Anionic- and lipophilic-mediated surface binding inhibitors of human leukocyte elastase

Article abstract of DOI:10.1021/jm970251r

We report the synthesis of a series of diphenylmethane-based oligomers containing anionic and lipophilic functionalities that are potent inhibitors of human leukocyte elastase (HLE). The enzyme inhibition is regulated by the size of the oligomer, as well

Full text of DOI:10.1021/jm970251r

3408
J . Med. Chem. 1997, 40, 3408-3422
An ion ic- a n d Lip op h ilic-Med ia ted Su r fa ce Bin d in g In h ibitor s of Hu m a n
Leu k ocyte Ela sta se
J ohn Regan,*,† Daniel McGarry,† J oseph Bruno,† Daniel Green,† J ack Newman,‡ Chin-Yi Hsu,‡ J ane Kline,‡
J effrey Barton,† J effrey Travis,‡ Yong Mi Choi,† Francis Volz,† Henry Pauls,† Richard Harrison,‡
Asher Zilberstein,‡ Shmuel A. Ben-Sasson,§ and Michael Chang†
Departments of Medicinal Chemistry and Inflammation Biology, Rhoˆne-Poulenc Rorer, 500 Arcola Drive,
Collegeville, Pennsylvania 19426, and Department of Experimental Medicine and Cancer Research, Hebrew University,
Hadassah Medical School, J erusalem, Israel
Received April 14, 1997X
We report the synthesis of a series of diphenylmethane-based oligomers containing anionic
and lipophilic functionalities that are potent inhibitors of human leukocyte elastase (HLE).
The enzyme inhibition is regulated by the size of the oligomer, as well as, the number of charges.
Lipophilicity is an important element in determining potency and specificity against other basic
enzymes. Compounds whose scaffolds contain three phenoxyacetic acid groups and three alkyl
ethers are competitive and specific inhibitors of HLE with Ki ) 20 nM. The mechanism of
action of this class of compounds is believed to involve multidendate interactions with the
surface of HLE near the active site which prevents substrate access to the catalytic site.
In tr od u ction
We have previously reported that certain linear
aromatic anionic polymers (e.g. 2, RG 13577, Mw
)
Human leukocyte elastase (HLE, EC 3.4.21.37) is a
highly basic, 30 kDa, serine protease contained within
azurophilic granules of polymorphonuclear leukocytes.1
The earliest recognized role of HLE was in the degrada-
tion of phagocytized proteins.2,3 More recently, it has
been suggested that HLE may be externalized in small
quantities and bound to surface “receptors” on leuko-
cytes during migration from the surface of the endo-
thelium into extravascular connective tissues.4,5 Upon
acute activation, it may also be exocytized along with
other granular contents. An imbalance between free
HLE and its natural inhibitors, R1-proteinase and R2-
macroglobulin in plasma6 and secretory leukocyte pro-
tease inhibitor on mucosal surfaces,7 has been suggested
as a causative factor in emphysema,8 chronic bronchi-
tis,9 acute respiratory distress syndrome,9 cystic fibro-
sis,10 and arthritis.11
Several types of chemical strategies have emerged to
identify agents which inhibit HLE.12 Active site di-
rected inhibitors have been constructed with reactive
functionalities appended onto substrate peptides as a
method to control the proteolytic process. Examples
include the transition state analog inhibitors such as
peptidyl trifluoromethyl ketones13 (e.g. 1, ICI 200,355,14
and analogs15), peptidyl ketobenzoxazoles,16 peptidyl
aldehydes,17 peptidyl boronic acid esters,18 peptidyl
R-keto amides,19 and peptidyl R,R-difluoro-â-keto amides
and ketones.20 HLE inactivation through active site
acylation has been accomplished by â-lactams,21 isox-
azolines,22 saccharins,23 and benzisothiazolones.24 Also
reported as inhibitors of HLE are the chemical classes
of phenylbutyrates,25 oligonucleotides,26 polyanionic
chelators,27 heparin derivatives,28 peptidyl carbam-
ates,29 sulfonamidobenzoylglycines,30 isocoumarins,31
and biphenyldisulfonic acid copolymer.32
1000-5000) can modulate the activity of biological
systems via charge-charge interactions of the polymer
with basic residues in proteins and produce inhibitory
effects,33 including the inhibition of HLE (IC50 ) 40-
130 ng/mL).34 It was our belief that these polymers
share a common bioactive motif. That is, a certain
fragment length, composed of repeating monomer units
containing anionic charges, is responsible for the ob-
served biological activity. As a consequence, the re-
mainder of the polymer backbone does not contribute
to the biological efficacy. With this premise in mind and
coupled with our ability to assemble discrete anionic
oligomers contained in a polymeric mixture,35 we un-
dertook the task of establishing whether small, well-
defined anionic oligomeric compounds could also provide
potent charge-mediated inhibition of HLE.36 From the
diversity of our polymer scaffolds33a we decided that the
manifold for carrying the anionic charges should be
constructed of phenyl rings linked in a para relationship
by methylene groups, as illustrated by diphenylmethane-
based oligomer 3a ,b.
In addition to its pathophysiological significance,8-11
HLE was chosen as a target for this study due to its
unique molecular surface topology. The high basicity
of HLE is due to 18 arginine residues on its surface
which are balanced by only 6 acidic residues. Ap-
proximately 40% of the total amino acid side chains are
hydrophobic and accessible to bulk water molecules.37
Four Arg residues, Arg 36, Arg 147, Arg 177, and Arg
217, are located adjacent to the active site, span a total
of approximately 40 Å, and are separated by hydropho-
bic amino acids (e.g. Leu 35, Phe 41, Val 62, Val 99,
Leu 143, Ile 151, Phe 215, Val 216). Thus, HLE
represents an attractive chemical landscape from which
to explore the hypothesis that potent and specific
enzyme inhibition is attainable with a synthetic mol-
ecule bound, not in, but rather, adjacent to the active
site by a combination of anionic and lipophilic interac-
tions.38,39 For a compound to be an effective and specific
inhibitor of HLE, it should be designed to chemically
* Address correspondence to present address: Research and Devel-
opment Center, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury
Road, Ridgefield, CT 06877.
† Medicinal Chemistry.
‡ Inflammation Biology.
§ Hebrew University.
X Abstract published in Advance ACS Abstracts, September 1, 1997.
S0022-2623(97)00251-3 CCC: $14.00 © 1997 American Chemical Society

Inhibitors of Human Leukocyte Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3409
sequentially 16, 17, and 18 in 92% overall yield from
15. Iodination40 (I2, morpholine) of phenol 18 provided
19 as the sole regioisomer in 84% yield, and 19 was
converted to benzyl phenyl ether 20 using NaH and
benzyl bromide. Alcohol 16 was transformed to benzyl
bromide 21 in 65% isolated yield with NBS and tri-
phenylphosphine. Treatment of 21 with freshly acti-
vated Zn powder41 followed by aryl iodide 20 and
catalytic [(Ph)3P]4Pd provided dimer 22 in 88% yield.
Processing of 22 to dimers 25 and 27 was accomplished
with similar reaction conditions as those described
above. That is, 22 was hydrolyzed (NaOMe), iodinated
(I2, morpholine), and alkylated (NaH, benzyl bromide)
to give sequentially 23, 24, and 25 in 80% overall yield.
Also, 22 was treated with tetra-n-butylammonium
fluoride (TBAF), and the resulting alcohol, 26, was
converted (NBS, Ph3P) to benzyl bromide 27 in 81%
overall yield. The conversion of the dimer aryl iodide,
25, and the benzylic zinc species41 derived from dimer
27 into tetramer 28 was accomplished by Pd(0)-medi-
ated coupling in 89% yield. Saponification of the four
nitriles into carboxylic acids, removal of the benzoate
ester, and silyl ether hydrolysis of 28 by the action of
KOH in hot methanol and THF provided 4 in 45% yield.
The preparation of pentamer 5 is outlined in Scheme
3. Tetramer 28 was transformed into an aryl iodide 31
in 76% overall yield by exposure to NaOMe at low
temperature to provide phenol 29, I2 and morpholine40
to give 30 and subsequently NaH and benzyl bromide.
Coupling of the benzylic zinc intermediate derived from
21 with 31, mediated by Pd(0), furnished pentamer 32
in 79% yield. Hot KOH in methanol saponified the five
nitriles to carboxylic acids, benzoate ester to phenol, and
silyl ether to hydroxmethyl and gave 5 in 53% yield.
match, as closely as possible, the electronic features near
the active site. That is, the target molecule should
possess appropriately positioned carboxylic acids to bind
with the arginine’s guanidinium groups and also hy-
drophobic groups to interact at the hydrophobic do-
mains. Other important features would include a
flexible backbone that could conformationally adapt to
the surface topology and be resistant to proteolytic
degradation. It would be anticipated that compounds
of this description could tightly adhere to the surface
of HLE via multiple interactions, sterically prevent
access of the peptide substrate to the catalytic site, and,
thereby, halt the proteolytic process. Tetramer 4,
pentamer 5, and hexamers 6 and 7a -f were designed
to fulfill these criteria. Thus, the construction and in
vitro inhibition of these compounds is the subject of this
report.
The construction of hexamer 6 is shown in Scheme 4.
The hexamer scaffold, 33, was obtained in 55% yield
from the coupling of the benzylic zinc derivative of 2741
and tetramer 31 mediated by Pd(0). Conversion of 33
to target molecule 6 was accomplished by the action of
KOH in hot methanol and THF. The hydrolysis of the
six nitriles to carboxylic acids, the benzoate ester to
phenol, and the silyl ether to hydroxy methyl occurred
in 88% yield.
The construction of hexamer 7f, whose scaffold is
composed of three phenoxyacetic acid units alternating
with three cyclohexylmethyl aryl ethers, is described in
Scheme 5. Conversion of phenol 14 to aryl iodide 37
was readily accomplished in four steps and in 22%
overall yield by treatment of 14 with NaH and MEM
chloride (34), reduction of the aldehyde with NaBH4,
protection of the benzylic alcohol as a TBDPS silyl ether,
benzoate ester hydrolysis (NaOMe) (35), iodination (N-
iodosuccinamide/morpholine40) (36), and alkylation of
the phenol with methyl iodide (37). Dimer 38 was
obtained in 73% yield from the Pd(0)-mediated coupling
of the benzylic zinc species from 2141 and aryl iodide
37. The TBDPS protecting group of 38 was removed
(TBAF) to give alcohol 39, which was transformed (NBS/
Ph3P) to benzyl bromide 40 in 48% yield. In addition,
38 was converted to aryl iodide 43 by sequential
treatment with NaOMe (41, 86%), I2 and morpholine
(42, 85%), and NaH and MeI (99%). Tetramer 44 was
produced from aryl iodide 43 and the benzylic zinc
intermediate from 40 with the use of Pd(0) in 28% yield.
The conversion of 44 to aryl iodide 47 was realized using
Ch em istr y
The retrosynthetic strategy applied to assemble hex-
amer 6 is outlined in Scheme 1. Palladium-mediated
coupling of dimer benzylic zinc 8 with tetramer aryl
iodide 9 would provide the hexamer scaffold. Tetramer
9 would be assembled from the coupling of dimers 8 and
10. Monomers 11 and 12 would serve as precursors for
both dimers 8 and 10.
The synthesis of tetramer 4 is described in Scheme
2. Treatment of 2,5-dihydroxybenzaldehyde (13) with
benzoic anhydride followed by monoester hydrolysis by
K2CO3 in methanol produced 14. Alkylation of 14 with
bromoacetonitrile furnished phenoxyacetonitrile 15 in
73% overall yield. Sodium borohydride reduction of
aldehyde 15, protection of the alcohol as the tert-
butyldiphenylsilyl (TBDPS) ether and benzoate ester
hydrolysis using NaOMe at low temperature gave

