Synthesis and evaluation of esters and carbamates to identify critical functional groups for esterase-specific metabolism

Article abstract of DOI:10.1016/S0968-0896(03)00302-X

In an effort to develop novel prodrugs for viral directed enzyme prodrug therapy (VDEPT) approaches to chemotherapy, eleven esters and carbamates of o-nitrophenol, p-nitrophenol, and β-naphthol were synthesized and characterized as substrates for rabbit (rCE) and human liver (hCE1) carboxylesterases. All of the esters of o-, p-nitrophenols, and β-naphthols showed moderate hydrolysis by both rCE and hCE1. Esters of β-naphthols exhibited higher hydrolysis rates compared to esters of p-nitrophenols by rCE. Of the carbamates, 4-benzyl-piperazine-1-carboxylic acid 2-nitrophenol showed preferential hydrolysis by rCE compared to hCE1 with a V_max of 54.4 μmoles/min/mg, and a K_m value of 1071 μM. Substrate metabolism by a specific CE or inhibition of CEs by each compound depended on several factors, including the types of functional groups and linking moieties.

Full text of DOI:10.1016/S0968-0896(03)00302-X

Bioorganic & Medicinal Chemistry 11 (2003) 3237–3244
Synthesis and Evaluation of Esters and Carbamates to Identify
Critical Functional Groups for Esterase-specific Metabolism
Kyoung Jin P. Yoon,a Christopher L. Morton,a Philip M. Potter,a
Mary K. Danksa and Richard E. Leeb,*
aDepartment of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN, USA
bDepartment of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, USA
Received 27 December 2002; revised 18 April 2003; accepted 3 May 2003
Abstract—In an effort to develop novel prodrugs for viral directed enzyme prodrug therapy (VDEPT) approaches to chemo-
therapy, eleven esters and carbamates of o-nitrophenol, p-nitrophenol, and b-naphthol were synthesized and characterized as sub-
strates for rabbit (rCE) and human liver (hCE1) carboxylesterases. All of the esters of o-, p-nitrophenols, and b-naphthols showed
moderate hydrolysis by both rCE and hCE1. Esters of b-naphthols exhibited higher hydrolysis rates compared to esters of
p-nitrophenols by rCE. Of the carbamates, 4-benzyl-piperazine-1-carboxylic acid 2-nitrophenol showed preferential hydrolysis by
rCE compared to hCE1 with a Vmax of 54.4 mmoles/min/mg, and a Km value of 1071 mM. Substrate metabolism by a specific CE or
inhibition of CEs by each compound depended on several factors, including the types of functional groups and linking moieties.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
drug catalysis or they are not expressed at high levels in
tissues exposed to this prodrug. A rabbit liver CE (rCE)
is the most efficient enzyme thus far identified in the
activation of CPT-11.3,6 Human liver microsomal CE 1
(hCE1) is one of the least efficient enzymes in activating
this prodrug.6
Carboxylesterases (CEs) belong to a group of serine
esterases found in virtually all animals and mammalian
tissues.1 Mammalian CEs are usually associated with
metabolism of water-soluble drugs and xenobiotics.2À4
CPT-11, a water-soluble camptothecin analogue used
clinically for its anti-tumor activity, is activated by
esterases.
The ability of CE to metabolize CPT-11 depends on the
access of the drug to the catalytic site of the enzyme,
and this access is restricted by the size of the entrance to
gorge containing the active site.7 The entrance to the
gorge containing the active site of rCE is larger than
that of hCE1(3.2 and 2.9 A for rCE and hCE1, respec-
tively), and rCE is 100- to 1000-fold more efficient than
hCE1 at converting CPT-11 to SN-38. Therefore it
should be possible to synthesize drugs that have access
to the catalytic site of rCE, but not hCE1, for use in
VDEPT applications.
CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino] car-
bonyloxy camptothecin, irinotecan) is a carbamate pro-
drug that, when activated, inhibits topoisomerase I (Fig.
1). CPT-11 has a piperidino side chain at the C-10
position that is cleaved by CEs present in human liver
and intestine. The cleavage of this side chain by CEs
yields SN-38 (7-ethyl-10-hydroxycamptothecin), the
active form of this drug.5 However, CPT-11 is activated
very inefficiently by human CEs (hCEs). Depending on
the schedule of administration, only 0.1–5% of this
prodrug is converted to the active drug SN-38 in vivo,5
possibly because either human CEs are inefficient at
Viral directed enzyme prodrug therapy (VDEPT) is a type
of gene therapy that uses a viral vector to deliver a cDNA
encoding an enzyme that converts a nontoxic prodrug to a
cytotoxic drug.8,9 The principal advantage of VDEPT
compared to conventional chemotherapy is its potential to
be tumor-specific. Our laboratories recently characterized
a VDEPT system using adenovirus to deliver the cDNA
*Corresponding author. Tel.: +1-901-448-6018; fax: +1-901-448-
6828; e-mail: relee@utmem.edu
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00302-X

