-activity relationship for inhibition of human leukocyte elastase (HLE), a serine protease involved in several degenerative lung and tissue diseases. The most potent IC₅₀ values were obtained with neutral hydrophobic 7α-methoxy cephalosporanic acid derivatives. Tryptophanyl-9-fluorenylmethyl ester and N-benzhydryl piperazine derivatives of 7α-methoxy cephalosporanic acid represent two novel HLE inhibitors, with length of action persisting beyond 24 h.

Full text of DOI:10.1139/v01-062

377
Synthesis and evaluation of novel -lactam
inhibitors of human leukocyte elastase
K.P. Koteva, André M. Cantin, W.A. Neugebauer, and E. Escher
Abstract: A series of β -lactam derivatives were synthesized and tested to determine the structure–activity relationship
for inhibition of human leukocyte elastase (HLE), a serine protease involved in several degenerative lung and tissue
diseases. The most potent IC50 values were obtained with neutral hydrophobic 7α-methoxy cephalosporanic acid deriva-
tives. Tryptophanyl-9-fluorenylmethyl ester and N-benzhydryl piperazine derivatives of 7α-methoxy cephalosporanic
acid represent two novel HLE inhibitors, with length of action persisting beyond 24 h.
Key words: β -lactams, neutrophil elastase, protease inhibitors, peptides.
Résumé : Une série de dérivés de molécules β -lactames fut synthétisée et analysée afin de déterminer la relation struc-
ture-activité en tant qu’inhibiteurs de l’élastase neutrophilique humaine (ENH), une protéase impliquée dans plusieurs
maladies pulmonaires et tissulaires. Les valeurs inhibitrices (IC50) les plus puissantes furent obtenues avec des dérivés
neutres hydrophobes de l’acide 7α-methoxy cephalosporanique. Les dérivés tryptophanyl-9-fluorenylmethyl ester et N-
benzhydrylpiperazine de l’acide 7α-methoxy cephalosporanique représentent deux nouveaux inhibiteurs de l’ENH, dont
l’effet persiste plus de 24 heures.
Mots clés : β -lactames, élastase neutrophilique, inhibiteurs de protéases, peptides.
Koteva et al. 387
Introduction
brosis (CF) (3–5) are characterized by an excessive destruc-
tion of connective tissue of the lung and joints. Neutrophil
elastase is believed to participate in this process (6). Al-
though α1-PI is present in the CF-lung, it is inactive in most
patients, apparently due to oxidative and proteolytic pro-
cesses (7, 8). Myeloperoxidase present in CF respiratory se-
cretions converts Cl– to OCl– in the presence of hydrogen
peroxide, inducing a large oxidant burden in the airways (9,
10). Moreover, bronchial secretions from CF-patients can di-
rectly inactivate α1-PI through elastase-mediated proteolysis
(11). Therefore, individuals with CF have a functional defi-
ciency of airway α1-PI (11, 12). They are especially suscep-
tible to the early development of lung disease. It is
reasonable to expect that a selective inhibitor of HLE might
be useful for the treatment of emphysema, rheumatoid ar-
thritis, CF, and other diseases. This provides a rationale for
developing such inhibitors of low molecular weight for the
evaluation of their efficacy to supplement natural inhibitors
in diseases involving HLE (13). Several classes of HLE in-
hibitors have been reported in the past, including N-
arylbenzylisothiazolinone 1,1-dioxide (14), fatty acids (15),
sulfonyl fluorides (16), 4H-3,1-benzoxazin-4-one, 4-
chloroquinazolines (17), azapeptide p-nitrophenyl esters
(18), and cephalosporin derivatives. The discovery that neu-
tral cephalosporins are inhibitors of mammalian serine pro-
teinases led Zimmerman and co-workers (19) to evaluate the
activity of clavulanic acid and its benzyl esters as inhibitors
of HLE. The free acid, known to inhibit the bacterial β-
lactamase, was inactive against HLE, but its benzyl ester
had good activity against HLE. This observation prompted
several investigators to select a large number of β-lactams
for evaluation as inhibitors of HLE and other mammalian
serine proteases (20–28).
Elastin is a proteinaceous part of the extracellular matrix,
commonly called connective tissue. It gives coherence and
elasticity to many tissues, such as lung, yellow tendon, and
cartilage of joints. Elastin remodeling is controlled by a fine
balance between continuous elastin synthesis and breakdown
by neutrophil and tissue elastases. Human leukocyte
(neutrophil) elastase (HLE, EC 3.4.21.37) secreted by
polymorphonuclear neutrophils (PMN) has been considered
over two decades as one of the most interesting therapeutic
targets in several lung diseases (1). One of the main func-
tions of HLE is the degradation of structural proteins, such
as elastin, fibronectin, and collagen (2). Under normal physi-
ological conditions, the lungs are protected from excessive
HLE elastolytic activity by endogenous inhibitors such as
α1-protease inhibitor (α1-PI) and α2-macroglobulin (α2-M).
However, the rate of elastin (and collagen) degradation is
greatly enhanced under a variety of clinical conditions. In
particular, pulmonary emphysema, rheumatoid arthritis,
acute respiratory distress syndrome (ARDS), and cystic fi-
Received September 15, 2000. Published on the NRC
Research Press Web site on May 3, 2001.
K.P. Koteva, W.A. Neugebauer, and E. Escher. Department
of Pharmacology, Faculty of Medicine, University of
Sherbrooke, 3001, 12ème Avenue Nord, Sherbrooke, QC J1E
5N4, Canada.
André M. Cantin.1 Pulmonary Research Unit, Faculty of
Medicine, University of Sherbrooke, 3001, 12ème Avenue
Nord, Sherbrooke, QC J1E 5N4, Canada.
1Corresponding author (telephone: (819) 346-1110 ext: 14893;
fax: (819) 564-5377; e-mail: acanti01@courrier.usherb.ca).
Can. J. Chem. 79: 377–387 (2001)
© 2001 NRC Canada
DOI: 10.1139/cjc-79-4-377

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Can. J. Chem. Vol. 79, 2001
Scheme 1.
Our objective was to develop a series of novel HLE syn-
thetic inhibitors, including hydrophobic residues covalently
linked to β-lactam structures (dibromopenicillanic acid, sul-
bactam, and 7α-methoxycephalosporin sulfone). Our first
approach was to choose three different N-substituted pipera-
zines: N-methylpiperazine, N-benzylpiperazine, and N-benz-
hydrylpiperazine (N-Bhpip). They were covalently linked to
6,6-dibromopenicillanic acid-(S,S)-dioxide (5–7) and penicillanic
acid-(S,S)-dioxide (sulbactam) (8–10) (Scheme 1). From this
group, N-Bhpip was detected as the most potent substituent.
