Serendipitous discovery of an unexpected rearrangement leads to two new classes of potential protease inhibitors

Article abstract of DOI:10.1016/j.bmc.2004.08.044

The pathogenesis of a range of human diseases arises from the aberrant activity of proteolytic enzymes. Agents capable of selectively modulating the activity of these enzymes are of potential therapeutic value. Thus, there is a continuing need for the des

Full text of DOI:10.1016/j.bmc.2004.08.044

Bioorganic & Medicinal Chemistry 12 (2004) 6249–6254
Serendipitous discovery of an unexpected rearrangement leads
to two new classes of potential protease inhibitors
Jiaying Zhong,a Zhong Lai,a Christopher S. Groutas,a Tzutshin Wong,a Xiangdong Gan,a
Kevin R. Alliston,a David Eichhorn,a John R. Hoidalb and William C. Groutasa,*
aDepartment of Chemistry, Wichita State University, Wichita, KS 67260, USA
bDepartment of Medicine, University of Utah Health Sciences Center, Salt Lake City, UT 84132, USA
Received 13 July 2004; revised 30 August 2004; accepted 30 August 2004
Available online 2 October 2004
Abstract—The pathogenesis of a range of human diseases arises from the aberrant activity of proteolytic enzymes. Agents capable of
selectively modulating the activity of these enzymes are of potential therapeutic value. Thus, there is a continuing need for the design
of scaffolds that can be used in the development of new classes of protease inhibitors. We describe herein the serendipitous discovery
of an unexpected rearrangement that leads to the formation of two novel templates that can be used in the design of protease
inhibitors.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
We have recently described the design of a new class of
transition state inhibitors of serine and cysteine pro-
teases (Fig. 1, structure (I)) that has the distinct advan-
tage of exploiting favorable binding interactions with
both the S and S0 subsites8 of a target protease.9,10
The exploitation of multiple binding interactions with
the S and S0 subsites is of paramount importance in
enhancing potency and, more importantly, attaining
maximal selectivity among proteases that have the same
primary substrate specificity. Thus, inhibitors based on
structure (I) could, in principle, exploit the subtle differ-
ences that exist in some of the subsites of these
proteases.
A hallmark of inflammatory disease is the activation
and recruitment of various cell types to inflammatory
sites. For instance, chronic obstructive pulmonary dis-
ease (COPD) is characterized by an influx of macro-
phages, neutrophils, and T lymphocytes to the lungs.1
Likewise in asthma, an inflammatory disease of the air-
ways, mast cells, eosinophils, and neutrophils are the
major effector cells.2 The recruitment of these cells is
accompanied by the extracellular release of pro-inflam-
matory mediators, including a range of serine, cysteine,
and metallo-proteases.1,2 Besides their degradative ac-
tion, the secreted proteases play a prominent role in
attenuating the inflammatory response through various
actions, including the activation of signaling path-
ways.3–5 While many questions remain regarding the
precise role that each protease plays in a particular dis-
ease state, it is generally believed that selective inhibition
of the proteases associated with inflammatory diseases
such as COPD(neutrophil elastase, proteinase 3) and
asthma (tryptase) may not only lead to new therapeutic
agents, but also shed light on the molecular mechanisms
underlying the pathophysiology of inflammatory
disease.6,7
S1
O
(a)
(L)
P1
R2
P1
COOCH3
NH2
X
R4
R3
N
SO2
N
Sn-S2
Sn'
(I)
(X=H,CF3,COOR,CONHR,
heterocycle, etc)
(b)
O
S
HO O-Ser-E
P1
P1
R2
X
X
+
E-Ser-OH
N
NR3R4
N
NR3R4
R2
S
O
O
Keywords: Protease inhibitors; New scaffolds; Rearrangement.
*
O
O
Corresponding author. Tel.: +1 316 978 7374; fax: +1 316 978
3431; e-mail: bill.groutas@wichita.edu
Figure 1. Design and mechanism of action of (I).