3410 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21
Sch em e 1. Retrosynthetic Strategy for the Synthesis of 6
Regan et al.
reaction conditions similar to those described for the
preparation of 43. That is, benzoate hydrolysis (NaOMe)
gave 45, iodination (I2/morpholine) provided 46, and
alkylation of the phenol (NaH/MeI) furnished 47 in 85%
overall yield. Coupling of the benzylic zinc derivative
of 40 and 47, using Pd(0) as catalyst, provided hexamer
48 in 68% yield. Conversion of the TBDPS ether in 48
to a methyl group was accomplished in 43% overall yield
by exposure of 48 to TBAF to give alcohol 49, bromi-
nation with NBS and triphenylphosphine to furnish 50,
and tri-n-butyltin hydride/AIBN reduction to provide 51.
Benzoate hydrolysis (NaOMe) of 51 produced phenol 52,
which was methylated (NaH, MeI) and gave 53 in 73%
yield for the two steps. Removal of the three MEM
protecting groups from the phenols in 53 was ac-
complished in 95% yield with PPTS and furnished 54.
Alkylation of the phenolic hydroxylic groups in 54 with
cyclohexylmethyl bromide (K2CO3, DMF) gave 55 in
44% yield. The three nitriles in 55 were hydrolyzed
with KOH and produced triacid 7f in 56% yield.
The second objective was to discern the effect of the
oligomer scaffold size on the in vitro inhibition of HLE.
Examples of other anionic-mediated inhibitors that are
size sensitive include sulfated saccharides as antipro-
liferative agents42 and the platelet aggregation inhibitor
aurintricarboxylic acid polymer.43 These two classes of
compounds have demonstrated that a minimum molec-
ular size is required for biological activity, and once this
has been achieved, further increases in chain size do
not significantly improve efficacy. Table 1 summarizes
the relative abilities of compounds 1 and 4-6 to
competitively inhibit HLE in vitro vs the substrate Suc-
Ala-Ala-Pro-Phe-pNA in HEPES buffer containing 200
mM NaCl at pH 7.5. Tetramer 4 was substantially less
potent than pentamer 5 and hexamer 6 (Ki ) 8300, 130,
and 120 nM, respectively). No improvement in biologi-
cal efficacy was observed with octamers or hexadecam-
ers from a similar class of compounds (data not shown).
In addition, no inhibitory potency was observed with the
phenoxyacetonitrile precursors of these compounds (i.e.
32). Thus, a minimum scaffold size consisting of five
phenoxyacetic acid units is needed to effectively span
the relevant surface of HLE and bind with the guani-
dinium groups to retard the proteolytic activity. It is
worthwhile to note that calix[8]arene 56,44 despite an
appropriate number of phenoxyacetic acids, displayed
modest inhibitory activity against HLE with Ki ) 1300
nM (Table 1). The cyclic array of charges of 56 is
capable of only spanning approximately the same
Resu lts a n d Discu ssion
Our first program goal was to establish the mecha-
nism by which these compounds inhibit HLE. Figure
1 shows the inhibition of 6 vs the substrate pGlu-Pro-
Val-pNA (S-2484) at pH 7.0 with 120 mM NaCl in
HEPES buffer. Kinetic analysis indicates that 6 is a
competitive inhibitor (Ki ) 190 nM) since it increases
the Km values without affecting Vm.

Inhibitors of Human Leukocyte Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3411
Sch em e 2. Synthesis of Tetramer 4a
a
Abbreviations: Bn ) CH2Ph, Bz ) C(O)Ph, TBDPS ) Si(t-Bu)Ph2. Reagents: (a) (1) (PhCO)2O/Et3N/CH2Cl2, (2) K2CO3/MeOH/25 °C;
(b) BrCH2CN/K2CO3/CH3CN/80 °C; (c) NaBH4/THF/diglyme/-70 °C; (d) t-Bu(Ph)2SiCl/imidazole/DMAP/CH2Cl2; (e) NaOMe/MeOH/-50
to -30 °C; (f) I2/morpholine/CH2Cl2; (g) NaH/PhCH2Br/DMPU/THF; (h) NBS/(Ph)3P/THF; (i) 21/Zn/THF/0-5 °C then 20/[(Ph)3P]4Pd/
THF/60 °C; (j) (n-Bu)4NF/MeCO2H/THF/25 °C; (k) 27/Zn/THF/0-5 °C then 25/[(Ph)3P]4Pd/THF/69 °C; (l) KOH/MeOH/THF/reflux.
distance as a tetramer, such as 4. Also, 56 may not be
properly aligned or flexible enough to productively bind
to the enzyme surface. Taken together, these results
are consistent, although not conclusive, with our pro-
posed mechanism of HLE inhibition by electostatic and
hydrophobic interactions with the enzyme surface.
The third goal of the program was the identification
of target molecules which would carry fewer charged
groups and include other hydrophobic moieties. The
rationale for pursuing this strategy was the belief that
compounds devoid of noninteracting carboxylic acids and
with an array of anionic charge and lipophilicity that
better matched the cationic charge and lipophilic surface
character of HLE might prove to be superior inhibitory
agents. Hydrophobic amino acids adjacent to the Arg
residues near the active site are exposed to bulk solvent,
and therefore, lipophilic groups attached to the oligomer
scaffold would be expected to interact at these sites and
improve efficacy. As an initial venture that was guided
by the requirements of the surface topology of HLE,
analogs were prepared wherein alternating acetic acids
on the backbone of 6 were replaced with ethers of
varying lipophilicity (i.e. 3b). In addition, for ease of
synthesis, methyl ethers were substituted for the benzyl
ethers of 6. As a result, the scaffolds consisted of three
phenoxyacetic acid units linked with three phenols (7a )
or three phenolic ethers (7b-f). The competitive in vitro
inhibitory activity of 7a -f against HLE is shown in
Table 2. Compounds 7a and 7b containing alternating
phenoxyacetic acids with phenol and phenolic MEM
ethers, respectively, were weakly inhibitory (Ki ) 1000-
1300 nM). In vitro potency was improved for those
compounds endowed with additional lipophilic charac-
ter. For example, the benzyl, 7c, and phenylpropyl, 7d ,
derivatives showed a 5-10-fold enhancement in activity
with Ki values of 100-200 nM. Compounds 7e and 7f,
containing n-hexyl and cyclohexylmethyl phenyl ethers,
respectively, were the most potent with Ki values of 20
nM. The alkyl ethers analogs were somewhat more
potent than the alkyl aryl ethers45 as seen in 7e and 7f
vs 7c and 7d . Thus, increases in inhibitory activity
against HLE by 7e and 7f were secured, in addition to
the methyl to benzyl ether exchange, by deleting three
nonproductively binding acetic acid groups from 6 and
replacing them with aliphatic ethers. A 65-fold im-
provement in in vitro potency was observed between 7a
and 7f and supports the importance of the hydrophobic
interactions for these compounds. Molecules with this
motif apparently provide a better match with the
electronic properties on the surface of HLE, and this
improved binding is reflected in enhanced biological
activity.46,47
In addition to the evaluation of 4-6 and 7a -f against
HLE, the compounds were evaluated in vitro for their

3412 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21
Sch em e 3. Synthesis of Pentamer 5a
Regan et al.
Con clu sion s
We have shown that competitive inhibition of HLE
can be effectively accomplished by the ability of an
anionic and hydrophobic-based oligomer to bind to the
enzyme via charge-charge interactions with the basic
amino acid residues and in conjuction with hydrophobic
interactions on the surface. We believe the inhibition
is occurring as a result of multiple interactions of the
oligomer with the basic residues that are adjacent to
the active site and with surrounding hydrophobic resi-
dues.49 Consequently, the substrate is denied access to
the enzyme. This type of HLE inhibition requires a
minimum size of the oligomer scaffold as seen by the
activity of 4 vs 5. Furthermore, an increase in the
number of charges does not provide additional inhibitory
potency as observed with 5 vs 6 and 56. Non-enzyme-
interacting anionic charges can be deleted from the
oligomer manifold and potency retained. Hydrophobic
interactions with the enzyme surface were realized with
aliphatic ethers appended to the oligomer and produced
improvements in HLE inhibition as shown with 7a vs
7f. Moreover, specificity versus other proteins and basic
enzymes was obtained through the construction of
oligomers that are endowed with functional groups and
a charge distribution pattern that are complimentary
to the surface of HLE (compare 7b with 7f). Thus, the
surface topology and electronic properties of HLE have
been shown to be an exploitable physiological feature
from which potent and specific inhibitors can be de-
signed.
Further application of this principle can be applied
to any protein (enzyme or receptor) whose solvent
accessable domains are composed of charged and hy-
drophobic amino acid residues. These regions, if bound
to a synthetic inhibitor, should be capable of producing
a biologically modulating affect by sterically preventing
protein-ligand or protein-protein interactions. It is
also our belief that every protein’s surface is a unique
composition of charged, hydrophobic, and hydrophilic
domains. Biological specificity for a single protein can
be achieved with a compound with electronically com-
plimentary functionality attached to a scaffold capable
of adapting to the topology of the protein surface.
Therefore, as an aid to future drug discovery efforts
compounds with protein-surface-matched electronics
and topology can be considered for synthesis.
a
Abbreviations: Bn ) CH2Ph, Bz ) C(O)Ph, TBDPS ) Si(t-
Bu)Ph2. Reagents: (a) NaOMe/MeOH/-50 to -22 °C; (b) I2/
morpholine/CH2Cl2; (c) NaH/PhCH2Br/DMPU/THF; (d) 21/Zn/
THF/0-5 °C then 31/[(Ph)3P]4Pd/THF/60 °C; (e) KOH/MeOH/THF/
reflux.
inhibition against other basic enzymes. Our goal was
to determine if the geometric array of anionic charges
on the oligomers was specific only for the positive
charges on HLE. Cathepsin G, a serine protease also
found in human neutrophils, is more basic than HLE.
This enzyme has been shown to be inhibited by heparin
fragments28a and a biphenyldisulfonic acid copolymer.32
Thrombin, a serine protease, contains a heparin binding
domain on its surface and can interact with heparin.48
The selectivity of 4-6 and 7a ,c-f for HLE versus
cathepsin G and thrombin was between 4 and >750 as
shown in Table 3. This data suggested that the molec-
ular size or electronic or topological properties of these
oligomers do not provide the satisfactory matches that
are required for productive binding to these other basic
enzymes. Compound 7b, containing three MEM ethers,
was a more potent inhibitor of thrombin (Ki ) 240 nM)
than HLE (Ki ) 1 µM).
To further explore the issue of biological specificity,
hexamer 6 was evaluated for its ability to inhibit HLE
in vitro in the presence of physiological concentrations
of various purified human plasma proteins. Albumin,
γ-globulin, fibrinogen, and fibronectin were individually
incubated with 6. The rate of HLE hydrolysis of the
substrate MeO-Suc-Ala-Pro-Val-pNA by 6 was not
changed when the plasma protein was present in
reaction medium. These results suggest that 6 has a
higher binding affinity for HLE than albumin, γ-glob-
ulin, fibrinogen, and fibronectin.
Exp er im en ta l P r oced u r es
Melting points were determined with a Thomas-Hoover
capillary melting point apparatus and are uncorected. NMR
spectra were measured on a Varian EM-390 or Bruker AC-F
300 NMR spectrometer using tetramethylsilane as an internal
standard. Chemical shifts are reported in parts per million
(δ) from the tetramethylsilane resonance in the indicated
solvent. Mass spectra were run on a Varian MATT 112. TLC
analytical separations were conducted with E. Merck silica gel
F-254 plates of 0.25 mm thickness and were visualized with
UV or I2. Flash chromatographies were run according to the
procedure of Still et al. (EM Science Kieselgel 60, 70-230
mesh) or on a Waters Prep LC/System 500 using Prep Pak-
500 silica cartridges in the indicated solvent. The com-
mercially available starting materials and anhydrous solvents
were used as obtained. All buffer salts and substrates were
obtained from Sigma and were of the highest purity avaliable.
Human R-thrombin was obtained from Enzyme Research
Laboratories (South Bend, Indiana). HLE was purchased from
Elastin Products (St. Louis, Mo).