Based on the knowledge that tert-butyl 7α-methoxycephalo-
sporanate 1,1-dioxide is a potent inhibitor of HNE, we hy-
pothesized that replacing the tert-butyl group with
hydrophobic amino acids would yield novel potent HNE in-
hibitors. With a 4-mer combinatorial peptide library, we have
shown earlier that hydrophobic amino acids have definite
anti-elastase effect (29). The selected amino acids and amino
acid esters were covalently linked to 7α-methoxy cephalo-
sporin sulfone (20–31) and tested for anti-elastase effect.
Molecular ion peaks for compounds 5–10 were obtained. The
MS of products 19–25, except product 22, revealed no molecu-
lar ion peak. However, a characteristic homologue [M]+ ion se-
ries, subsequently missing – SO2, – OCH3, and – CO-
CH3COO, could be assigned. Chromatography generally refers
to flash silica gel 60 200–400 mesh (SiliCycle, Quebec, QC,
Canada) and TLC plates (Merck Kieselgel 60 F254) with detec-
tion using UV or ninhydrine. Thin-layer chromatograms were
developed in the following solvent systems: (a)
CHCl3:MeOH:AcOH, 95:5:3; (b) BuOH:AcOH:H2O, 5:2:3; (c)
BuOH:pyridine:H2O, 4:1:1; (d) EtOAc:BuOH:AcOH:H2O,
2:1:1:1; (e) CHCl3:MeOH:AcOH, 5:3:1; (f) EtOAc:hexane,
8:2; (g) MeOH:CHCl3, 8:2; (h) CHCl3:MeOH, 7:3; or (i)
CHCl3:MeOH, 5:5. HPLC wt% analysis was performed on a
Waters System 600E Controller at the following conditions:
column C18 Bondpack (3.9 × 300 mm), eluent 95:5 (A:B
isocratic at 1.0 mL min–1, A = H2O (0.05% TFA v/v), B =
CH3CN (0.05% TFA v/v)), UV detector at 254 nm.
Dimethylformamide (DMF) was distilled over ninhydrine. Dry
dichloromethane (DCM) was obtained after filtration over ba-
sic alumina. Dicyclohexylcarbodiimide (DCC) was used as 8%
solution in DCM. 6,6-Dibromopenicillanic acid-(S,S)-dioxide
was prepared according to refs. 30 and 31. 6-Aminopenicillanic
acid (6-APA) and penicillanic acid-(S,S)-dioxide (sulbactam)
were purchased from Sigma-Aldrich (ON, Canada). 7-
Aminocephalosporanic acid (7-ACA) was purchased from
Fluka (Ronkonkoma, NY). Abbreviations for amino acids fol-
low the recommendations of the IUPAC-IUB Commission on
Biochemical Nomenclature (32). Other abbreviations are de-
scribed as follow: tert-butyloxycarbonyl (Boc), 9-
fluorenylmethyl ester (OFm), benzyl ester (OBz), tert-butyl es-
ter (O-t-Bu), o-nitrophenyl ester (ONp), 9-aminofluorene
(NFm), 7α-methoxycephalosporanic acid 1,1-dioxide (CA).
Experimental
General methods: chemistry
Melting points were measured on a Thomas–Hoover capil-
lary melting point apparatus and were uncorrected. FT-IR spec-
tra were recorded by using PerkinElmer Model 1600
1
spectrophotometer in chloroform. H NMR spectra were re-
corded on a Bruker 300 spectrophotometer in DMSO-d6 or
CDCl3 referenced to tetramethylsilane as an internal standard at
0.00 ppm. MS spectra were recorded on a high resolution mass
spectrophotometer Z AB-IF (electron ionization, 70 eV) and
Voyager Biospectrometry WorkstationTM based on matrix as-
sisted laser desorption ionization mechanism (MALDI-TOF).
© 2001 NRC Canada

Koteva et al.
379
(2S)-6,6-Dibromo-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]
heptane-2-(4′-benzhydrylpiperazine-4′-yl-1′-amido)-4,4-dioxide
(7): Compound 7 was obtained using method A. Yield: 45%.
EI-MS m/z (C25H27Br2N3O4S, MW 625.38): 625. FT-IR (cm–1):
2982, 2808, 2760, 1817, 1757, 1690, 1662, 1599, 1551. H
NMR (DMSO-d6) δ: 7.35 (m, 10H), 4.20 (t, J = 8.2 Hz, 2H),
Synthesis of the compounds 5–10 (Scheme 1)
Method A
To a stirred solution of dibromopenicillanic acid-(S,S)-dioxide
(0.5 g, 1.2 mmol) in DCM (1 mL) was added DCC (1.5 mL,
0.6 mmol) at 0°C. The reaction mixture was then stirred for
20 min. After the filtration of dicyclohexylurea, the solution
was used immediately in the next step. N-Methylpiperazine
(0.55 g, 6.0 mmol) was added and the reaction was carried
out at –5 to 0°C for 2 h. Then ice cold water was added and
the mixture was stirred for 15 min. The organic layer was
separated and the aqueous phase was extracted three times
with DCM. The combined DCM extracts were washed with
water, citric acid (10%), and brine. After drying over anhy-
drous sodium sulfate, the solvent was evaporated off under
reduced pressure.
1
3.61 (m, 8H), 2.38 (s, 3H), 1.59 (s, 3H), 1.42 (s, 3H).
(2S)-3,3-Dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
(4′-methylpiperazine-4′-yl-4′-amido)-4,4-dioxide (8): Com-
pound 8 was obtained according to method C. Yield: 18%.
EI-MS m/z (C13H21N3O4 S, MW 315.39): 315. FT-IR (cm–1):
1
3460, 1790, 1667. H NMR (DMSO-d6) δ: 3.4–3.8 (m, 8H),
2.4–2.6 (m, 3H), 1.58 (s, 3H), 1.37 (s, 3H).
(2S)-3,3-Dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-(4′-benzylpiperazine-4′-yl-1′-amido)-4,4-dioxide (9): The
synthesis of compound 9 was carried out using method A.
Yield: 20%. EI-MS m/z (C19H25N3O4S, MW 391.49): 391.
Method B
1
FT-IR (cm–1): 3465, 1789, 1666. H NMR (DMSO-d6) δ:
The synthesis was carried out using the method of mixed
anhydrides. For this purpose 1-ethoxycarbonyl-2-ethoxy-1,
2-dihydroquinoline (EEDQ) (33) was used as a coupling re-
agent. N-Benzylpiperazine (0.7 g, 4 mmol) was added to a
DMF solution of 6,6-dibromopenicillanic acid-(S,S)-dioxide
(1.5 g, 4 mmol) followed by EEDQ (1.1 g, 4 mmol). The re-
action mixture was then stirred for 48 h at room temp. Then
ethyl acetate was added and the organic solution was ex-
tracted with water, citric acid (10%), and sodium bicarbon-
ate (1 M). After drying over anhydrous sodium sulfate, the
ethyl acetate solution was concentrated under vacuum and
purified.
7.2–7.4 (m, 5H), 4.88 (s, 1H), 4.73 (s, 1H), 3.4–3.8 (m, 8H),
2.4–2.6 (m, 3H), 1.61 (s, 3H), 1.38 (s, 3H).