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.044

6250
J. Zhong et al. / Bioorg. Med. Chem. 12 (2004) 6249–6254
P1
(L)
COOCH3
P1
R2
COOCH3
P1
R2
COOCH3
P1
R2
COOCH3
a
c
d
b
NH2
N
N
N
H
SO2NR3Boc
3
SO2NHBoc
2
1
P1
N R3
O
N
S
R2
O
OH
(II)
g
h
P1
R2
e
P1
R2
CHO
(7,P1=isobutyl,R2=benzyl,
R3=2-phenethyl)
N
N
SO2NR3Boc
4
SO2NR3Boc
5
h
O
O
f
P1
S
R2
N
N
O
R3
H
P1
CHO
(III)
N
(8,P1=isobutyl,R2=benzyl,
R3=2-phenethyl)
R2
SO2NHR3
6
Figure 2. Synthesis of (II) and (III). Reagents: (a) RCHO/NaBH(OAc)3/HOAc; (b) ClSO2N@C@O/t-BuOH/TEA; (c) R3OH/Ph3P/DEAD; (d)
LiBH4/THF; (e) (ClCO)2/DMSO/TEA; (f) TFA; (g) AlCl3/CH2Cl2; (h) p-TSA hydrate/CH2Cl2/THF/rt.
It was initially envisaged using the general sequence of
steps shown in Figure 2 for the synthesis of template
(I). During the course of synthetic studies leading to
(I), it was noted that aldehyde 5 could be readily trans-
formed into scaffold (II) or (III). We describe herein the
results of studies related to this unexpected rearrange-
ment and advance the notion that these observations
provide the framework for the design of two new poten-
tial classes of protease inhibitors.
410nm for 2min. A control (hydrolysis) containing
10lL of 7.0lL HLE, 930lL 0.1M HEPES buffer,
0.5M NaCl, pH7.25, and 50lL DMSO was run under
the same conditions. The remaining activity of HLE
was expressed as % remaining activity = (v/v0) · 100
and is the average of duplicate or triplicate
determinations.
2.1.2. Screening procedure/proteinase 3 (PR3). Ten
microliters of a 3.40lM solution of proteinase 3 in
0.1M phosphate buffer, pH6.50 were added to a cuvette
containing 930lL 0.1M HEPES buffer, pH7.25 con-
taining 0.5M NaCl, 10lL DMSO, and 10lL 1.70mM
inhibitor solution in DMSO (final inhibitor concentra-
tion 1.70lM at an [I]/[E] = 500). After the solution
was incubated for 0.5h at 25ꢁC, 20lL of 16mM 5,50-
dithio-bis(2-nitrobenzoic acid) in DMSO and 20lL of
6.49mM Boc-Ala-Ala-Nva-SBzl in DMSO were added
and the change in absorbance was monitored at
410nm for 2min. A control (hydrolysis) containing
930lL of 0.1M HEPES buffer/0.50M NaCl, pH7.25,
10lL PR 3, 20lL 5,50-dithio-bis(2-nitrobenzoic acid),
20lL Boc-Ala-Ala-Nva-SBzl, and 20lL DMSO was
also run under the same conditions. Determinations
were performed in duplicate or triplicate and remaining
activity was calculated as described for HLE.
2. Chemistry
Compounds 1–5 and 7–9 were synthesized as shown in
Figures 2 and 3.