Inhibitors of Human Leukocyte Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3413
Sch em e 4. Synthesis of Hexamer 6a
a
Abbreviations: Bn ) CH2Ph, Bz ) C(O)Ph, TBDPS ) Si(t-Bu)Ph2. Reagents: (a) 27/Zn/THF/0-5 °C then 31/[(Ph)3P]4Pd/THF/69 °C;
(b) KOH/MeOH/THF/reflux.
2-(H yd r oxym et h yl)-4-(b en zyloxy)-5-[4-[4-[4-[2-[[(h y-
d r oxyca r b on yl)m e t h yl]oxy]-5-h yr oxyb e n zyl]-3-(b e n -
zyloxy)-5-[[(h ydr oxycar bon yl)m eth yl]oxy]ben zyl]-3-(ben -
zyloxy)-5-[[(h ydr oxycar bon yl)m eth yl]oxy]ben zyl]-3-(ben -
zy lo x y )-5-[[(h y d r o x y c a r b o n y l)m e t h y l]o x y ]b e n zy l]-
p h en oxya cetic Acid (5). To a room temperature solution of
32 (0.100 g, 0.065 mmol) in 1 mL of 2:1 EtOH/H2O was added
potassium hydroxide (0.087 g, 1.56 mmol), and the solution
was stirred and warmed to 55-60 °C. Dioxane (∼200 µL) was
added to obtain complete solubility. The mixture was stirred
overnight and cooled to room temperature and the pH adjusted
to 1 with 1 N HCl. The resulting cloudy mixture was diluted
with ethyl acetate and extracted with half-saturated brine (5×)
followed once by brine. After sitting overnight the aqueous
layer was filtered to obtain 45 mg (53%) of product. 1H NMR
(DMSO-d6): δ 3.81 (m, 8H), 4.52 (m, 18H), 4.97 (s, 2H), 6.32
(br s, 1H), 6.46 (dd, J ) 12, 3 Hz, 1H), 6.62 (m, 2H), 6.70 (d,
J ) 6 Hz, 2H), 6.83 (s, 1H), 6.88 (s, 1H), 6.92 (s, 1H), 7.05 (br
s, 3H), 7.15-7.32 (m, 14H), 7.40 (t, J ) 9 Hz, 4H), 7.68 (br s,
1H). IR (KBr, cm-1): 3060 m, 2911 m, 1726 s, 1503 s, 1405
m, 1283 w, 1218 s, 1063 m, 730 w, 696 w. MS (FAB pNBA):
m/z 1279 (M + H+). Combustion analysis: C, H.
chromatography using a solution of 4% formic acid and 33%
THF in CHCl3 as the eluent. Concentration in vacuo of the
product-rich fractions and trituration of the residue with 50%
ether in CHCl3 provided the solid product in 13% yield. 1H
NMR (DMSO-d6): δ 6.85-6.35 (m, 13 H), 4.60 (s, 2 H), 4.45
(s, 4 H), 3.85-3.5 (m, 28 H with singlets, 3 H, at 3.71, 3.67,
3.66, 3.63, and 3.61), 2.08 (s, 3 H). MS (FAB-pNBA): m/z 1004
(M+). Combustion analysis: C, H.
2-[4-[4-[4-[4-[2-Meth oxy-4-m eth yl-5-[[(2-m eth oxyeth oxy)-
m et h yl]oxy]b en zyl]-2-m et h oxy-5-[[(h yd r oxyca r b on yl)-
m et h yl]oxy]b en zyl]-2-m et h oxy-5-[[(2-m et h oxyet h oxy)-
m e t h yl]oxy]b e n zyl]-2-m e t h oxy-5-[[(h yd r oxyc a r b on -
yl)m eth yl]oxy]ben zyl]-2-m eth oxy-5-[[(2-m eth oxyeth oxy)-
m eth yl]oxy]ben zyl]-4-m eth oxyp h en oxya cetic Acid (7b).
Prepared from 53, as described for the synthesis of 7f, in 91%
yield, mp 65-70 °C. 1H NMR (CDCl3): δ 6.95-6.65 (m, 11
H), 6.4 (m, 2 H), 5.10 (s, 2 H), 5.01 (s, 2 H), 4.95 (s, 2 H), 4.52
(s, 2 H), 4.39 (s, 4 H), 3.95-3.7 (m, 36 H) containing singlets
at 3.94, 3.89, 3.83, 3.80, 3.77, 3.76, 3.74), 3.65 (m, 2 H), 3.53
(m, 2 H), 3.41 (m, 2 H), 3.35 (s, 3 H), 3.30 (s, 3 H), 2.19 (s, 3
H). MS (FAB-pNBA): m/z 1257 (M + H), 1280 (M + Na).
Combustion analysis: C, H.
2-(Hyd r oxym eth yl)-4-(ben zyloxy)-5-[4-[4-[4-[4-[2-[[(h y-
d r oxyca r b on yl)m et h yl]oxy]-5-h yd r oxyb en zyl]-3-(b en -
zyloxy)-5-[[(h ydr oxycar bon yl)m eth yl]oxy]ben zyl]-3-(ben -
zyloxy)-5-[[(h ydr oxycar bon yl)m eth yl]oxy]ben zyl]-3-(ben -
zyloxy)-5-[[(h yd r oxyca r b on yl)m e t h yl]oxy]b e n zyl]-3-
(b en zyloxy)-5-[[(h yd r oxyca r b on yl)m et h yl]oxy]b en zyl]-
p h en oxya cetic Acid (6). A solution of 33 (0.703 g) in 15 mL
of 3 M KOH, 4 mL of methanol, and 4 mL of THF was heated
to reflux for 72 h, cooled to room temperature, acidified to pH
1 with aqueous HCl, and diluted with THF. The organic layer
was washed with brine and dried (MgSO4), and the volatiles
were removed in vacuo. The residue was precipitated from a
hot solution of THF by the addition of ethyl acetate and
2-[4-[4-[4-[2-M e t h o x y -4-(h y d r o x y m e t h y l)-5-(b e n -
zyloxy)ben zyl]-2-m eth oxy-5-[[(h ydr oxycar bon yl)m eth yl]-
oxy]ben zyl]-2-m eth oxy-5-(ben zyloxy)ben zyl]-2-m eth oxy-
5-(ben zyloxy)ben zyl]-4-h yd r oxyp h en oxya cetic Acid (7c).
Prepared in 35% yield by hydrolysis of the nitriles (KOH,
ethanol, dioxane, 40-50 °C, 14 h) and purification by flash
chromatography using a solution of 15% THF and 2% formic
acid in CHCl3 as the eluent. Concentration in vacuo of the
product-rich fractions and trituration of the residue with ethyl
acetate and CH2Cl2 gave the solid product, mp 185-9 °C. 1H
NMR (CDCl3): δ 7.35-7.2 (m, 15 H), 7.05-6.45 (m, 13 H), 4.87
(s, 2 H), 4.81 (s, 2 H), 4.77 (s, 2 H), 4.66 (s, 2 H), 4.35 (s, 2 H),
4.1-3.65 (m, 29 H containing singlets at 3.98, 3.92, 3.88, 3.79,
3.75, and 3.71). MS (FAB-pNBA): m/z 1265 (M + H).
Combustion analysis: C, H.
1
subsequent cooling and provided 0.539 g (88%) of product. H
NMR (DMSO-d6): δ 3.76 (s, 4H), 3.81 (s, 2H), 3.90 (s, 2H),
4.45 (s, 2H), 4.50 (s, 8H), 4.52 (s, 8H), 4.97 (s, 2H), 6.33 (d, J
) 2.6 Hz, 1 H), 6.45 (dd, J ) 8, 2.6 Hz, 1H), 6.60 (s, 1H), 6.62
(s, 2H), 6.70 (s, 1H), 6.71 (s, 1H), 6.83 (s, 1H), 6.86 (s, 1H),
6.93 (s, 1H), 7.0-7.5 (m, 21H). MS (FAB pNBA): m/z 1184
(M + H). Combustion analysis: C, H.
2-[4-[4-[4-[4-(2-Meth oxy-4-m eth yl-5-h yd r oxyben zyl)-2-
m et h oxy-5-[[(h yd r oxyca r b on yl)m et h yl]oxy]b en zyl]-2-
m eth oxy-5-h yd r oxyben zyl]-2-m eth oxy-5-[[(h yd r oxyca r -
bon yl)m eth yl]oxy]ben zyl]-2-m eth oxy-5-h yd r oxyben zyl]-
4-m eth oxyp h en oxya cetic Acid (7a ). Prepared from 7b as
described for the synthesis of 54 followed by NaOH hydrolyses
of the propyl esters (dioxane, ethanol, 1 N NaOH, 25 °C, 18
h). Purification of the material was accomplished by flash
2-[4-[4-[4-[4-[2-Meth oxy-4-m eth yl-5-[(3-p h en ylp r op yl)-
oxy]ben zyl]-2-m eth oxy-5-[[(h ydr oxycar bon yl)m eth yl]oxy]-
b e n zyl]-2-m e t h oxy-5-[(3-p h e n ylp r op yl)oxy]b e n zyl]-2-
m et h oxy-5-[[(h yd r oxyca r b on yl)m et h yl]oxy]b en zyl]-2-
m e t h o x y -5-[(3-p h e n y lp r o p y l)o x y ]b e n z y l]-4-m e t h -
oxyp h en oxya cetic Acid (7d ). Prepared from 54 by alkyla-
tion (1-bromo-3-phenylpropane, K2CO3, DMF, 85 °C, 18 h),
hydrolysis (KOH, dioxane, ethanol, 65 °C, 5 h), and purification
(flash chromatography using a solution of 11% THF and 2%
formic acid in CHCl3 as the eluent) in 50% overall yield. 1H
NMR (DMSO-d6) δ 7.3-7.05 (m, 15 H), 6.8-6.5 (m, 13 H), 4.59
(s, 2 H), 4.44 (s, 4 H), 3.85-3.50 (m, 34 H), 2.65 (m, 6 H), 2.14

3414 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21
Regan et al.
a
Sch em e 5. Synthesis of Hexamer 7f
a
Abbreviations: Bz ) C(O)Ph; MEM ) CH2O(CH2)2OMe; TBDPS ) Si(t-Bu)Ph2. Reagents: (a) NaH/MEM-Cl/THF/0-25 °C; (b) (1)
NaBH4/THF/-60 °C, (2) t-Bu(Ph)2SiCl/imidazole/CH2Cl2, (3) NaOMe/MeOH/THF/-25 °C; (c) NIS/morpholine/CH2Cl2; (d) NaH/MeI/DMPU/
THF/0-25 °C; (e) 21/Zn/THF/0-5 °C then 37/[(Ph)3P]Pd/THF/65 °C; (f) (n-Bu)4NF/CH3COOH/THF/25 °C; (g) NBS/(Ph)3P/THF; (h) NaOMe/
MeOH/THF/-55 °C; (i) I2/morpholine/CH2Cl2/0-25 °C; (j) 40/Zn/THF/0-5 °C then 43/[(Ph)3P]Pd/THF/70 °C; (k) 40/Zn/THF/0-5 °C then
47/[(Ph)3P]Pd/THF/70-80 °C; (l) (n-Bu)3SnH/AIBN/toluene/reflux; (m) PPTS/(Me)2CO/(Me)2CHOH/65 °C/3 days; (n) (c-C6H11)CH2Br/K2CO3/
DMF/85 °C; (o) KOH/EtOH/dioxane/65 °C/18 h.