(2S)-3,3-Dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-(4′-benzhydrylpiperazine-4′-yl-1′-amido)-4,4-dioxide (10):
Compound 10 was prepared using method C. Yield: 40%.
EI-MS m/z (C25H29N3O4S, MW 467.59): 467. FT-IR (cm–1):
1
3479, 1790, 1681, 1651. H NMR (DMSO-d6) δ: 7.30 (m,
10H), 4.91 (s, 1H), 4.68 (s, 1H), 3.4–3.8 (m, 8H), 2.4–2.6
(m, 3H), 1.62 (s, 3H), 1.42 (s, 3H).
Chemistry of 7-methoxy cephalosporanic acid-1,1-dioxide
tert-butyl-7β-aminocephalosporanate (12) (Scheme 2)
Dry dioxan was freed from peroxides by passage through
a column of neutral alumina. To 100 mL of this solvent was
added, by turns, ice cooled sulfuric acid (10 mL), 7-ACA
(11) (10 g, 0.036 mol), and isobutylene (78.8 mL, 0.73 mol).
The mixture was sealed in a pressurized bottle, stirred at
room temp for 2 h, and then poured into an excess of ice
cooled sodium bicarbonate solution (500 mL). Vigorous re-
lease of CO2 occurred. The pH of the aqueous layer was
checked during the quenching to maintain a basic pH (pH 8–9)
Extraction with ethyl acetate and evaporation of the solvent
gave a brown oil residue, which was then purified through a
column of silicagel G 60 A (100 g) with chloroform as the
eluent. Yield: 41%, mp 110°C. EI-MS m/z (C14H20N2O5S,
MW 328.39): 272 [M – C(CH3)3]+, 240 [M – CO-
CH3COO]+. FT-IR (cm–1): 3459, 2975, 1785, 1723, 1526. 1H
NMR (CDCl3) δ: 5.03 (d, J = 13.5 Hz, 1H), 4.94 (d, J =
5.2 Hz, 1H), 4.79 (d, J = 13.5 Hz, 1H), 4.76 (d, J = 5.2 Hz,
1H), 3.55 (d, J = 18.3 Hz, 1H), 3.35 (d, J = 18.3 Hz, 1H),
2.09 (s, 3H), 1.70 (br s, 2H, NH2), 1.54 (s, 9H).
Method C
To a DMF solution of 1-methylpiperazine (0.4 g,
4 mmol), N-hydroxybenzotriazole (HOBt) (0.65 g, 4 mmol),
2-(1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium tetra-
fluoroborate (TBTU) (1.37 g, 4 mmol), and N-methyl-
morpholine (NMM) (8 mmol) were added, followed by
sulbactam (1 g, 4 mmol). The reaction mixture was stirred at
room temp for 16 h. Then the mixture was diluted with wa-
ter (70 mL) and extracted three times with ethyl acetate
(100 mL). The organic layer was concentrated under reduced
pressure and the isolated product purified.
(2S)-6,6-Dibromo-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]
heptane-2-(4′-methylpiperazine-4′-yl-1′-amido)-4,4-dioxide (5):
Compound 5 was obtained according to method A and puri-
fied by preparative thin layer chromatography. Yield: 28%.
EI-MS m/z (C13H19Br2N3O4S, MW 473.19): 473. FT-IR
1
(cm–1): 3400, 1808, 1753, 1672. H NMR (DMSO-d6) δ: 3.6
(m, 8H) 2.4 (m, 3H), 1.2 (s, 3H), 1.0 (s, 3H).
tert-Butyl-7β-aminocephalosporanate-1,1-dioxide (13)
In a 250 mL three-necked round-bottomed flask, equipped
with a stirrer and device for external cooling–heating was dis-
solved 12 (5 g, 0.015 mol) in ethyl acetate (100 mL) at 25°C.
The solution was stirred quickly while solid sodium tungstate
dihydrate (0.5 g, 0.001 mol) and 30% hydrogen peroxide
(6.25 mL, 0.061 mol) were added. The temperature of this
biphasic mixture was maintained at 20–25°C with cooling for
2 h. The reaction mixture took on a yellow cast due to the
(2S)-6,6-Dibromo-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]
heptane-2-(4′-benzylpiperazine-4′-yl-1′-amido)-4,4-dioxide
(6): The synthesis was carried out using method B. Yield:
30%. EI-MS m/z (C19H23Br2N3O4S, MW 549.28): 550. FT-
1
IR (cm–1): 1802, 1734, 1649, 1633, 1570. H NMR (DMSO-
d6) δ: 7.60 (m, 5H), 4.72 (t, J = 6.1 Hz, 2H), 3.61 (m, 8H),
2.43 (s, 3H), 1.60 (s, 3H), 1.39 (s, 3H).
© 2001 NRC Canada

380
Can. J. Chem. Vol. 79, 2001
Scheme 2.
formation of the oxidizing species, sodium pertungstate. After
2 h, an additional amount of 30% hydrogen peroxide
(1.25 mL, 0.012 mol) was added. After 18 h, an additional
hydrogen peroxide amount was added and the batch was left
until completion (21 h). When the reaction was deemed com-
plete (TLC), the mixture was diluted with ethyl acetate
(150 mL) and cooled to 10°C. The reaction mixture was well
stirred while a solution of sodium sulfite (5 g, 0.037 mol) in
water (100 mL) was added slowly to decompose any remain-
ing hydrogen peroxide. After 10 min a test for residual perox-
ide was performed (starch iodide). The biphasic mixture was
further cooled to 5°C, and a cold (5°C) solution of aqueous
sodium bicarbonate solution (5%) was added to decompose
any oxime by-product (14). The reaction mixture was stirred
for 10 min at 5°C. Stirring was discontinued until the layers
were ready to be separated (30 min). The lower aqueous layer
was removed and discarded. The upper ethyl acetate product
layer was then washed with saturated sodium chloride solu-
tion (100 mL). The organic layer was dried over sodium sul-
fate (5 g), filtered, and cake washed with ethyl acetate (50 mL).
The combined filtrate and liquid phases from washing were
concentrated under vacuum to give an oily residue, which
was further purified through a silicagel G 60A column, using
chloroform–methanol (0 → 50% MeOH). Yield: 18.5%. EI-MS
© 2001 NRC Canada

Koteva et al.
381
m/z (C14H20N2O7S, MW 360.51): 273 [M – SO2 – C(CH3)3]+,
(C15H23NO8S, MW 377.42): 273 [M – COOC(CH3)3]+, 208
[M – SO2 – COOC(CH3)3]+, 180 [M – SO2 – COOC(CH3)3
– CO]+, 150 [M – SO2 – COOC(CH3)3 – CO – OCH3]+. FT-
IR (cm–1): 2978, 1797, 1723, 1693, 1513, 1369, 1157, 1128.