2.1. Biochemical studies
2.1.1. Screening procedure/human leukocyte elastase
(HLE). In a typical inhibition experiment, 10lL of
7.0lM HLE was incubated with 10lL of a 3.50mM
solution of inhibitor in DMSO ([inhibitor]/[enzyme] =
500), 40lL DMSO and 930lL 0.1M HEPES buffer/
0.5M NaCl, pH7.25 at 25ꢁC. After 20min, 10lL of
70mM methoxysuccinyl Ala-Ala-Pro-Val p-nitroanilide
was added and the amount of active enzyme was deter-
mined by monitoring the release of p-nitroaniline at
P1
R2
CHO
N
OH
SO2NHR3
6
P1
R2
COOCH3
P1
R2
COOCH3
P1
R2
a
b
c
N
N
N
SO2NR3Boc
3
SO2NHR3
9
SO2NHR3
10
P1
N R3
N
(P1=isobutyl,R2=bzl,
S
R2
R3=2-phenethyl)
O
O
7(II)
Figure 3. Formation of compound 7/template (II). Reagents: (a) CF3COOH/CH2Cl2; (b) LiBH4/THF; (c) (ClCO)2/DMSO/TEA.

J. Zhong et al. / Bioorg. Med. Chem. 12 (2004) 6249–6254
6251
3. Results and discussion
3.1. Synthesis
We have recently disclosed the design and use of amino
acid-derived sulfamide derivatives as potential inhibitors
of serine proteases (structure (I), Fig. 1a).9,10 Structure
(I) constitutes a new and versatile class of transition
state inhibitors (Fig. 1b) of serine and cysteine proteases
capable of exploiting favorable binding interactions with
the S and S0 subsites of a target protease. The general
synthetic scheme devised for the synthesis of derivatives
of (I) with X = R4 = H, envisioned the initial synthesis
of key aldehyde 5, followed by deblocking (Fig. 2).
NMR analysis of the crude product formed by treating
compound 5 with TFA/CH2Cl2 (4:1)/2h showed the
absence of the desired aldehyde 6. Several other methods
for removing the Boc group were then explored, includ-
ing silica gel/microwave11 and ammonium cerium(IV)-
nitrate/acetonitrile,12 leading to complex mixtures of
products. Surprisingly, treatment of compound 5 with
Figure 4. ORTEP drawing of compound 8.
compounds 7 and 8 is shown in Figure 5 where acid-cata-
lyzed removal of the Boc group is followed by an
intramolecular addition and loss of water to give com-
pound 7/scaffold (II). Acid-catalyzed hydration of 7 fol-
lowed by ring opening ultimately leads to compound 8/
scaffold (III).
13
anhydrous AlCl3 yielded compound 7 in high yield
(structure (II), P1 = isobutyl, R2 = bzl, R3 = 2-phen-
ethyl). Swern oxidation of alcohol 10 also yielded com-
pound 7 in quantitative yield (Fig. 3). To our
knowledge, this the first report describing the synthesis
of the 2,5-dihydro-[1,2,5]thiadiazole-1,1 dioxide scaffold
(II). Thus, this novel template can be readily assembled
in two ways from the readily available aldehyde 5 or
alcohol 10.
3.2. Biochemistry
The observations cited above, taken together with recent
studies related to the design of new scaffolds, suggested
that core structures (II) and (III) can be used in the
development of protease inhibitors. Specifically, in ear-
lier studies the 1,2,5-thiadiazolidin-3-one-1,1-dioxide
template was used in the design of protease inhibi-
tors15–23 and, more recently, the same template was used
as a prototype for the design of protease inhibitors
based on surrogate scaffolds such as the 4-imidazolidi-
none24 and cyclic sulfamide25–27 scaffolds. The structure
of template (II) suggested that it can be used for the
same purpose (Fig. 6a). Indeed, incubation of a 250-fold
excess of compound 7 with human leukocyte elastase led
Treatment of aldehyde 5 with p-toluenesulfonic acid
monohydrate14 led to the exclusive formation of 8
(Fig. 2, structure (III), P1 = isobutyl, R2 = bzl, R3 = 2-
phenethyl). The structure of compound 8 was confirmed
by single crystal X-ray analysis (Fig. 4).