Inhibitors of Human Leukocyte Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3415
Ta ble 1. In Vitro Inhibition of HLE by 1, 4-6, and 56
a
All compounds exhibited satisfactory spectral data consistent with their structures. Analytical results are within ( 0.4% of the
b
theoretical value. Inhibition of HLE at pH 7.5 in HEPES buffer containing 200 mM NaCl with the substrate Suc-Ala-Ala-Pro-pNA. See
the Experimental Section for details. c Twenty minute incubation of HLE and test compound.
Ta ble 2. In Vitro Inhibition of HLE by Hexamers 7a -f
HLE inhibb
Ki (nM)
compd R1
R2
R3
formulaa
7a
7b
7c
7d
7e
7f
Me
Me CH2O(CH2)2OMe
CH2Ph
Me (CH2)3Ph
Me (CH2)5Me
H
H
H
C54H56O18‚4HO
C66H80O24‚H2Oc
1300
1000
200
100
17
H
OH C74H72O19‚2H2Od
H
H
H
C81H86O18‚H2O
C72H92O18‚3H2O
C75H92O18‚H2O
F igu r e 1. HLE kinetics: compound 6 vs S-2484. Legend:
Inhibition kinetic analysis of HLE activity with compound 6.
Each reaction was performed in 600 µL of HEPES buffer
containing 15 nM HLE, compound 6 at the indicated concen-
trations, and the peptide substrate S-2484 at varying concen-
trations. The absorbance at 405 nm over a period of 5 min
was recorded. Shown are the double-reciprocal plots and the
determined apparent Vm and Km values at each inhibitor
concentration.
Me CH2-(cyclo)C6H11
20
a
All compounds exhibited satisfactory spectral data consistent
with their structures. Analytical results are within (0.4% of the
theoretical value. Inhibition of HLE at pH 7.5 in HEPES buffer
b
containing 200 mM NaCl with the substrate Suc-Ala-Ala-Pro-Phe-
pNA. See the Experimental Section for details. c Anal. C: calcd,
d
62.16; found, 62.6. Anal. C: calcd, 69.15; found, 68.59.
h) and purification (flash chromatography using a solution of
9% THF and 1% formic acid in CHCl3 as the eluent) in 57%
yield. 1H NMR (DMSO-d6): δ 6.85-6.5 (m, 13 H), 4.61 (s, 2
H), 4.47 (s, 4 H), 3.90-3.25 (m, 34 H, with singlets at 3.80,
3.73, 3.64, 3.62, 3.34), 2.10 (s, 3 H), 1.60 (m, 6 H), 1.24 (m, 18
H), 0.83 (m, 9 H). MS (FAB-pNBA): m/z 1245 (M + H).
Combustion analysis: C, H.
2-[4-[4-[4-[4-[2-Me t h o x y -4-m e t h y l-5-[(c y c lo h e x y l-
m et h yl)oxy]b en zyl]-2-m et h oxy-5-[[(h yd r oxyca r b on yl)-
m et h yl]oxy]b en zyl]-2-m et h oxy-5-[(cycloh exylm et h yl)-
(s, 3 H), 1.9 (m, 6 H). MS (FAB-pNBA): m/z 1347 (M + H).
Combustion analysis: C, H.
2-[4-[4-[4-[2-Meth oxy-4-m eth yl-5-(h exyloxy)ben zyl]-2-
m et h oxy-5-[[(h yd r oxyca r b on yl)m et h yl]oxy]b en zyl]-2-
m et h oxy-5-(h exyloxy)b en zyl]-2-m et h oxy-5-(h exyloxy)-
ben zyl]-4-m eth oxyp h en oxya cetic Acid (7e). Prepared
from 54 by alkylation (iodohexane, K2CO3, DMF, 85 °C, 18 h,
54%) followed by hydrolysis (KOH, ethanol, dioxane, 65 °C, 5

3416 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21
Regan et al.
Ta ble 3. Specificity of 4, 5, 6, and 7a -f
Ki (nM)
2-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-5-
h yd r oxyp h en oxya ceton itr ile (18). To a solution of 16 (29.1
g, 0.103 mol) in 300 mL of CH2CI2 were added imidazole (8.31
g, 0.125 mol), 4-(dimethylamino)pyridine (DMAP) (1.26 g, 10.3
mmol), and then tert-butylchlorodiphenylsilane (29.5 mL). The
mixture was stirred 1 h, diluted with ether, washed with H2O
and brine, and dried (MgSO4), and the volatiles were removed
in vacuo to provide 17. This material was dissolved in 400
mL of THF, cooled to -50 °C, and sodium methoxide (24.7 mL
of a 25% methanol solution) was added. The mixture was
stirred 2 h while warming to -30 °C and quenched with 2 N
HCl (53 mL) and ether. The organic layer was washed with
water and brine and dried (MgSO4). Removal of the volatiles
in vacuo, recrystallization of the residue with hexanes and
ethyl acetate, and purification of the mother liquor by flash
chromatography using 30% ethyl acetate in hexanes as the
eluent provided 42.9 g (99%) of solid product.
2-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
h yd r oxy-5-iod op h en oxya ceton itr ile (19). To a solution of
18 (42.9 g, 0.103 mol) and morpholine (22.4 mL, 0.258 mol) in
300 mL of CH2Cl2 at 0-5 °C was added I2 (24.7 g, 97.3 mmol).
The mixture was warmed to room temperature, stirred for 1.5
h, and quenched with 1 N HCl (155 mL) and ether. The
organic layer was washed with H2O, saturated sodium bisulfite,
and brine and dried (MgSO4). Removal of the volatiles in
vacuo provided a residue which was purified by flash chro-
matography using 35% ether in hexanes as the eluent.
Concentration in vacuo of the product-rich fractions furnished
46.8 g (84%) of the oily product.
4-(Ben zyloxy)-2-[[[(1,1-d im et h ylet h yl)d ip h en ylsilyl]-
oxy]m eth yl]-5-iod op h en oxya ceton itr ile (20). To a sus-
pension of NaH (60%, 3.62 g, 90.5 mmol) in 75 mL of
anhydrous THF at -20 °C was added a solution of 19 (46.8 g,
86.2 mmol), benzyl bromide (11.8 mL, 99.2 mmol), and DMPU
(63 mL) in 185 mL of THF. The mixure was warmed to room
temperature, stirred for 2.5 h, and quenched with 1 N HCl
(20 mL) and ether. The organic layer was washed with H2O
and brine and dried (MgSO4). Removal of the volatiles in
vacuo provided a residue which was purified by flash chro-
matography using 10% ethyl acetate in hexanes as the eluent.
Concentration in vacuo of the product-rich fractions gave 52.8
g (97%) of the oily product.
4-(B e n zo y lo x y )-2-(b r o m o m e t h y l)p h e n o x y a c e t o n i-
tr ile (21). To a solution of 16 (27.9 g, 98.5 mmol) and
triphenylphosphine (36.5 g, 0.138 mol) in 300 mL of anhydrous
THF was added portionwise N-bromosuccinimide (NBS) (21.1
g, 0.118 mol). After 1 h additional triphenylphosphine (4.3 g)
and NBS (2.1 g) were added. The mixture was stirred for 30
min, and the volatiles were removed in vacuo. The residue
was dissolved in CHCl3, applied to a plug of silica gel, and
eluted with 25% ethyl acetate in hexanes. Purification of the
filtrate by HPLC using 20% ethyl acetate in hexanes as the
eluent provided 22.2 g (65%) of the pale yellow solid, mp 87-8
°C. MS: m/z 347, 345 (M+).
compd
HLE
cathepsin Ga
thrombinb
4
5
6
7a
7b
7c
7d
7e
7f
8300
130
120
1300
1000
200
100
17
>15000
>15000
>15000
>15000
>15000
1500
>15000
>15000
>15000
>15000
750
>15000
>15000
240
>15000
1700
1400
20
>15000
a
Inhibition of cathepsin G at pH 7.5 in HEPES buffer contain-
ing 200 mM NaCl with the substrate Suc-Ala-Ala-Pro-Phe-pNA.
b
See the Experimental Section for details. Inhibition of thrombin
at pH 7.5 in HEPES buffer containing 200 mM NaCl with the
substrate Bz-Phe-Val-Arg-pNA. See the Experimental Section for
details.
oxy]ben zyl]-2-m eth oxy-5-[[(h ydr oxycar bon yl)m eth yl]oxy]-
ben zyl]-2-m eth oxy-5-[(cycloh exylm eth yl)oxy]ben zyl]-4-
m eth oxyp h en oxya cetic Acid (7f). A mixture of 55 (27 mg)
and KOH (70 µL of a 12.5 N solution) in ethanol (500 µL) and
dioxane (200 uL) was heated at 65 °C for 5 h, the volume was
reduced by 50% by removal of the volatiles in vacuo, and the
mixture was diluted with H2O, acidified with HCl, and
extracted with ethyl acetate. The combined organic extracts
were washed with H2O and brine and dried (MgSO4). Removal
of the volatiles in vacuo provided a residue which was purified
by flash chromatography using as the eluent a solution of 1%
formic acid and 2% THF in CHCl3. Concentration in vacuo of
the product-rich fractions gave 16 mg (56%) of tan solid. MS
(FAB-pNBA): m/z 1281 (M + H). Combustion analysis: C,
H.
5-(Ben zoyloxy)-2-h yd r oxyben za ld eh yd e (14). A solu-
tion of 2,5-dihydroxybenzaldehyde (13, 84.3 g, 0.610 mol),
benzoic anhydride (296 g, 1.28 mol), and triethylamine (137.3
g., 1.34 mol) in 800 mL of CH2Cl2 was stirred at room
temperature overnight and the volume reduced by ∼50% by
concentration in vacuo. The residue was diluted with ether
and ethyl acetate, washed with H2O and brine, and dried
(MgSO4). Removal of the volatiles in vacuo provided a residue
which was dissolved in 700 mL of anhydrous methanol, and
85 g of anhydrous K2CO3 was added. The mixture was stirred
2 h with occassional heating, diluted with H2O, and extracted
with ether. The combined organic layers were washed with 2
N HCl, saturated Na2HPO4, and brine and dried (MgSO4).
Removal of the volatiles in vacuo provided 145 g (98%) of the
tan solid which was used without further purification. MS:
m/z 242 (M+).
5-(Ben zoyloxy)-2-[(cyan om eth yl)oxy]ben zaldeh yde (15).
A mixutre of 14 (37.4 g, 0.154 mol), bromoacetonitrile (22.9
g., 0.185 mol), and K2CO3 (25.9 g, 0.185 mol) in 300 mL of
acetonitrile was heated at 80 °C for 10 h under
a
N2
4-(Be n zyloxy)-5-[2-[(cya n om e t h yl)oxy]-5-(b e n zolyl-
oxy)b en zyl]-2-[[[(1,1-d im et h ylet h yl)d ip h en ylsilyl]oxy]-
m eth yl]p h en oxya ceton itr ile (22). A suspension of Zn
powder (325 mesh, 12.6 g, 0.193 mol) and 0.8 mL of freshly
distilled 1,2-dibromoethane in 12 mL of anhydrous THF was
warmed to 80 °C for 1.5 min and cooled to 0-5 °C. To the
suspension was added 21 (17.4 g, 50.3 mmol) in 70 mL of
anhydrous THF over 2.75 h. After the addition was complete
the mixture was added via cannula to a mixture of 20 (30.0 g,
47.3 mmol) and tetrakis(triphenylphosphine)palladium (3.0 g).
The zinc was washed with 30 mL of THF, and the washings
were added to the mixture. The resulting solution was heated
at 60 °C for 4.25 h, cooled to room temperature, and diluted
with ether and 5% aqueous NH3. The organic layer was
washed with 5% aqueous NH3 and brine and dried (MgSO4),
and the volatiles were removed in vacuo. Purification of the
residue by flash chromatography using 40% ether, 10% CH2-
Cl2 in hexanes as the eluent provided 32.02 g (88%) of the
product, mp 100-1 °C. 1H NMR (CDCl3): δ 1.07 (s, 9H), 4.0
(s, 2H), 4.56 (s, 2H), 4.69 (s, 2H), 4.75 (s, 2H), 5.01 (s, 2H),
6.77 (s, 1H), 6.94 (d, J ) 9 Hz, 1H), 7.01 (d, J ) 2.8 Hz, 1H),
7.07 (dd, J ) 9, 2.8 Hz, 1H), 7.1-7.41 (m, 12H), 7.50 (t, J )
atmosphere, cooled to 0-5 °C, neutralized with 1 N HCl, and
diluted with ether. The organic layer was washed with H2O
and brine and dried (MgSO4), and the volatiles were removed
in vacuo. Recrystallization of the residue with hexanes and
ethyl acetate and purification of the mother liquor on HPLC
using 20% ethyl acetate in hexanes as the eluent provided 32.0
g (74%) of the solid, mp 100-1 °C. MS: m/z 281 (M+). 1H
NMR (CDCl3): δ 4.92 (s, 2H), 7.13 (d, J ) 8.9 Hz), 7.46-7.52
(m, 3H), 7.63 (t, J ) 7.6 Hz, 2H), 7.72 (d, J ) 3 Hz, 1H), 8.15
(d, J ) 7.1 Hz, 2H) 10.39 (s, 1H).
4-(Ben zoyloxy)-2-(h yd r oxym et h yl)p h en oxya cet on i-
tr ile (16). To a -70 °C solution of 15 (32.0 g, 0.114 mol) in
300 mL of anhydrous THF was added NaBH4 (114 mL of 0.5
M in diglyme, 56.9 mmol). The mixture was stirred 5 h and
quenched with 60 mL of 2 N HCl and ther. The organic layer
was washed with H2O and brine and dried (MgSO4), and the
voltiles were removed in vacuo. Recrystallization of the
residue with hexanes-ethyl acetate and purification of the
mother liquor on HPLC using 43% ethyl acetate in hexanes
as the eluent provided 30 g (93%) of the white solid. MS: m/z
283 (M+).