1H NMR (CDCl3) δ: 5.20 (d, J = 14.1 Hz, 1H), 4.99 (d, J =
5.0 Hz, 1H), 4.88 (d, J = 12.0 Hz, 1H), 4.34 (d, J = 13.2 Hz,
1H), 3.99 (d, J = 18.8 Hz, 1H), 3.70 (d, J = 18.8 Hz, 1H),
3.60 (s, 3H), 2.10 (s, 3H), 1.55 (s, 9H).
196 [M – SO2 – COOC(CH3)3]+. FT-IR (cm–1): 3360, 2978,
1
1798, 1731, 1643, 1359, 1154, 1128. H NMR (CDCl3) δ:
5.12 (d, J = 14.0 Hz, 1H), 4.91 (d, J = 5.2 Hz, 1H), 4.79 (d, J =
14.0 Hz, 1H), 4.76 (d, J = 4.9 Hz, 1H), 3.66 (d, J = 18.6 Hz,
1H), 2.33 (br s, 2H), 2.09 (s, 3H), 1.54 (s, 9H).
tert-Butyl-7-diazocephalosporanate-1,1-dioxide (15)
A 500 mL three-necked round-bottomed flask equipped
with a mechanical agitator and nitrogen bubbler was charged
with 13 (0.5 g, 0.0014 mol) in ethyl acetate (15 mL). A
freshly prepared solution of isopropyl nitrite (0.16 g, 20 mmol)
in DCM (33) was added to the stirred solution at
15–20°C, followed by a catalytic amount of TFA. The batch
temperature rose to 31°C over 15 min and the reaction mix-
ture was stirred for 1 h at this temperature. When the
diazotization was complete, the batch was diluted with ethyl
acetate and concentrated under reduced pressure at 30°C or
less to the previous volume (NB: in isolated form the diazo
compound may be explosive, however, previous investigators
have suggested that this material may be handled safely as a
dilute solution in ethyl acetate. Therefore, it was important
that the product did not crystallize near the neck of the flask
during concentration. The above flash concentration re-
moved most of the excess unreacted isopropyl nitrite,
isopropyl alcohol by-product, and methylene chloride.). Af-
terwards, the batch was diluted with fresh ethyl acetate and
used immediately in the next step.
7α-Methoxycephalosporanic acid-1,1-dioxide (18)
A solution of 16 (50 mg, 0.1 mmol) and thioanisol
(0.8 mL) in TFA (4 mL) was stirred at 0°C for 30 min and
was then evaporated in vacuo. Two additional aliquots of
DCM were added and then evaporated to remove most of the
residual TFA. The compound was used directly in the next
amidation step.
Synthesis in solution of H-Trp-Trp-Trp-Trp-OH (Boc-
Trp-OFm) (34), Boc-strategy (Scheme 3)
9-Fluorenylmethanol (5.07 g, 0.0258 mol) and imidazol
(2.35 g, 0.0345 mol) were added to tetrahydrofurane
(40 mL). The suspension was stirred at room temp and t-
Boc-Trp-ONp (33) (10 g, 0.23 mol) was added. Stirring was
continued for 18 h. The solvent was then removed under re-
duced pressure. The residue was dissolved in ethyl acetate,
and the solution washed with 2% citric acid solution, sodium
bicarbonate solution (0.5 N), and water. The ethyl acetate
solution was dried over anhydrous sodium sulfate and evapo-
rated to dryness. The compound was purified by flash chro-
matography silicagel G 60A, using chloroform as an eluent.
Yield 80%. Rfa: 0.55, Rfb: 0.84, tR: 13.6 min. EI-MS m/z
(C30H30N2O4, MW 482.58): 482, 178, 130. FT-IR (cm–1):
3409, 3328, 3007, 2979, 2930, 1736, 1699, 1664, 1617, 1590.
tert-Butyl-7α-methoxycephalosporanate-1,1-dioxide (16)
Simultaneously to the preparation of the diazocephalo-
sporanate (15), rhodium octanoate dimer (20 mg,
0.02 mmol) was dissolved in methanol (1.7 mL) and diluted
with ethyl acetate (13 mL). The solution was let to cool. The
mixture from the previous step, containing 15, was cooled to
–5°C and vacuum was purged three times with nitrogen. Be-
fore the reaction with the compound 15, triethylamine
(0.05 mL) was added to the catalyst mixture, and the solu-
tion was stirred for 5 min. The color changed from blue
green to purple, probably due to ligand exchange and (or)
complexation of the rhodium octanoate dimer with
triethylamine. The catalyst solution was added to the solu-
tion of diazocompound for about 1 h. The reaction was car-
ried out in an open flask to eliminate nitrogen gas. After 1 h,
the batch was concentrated in vacuo, washed with deionized
water, containing sodium chloride and phosphoric acid, and
then washed with a solution of 10% sodium chloride. The
ethyl acetate layer was dried over anhydrous sodium sulfate
and then passed through a bed of silicagel G 60A. The
silicagel was then washed with ethyl acetate. The batch and
wash were combined and concentrated in vacuo to 40 mL.
Hexane (8 mL) was slowly added, the crystallization oc-
curred progressively. The crystallization mixture was further
concentrated to 5 mL, diluted with hexane (12 mL) over 4 h,
let with stirring 4 h at 25°C, and then for 24 h at 0–5°C. The
batch was filtered and cake washed with 6 mL of hexane.
The residue was dried completely in vacuo. The major prod-
uct fraction contained 220 mg (24%) of a white crystalline
solid, mp 130 to 131°C (EtOAc–hexane), and was identified
as tert-butyl-7α-methoxycephalosporanate-1,1-dioxide (16)
on the basis of its spectral characteristics. EI-MS m/z
H-Trp-OFm·HCl (35): Boc-Trp-OFm (34) (2.5g, 5 mmol)
was dissolved in ether and HCl gas was passed through the
solution until precipitation was observed. The amino acid es-
ter hydrochloride was filtered, washed several times with
hexane and dried. Yield 95%, mp 200°C. Rfa: 0.12, Rfb: 0.80,
tR: 15.4 min. EI-MS m/z (C25H22N2O2, MW 382.47): 382,
178, 152, 130.
Boc-Trp-Trp-OFm (36): To a solution of 35 (0.650 g,
1.5 mmol) and HOBt (0.3 g, 1.9 mmol) in DMF (15 mL)
was added NMM (0.2g, 1.9 mmol) followed by 33 (0.8 g,
1.9 mmol). The solution was stirred for 4 h at room temp. A
10-fold excess of water was then added, and the obtained
suspension was extracted with ethyl acetate. The organic
layer and extracts were combined, washed with 0.1% so-
dium bicarbonate solution, and evaporated under vacuum.
The process was followed by TLC and HPLC. The product
was further purified by flash chromatography, using silicagel
G 60A and CHCl3–MeOH (0 → 50% MeOH) as the eluent.
After the purification step, the obtained compound was 1g
(91%), mp 88–90°C. Rfa: 0.60, Rfb: 0.83, tR: 20 min. EI-MS
m/z (C41H40N4O5, MW 668.80): 669, 569. FT-IR (cm–1):
3409, 3328, 3007, 2977, 2931, 1736, 1697, 1664, 1617, 1591.