Treatment of compound 7 with p-toluenesulfonic acid
(p-TSA) monohydrate under the same conditions gave
compound 8, suggesting that compound 7 is an inter-
mediate in the reaction leading to the formation of 8
from 5. A plausible mechanism for the formation of
O
O
O
P1
H+
H
O
P1
R2
P1
H
O
H
O
N
H
R2
SO2
N
R3
O
N
N
SO2
N
O
R2
SO2
N
O H
+
R3
R3
H+
O
H
HO
H+
O
H
N R3
H
P1
P1
R2
-H2O
P1
R2
P1
R2
N R3
H
N R3
O
N
O
S
N
R2
N
H
N
S
O
S
+
SO2
N
R3
O
O
O
(II)
H
O
P1
P1
R3
N
N R3
O
N
S
O
O
SO2NHR2
R2
(III)
Figure 5. Postulated mechanism for the formation of scaffolds (II) and (III).

6252
J. Zhong et al. / Bioorg. Med. Chem. 12 (2004) 6249–6254
was removed and the crude product was purified by
flash chromatography (ethyl ether/hexane) to give com-
pound 1 (1.87g, 66% yield). H NMR (CDCl3): d 0.88
(dd, 6H), 1.47 (t, 2H), 1.70–1.85 (m, 2H), 3.30 (t, 1H),
3.61 (d, 1H), 3.71 (s, 3H), 3.82 (d, 1H), 7.19–7.20 (m,
5H).
(a)
S1
1
P1
R2
Sn'
N R3
O
N
S
O
Sn-S2
4.3. Compound 2
(b)
S1
A solution of N-chlorosulfonyl isocyanate (2.10g;
14.8mmol) in dry methylene chloride (25mL) was
cooled in an ice bath. A solution of t-butyl alcohol
(1.10g; 14.8mmol) in dry methylene chloride (10mL)
was added dropwise with stirring. After stirring for
15min at 0ꢁC, the resulting mixture was added dropwise
to a solution of compound 1 (3.27g; 14.8mmol) and tri-
ethylamine (1.50g; 14.8mmol) in dry methylene chloride
(30mL) kept in an ice bath. The ice bath was removed
and the reaction mixture was stirred for 2.5h. The reac-
tion mixture was then washed with water (2 · 25mL)
and the organic phase was separated and dried over
anhydrous sodium sulfate. The solvent was removed
and the crude product was purified by flash chromato-
graphy (ethyl ether/hexane) to give compound 2
O
O
S2'-Sn'
P1
S
R2
N
R3
N
H
O
S1'
Figure 6. Potential binding interactions of (II) and (III) with the S and
S0 subsites.
to weak inhibition (20% inhibition) of the enzyme. Com-
pound 7 was devoid of any inhibitory activity toward
human leukocyte proteinase 3. Structure–activity rela-
tionship (SAR) studies with template (II) aimed at
optimizing potency are currently in progress. Likewise,
template (III) is a potential class of transition state
inhibitors (Fig. 6b). The results of SAR studies with
template (III) will be reported in due course.28
1
(1.87g; 66% yield). H NMR (CDCl3): d 0.53 (d, 3H),
0.83 (d, 3H), 1.38–1.61 (m, 12H), 3.69 (s, 3H), 4.52 (d,
1H), 4.65 (t, 1H), 4.92 (d, 1H), 7.10–7.52 (m, 5H).
4.4. Compound 3
4. Experimental
4.1. General
A solution of compound 2 (41.45g; 100.0mmol) in dry
THF (260mL) was treated with triphenyl phosphine
(52.99g; 20.0mmol), followed by 2-phenyl ethanol
(12.20g; 100.0mmol) and diethyl azodicarboxylate
(31.2mL; 200.0mmol). The reaction mixture was stirred
for 3h at room temperature. Removal of the solvent
yielded a crude product, which was purified by flash
chromatography on silica gel (ethyl ether/hexane) to
give compound 3 (45.67g; 88% yield). 1H NMR
(CDCl3): d 0.61 (d, 3H), 0.89 (d, 3H), 1.40–1.59 (m,
12H), 2.88 (t, 2H), 3.55–3.64 (m, 5H), 4.57–4.68 (m,
2H), 4.91 (d, 1H), 7.18–7.50 (m, 10H).