Inhibitors of Human Leukocyte Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3417
7.5 Hz, 2H), 7.60-7.67 (m, 5H), 8.15 (dd, J ) 8.1, 1.4 Hz, 2H).
13C NMR (CDCl3): δ 19.98, 27.5, 31.1, 54.8, 55.2, 61.2, 71.1,
112.3, 113.3, 115.9, 121.1, 125.5, 128.1, 128.3, 128.4, 128.5,
129.19, 129.23, 130.4, 130.8, 131.9, 134.0, 134.3, 136.2, 137.7,
146.6, 147.5, 153.0, 153.1, 166.0. IR (KBr, cm-1): 3070 w, 2922
m, 2851 m, 1722 s, 1509 m, 1495 s, 1380 m, 1281 s, 1200 s,
1077 s, 706 s. MS (FAB-pNBA): m/z 771 (M - H+). Combus-
tion analysis: C, H.
5-(b en zyloxy)-2-(cya n om et h yloxy)b en zyl]p h en oxya ce-
ton itr ile (28). A mixture of Zn dust (3.2 g, 48.9 mmol) and
1,2-dibromoethane (200 µL) in 3 mL of anhydrous THF was
heated to reflux for 2 min and cooled to 0-5 °C, and a solution
of 27 (4.5 g, 7.54 mmol) in 20 mL of THF and 4 mL of DMF
was added dropwise over the course of 1.1 h. The mixture was
stirred 15 min, and the supernatant was added to a mixture
of 25 (4.4 g, 4.98 mmol) and [(Ph)3P]4Pd] (0.36 gm). The
mixture was heated to 69 °C for 2.75 h, cooled to room
temperature, and diluted with ether and 5% aqueous am-
monia. The organic layer was dried (MgSO4), and the volatiles
were removed in vacuo. Purification of the residue by flash
chromatography using 30% ethyl acetate, 30% CH2Cl2 in
hexanes as the eluent and concentration in vacuo of the
product-rich fractions furnished 5.62 g (89%) of product.
2-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
(ben zyloxy)-5-[4-[4-[2-[(cya n om eth yl)oxy]-5-h yd r oxyben -
zyl]-5-(ben zyloxy)-2-[(cya n om et h yl)oxy]b en zyl]-5-(b en -
zyloxy)-2-[(cya n om et h yl)oxy]b en zyl]-5-(b en zyloxy)-2-
[(cya n om et h yl)oxy]b en zyl]p h en oxya cet on it r ile (29). A
solution of 28 (4.9 g, 3.86 mmol) and sodium methoxide (840
µL of a 25% methanol solution) in 15 mL of THF was stirred
at -50 to -22 °C during the course of 2 h and quenched with
aqueous HCl (3.9 mL of a 1 N solution) and 20% CH2
ether. The organic layer was washed with H2O and brine and
dried (MgSO4), and the volatiles were removed in vacuo.
Purification of the residue by flash chromatography using 30%
ethyl acetate, 30% CH2Cl2 in hexanes as the eluent and
concentration in vacuo of the product-rich fractions gave 4.3
g (96%) of the product.
2-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
(b en zyloxy)-5-[4-[4-[2-[(cya n om et h yl)oxy]-4-iod o-5-h y-
dr oxyben zyl]-5-(ben zyloxy)-2-[(cyan om eth yl)oxy]ben zyl]-
5-(b e n zyloxy)-2-[(cya n om e t h yl)oxy]b e n zyl]p h e n oxy-
a ceton itr ile (30). To a 0-5 °C solution of 29 (3.7 g, 3.18
mmol) in 15 mL of CH2Cl2 were added morpholine (688 µL,
7.87 mmol) and then I2 (0.802 g, 3.16 mmol). The mixture
was warmed to room temperature, stirred 2.5 h, and diluted
with 20% CH2Cl2 in ether and water. The organic layer was
washed with H2O and brine and dried (MgSO4), and the
volatiles were removed in vacuo. Purification of the residue
by flash chromatography using 20% ethyl acetate and 30%
CH2Cl2 in hexanes as the eluent and concentration in vacuo
of the product-rich fractions furnished 3.41g (83%) of product.
2-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
(ben zyloxy)-5-[4-[4-[2-[(cya n om eth yl)oxy]-4-iod o-5-(ben -
zyloxy)ben zyl]-5-(ben zyloxy)-2-[(cya n om eth yl)oxy]ben -
z y l ]-5 -(b e n z y l o x y )-2 -[(c y a n o m e t h y l )o x y ]b e n z y l ]-
p h en oxya ceton itr ile (31). To a suspension of NaH (60%,
109 mg, 2.73 mmol) in 2 mL of anhydrous THF at -20 °C was
added a solution of 30 (3.4 g, 2.62 mmol) and benzyl bromide
(0.4 mL) in 2 mL of DMPU and 6 mL of THF. The mixture
was warmed to room temperature, stirred for 2 h, and diluted
with ether and aqueous HCl (5 mL of a 0.1 M solution). The
4-(Ben zyloxy)-5-[2-[(cya n om eth yl)oxy]-5-h yd r oxyben -
zyl]-2-[[[(1,1-d im et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-
p h en oxya ceton itr ile (23). To a -50 °C solution of 22 (16.21
g) in 80 mL of THF was added sodium methoxide (4.5 mL of
a 25% solution in methanol). The mixture was stirred 50 min
while warming to -30 °C and quenched with ether and 1 N
HCl (21 mL). The organic layer was washed with H2O and
brine and dried (MgSO4). Removal of the volatiles in vacuo
provided a residue which was purified by flash chromatogra-
phy using 60% ether in hexanes as the eluent. Concentration
in vacuo of the product-rich fractions yielded 13.8 g (98%) of a
viscous oil. 1H NMR (CDCl3): δ 1.09 (s, 9H), 3.92 (s, 2H), 4.50
(s, 2H), 4.54 (s, 2H), 4.75 (s, 2H), 5.00 (s, 2H), 5.13 (s, 1H)
6.58 (d, J ) 2.9 Hz, 1H), 6.62 (dd, J ) 8.6, 2.9 Hz, 1H), 6.70
(s, 1H), 6.76 (d, J ) 8.6 Hz, 1H), 7.24 (s, 1H), 7.28-7.42 (m,
11H), 7.66 (dd, J ) 7.6, 1.4 Hz, 4H). 13C NMR (CDCl3): δ 20.0,
27.5 30.6, 55.2, 55.5, 61.2, 71.1, 112.4, 114.4, 115.0, 116.2,
116.4, 119.0, 128.2, 128.4, 128.5, 128.6, 129.2, 130.4, 130.5,
132.0, 134.0, 136.2, 137.7, 147.4, 149.3, 152.0, 153.0.
4-(Be n zyloxy)-5-[2-[(cya n om e t h yl)oxy]-4-iod o-5-h y-
d r oxyb e n zyl]-2-[[(1,1-d im e t h yle t h yl)d ip h e n ylsiloxy]-
m eth yl]p h en oxya ceton itr ile (24). To a solution of 23 (4.10
g, 6.14 mmol) in 20 mL of CH2Cl2 at 0-5 °C were added
morpholine (1.3 mL, 14.9 mmol) and then I2 (1.6g, 6.30 mmol).
The mixture was warmed to room temperature, stirred 2.5 h,
and diluted with ether and aqueous HCl (9.5 mL of a 1 N
solution). The organic layer was washed with H2O and brine
and dried (MgSO4) and the volatiles were removed in vacuo.
Purification of the residue by flash chromatography using 40%
ethyl acetate in hexanes as the eluent provided 4.0 g (82%) of
product.
4-(Ben zyloxy)-5-[2-[(cya n om et h yl)oxy]-4-iod o-5-(ben -
zyloxy)ben zyl]-2-[[[(1,1-dim eth yleth yl)diph en ylsilyl]oxy]-
m eth yl]p h en oxya ceton itr ile (25). To a suspension of NaH
(60%, 0.20 g, 5.0 mmol) in 5 mL of anhydrous THF at -20 °C
was added a solution of 24 (4.0 g, 5.04 mmol), DMPU (3.8 mL),
and benzyl bromide (0.77 mL) in 12 mL of THF. The mixture
was warmed to room temperature, stirred for 2.75 h, and
quenched with ether and aqueous HCl (5 mL of a 0.1 N
solution). The organic layer was washed with water and dried
(MgSO4). Removal of the volatles in vacuo provided a residue
which was purified by flash chromatography using 40% ethyl
acetate in hexanes as the eluent. Concentration in vacuo of
the product-rich fractions furnished 4.4 g (99%) of the product.
4-(Ben zoyloxy)-5-[2-[(cyan om eth yl)oxy]-5-(ben zoyloxy)-
ben zyl]-2-(h yd r oxym eth yl)p h en oxya ceton itr ile (26). A
solution of 22 (40.9 g, 53.0 mmol), acetic acid (30 mL of a 2.0
M solution in THF), and tetra-n-butylammonium fluoride
(TBAF) (60 mL of a 1.0 M solution in THF) in 120 mL of THF
was stirred at room temperatue 2.5 h and diluted with 20%
CH2Cl2 in ether and H2O. The organic layer was washed with
H2O and brine and dried (MgSO4). Removal of the volatiles
in vacuo provided a residue which was purified by flash
chromatography using 20% hexanes in ether followed by 20%
CH2Cl2 in ether as the eluents. Concentration in vacuo of the
product-rich fractions gave 26.0 g (92%) of the product, mp
115-6 °C.
Cl2 in
organic layer was washed with H2
O and brine and dried
(MgSO4). Removal of the volatiles in vacuo provided a residue
which was purified by flash chromatography using 20% ethyl
acetate and 20% CH2Cl2 in hexanes as the eluent. Concentra-
tion in vacuo of the product-rich fractions furnished 3.46 g
(95%) of product.
2-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
(b e n zyloxy)-5-[4-[4-[4-[2-[(cya n om e t h yl)oxy]-5-(b e n -
zoyloxy)ben zyl]-3-(ben zyloxy)-5-[(cya n om eth yl)oxy]ben -
zyl]-3-(ben zyloxy)-5-[(cya n om et h yl)oxy]b en zyl]-3-(b en -
z y l o x y ) -5 -[ ( c y a n o m e t h y l ) o x y ] b e n z y l ] p h e n o x y -
a ceton itr ile (32). A suspension of Zn powder (325 mesh, 3.03
g, 0.046 mol) and 0.2 mL of freshly distilled 1,2-dibromoethane
in 7 mL of anhydrous THF was warmed to 80 °C for 1.5 min
and cooled to 0-5 °C. To the suspension was added 21 (3.46
g, 10 mmol) in 10 mL of anhydrous THF over 1 h. After the
addition was complete, 4.16 mL of the supernatant was added
via syringe to a mixture of 31 (1.0 g, 0.8 mmol) and tetrakis-
(triphenylphosphine)palladium (37 mg). The resulting solution
was heated at 60 °C for 3 h, cooled to room temperature, and
diluted with ether and 5% aqueous NH3. The organic layer
was washed with 5% aqueous NH3 and brine and dried
4-(Ben zyloxy)-5-[2-[(cya n om eth yl)oxy]-5-(ben zoyloxy)-
ben zyl]-2-(br om om eth yl)p h en oxya ceton itr ile (27). To a
solution of 26 (2.81 g, 5.26 mmol) in 35 mL of anhydrous THF
were added triphenylphosphine (1.77 g, 6.76 mmol) and NBS
(1.11 g, 6.23 mmol). The mixture was stirred 25 min, and the
volatiles were removed in vacuo. Purification of the residue
by flash chromatography using CH2Cl2 as the eluent provided
2.76 g (88%) of the product.
2-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
(b e n zyloxy)-5-[4-[4-[2-[(cya n om e t h yl)oxy]-5-(b e n zoyl-
oxy)ben zyl]-5-(ben zyloxy)-2-[(cya n om eth yl)oxy]ben zyl]-