H-Trp-Trp-OFm·HCl (37): The deprotection was carried out
using the procedure already described for 35. Yield 78%, mp
164 to 165°C. Rfa: 0.15, Rfb: 0.78, tR: 16.5 min. EI-MS m/z
(C36H32N4O3, MW 568.68): 569. FT-IR (cm–1): 3409, 3007,
1736, 1697, 1664, 1516.
© 2001 NRC Canada

382
Can. J. Chem. Vol. 79, 2001
Scheme 3.
Boc-Trp-Trp-Trp-OFm (38): The synthesis was carried out
using the method described above for 36. Yield 90%, mp
93–95°C. Rfa: 0.64, Rfb: 0.80, tR: 21.5 min. EI-MS m/z
(C52H50N6O6, MW 855.01): 855, 475, 374, 146. FT-IR (cm–1):
3404, 3318, 2928, 1736, 1690, 1663, 1509.
Solid phase synthesis of H-Trp-Trp-Trp-Trp-OH, Fmoc-
strategy
Synthesis was performed using Fmoc-Trp(Boc)-Wang
resin, substitution Fmoc-Trp-(Boc)-OH (0.4 mmol g–1, 2.5
eq), DCC (2.5 eq), and HOBt (5 eq), and NMM as a base.
The resin cleavage was carried out with TFA–ethanedithiol–
water (95:2.5:2.5) for 1 h at room temp. The following com-
pounds were synthesized:
H-Trp-Trp-Trp-OFm·HCl (39): The Boc-deprotection was
carried out according to the method described above. Yield
70%. Rfa: 0.04, Rfb: 0.81, tR: 17.4 min. EI-MS m/z
(C47H42N6O4, MW 754.90): 755.
H-Trp-Trp-Trp-OH (39): EI-MS m/z (C33H35N6O4, MW
576.66): 577.
Boc-Trp-Trp-Trp-Trp-OFm (40): The reaction was carried
out according to the method described above. After 16 h wa-
ter was added and then the extraction with ethyl acetate was
done. After purification, the peptide fractions were pooled
together and submitted to TLC, HPLC, and EI-MS analysis.
H-Trp-Trp-Trp-Trp-OH (41): EI-MS m/z (C44H42N8O5, MW
762.88): 763.
7α-Methoxycephalosporanic acid-1,1-dioxide derivatives
(19–31) (Scheme 2)
The first and major product was Boc-(Trp)4-OFm (M+
=
1041, MW 1040), and the second one was tert-butylated
Boc-(Trp)4-OFm (M+ = 1097, MW 1096). Rfa: 0.30, Rfb:
0.70, tR: 22 min. EI-MS m/z (C63H60N8O7, MW 1041.23):
1040, 941, 740, 569, 474, 375.
CA-Bhpip (19)
To a solution of 18 (42.5 mg, 0.1 mmol) in DMF (3 mL)
was added NMM to pH 8, followed by HOBt (19 mg,
© 2001 NRC Canada

Koteva et al.
383
0.1 mmol) and DCC (0.5 mL, 0.1 mmol). The mixture was
stirred over 30 min at 0°C. N-Bhpip (44 mg, 0.17 mmol)
was added and the stirring continued overnight. When the
reaction was complete DCM was added, followed by water,
and the layers were allowed to separate. The organic layer
was washed with citric acid solution (2%), water, and brine.
After drying over anhydrous sodium sulfate the solvent was
removed under reduced pressure. The compound was then
purified by column chromatography on silicagel G60 A using
chloroform–methanol (0 → 50% MeOH) as a mobile phase.
EI-MS m/z (C28H31N3O7S, MW 553.64): 458 [M – SO2 –
CH3CO]+, 208 [M – SO2 – CH3CO – Bhpip]+. FT-IR (cm–1):
MW 879.95): 783 [M – SO2 – OCH3]+. FT-IR (cm–1): 1790,
1771, 1742, 1670, 1653, 1637, 1593, 1532, 1510.
CA-(Trp)4-OH (25): Yield: 10%. EI-MS m/z (C55H55N9O12S,
MW 1066.17): 969 [M – SO2 – OCH3]+. FT-IR (cm–1):
2926, 2852, 1794, 1774, 1736, 1701, 1666, 1654, 1560,
1523, 1509.
CA-Trp-O-t-Bu (26): Yield: 12%. EI-MS m/z (C26H31N3O9S,
MW 561.62): 563, 228 [M – CH3 – OCOCH2 – Trp-O-t-
Bu]+, 136 [M – CH3OCOCH2 – Trp-O-t-Bu – SO2]+. FT-IR
1
(cm–1): 3370, 1787, 1735, 1679, 1508, 1458, 1385, 1157. H
NMR (DMSO-d6) δ: 7.18 (m, 4H), 6.80 (d, J = 2.5 Hz, 1H),
5.19 (d, J = 1.6 Hz, 1H), 4.81 (t, J = 6.4 Hz, 1H), 4.75 (s,
2H), 4.40 (d, J = 2.5 Hz, 1H), 4.08 (s, 2H), 3.32 (s, 3H),
2.12 (s, 3H), 1.45 (s, 9H).
1
3387, 2936, 1791, 1735, 1682, 1522. H NMR (CDCl3) δ:
7.60 (m, 10H), 5.20 (d, J = 1.6 Hz, 1H), 4.99 (d, J =
14.0 Hz, 1H), 4.88 (d, J = 1.6 Hz, 1H), 4.34 (d, J = 14.0 Hz,
1H), 3.99 (d, J = 19.0 Hz, 1H), 3.4–3.8 (m, 8H), 2.10 (s,
3H).
CA-Trp-OBz (27): Yield: 10%. EI-MS m/z (C29H29N3O9S,
MW 595.63): 596, 484 [M – OCH3 – SO2 – CH3]+, 277 [M
– CO – Trp-OBz]+, 137 [M – CO – Trp-OBz – SO2
–
CA-Trp-OFm (20)
CH2OCOCH3]+. FT-IR (cm–1): 3378, 1795, 1741, 1666,
To a solution of 35 (47 mg, 0. 1mmol), HOBt (19 mg,
0.1 mmol), and TBTU (40 mg, 0.1mmol) in DMF (2 mL)
was added NMM to pH 8 followed by 18 (40 mg, 0.1 mmol).
The reaction was checked using TLC. After 7 h the reaction
was stopped and 10-fold excess of water was added. The so-
lution was then extracted with ethyl acetate. The ethyl ace-
tate layer was dried over anhydrous sodium sulfate and the
solvent was removed under reduced pressure. EI-MS m/z
(C36H33N3O9S, MW 683.74): 273 [M – CO – Trp-OFm]+,
208 [M – SO2 – CO – Trp-OFm]+. FT-IR (cm–1): 1797, 1789,
1
1571, 1135. H NMR (DMSO-d6) δ: 7.20 (m, 5H), 7.18 (m,
4H), 6.80 (d, J = 2.8 Hz, 1H), 5.21 (d, J = 1.4 Hz, 1H),
4.81 (t, J = 7.1 Hz, 1H), 4.75 (s, 2H), 4.42 (d, J = 2.5 Hz,
1H), 4.08 (s, 2H), 3.33 (s, 3H), 2.07 (s, 3H).