Melting points were recorded on a Mel-Temp apparatus
1
and are uncorrected. The H and 13C NMR spectra of
the synthesized compounds were recorded on a Varian
XL-300 or XL-400 spectrometers. Human leukocyte ela-
stase and Boc-Ala-Ala-Nva-SBzl were purchased from
Elastin Products Co., Owensville, MO. Methoxysuccinyl
Ala-Ala-Pro-Val p-nitroanilide and 5,50-dithio-bis(2-
nitrobenzoic acid) were purchased from Sigma Chemi-
cals Co., St Louis, MO. Silica gel (230–450 mesh) used
for flash chromatography was purchased from Sorbent
Technologies (Atlanta, GA). Thin layer chromatogra-
phy was performed using Analtech silica gel plates.
The TLC plates were visualized using iodine vapor
and/or UV light. A Hewlett–Packard diode array UV/
vis spectrophotometer was used in the enzyme assays
and inhibition studies.
4.5. Compound 4
To a solution of compound 3 (45.63g; 87.98mmol) in
dry THF (170mL) was added dropwise a solution of
2M lithium borohydride in THF (44mL, 88mmol), fol-
lowed by the dropwise addition of absolute ethanol
(320mL). The reaction mixture was stirred for 28h at
room temperature and was then cooled in an ice bath
and neutralized dropwise with 5% aqueous HCl solution
to pH4 with stirring. The solvent was removed to near
dryness and water (200mL) was added. The resulting
mixture was extracted with ethyl acetate (3 · 300mL)
and the organic extracts were combined and dried over
anhydrous sodium sulfate. The solvent was removed
and the crude product was purified by flash chromato-
graphy (ethyl ether/hexane) to give compound 4
(40.12g; 93% yield). 1H NMR (CDCl3): d 0.87 (dd,
6H), 1.19 (m, 1H), 1.30 (m, 1H), 1.40 (s, 9H), 1.61 (m,
1H), 2.88 (t, 2H), 3.50–3.70 (m, 4H), 4.07 (m, 1H),
4.46 (dd, 2H), 7.15–7.48 (m, 10H).
4.2. Compound 1
To (DL) leucine methyl ester hydrochloride (2.18g;
12.0mmol) and benzaldehyde (1.52g; 14.3mmol) in
dry 1,2-dichloroethane (30mL) was added acetic acid
(0.96g; 16.0mmol), followed by sodium (triacet-
oxy)borohydride (3.55g; 16.8mmol). The reaction mix-
ture was stirred for 3.5h at room temperature and was
then neutralized dropwise with 10% aqueous NaOH
solution to pH9–10 with stirring. The organic phase
was separated and the aqueous phase was extracted with
ethyl ether (3 · 25mL). The combined organic phase
was dried over anhydrous sodium sulfate. The solvent

J. Zhong et al. / Bioorg. Med. Chem. 12 (2004) 6249–6254
6253
4.6. 1-Benzyl-1-[(1-formyl-3-methylbutyl)]-3-phenethyl-3-
(carbamic acid t-butyl ester)sulfamide 5
purified by flash chromatography (ethyl ether/hexane)
to give compound 7 (1.00g; 100% yield).
A solution of 2M oxalyl chloride in methylene chloride
(2.44mL; 4.88mmol) kept in an acetone–dry ice bath
was added dropwise a solution of dry DMSO (0.51mL;
3.25mmol) in dry methylene chloride (4mL) over a per-
iod of 10min. After the solution was stirred for 0.5h, the
reaction mixture was treated with a solution of com-
pound 4 (1.59g; 3.25mmol) in dry methylene chloride
(8mL). Stirring was continued for an additional 0.5h.