3418 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21
Regan et al.
(MgSO4), and the volatiles were removed in vacuo. Purifica-
tion of the residue by flash column using 35-50% ethyl acetate
in hexanes as the eluent provided 870 mg (79%) of the product.
1H NMR (CDCl3): δ 1.08 (s, 9H), 3.92 (m, 8H), 4.48 (s, 2H),
4.54 (s, 2H), 4.61 (m, 8H), 4.68 (s, 2H), 4.73 (s, 2H), 4.98 (s,
2H), 6.72 (m, 6H), 6.90 (d, J ) 9 Hz, 1H), 6.97 (d, J ) 3 Hz,
1H), 7.06 (dd, J ) 9, 3 Hz, 1H), 7.15-7.55 (m, 30H), 7.65 (d,J
) 9 Hz, 6H), 8.12 (d, J ) 9 Hz, 2H). MS (FAB-pNBA): m/z
1526 (M + H).
2-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
(b en zyloxy)-5-[4-[4-[4-[4-[2-[(cya n om et h yl)oxy]-5-(b en -
zoyloxy)ben zyl]-3-(ben zyloxy)-5-[(cya n om eth yl)oxy]ben -
zyl]-3-(ben zyloxy)-5-[(cya n om et h yl)oxy]b en zyl]-3-(ben -
zyloxy)-5-[(cyan om eth yl)oxy]ben zyl]-3-(ben zyloxy)-5-[(cy-
a n om eth yl)oxy]ben zyl]p h en oxya ceton itr ile (33). A mix-
ture of Zn dust (3.2 g, 48.9 mmol) and 1,2-dibromoethane (200
µL) in 2 mL of anhydrous THF was heated at reflux for 2 min
and cooled to 0-5 °C.
A solution of 27 (0.85 g, 1.42 mmol) in 4 mL of THF was
added dropwise over 30 min. The mixture was stirred for 15
min, and the supernatant was added to a mixture of 31 (1.38
g, 0.996 mmol) and [(Ph)3P]4Pd (0.11 mg). The mixture was
heated at 60 °C for 1.25 h, cooled to room temperature, and
diluted with THF, ether, and 5% aqueous NH3. The organic
layer was washed with brine and dried (MgSO4). Removal of
the volatiles in vacuo provided a residue which was dissolved
in hot ethyl acetate, cooled, filtered, and provided 0.983 g (55%)
of product.
2-[[(2-Meth oxyeth oxy)m eth yl]oxy]-5-(ben zoyloxy)ben -
za ld eh yd e (34). To a suspension of NaH (60%, 13.0 g, 0.321
mol) in 20 mL of anhydrous THF under an Ar atmosphere at
0-5 °C was added a solution of 14 (67.67 g, 0.279 mol), (2-
methoxyethoxy)methyl chloride (90%, 46.4 g, 0.335 mol), and
DMPU (30 mL) in 400 mL of THF. The mixture was slowly
warmed to room temperature, stirred for 90 min, and quenched
with aqueous HCl (330 mL of a 1 N solution) and ether. The
organic layer was washed with H2O and brine and dried
(MgSO4). Removal of the volatiles in vacuo provided a residue
which was purified by HPLC using 25% ethyl acetate in
hexanes as the eluent. Concentration in vacuo of the product-
rich fractions provided 75.5 g (82%) of the pale yellow solid.
MS: m/z 300 (M+).
3-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
[[(2-m eth oxyeth oxy)m eth yl]oxy]p h en ol (35). To a -60 °C
solution of 34 (75.52 g, 0.229 mol) in 250 mL of anhydrous
THF was added dropwise NaBH4 (230 mL of 0.5 M in diglyme,
0.115 mol). The mixture was stirred under a N2 atmosphere
for 90 min, and additional NaBH4 (23 mL) was added. After
45 min of stirring, the reaction was quenched by the addition
of aqueous HCl (250 mL of a 1 N solution). The mixture was
diluted with H2O and extracted with ether. The combined
organic extracts were washed with H2O and brine and dried
(MgSO4). Removal of the volatiles in vacuo provided a residue
which was dissolved in 300 mL of CH2Cl2 and added to an
aged (15 min) mixture of imidazole (32 g, 0.458 mol) and tert-
butyldiphenylsilyl chloride (62.95 g, 0.229 mol) in 500 mL of
CH2Cl2. The mixture was stirred for 90 min, and approxi-
mately 50% of the volatiles removed in vacuo. The residue
was dissolved in ether, washed with 0.5% aqueous HCl, water,
and brine, and dried (MgSO4). Removal of the volatiles in
vacuo provided a residue which was dissolved in 500 mL of
THF and cooled to -25 °C. Sodium methoxide (53 mL of a
25% methanol solution, 0.229 mol) was added, and the mixture
was stirred for 30 min and quenched with aqueous HCl (250
mL of a 1 N solution) and ether. The organic layer was washed
with H2O and brine and dried (MgSO4). Removal of the
volatiles in vacuo provided a residue which was purified by
HPLC using 20% ethyl acetate in hexanes as the eluent.
Concentration in vacuo of the product-rich fractions gave 68.6
g (64%) of the oily product. MS: m/z 465 (M - H).
additional morpholine (7 mL) and NIS (8.75 g) were added.
After 1 h the mixture was diluted with ether, washed with 1
N HCl, H2O, aqueous Na2S2O5, H2O, and brine, and dried
(MgSO4). Removal of the volatiles in vacuo provided a residue
which was purified by HPLC using 11% ethyl acetate in
hexanes as the eluent. Concentration in vacuo of the product-
rich fractions gave 70.8 g (81%) of the product. MS (FAB-
pNBA): m/z 593 (M - H).
3-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
[[(2-m eth oxyeth oxy)m eth yl]oxy]-6-iod oa n isole (37). To
a suspension of NaH (60%, 4.75 g, 0.119 mol) in 20 mL of
anhydrous THF at 0-5 °C was added a solution of phenol 36
(70.3 g, 0.119 mol), iodomethane (33.8 g, 0.238 mol), and
DMPU (20 mL) in 230 mL of THF. The mixture was warmed
to room temperature, stirred for 90 min, quenched with
aqueous HCl (125 mL of a 1 N solution), and extracted with
ether. The organic extracts were washed with H2O and brine
and dried (MgSO4). Removal of the volatiles in vacuo provided
a residue which was purified by HPLC using 5% ethyl acetate
in hexanes as the eluent. Concentration in vacuo of the
product-rich fractions gave 37.0 g (51%) of the gummy product.
MS: FAB 608 (M+). 1H NMR (CDCl3): δ 7.65 (m, 4 H), 7.35
(m, 7 H), 7.22 (d, 1 H), 5.05 (s, 2 H), 4.75 (s, 2 H), 3.82 (s, 3 H),
3.65 (m, 2 H), 3.5 (m, 2 H), 3.35 (s, 3 H), 1.1 (s, 9 H).
2-[(2-Met h oxy-4-[[[(1,1-d im et h ylet h yl)d ip h en ylsilyl]-
oxy]m eth yl]-5-[[(2-m eth oxyeth oxy)m eth yl]oxy]ben zyl]-
4-(ben zoyloxy)p h en oxya ceton itr ile (38). A mixture of zinc
(70.0 g, 1.07 mol) and 1,2-dibromoethane (5.02 g, 0.027 mol)
in 30 mL of anhydrous THF was heated at 75 °C for 5 min
and cooled to 0-5 °C, and a solution of 21 (37.0 g, 0.107 mol)
in 60 mL of THF was added dropwise. After the addition was
complete (4.5 h), the supernatant was added to a solution of
37 (65.0 g, 0.107 mol) and (Ph3P)4 Pd (6.18 g, 5.35 mmol) in
60 mL of degassed THF. The mixture was heated at 65 °C
for 5 h, cooled to room temperature, and diluted with ether
and 5% NH4OH solution. The organic layer was washed with
H2O and brine and dried (MgSO4). Removal of the volatiles
in vacuo provided a residue which was purified by HPLC using
25% ethyl acetate in hexanes as the eluent. Concentration in
vacuo of the product-rich fractions furnished 58.4 g (73%) of
the orange gum. 1H NMR (CDCl3): δ 8.15 (d, 2 H), 7.8-7.3
(m, 12 H), 7.24 (d, 2 H), 7.1-6.9 (m, 4 H), 5.05 (s, 2 H), 4.82
(s, 2 H), 4.80 (s, 2 H), 3.95 (s, 2 H), 3.8 (s, 3 H), 3.65 (m, 2 H),
3.45 (m, 2 H), 3.30 (s, 3 H), 1.1 (s, 9 H). MS (FAB-pNBA):
m/z 745 (M+).
2-[(2-Me t h o x y -4-(h y d r o x y m e t h y l)-5-[[(2-m e t h o x y -
eth oxy)m eth yl]oxy]ben zyl]-4-(ben zoyloxy)p h en oxya ce-
ton itr ile (39). A solution of 38 (34.4 g, 46.2 mmol), tetrabu-
tylammonium fluoride (TBAF) (69 mL of a 1.0 M THF solution,
69.3 mmol), and acetic acid (4.45 g, 74.0 mmol) in 100 mL of
anhydrous THF was stirred for 3 h, and additional acetic acid
(900 µL) and TBAF (14 mL) were added. After 1 h the mixture
was diluted with ether and H2O. The organic layer was
washed with brine and dried (MgSO4). Removal of the
volatiles in vacuo provided a residue which was purified by
HPLC using 50% ethyl acetate in hexanes as the eluent.
Concentration in vacuo of the product-rich fractions gave 19.3
g (82%) of the orange gum. MS: FAB 507 (M+). 1H NMR
(CDCl3): δ 5.18 (s, 2 H), 4.78 (s, 2 H), 4.65 (s, 2 H), 3.95 (s, 2
H), 3.75 (m, 5 H), 3.5 (m, 2 H), 3.3 (s, 3 H), 1.6 (bs, 1 H).
2-[2-Meth oxy-4-(br om om eth yl)-5-[[(2-m eth oxyeth oxy)-
m e t h yl]oxy]b e n zyl]-4-(b e n zoyloxy)p h e n oxya ce t on i-
tr ile (40). To a mixture of 39 (19.3 g, 38 mmol) and
triphenylphosphine (16.1 g, 60.8 mmol) in 140 mL of THF was
added portionwise NBS (9.50 g, 53.2 mmol). The mixture was
stirred for 90 min, and the volatiles were removed in vacuo.
Purification of the CH2Cl2 soluble residue by flash chroma-
tography using 33% ethyl acetate in hexanes as the eluent
provided 12.8 g (59%) of the tan solid. 1H NMR (CDCl3): δ
5.25 (s, 2 H), 4.75 (s, 2 H), 4.5 (s, 2 H), 3.9 (s, 2 H), 3.82 (m, 2
H), 3.75 (s, 3 H), 3.5 (m, 2 H), 3.32 (s, 3 H). MS (FAB-pNBA):
m/z 571, 569 (M+).
3-[[[(1,1-Dim et h ylet h yl)d ip h en ylsilyl]oxy]m et h yl]-4-
[[(2-m eth oxyeth oxy)m eth yl]oxy]-6-iod op h en ol (36). To
a solution of 35 (68.6 g, 0.147 mol) and morpholine (25.6 g,
0.294 mol) in 600 mL of anhydrous CH2Cl2 at 0-5 °C was
added portionwise N-iodosuccinamide (NIS) (35 g, 0.147 mol).
The mixture was warmed to room temperature over 2 h, and
2-[2-Meth oxy-4-[[[(1,1-dim eth yleth yl)diph en ylsilyl]oxy]-
m eth yl]-5-[[(2-m eth oxyeth oxy)m eth yl]oxy]ben zyl]-4-h y-
d r oxyp h en oxya ceton itr ile (41). A -55 °C solution of 38
(24.08 g, 32.3 mmol) and sodium methoxide (7.4 mL of a 25%