CA-Pro-OFm (28): Yield: 8%. EI-MS m/z (C30H30N2O9S,
MW 594.64): 595, 564 [M – OCH3]+, 286 [M – Pro-OFm]+.
FT-IR (cm–1): 3410, 2960, 1794, 1550. 1H NMR (DMSO-d6)
δ: 7.3–7.8 (m, 8H) 5.21 (d, J = 1.4 Hz, 1H), 4.75 (s, 2H),
4.70 (d, J = 12.8 Hz, 2H), 4.46 (d, J = 15.8 Hz, 1H), 4.21 (t,
J = 7.0 Hz, 1H), 4.08 (s, 2H), 3.32 (t, J = 6.3 Hz, 2H), 3.24
(s, 3H), 2.09 (s, 3H), 1.87 (t, J = 7.5 Hz, 2H), 1.60 (m, 2H).
1
1769, 1745, 1738, 1731, 1713, 1681, 1667, 1573. H NMR
(DMSO-d6) δ: 7.2–7.8 (m, 12H), 6.82 (d, J = 2.5 Hz, 1H),
5.18 (d, J = 1.6 Hz, 1H), 4.80 (s, 2H), 4.67 (d, J = 12.8 Hz,
2H), 4.42 (s, 2H), 4.09 (s, 2H), 3.24 (s, 3H), 2.09 (s, 3H).
CA-Phe-OFm (29): Yield: 10%. EI-MS m/z (C34H32N2O9S,
MW 644.70): 645, 614 [M – OCH3]+. FT-IR (cm–1): 3401,
1
CA-Trp-OH (21): The compound was obtained after
deprotection of carboxyl function with 15% piperidine in
DMF. EI-MS m/z (C22H25N3O9S, MW 507.52): 369 [M –
SO2 – CH3CO – OCH3]+, 183 [M – SO2 – CH3CO – OCH3 –
CO – Trp]+. FT-IR (cm–1): 2925, 2854, 1789, 1734, 1714,
1667, 1557, 1519.
1790, 1731, 1679, 1391, 1185. H NMR (DMSO-d6) δ: 7.8–
7.1 (m, 13H), 5.22 (d, J = 1.4 Hz, 1H), 4.81 (t, J = 7.5 Hz,
1H), 4.75 (s, 2H), 4.40 (d, J = 12.8 Hz, 1H), 4.13 (s, 2H),
3.3 (s, 3H), 2.11 (s, 3H).
CA-Ala-OBz (30): Yield: 12%. EI-MS m/z (C20H22N2O9S,
MW 466.47): 466, 407 [M – COOCH3]+, 343 [M – COOCH3 –
SO2]+, 233 [M – COOCH3 – SO2 – OBz]+, 135 [M – COOCH3 –
SO2 – CO – Ala-OBz]+. FT-IR (cm–1): 3370, 2983, 1786,
CA-(Trp)2-OFm (22): The synthesis was carried out accord-
ing to the method described above. Yield: 31%. EI-MS m/z
(C47H43N5O10S, MW 869.96): 870. FT-IR (cm–1): 2925,
2854, 1789, 1729, 1714, 1667, 1557, 1519.
1
1739, 1662, 1384, 1227, 1156. H NMR (DMSO-d6) δ: 7.4–
7.6 (m, 5H), 5.22 (d, J = 1.2 Hz, 1H), 4.75 (d, J = 12.8 Hz,
2H), 4.43 (d, J = 12.0 Hz, 1H), 4.07 (d, J = 5.8 Hz, 2H),
4.08 (s, 2H), 3.24 (d, J = 18.6 Hz, 3H), 2.10 (s, 3H).
CA-Trp-Trp-OH (23): Yield 56%. EI-MS m/z (C33H33N5O10S,
MW 691.72): 413 [M – SO2 – Trp – OCH3]+, 199 [M – SO2
– OCH3 – CO – Trp]+. FT-IR (cm–1): 2925, 2854, 1789,
1734, 1714, 1667, 1557, 1519.
CA-Gly-OBz (31): Yield: 30%. EI-MS m/z (C19H20N2O9S,
MW 452.44): 452, 355 [M – COOCH3 – SO2]+, 261 [M –
COOCH3 – SO2 – OBz]+, 107 (OBz), 135 [M – COOCH3 –
SO2 – CO – Gly-OBz]+. FT-IR (cm–1): 3408, 1786, 1738,
CA-(Trp)3-OH (24): 7α-Methoxycephalosporanic acid-1,1-
dioxide (18) (37 mg, 0.1 mmol) in DMF was added to pep-
tide-resin (250 mg, 0.4 mmol g–1), followed by DCC
(0.65 mL, 0.1 mmol), HOBt (17 mg, 0.1 mmol), and a drop
of NMM. The synthesis was carried out overnight. After
washing and drying, the resin was cleaved with a mixture of
TFA–thioanisol–water (3.5:25:0.25) for 1 h at room temp.
The peptide was further precipitated in ice cooled ether
(200 mL). The solvent was then evaporated and the product
lyophilised (purity 98%, HPLC). EI-MS m/z (C44H45N7O11S,
1
1710, 1665, 1385, 1134. H NMR (DMSO-d6) δ: 7.4–7.6
(m, 5H), 5.22 (d, J = 1.2 Hz, 1H), 4.75 (d, J = 12.8 Hz, 2H),
4.4 (d, J = 12.8 Hz, 1H), 4.13 (d, J = 6.9 Hz, 2H), 4.08 (s,
2H), 3.24 (d, J = 18.6 Hz, 3H), 2.10 (s, 3H).
CA-NFm (32): Yield: 40%. EI-MS m/z (C24H22N2O7S, MW
482.51): 451 [M – OCH3]+. FT-IR (cm–1): 3411, 2874, 1788,
1
1726, 1665, 1368, 1187. H NMR (DMSO-d6) δ: 7.3–7.8
(m, 8H), 5.21 (s, 1H), 4.75 (s, 2H), 4.46 (s, 1H), 4.42 (d, J =
12.8 Hz, 1H), 3.35 (s, 3H), 2.01 (s, 3H).
© 2001 NRC Canada

384
Can. J. Chem. Vol. 79, 2001
Table 1. Physico-chemical data and anti-elastase activity of penicillin amides 5–10.