A solution of dry triethylamine (1.4mL; 10mmol) in
methylene chloride (4mL) was then added, the ace-
tone–dry ice bath was removed, and the reaction mix-
ture was stirred for 15min. Water (10mL) was added
and the organic phase was separated and washed with
10% aqueous KHSO4 (10mL), saturated aqueous NaH-
CO3 (10mL), and brine (10mL), and then dried over
anhydrous sodium sulfate. After the solvent was re-
moved, the resulting crude product was purified by flash
chromatography (ethyl ether/hexane) to give compound
5 (1.23g; 83% yield). 1H NMR (CDCl3): d 1.88 (dd, 6H),
1.42 (m, 10H), 1.65 (m, 2H), 2.95 (t, 2H), 3.76 (m, 2H),
4.24 (m, 1H), 4.50 (d, 1H), 4.75 (d, 1H), 7.18–7.41 (m,
10H), 9.36 (s, 1H).
4.8. 3-Benzyl-1-[(4-methyl-2-oxo-pentyl)]-1-phenethyl
sulfamide 8
(a) Compound 5 (0.72g; 1.47mmol) was added in a solu-
tion of p-toluenesulfonic acid monohydrate (3.80g;
19.68mmol) in dry THF (7.5mL) and dry methylene
chloride (4mL). The reaction mixture was stirred for
40h at room temperature. The solvent was removed to
near dryness and ethyl acetate (50mL) was added. The
resulting solution was washed with saturated aqueous
NaHCO3 (3 · 30mL), brine (15mL) and then dried over
anhydrous sodium sulfate. The solvent was removed
and the crude product was purified by flash chromato-
graphy (ethyl ether/hexane) to give compound 8 (0.40
1
g; 70% yield). H NMR (CDCl3): d 1.89 (d, 6H), 2.04
(m, 1H), 2.16 (d, 2H), 2.83 (t, 2H), 3.42 (t, 2H), 3.95
(s, 2H), 4.24 (d, 2H), 5.10 (t, 1H), 7.12–7.39 (m, 10H).
(b) Compound 7 (0.23g; 0.62mmol) was added in a
solution of p-toluenesulfonic acid monohydrate (1.61g;
8.30mmol) in dry THF (3.2mL) and dry methylene
chloride (1.7mL). The reaction mixture was stirred for
40h at room temperature. The solvent was removed to
near dryness and ethyl acetate (50mL) was added. The
resulting solution was washed with saturated aqueous
NaHCO3 (3 · 30mL), brine (15mL) and then dried over
anhydrous sodium sulfate. The solvent was removed to
give crude compound 8, which was purified by flash
chromatography (0.21g; 88% yield).
4.7. 2-Benzyl-3-isobutyl-5-phenethyl-2,5-dihydro-
[1,2.5]thiadiazole-1,1 dioxide 7 (II, P1 = isobutyl,
R2 = benzyl, R3 = 2-phenethyl)
(a) A solution of compound 5 (0.61g; 1.25mmol) in dry
methylene chloride (6mL) was kept in an ice bath.
Anhydrous AlCl3 (0.17g; 1.25mmol) was added in one
portion, the ice bath was removed, and the reaction
mixture was stirred for 3h at room temperature. The
solution was neutralized dropwise with 10% aqueous
NaHCO3 solution to pH8–9 while stirring, and the
resulting solution was extracted with ethyl acetate
(3 · 60mL). The organic extracts were washed with
brine (2 · 40mL) and dried over anhydrous sodium sul-
fate. Removal of the solvent left a crude product that
was purified using flash chromatography. 1H NMR
(CDCl3): d 0.79 (d, 6H), 1.55 (m, 1H), 1.84 (d, 2H),
3.01 (t, 2H), 3.55 (t, 2H), 4.98 (s, 2H), 5.47 (s, 1H),
7.19–7.38 (m, 10H).
4.9. Compound 9
A solution of compound 3 (1.90g; 3.66mmol) in dry
methylene chloride (2.5mL) was treated with trifluoro-
acetic acid (10mL). The reaction mixture was stirred
for 2h at room temperature and the solvent was re-
moved on a rotary evaporator to yield a crude product,
which was purified by flash chromatography (ethyl
ether/hexane) to give compound 9 (1.48g; 97% yield).