Inhibitors of Human Leukocyte Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3419
methanol solution, 32.3 mmol) in 120 mL of anhydrous THF
was stirred under Ar for 30 min, and additional sodium
methoxide (0.75 mL) was added. After 20 min the mixture
was quenched with aqueous HCl (40 mL of 1 N solution) and
ether. The organic layer was washed with brine and dried
(MgSO4). Removal of the volatiles in vacuo and purification
of the residue by HPLC using 33% ethyl acetate in hexanes
as the eluent provided 17.74 g (86%) of the tan solid. MS
(FAB-pNBA): m/z 641 (M+).
product-rich fractions gave a quantitative yield of yellow gum
45. To this material and morpholine (0.767 g, 8.80 mmol) in
15 mL of anhydrous CH2Cl2 at 0-5 °C was added dropwise a
solution of I2 (1.02 g, 4.0 mmol) in 45 mL of CH2Cl2. The
mixture was warmed to room temperature, stirred for 3 h,
diluted with ether, washed with 1 N HCl, H2O, saturated
Na2S2O5, and brine, and dried (MgSO4). Removal of the
volatiles in vacuo, purification of the residue by flash chro-
matography using 50% ethyl acetate in hexanes as the eluent,
and concentration of the product-rich fractions gave 3.92 g
(86%) of the tan solid 46. This material, iodomethane (0.952
g, 6.70 mmol), and DMPU (2 mL) in 24 mL of anhydrous THF
was added to a suspension of NaH (60%, 0.135 g, 3.35 mmol)
in 1 mL of THF at 0-5 °C. The mixture was warmed to room
temperature, stirred for 2 h, acidified with aqueous HCl (5
mL of a 1 N solution), and diluted with ether and H2O. The
organic layer was washed with brine and dried (MgSO4).
Removal of the volatiles in vacuo and purification of the
residue by flash chromatography using 50% ethyl acetate in
hexanes as the eluent and concentration in vacuo of the
product-rich fractions furnished 3.93 g (99%) of a yellow gum.
1H NMR (CDCl3): δ 7.7 (d, 4 H), 7.7-7.15 (m, 8 H), 6.89 (s, 1
H), 6.84 (s, 1 H), 6.72 (s, 1 H), 6.71 (s, 1 H), 6.70 (s, 1 H), 6.6
(s, 1 H), 5.1 (s, 2 H), 5.00 (s, 2 H), 4.79 (s, 2 H), 4.69 (s, 2 H),
4.59 (s, 2 H), 3.89 (s, 4 H), 3.86 (s, 2 H), 3.80 (s, 3 H), 3.77 (s,
3 H), 3.74 (m, 2 H), 3.73 (s, 3 H), 3.70 (s, 3 H), 3.61 (m, 2 H),
3.44 (m, 2 H), 3.39 (m, 2 H), 3.34 (s, 3 H), 3.28 (s, 3 H), 1.1 (s,
9 H). MS (FAB-pNBA): m/z 1185 (M + H).
2-[4-[4-[4-[4-[2-Meth oxy-4-[[[(1,1-d im eth yleth yl)d ip h e-
n ylsilyl]oxy]m eth yl]-5-[[(2-m eth oxyeth oxy)m eth yl]oxy]-
b e n zy l]-2-m e t h o x y -5-[(c y a n o m e t h y l)o x y ]b e n zy l]-2-
m et h oxy-5-[[(2-m et h oxyet h oxy)m et h yl]oxy]b en zyl]-2-
m eth oxy-5-[(cya n om eth yl)oxy]ben zyl]-2-m eth oxy-5-[[(2-
m eth oxyeth oxy)m eth yl]oxy]ben zyl]-4-(ben zoyloxy)p h e-
n oxya ceton itr ile (48). A mixture of Zn dust (1.43 g, 21.8
mmol) and 1,2-dibromoethane (0.103 g, 0.545 mmol) in 2 mL
of anhydrous THF was heated at reflux for 3 min and cooled
to 0-5 °C. A solution of 40 (1.25 g, 2.18 mmol) in 5 mL of
THF was added dropwise over 90 min. After 15 min of stirring
the supernatant was added to a mixture of 47 (1.80 g, 1.53
mmol) and (Ph3P)4 Pd (0.13 g) in 2 mL of THF. The mixture
was heated at 70-80 °C overnight, cooled to room tempera-
ture, and diluted with ether and 5% NH4OH. The organic
layer was washed with H2O and brine and dried (MgSO4).
Removal of the volatiles in vacuo provided a residue which
was purified by flash chromatography using 50% ethyl acetate
in hexanes as the eluent. Concentration in vacuo of the
product-rich fractions gave 1.60 g (68%) of the tan solid. MS
(FAB pNBA): m/z 1543 (M+).
2-[2-Meth oxy-4-[[[(1,1-dim eth yleth yl)diph en ylsilyl]oxy]-
m eth yl]-5-[[(2-m eth oxyeth oxy)m eth yl]oxy]ben zyl]-4-h y-
d r oxy-5-iod op h en oxya ceton itr ile (42). To a cold (0-5 °C)
solution of 41 (17.74 g, 27.6 mmol) and morpholine (5.30 g,
60.8 mmol) in 60 mL of anhydrous CH2Cl2 was added a
solution of I2 (7.02 g, 27.6 mmol) in 210 mL of CH2Cl2. The
mixture was stirred 2 h, warmed to room temperature, stirred
for 2 h, and diluted with ether and aqueous HCl (80 mL of 1
N solution). The organic layer was washed with H2O, satu-
rated Na2S2O4, and brine and dried (MgSO4). Removal of the
volatiles in vacuo and purification of the residue by HPLC
using 25% ethyl acetate in hexanes as the eluent provided
17.93 g (85%) of the tan solid. MS (FAB-pNBA): m/z 767 (M+).
2-[2-Meth oxy-4-[[[(1,1-dim eth yleth yl)diph en ylsilyl]oxy]-
m eth yl]-5-[[(2-m eth oxyeth oxy)m eth yl]oxy]ben zyl]-4-m eth -
oxy-5-iod op h en oxya cet on it r ile (43). To a suspension of
NaH (60%, 0.935 g, 23.4 mmol) in 10 mL of anhydrous THF
at 0-5 °C was added 42 (17.93 g, 23.4 mmol), DMPU (10 mL),
and iodomethane (6.63 g, 46.7 mmol) in 100 mL of THF. The
mixture was warmed to room temperature, stirred for 1 h, and
quenched with ether and aqueous HCl (30 mL of a 1 N
solution). The organic layer was washed with H2O and brine
and dried (MgSO4). Removal of the volatiles in vacuo and
purification of the residue by HPLC using 20% ethyl acetate
in hexanes as the eluent provided 18.1 g (99%) of the brown
gum. 1H NMR (CDCl3): δ 7.7 (dd, 4 H), 7.4 (m, 6 H), 7.2 (s, 1
H), 6.82 (s, 1 H), 6.7 (s, 1 H), 5.05 (s, 2 H), 4.78 (s, 2 H), 4.65
(s, 2 H), 3.85 (s, 2 H), 3.80 (s, 3 H), 3.75 (s, 3 H), 3.6 (m, 2 H),
3.4 (m, 2 H), 3.3 (s, 3 H), 1.1 (s, 9 H). MS (FAB-pNBA): m/z
781 (M+).
2-[4-[4-[2-Meth oxy-4-[[[(1,1-d im eth yleth yl)d ip h en ylsi-
lyl]oxy]m et h yl]-5-[[(2-m et h oxyet h oxy)m et h yl]oxy]b en -
zyl]-3-[(cya n om eth yl)oxy]-6-m eth oxyben zyl]-2-m eth oxy-
5-[[(2-m e t h o x y e t h o x y )m e t h y l]o x y ]b e n z y l]-4-(b e n -
zoyloxy)p h en oxya ceton itr ile (44). A mixture of Zn dust
(9.2 g, 0.14 mol) and 1,2-dibromoethane in 5 mL of anhydrous
THF was heated at reflux for 3 min and cooled to 0-5 °C, and
a solution of 40 (8.00 g, 14.0 mmol) in 25 mL of anhydrous
THF was added dropwise over 3 h. The supernatant was
added to a degassed solution of 43 (11.0 g, 14.0 mmol) and
(Ph3P)4 Pd (0.81 g, 0.70 mmol) in 25 mL of THF. The mixture
was heated at 70 °C overnight, cooled to room temperature,
diluted with 5% NH4OH, and extracted with ether. The
combined organic extracts were washed with brine and dried
(MgSO4). Removal of the volatiles in vacuo and purification
of the residue by HPLC using 50% ethyl acetate in hexanes
as the eluent provided 4.54 g (28%) of the white solid product.
1H NMR (CDCl3): δ 8.15 (d, 2 H), 7.70 (dd, 4 H), 7.6-6.6 (m,
18 H), 5.15 (s, 2H), 5.0 (s, 2 H), 4.78 (s, 4 H), 4.55 (s, 2 H),
3.95 (s, 2 H), 3.90 (s, 2 H), 3.88 (s, 2 H), 3.80 (s, 3 H), 3.75 (m,
2 H), 3.70 (s, 3 H), 3.65 (s, 3 H), 3.60 (m, 2 H), 3.45 (m, 2 H),
3.40 (m, 2 H), 3.35 (s, 3 H), 3.28 (s, 3 H), 1.1 (s, 9 H). MS:
FAB 1145 (M + H). TLC (4:3 hexanes:ethyl acetate): Rf 0.24.
Also recovered was 4.4 g (40%) of 43.
2-[4-[4-[4-[4-[2-Meth oxy-4-(h ydr oxym eth yl)-5-[[(2-m eth -
oxyet h oxy)m et h yl]oxy]b en zyl]-2-m et h oxy-5-[[(2-m et h -
o x y e t h o x y )m e t h y l]o x y ]b e n zy l]-2-m e t h o x y -5-[(c y a n -
o m e t h y l)o x y ]b e n zy l]-2-m e t h o x y -5-[[(2-m e t h o x y e t h -
oxy)m eth yl]oxy]ben zy]-4-(ben zoyloxy)p h en oxya ceton i-
tr ile (49). In a manner similar to the preparation of 26, 48
(3.35 g, 2.17 mmol) was treated with TBAF (3.26 mmol) and
acetic acid (3.47 mmol) in 10 mL of THF for 2 h at room
temperature. Workup and purification by flash chromatog-
raphy using 25% hexanes in ethyl acetate as the eluent gave
2.00 g (71%) of white solid. 1H NMR (CDCl3): δ 8.15 (dd, 2
H), 7.61 (t, 1 H), 7.49 (t, 2 H), 7.1-6.95 (m, 3 H), 6.71 (bs, 3
H), 6.7-6.6 (m, 7 H), 5.16) (s, 2 H), 5.15 (s, 2 H), 5.11 (s, 2 H),
4.79 (s, 2 H), 4.65 (s, 2 H), 4.57 (s, 2 H), 4.55 (s, 2 H), 3.92 (s,
2 H), 3.89 (b s, 6 H), 3.85 (s, 2 H), 3.80 (s, 3 H), 3.75 (s, 6 H),
3.74 (s, 3 H), 3.72 (s,3 H), 3.5 (m, 6 H), 3.32 (s, 3 H), 3.31 (s,
3 H), 3.30 (s, 3 H). MS (FAB-pNBA): m/z 1305 (M+).
2-[4-[4-[2-Meth oxy-4-[[[(1,1-d im eth yleth yl)d ip h en ylsi-
lyl]oxy]m et h yl]-5-[[(2-m et h oxyet h oxy)m et h yl]oxy]b en -
zyl]-3-[(cya n om eth yl)oxy]-6-m eth oxyben zyl]-2-m eth oxy-
5-[[(2-m eth oxyeth oxy)m eth yl]oxy]ben zyl]-4-m eth oxy-5-
iod op h en oxya ceton itr ile (47). A solution of 44 (4.50 g, 3.93
mmol) and sodium methoxide (0.92 mL of a 25% sodium
methoxide in methanol solution) in 30 mL of anhydrous THF
at -50 °C was stirred for 60 min, quenched with aqueous HCl
(5 mL of 1 N solution) and H2O, and extracted with ether. The
combined organic extracts were washed with brine and dried
(MgSO4). Removal of the volatiles in vacuo, purification of the
residue by flash chromatography with 50% ethyl acetate in
hexanes as the eluent, and concentration in vacuo of the
2-[4-[4-[4-[4-[2-Meth oxy-4-(br om om eth yl)-5-[[(2-m eth -
oxyeth oxy)m eth yl]oxy]ben zyl]-2-m eth oxy-5-[(cya n om e-
t h y l)o x y ]b e n zy l]-2-m e t h o x y -5-[[(2-m e t h o x y e t h o x y )-
m eth yl]oxy]ben zyl]-2-m eth oxy-5-[(cyan om eth yl)oxy]ben -
zyl]-2-m eth oxy-5-[[(2-m eth oxyeth oxy)m eth yl]oxy]ben zyl]-
4-(ben zoyloxy)p h en oxya ceton itr ile (50). In a manner
similar to the preparation of 27, 49 (1.95 g, 1.49 mmol) was
treated with NBS (2.1 mmol) and triphenylphosphine (2.4
mmol) in 10 mL of THF, purified by flash chromatography