R
R1
% yield
mp (°C)
tr (min)
Rfa
IC50
Formula
f
5
6
7
8
9
CH3
Br
Br
Br
H
H
H
28
30
45
18
20
40
oil
oil
oil
oil
oil
oil
14.7
10.5
15.2
—
—
—
b0.59, 0.66
>40
inactive
>40
>40
inactive
40
C13H19Br2N3O4S
C19H23Br2N3O4S
C25H27Br2N3O4S
C13H21N3O4S
C19H25N3O4S
C25H29N3O4S
f
CH2C6H5
CH(C6H5)2
CH3
CH2C6H5
CH(C6H5)2
a0.09, 0.64
i
b0.57, 0.66
f
a0.21, 0.48
f
a0.15, 0.50
b
10
a0.20, 0.51
aTLC solvent systems are described in General methods section.
1
micro-crystalline solids or oils. FT-IR, EI-MS, and H NMR
spectroscopy confirmed the structures.
Elastase inhibition assay
Neutrophil elastase (HNE) activity was determined with
the specific human neutrophil elastase substrate N-methoxy-
succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide (MeO-
Succ-Ala-Ala-Pro-Val-pNA, Sigma, St. Louis) as described
in ref. 12. HNE inhibitory capacity was determined by incu-
bating 20 nM HNE with 0–1 µM inhibitor for 1–24 h at
room temp. Residual elastase activity was assayed with
MeO-Succ-Ala-Ala-Pro-Val-pNA by measuring the change
in absorbance 1 min at 410 nm in a spectrophotometer
(model DU7, Beckman Instruments Inc., Montreal, Qc).
Synthesis of 7α-methoxy-cephalosporanic acid-1,1-dioxide
(18) (Scheme 2)
The synthesis was carried out according to Scheme 2. 7-
ACA (11) was used as a starting material. It was apparent
that any synthesis of 18 must involve two key chemical
transformations, i.e., oxidation of 1-sulfide to the corre-
sponding 1-sulfone and conversion of the 7β-amino group to
the 7α-methoxy group. For this reason our preparation fol-
lowed the procedure described by Blackock et al. (34). Fol-
lowing Scheme 2, the carboxylic function of 11 was
protected by esterification with isobutylene, using the
method of Stedmann (35). Compound 12 was then oxidized
to tert-butyl-7-aminocephalosporanate-1,1-dioxide (13). Di-
rect oxidation of 7-ACA tert-butyl ester to the corresponding
sulfone caused some unique problems due to the potential
for N-oxidation of the 7-amino group. As in step one, we be-
lieved that protection from N-oxidation might come via
protonation of the amine prior to the oxidation step. Our ini-
tial attempts to oxidize compound 12 using m-chloroperoxy-
benzoic acid (m-CPBA) were unsuccessful. However, using
sodium tungstate as a catalyst and 30% hydrogen peroxide
solution we managed to obtain the sulfone, but in very low
yield (18.5%). Some N-oxidation occurred to form 20% of a
nearly insoluble and crystalline by-product, 7-hydroxyimino
sulfone derivative (14) of 7-ACA tert-butyl ester. Synthesis
of tert-butyl-7α-methoxycephalosporanate-1,1-dioxide (16)
was the next step. For the diazotization–methoxylation pro-
cedure we followed the method previously described (34).
The use of isopropyl nitrite as the diazotizing reagent facili-
tated the workup, as the by-product (isopropyl alcohol) reac-
tion could be readily removed from the reaction mixture by
vacuum replacement concentration. The fast reaction rate of
methoxylation ensured a higher insertion yield, but slowing
down the rate with a reduced methanol and catalyst charge
increased stereoselectivity. For this reason cold (0°C) ethyl
acetate solution, containing rhodium octanoate dimer, methanol,
and thriethylamine was added slowly over 1 h to the cold so-
lution of the diazocomponent. tert-Butyl-7β-methoxy-
cephalosporanate (17) was also obtained as a by-product.
Results and discussion
Chemistry
Penicillin derivatives
The commercially available 6-APA, (1) (Scheme 1) was
first converted to 6,6-dibromopenicillanic acid (2) using a
diazotization–halogenation procedure (30). A high-yield
conversion of 1 to 2 was achieved since the diazo intermedi-
ate, once formed, was more easily brominated. The biphase
of the diazotization–bromination reaction exploited the solu-
bility of bromine and the 6,6-dibromopenicillanic acid inter-
mediate in organic solvents and the solubility of hydrogen
bromide in water. Following Scheme 1, 1 was added to a
cooled DCM–sulfuric acid mixture, containing sodium ni-
trite and bromine, and was converted to the desired 6,6-
dibromopenicillanic acid (2) in high yield (80%). 6,6-
Dibromopenicillanic acid (2), once formed, was converted in
a very high yield (more than 90%) to the corresponding
sulfone (3) by a potassium permanganate oxidation (31).
The synthesis of the final compounds 5–10 was achieved us-
ing three different methods: (A) method of symmetrical an-
hydrides, using dicyclohexylcarbodiimide (DCC) as
a
coupling reagent, (B) method of mixed anhydrides with the
use of EEDQ (33) as a coupling reagent, and (C) amidation
via HOBt active ester procedure, using the activating agent
TBTU or with DCC. The compounds having formula 5–10
obtained after synthesis were 93–99% pure as evidenced by
HPLC and TLC analysis (Table 1). They were isolated as
© 2001 NRC Canada

Koteva et al.
385
Table 2. Physico-chemical data and antielastase activity of cephalosporin amides 20–31.
%
yield
Rfa
tR (min)
IC50
0.8
>40
15
>40
>40
>40
<40
10
>40
<40
>40
<40
AA
R
mp (°C)
Formula
c
20
21
22
23
24
25
26
27
28
29
30
31
Trp
Trp
Fm
H
Fm
H
H
H
t-Bu
Bz
Fm
Fm
Bz
Bz
24
42
37
98
31
56
10
10
8
foam
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
oil
b0.65, 0.84
8.7
—
C36H33N3O9S
C22H25N3O9S
C47H43N5O10S
C33H33N5O10S
C44H45N7O11S
C35H55N9O12S
C26H31N3O9S
C29H29N3O9S
C30H30N2O9S
C34H32N2O9S
C20H22N2O9S
C19H20N2O9S
a0.01, 0.66
b
b
(Trp)2
(Trp)2
(Trp)3
(Trp)4
Trp
Trp
Pro
Phe
Ala
a0.05, 0.60
10.0
13.7
17.9
18.1
15.2
11.4
12.7
9.6
b0.72, 0.80
c
a0.09, b0.67
b0.95, c0.80
a0.10, 0.60
b
a0.11, b0.58
d0.76, 0.41
e
b
10
12
30
a0.06, 0.62
b0.07, h0.20
10.9
10.7
h
Gly
a0.05, 0.17
aTLC solvent systems are described in General methods section.
7α-Methoxycephalosporanic acid derivatives (19–32)
(Scheme 2)
The free acid 18 was obtained by cleavage of tert-butyl ester
group with TFA–thioanisol. Compound 18, a light yellow
oil, was used without isolation in the next amidation step.