1H NMR (CDCl3): d 1.62 (d, 3H), 1.82 (d, 3H), 1.43–
1.63 (m, 3H), 3.79 (t, 2H), 4.25 (m, 2H), 4.43 (t, 1H),
4.57 (d, 1H), 7.15–7.42 (m, 10H).
(b) To a solution of 2M oxalyl chloride in methylene
chloride (1.92mL; 3.84mmol) kept in an acetone–dry
ice bath was added dropwise a solution of dry DMSO
(0.37mL; 5.12mmol) in dry methylene chloride (3mL)
over a period of 7min. After the solution was stirred
for 0.5h, the reaction mixture was treated with a solu-
tion of compound 10 (1.00g; 2.56mmol) in dry methyl-
ene chloride (6mL) and the reaction mixture was stirred
for an additional 0.5h. A solution of dry triethylamine
(1.1mL; 7.7mmol) in methylene chloride (3mL) was
added, the acetone–dry ice bath was removed, and the
reaction mixture was stirred for 15min. Water (8mL)
was added and the organic phase was separated and
washed with 10% aqueous KHSO4 (8mL), saturated
aqueous NaHCO3 (8mL), and brine (8mL). The solu-
tion was dried over anhydrous sodium sulfate and the
solvent removed to give a crude product, which was
4.10. Compound 10
A solution of compound 9 (1.75g; 4.53mmol) in dry
THF (7mL) was added dropwise a solution of 2M lith-
ium borohydride in THF (2.3mL, 4.53mmol), followed
by the dropwise addition of absolute ethanol (14mL).
The reaction mixture was stirred for 28h at room tem-
perature and was then cooled in an ice bath and neutral-
ized dropwise with 5% aqueous HCl solution to pH4
with stirring. The solvent was removed to near dryness
and water (15mL) was added. The resulting mixture
was extracted with ethyl acetate (3 · 30mL) and the
organic phase was combined and dried over anhydrous
sodium sulfate. The solvent was removed and the crude
product was purified by flash chromatography (ethyl
ether/hexane) to give compound 10 (1.57g; 89% yield).
1H NMR (CDCl3): d 1.68 (d, 3H), 1.84 (d, 3H), 1.09

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(m, 1H), 1.30 (m, 1H), 1.54 (m, 1H), 2.79 (t, 2H), 3.26 (t,
2H), 3.53 (dd, 1H), 3.62 (dd, 1H), 3.93 (m, 1H), 4.26 (d,
1H), 4.40 (d, 1H), 7.15–7.47 (m, 10H).
Acknowledgements
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Financial support of this work by the National Insti-
tutes of Health (HL57788 to W.C.G. and HL66543
and HL72903 to J.R.H.) and Supergen, Inc. is gratefully
acknowledged.
16. Groutas, W. C.; Kuang, R.; Venkataraman, R.; Epp, J. B.;
Ruan, S.; Prakash, O. Biochemistry 1997, 36, 4739–4750.
17. Groutas, W. C.; Kuang, R.; Epp, J. B.; Venkataraman, R.;
Truong, T. M.; Ruan, S. Bioorg. Med. Chem. 1998, 6, 661–
667.
18. Kuang, R.; Venkataraman, R.; Ruan, S.; Groutas, W. C.
Bioorg. Med. Chem. Lett. 1998, 8, 539–544.
References and notes
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23. Groutas, W. C.; Epp, J. B.; Kuang, R.; Ruan, S.; Chong,
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S1, S2, S3, . . . ,Sn and S01; S20 ; S30 ; . . . ; S0n correspond to the
enzyme subsites on either side of the scissile bond. Each
subsite accommodates a corresponding amino acid side
chain designated P1, P2, P3, . . . ,Pn and P01; P20 ; P30 ; . . . ; Pn0 of
the substrate (or inhibitor). S1 is the primary specificity
site, and P1–P01 is the scissile bond.
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