3420 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21
Regan et al.
using 44% hexanes in ethyl acetate as the eluent, and gave
1.5 g (73%) of product. MS (FAB-pNBA): m/z 1369 (M + H).
2-4-[4-[4-[4-(2-Meth oxy-4-m eth yl-5-[[(2-m eth oxyeth oxy)-
m eth yl]oxy]ben zyl]-2-m eth oxy-5-[(cyan om eth yl)oxy]ben -
zyl]-2-m eth oxy-5-[[(2-m eth oxyeth oxy)m eth yl]oxy]ben zyl]-
2-m et h oxy-5-[(cya n om et h yl)oxy]b en zyl]-2-m et h oxy-5-
[[(2 -m e t h o x y e t h o x y )m e t h y l ]o x y ]b e n z y l ]-4 -(b e n -
zoyloxy)p h en oxya ceton itr ile (51). A mixture of 50 (0.90
g, 0.66 mmol), tri-n-butyltin hydride (0.24 g, 0.792 mmol), and
AIBN (12 mg) in 5 mL of anhydrous toluene was heated at
reflux for 90 min, and the volatiles were removed in vacuo.
Purification of the residue by flash chromatography using 44%
hexanes in ethyl acetate as the eluent and concentration in
vacuo of the product-rich fractions provided 0.7 g (82%) of
white solid. 1H NMR (CDCl3): δ 5.15 (s, 2 H), 5.13 (s, 2 H),
5.11 (s, 2 H), 4.79 (s, 2 H), 4.57 (s, 2 H), 4.54 (s, 2 H), 3.76 (s,
3 H), 3.72 (s, 3 H), 3.71 (s, 3 H), 3.69 (s, 3 H), 3.68 (s, 3 H),
3.34 (s, 3 H), 3.32 (s, 3 H), 3.30 (s, 3 H), 2.21 (s, 3 H). MS
(FAB-pNBA): m/z (M + H).
2-[4-[4-[4-[4-[2-Meth oxy-4-m eth yl-5-[[(2-m eth oxyeth oxy)-
m eth yl]oxy]ben zyl]-2-m eth oxy-5-[(cyan om eth yl)oxy]ben -
zyl]-2-m eth oxy-5-[[(2-m eth oxyeth oxy)m eth yl]oxy]ben zyl]-
2-m et h oxy-5-[(cya n om et h yl)oxy]b en zyl]-2-m et h oxy-5-
[[(2-m e t h o x y e t h o x y )m e t h y l]o x y ]b e n zy l]-4-h y d r o x y -
p h en oxya ceton itr ile (52). In a manner similar to the
preparation 29, 51 (0.91 g, 0.71 mmol) was treated with sodium
methoxide (0.71 mmol) in methanol and THF at -50 °C.
Workup and purification by flash chromatography using 40%
hexanes in ethyl acetate as eluent gave 0.66 g (80%) of the
white solid. MS (FAB-pNBA): m/z 1186 (M + H).
2-[4-[4-[4-[4-[2-Meth oxy-4-m eth yl-5-[[(2-m eth oxyeth oxy)-
m eth yl]oxy]ben zyl]-2-m eth oxy-5-[(cyan om eth yl)oxy]ben -
zyl]-2-m eth oxy-5-[[(2-m eth oxyeth oxy)m eth yl]oxy]ben zyl]-
2-m et h oxy-5-[(cya n om et h yl)oxy]b en zyl]-2-m et h oxy-5-
[[(2-m e t h oxye t h oxy)m e t h yl]oxy]b e n zyl]-4-m e t h oxy-
p h en oxya ceton itr ile (53). To NaH (60%, 23 mg, 0.557
mmol) was added a solution of 52 (0.66 g, 0.557 mmol),
iodomethane (0.158 g, 1.11 mmol), and DMPU (300 µL) in 5
mL of anhydrous THF. The mixture was stirred for 1 h,
acidified with aqueous HCl (1 mL of a 1 N solution), and
diluted with ether and H2O. The organic layer was washed
with brine and dried (MgSO4). Removal of the volatiles in
vacuo provided a residue which was purified by flash chro-
matography using 40% hexanes in ethyl acetate as the eluent.
Concentration in vacuo of the product-rich fractions gave 0.61
g (91%) of the gummy product. 1H NMR (CDCl3): δ 6.95-
6.65 (m, 13 H), 5.13 (s, 4 H), 5.12 (s, 2 H), 4.69 (s, 2 H), 4.58
(s, 2 H), 4.57 (s, 2 H), 3.88 (s, 6 H), 3.85 (s, 4 H), 3.77 (s, 3 H),
3.74 (s, 3 H), 3.73 (s, 3 H), 3.72 (s, 3 H), 3.71 (s, 3 H), 3.70 (s,
3 H), 3.5 (m, 6 H), 3.34 (s, 3 H), 3.33 (s, 3 H), 3.31 (s, 3 H),
2.22 (s, 3 H). MS (FAB-pNBA): m/z 1200 (M + H).
2-[4-[4-[4-[4-(2-Meth oxy-4-m eth yl-5-h yd r oxyben zyl)-2-
m eth oxy-5-[(cya n om eth yl)oxy]ben zyl]-2-m eth oxy-5-h y-
d r oxyben zyl]-2-m eth oxy-5-[(cya n om eth yl)oxy]ben zyl]-2-
m e t h o x y -5 -h y d r o x b e n z y l ] -4 -m e t h o x y -p h e n o x y -
a ceton itr ile (54). A mixture of 53 (0.405 g, 0.338 mmol) and
PPTS (0.312 g, 1.21 mmol) in acetone (2 mL) and 2-propanol
(6 mL) was heated at 65 °C for 3 days, cooled to room
temperature, diluted with ethyl acetate, washed with H2O and
brine, and dried (MgSO4). Removal of the volatiles in vacuo
provided a residue which was purified by flash chromatogra-
phy using 50% ethyl acetate in hexanes as the eluent.
Concentration in vacuo of the product-rich fractions gave 0.30
g (95%) of white solid. MS (FAB-pNBA): m/z 936 (M + H).
2-[4-[4-[4-[4-[2-Meth oxy-4-m eth yl-5-[(cycloh exylm eth y-
l)oxy]ben zyl]-2-m eth oxy-5-[(cya n om eth yl)oxy]ben zyl]-2-
m et h oxy-5-[(cycloh exylm et h yl)oxy]b en zyl]-2-m et h oxy-
5-[(cya n om et h yl)oxy]b en zyl]-2-m et h oxy-5-[(cycloh ex-
ylm eth yl)oxy]ben zyl]-4-m eth oxyph en oxyaceton itr ile (55).
A mixture of 54 (47 mg, 0.0502 mmol), cyclohexylmethyl
bromide (135 µL, 0.905 mmol), and K2CO3 (255 mg, 1.81 mmol)
in anhydrous DMF (0.5 mL) was heated at 85 °C overnight,
cooled to room temperature, diluted with ether, washed
thoroughly with H2O and brine, and dried (MgSO4). Removal
of the volatiles in vacuo provided a residue, which was purified
by flash chromatography using 25% ethyl acetate in hexanes
as the eluent. Concentration in vacuo of the product-rich
fraction gave 27 mg (44%) of a tan solid. 1H NMR (CDCl3): δ
6.9 (m, 1 H), 6.75-6.55 (m, 12 H), 4.68 (s, 2 H), 4.56 (s, 4 H),
3.88 (m, 6 H), 3.8-3.6 (m, 28 H), 2.20 (s, 3 H), 1.9-1.0 (m, 33
H). MS (FAB-pNBA): m/z 1223 (M+).
Ki Deter m in a tion s. All assays are based on the ability of
the test compound to inhibit the enzyme-catalyzed hydrolysis
of a peptide p-nitroanalide substrate. In a typical Ki deter-
mination substrate is prepared in DMSO and diluted into an
assay buffer consisting of 50 mM HEPES and 200 mM NaCl
at pH 7.5. The final concentration for each of the substrates
is listed below. All substrate concentrations are at least 10-
fold lower than the experimentally determined value for Km.
Test compounds are prepared as a 0.16 mg/mL solution in
DMSO. Dilutions are prepared in DMSO, yielding eight final
concentrations encompassing a 200-fold concentration range.
Enzyme solutions are prepared at the concentrations listed
below in assay buffer.
Inhibition kinetic studies of HLE activity with the test
compound at a fixed concentration has been performed in the
presence of varying concentrations of the peptide substrate.
The velocity of the reaction was calculated as the increase of
the absorbance at 405 nm/min. Double-reciprocal plots of the
data were used to determine the apparent Vm and Km values
and therefore to analyze the mode of inhibition of the HLE
activity by the compound. Figure 1 shows the result with 6.
In a typical Ki determination, into each well of a 96-well
plate were pipetted 280 µL of substrate solution and 10 µL of
inhibitor solution, and the plate was allowed to thermally
equilibrate at 37 °C in a Molecular Devices plate reader for
>10 min. Reactions were initiated by the addition of a 10 µL
aliquot of enzyme, and the absorbance increase at 405 nm is
recorded for 15 min. Data corresponding to more than 90%
of inhibition of substrate hydrolysis was used in the calcula-
tions. Using the protocol of I. H. Segel the ratio of the velocity
(rate of the change in absorbance as a function of time) for a
sample containing inhibitor is divided by the velocity of a
sample containing no inhibitor and is plotted as a function of
inhibitor concentration. The data is fit to the equation for a
competitive inhibitor
vi/vc ) Ki/(Ki + I) when [S] , Km
and from the fit, values for Ki are determined (SD ) (0.90).
Ela sta se. Elastase activity was assesed as the ability to
hydrolyze the substrate Suc-Ala-Ala-Pro-Phe-pNA. Substrate
solutions were prepared at a concentration of 10 µM (10 µM
< Km ) 200 µM). The final DMSO concentration was 0.3%.
Purified human leukocyte elastase was diluted into assay
buffer. Final reagent concentrations were [elastase] ) 3 nM,
[Suc-Ala-Ala-Pro-Phe-pNA] ) 10 µM.
Th r om bin . Thrombin activity was assesed as the ability
to hydrolyze the substrate Bz-Phe-Val-Arg-pNA. Substrate
solutions were prepared at a concentration of 60 µM (60 µM
, Km ) 1.2 mM). The final DMSO concentration was 0.3%.
Purified human R-thrombin was diluted into assay buffer to
a concentration of 450 nM. Final reagent concentrations were
[thrombin] ) 30 nM, [Bz-Phe-Val-Arg-pNa] ) 45 µM.
Ca th ep sin G. Cathepsin G activity was assesed as the
ability to hydrolyze the substrate Suc-Ala-Ala-Pro-Phe-pNA.
Substrate solutions were prepared at a concentration of 22 µM
(22 µM , Km ) 240 µM) in assay buffer. The final DMSO
concentration was 0.3%. Purified Cathepsin G was diluted
into assay buffer to a concentration of 225 nM. Final reagent
concentrations were [cathepsin G] ) 15 nM, [Suc-Ala-Ala-Pro-
Phe-pNA] ) 22 µM.
Ack n ow led gm en t. The authors thank the Analyti-
cal Department at Rhoˆne-Poulenc Rorer for mass spec-
tral data, D. Cheney for molecular modeling suppport,
and B. Schwartz and B. J . Kulp for manuscript prepa-
ration. Alain Schreiber, M.D., is thanked for his
enthusiasm and support for this program.

Inhibitors of Human Leukocyte Elastase
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 21 3421
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Inhibitors of HumanNeutrophil Elastase. 3. In Vitro and in Vivo
Potency of a Series of Peptidyl R-Ketobenzoxazoles. J . Med.
Chem. 1995, 38, 3972-3982.
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