The synthesis of the compounds with the formula 19–32
was achieved in low to moderate yield using the method of
active esters. The predicted structures were confirmed by
FT-IR, NMR, and EI-MS spectroscopy. Physico-chemical
data for the compounds from this series are shown in Ta-
bles 2 and 3.
Synthesis in solution of H-Trp-Trp-Trp-Trp-OH, Boc-strategy
(Scheme 3)
A method for the synthesis of tetra-tryptophanyl-9-
fluorenylmethyl ester was developed. From numerous meth-
ods proposed for the preparation of amino acids esters, we
selected the imidazol-catalyzed transesterification of a com-
mercially available protected amino acid active ester, Boc-
Trp-ONp (33). Following Scheme 3, in the first step the o-
nitrophenylester group was substituted with the 9-
fluorenylmethyl ester group using the method of Bednarek
and Bodansky (36). The tert-butyloxycarbonyl amino protec-
tion of 34, 36, 38, and 40 could be cleaved either by TFA or
by gaseous HCl as the fluorenylmethyl ester group remains
unaffected under these conditions. For the synthesis we
chose gaseous HCl Boc-cleavage. The next tryptophanyl res-
idue was then attached using Boc-Trp-ONp ester. The cou-
pling was carried out using HOBt–active ester in situ
formation in the presence of NMM as a base. It is interesting
to note that the fluorenylmethyl group contributes to the sol-
ubility of the protected peptides in organic solvents. Thus,
fluorenylmethyl esters might offer some advantages in pep-
tide synthesis, particularly because they are readily remov-
able under mild conditions (15% piperidine in DMF).
In vitro activity
In vitro inhibition of HLE activity by the target com-
pounds is shown in Tables 1, 2, and 3 as nominal IC50 values
(µM). From the results in Table 1, it can be seen that the
compounds prepared on the basis of the penicillin structures
(dibromopenicillanic acid and sulbactam) are very poor
time-dependant inhibitors of HLE. From this series of com-
pounds, we chose N-Bhpip as the most promising substituent
and it was determined that compound 19, on the basis of
cephalosporin structures, had a strong anti-elastase effect
(IC50 = 0.8 µM) (Table 3). Substitution of N-Bhpip with 9-
aminofluorene gave compound 32, which exhibited poor
anti-elastase activity. Biological data for the compounds
with amino acids and amino acid esters covalently linked to
the cephalosporin (structures 20–31) are shown in Table 2.
Replacement of the tert-butyl ester group in 16 with
tryptophanyl-9-fluorenylmethyl ester gave compound 20,
possessing the same anti-elastase activity as 19. The free
carboxylic group on 21 gave a diminished anti-elastase ef-
fect. The addition of more tryptophanyl residues gave poor
HLE inhibitors (22–25), as demonstrated by the absence of
activity in the compound with up to four tryptophanyl resi-
dues. Both the tryptophanyl residue and the Fm ester group
were found to be essential for anti-HLE activity. Replace-
ment of the Fm ester group in 20 with t-Bu (26) and benzyl
(Bz) (27) ester groups gave poor anti-elastase inhibitors.
Furthermore, the importance of tryptophanyl was
Solid phase synthesis of H-Trp-Trp-Trp-Trp-OH, Fmoc-
strategy
Synthesis was performed using Fmoc-Trp(Boc)-Wang
resin, substitution Fmoc-Trp(Boc)-OH (0.4 mmol g–1, 2.5
eq), DCC (2.5 eq), HOBt (5 eq), and MMM as a base. The
cleavage from the resin was carried out with TFA–
ethanedithiol–water (95:2.5:2.5) for 1 h at room temp. The
final product (41) was purified by HPLC.
© 2001 NRC Canada

386
Can. J. Chem. Vol. 79, 2001
Table 3. Physico-chemical data and antielastase activity of cephalosporin sulfones.
R
% yield
mp (°C)
Rfa
tR (min)
IC50
Formula
f
16
19
32
O-t-Bu
Bhpip
NHFm
24
42
30
130 to 131
foam
oil
a0.53, 0.55
—
10.9
—
1.0
0.8
<40
C15H23NO8S
C28H31N3O7S
C24H22N2O7S
b
a0.15, 0.54
g
f0.29, 0.73
aTLC solvent systems are described in General methods section.
6. E.J. Campbell, R.M. Senior, J.A. McDonald, and D.L. Cox. J.
Clin. Invest. 70, 845 (1982).
7. A.B. Cohen and H.L. James. Am. Rev. Respir. Dis. 126, 25
(1982).
8. P.M. Henson and R.B. Johnston Jr. J. Clin. Invest. 79, 669
(1987).
9. J.R. Mohammed, B.S. Mohammed, L.J. Pawluk, D.M. Bucci,
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demonstrated by the substitution of tryptophanyl 9-
fluorenylmethyl ester with prolyl-9-fluorenylmethyl ester
and phenylalanyl-9-fluorenylmethyl ester yielding com-
pounds 28 and 29, respectively, with reduced anti-elastase
activity. Finally, the substitution of tryptophanyl-9-
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benzyl ester (30) and glycyl benzyl ester (31) gave the same
results.
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Conclusion
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We have demonstrated that replacement of t-butyl ester
group in 7α-methoxycephalosporanic acid 1,1-dioxide with
tryptophanyl-9-fluorenylmethyl ester (compound 20) yields
a strong HLE inhibitor with a length of action beyond 24 h.
Deprotection of the carboxylic function resulted in loss of
activity. Replacement of tryptophan with tetra-tryptophan or
other amino acids, as well as replacement of tryptophanyl-9-
fluorenylmethyl ester with other tryptophanyl-esters led to
compounds with diminished anti-elastase effect. However,
replacement of tryptophanyl-9-fluorenylmethyl ester with
the hydrophobic N-Bhpip gave compound 19 having the
same inhibitory effect as 20, both of which are novel HLE
inhibitors.
19. J.B. Doherty, B.M. Ashe, L.W. Argenbright, P.L. Barker,
R.J. Bonney, G.O Chandler, M.E. Dahlgren, C.P Dorn, P.E.
Finke, R.A. Firestone, D. Fletcher, W.K. Hagmann, R.
Mumford, L. O’Grady, A.L. Maycock, J.M. Pisno, S.K. Shah,
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G.O. Chandler, M.E. Dahlgren, P. Davies, C.P. Dorn, Jr., P.E.
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Acknowledgements
This study was supported by a grant from the Canadian
Cystic Fibrosis Foundation (CCFF). Dr. Koteva was a fellow
of the CCFF and Dr. Escher is holder of a J.C. Edwards
chair of cardiovascular research. The authors thank Diane
Cloutier for expert technical assistance, Micheline Poulin for
excellent secretarial assistance, and Maud Deraët for proof-
reading of this contribution.
21. P.E. Finke, B.M. Ashe, W.B. Knight, A.L. Maycock, M.A.
Navia, S.K. Shah, K.R. Thompson, D.J. Underwood, H. Weston,
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H. Weston, P.E Finke, W.K. Hagmann, S.K. Shah, and J.B.
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