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K. J. P. Yoon et al. / Bioorg. Med. Chem. 11 (2003) 3237–3244
Figure 1. Structure and hydrolysis of CPT-11 by carboxylesterases.
encoding a rCE that efficiently activates CPT-11.10À13
We now intend to characterize functional groups and
linkages specific for activation by rCE, to design addi-
tional prodrugs for this VDEPT approach. Our long
range goal is to develop tumor-specific combination
chemotherapy.
Scheme 1. Synthetic scheme for carbamates. This specific synthesis
pictured used p-NP and 4-benzyl piperidine as starting materials. All
other carbamate-linked derivatives were prepared using this same
general scheme. Starting material (a) used included p-nitrophenol 1,
o-nitrophenol 3, and b-naphthol 4. The variety of secondary amines
(b) used for coupling included 4-benzyl piperidine, 1-benzyl piper-
azine, 1-(4-chlorobenzylhydryl) piperazine, and 1-(2-furosyl) piper-
azine. Reagents: (i) dichloromethane, 1 h, Ar (ii) pyridine, 12 h, Ar.
Toward achieving this goal, we determined the specifi-
city of substrates for rCE by using compounds of dif-
ferent chemical classes, and by varying the two main
structural features that influence the activity of mam-
malian CEs: the functional and linking groups of the
compounds.
linking groups that make general CE substrates selective
for rCE. The esters of two p-, and one o-NP (com-
pounds 11–13), and two b-NAP (compounds 14–15)
were synthesized and tested for their enzymatic activa-
tion by both rCE and hCE1. Table 1 shows the ability
of rCE and hCE1 to hydrolyze compounds 11–13. Vmax
values for these compounds ranged from 0.95 to 8.51
mmol/min/mg for hCE1, and from 1.21 to 12.61 mmol/
min/mg by rCE (Table 1). All of the NP benzoate
(11–13) were hydrolyzed slightly faster by rCE (1.3, 1.5,
and 1.5-fold for 11, 12, and 13, respectively) than by
hCE1. Of these esters, o-NP benzoate (13) had the
highest Vmax values and also showed the greatest differ-
ence (ꢀ2.7-fold) in Km (rCE>hCE1) for both enzymes.
However, the overall conclusion from these data was
that all three ester-linked nitrophenol derivatives were
readily activated by both rCE and hCE1, indicating that
a single ring structure containing varied ester-linked side
chains did not provide selectivity for rCE compared to
hCE1. Therefore, we next evaluated two member ring
structures, also having ester-linked side chains.
We synthesized a series of esters and carbamates con-
taining o- or p-nitrophenol or b-naphthol as the parent
moiety and tested them as substrates for hCE1 and rCE.
The purpose of this study was to determine the feasi-
bility of synthesizing compounds as CE substrates and
to identify critical residues that confer preferential acti-
vation by specific CEs. This was accomplished by (i) the
synthesis of compounds having a variety of secondary
amines coupled to different sizes of parent molecules,
coupled to side chains of various sizes by carbamate or
ester linkages, and (ii) kinetic studies of the synthesized
compounds with rCE and hCE1.
Results and Discussion
Synthesis of esters and carbamates of o- and p- nitro-
phenol (NP) and ꢀ-naphthol (NAP). A series of esters
of o- and p-NP, and b-NAP were prepared in a single step
reaction of o- or p-NP, or b-NAP with benzoyl chloride
(or trimethoxy benzoyl chloride) in triethylamine.14 The
synthesis of the carbamate-linked derivatives was
accomplished using triphosgene,15 with subsequent cou-
pling to various amines16 (Scheme 1). Secondary amines
were selected based on a previous study by Tsuji et al.
who showed that benzyl piperazine derivatives were pre-
ferentially activated by a rat serum CE.17
Hydrolysis of esters of ꢀ-naphthol by CEs. The benzoic
acid naphthalen-2-yl-ester (14), showed
a slightly
greater Vmax value for rCE compared to hCE1
(2.5-fold), indicating a slightly faster turnover rate by
rCE. This is consistent with the kcat/Km values of 34.1
for rCE and 26 for hCE1, and indicates a better sub-
strate efficiency of about 1.3-fold (Table 2). On the
other hand, 3,4,5-trimethoxy-benzoic acid naphthalen-
2-yl-ester (15), exhibited very similar Vmax values, and
Km values for both rCE and hCE1, again indicating
that, similar to compounds 11–13, an ester linkage did
not confer sufficient selectivity for rCE compared to
hCE1 to be considered for future prodrug development.
Therefore, since the dipiperidino side chain of CPT-11 is
Hydrolysis of esters of o- and p-nitrophenol (NP) by
CEs. One of our specific aims was to identify critical

K. J. P. Yoon et al. / Bioorg. Med. Chem. 11 (2003) 3237–3244
3239
Table 1. Kinetic parameters for the metabolism of three esters of nitrophenol by rCE and hCE1
b
c
Enzyme
Compda
Structure
Km
(mM)
Vmax
(mmol/min/mg)
Curve fit
(R2)
k
cat/Km
(minÀ1 mMÀ1
)
hCE1
11
12
13
11
12
13
184.5Æ51.0
0.95Æ0.09
0.9909
0.9652
0.9970
0.9945
0.9980
0.9994
11.1
11.9
187.8
9.4
311.9Æ238.8
98.1Æ12.9
1.71Æ0.37
8.51Æ0.24
1.21Æ0.23
2.55Æ0.41
12.61Æ0.32
rCE
431.8Æ168.0
509.9Æ154.8
272.6Æ22.4
16.7
154.2
aCompound synthesized.
b,cKm and Vmax values were presented as meanÆS.E.M. of triplicate experiments. Values were calculated from non-linear regression fit by using
GraphPad Prism.
Table 2. Kinetic parameters for the metabolism of two esters of b-naphthol by rCE and hCE1
a
b
Enzyme
Compd
Structure
Km
(mM)
Vmax
(mmol/min/mg)
Curve fit
(R2)
kcat/Km
(minÀ1 mMÀ1
)
hCE1
14
15
14
15
1200Æ485.5
14.44Æ2.18
0.9232
0.9121
0.8634
0.9980
26.0
918.9Æ738.7
3593Æ3345
1180Æ809
16.67Æ7.84
36.80Æ18.52
15.46Æ6.69
39.3
34.1
43.7
rCE
a,bKm, and Vmax values were presented as meanÆS.E.M. of triplicate experiments. Values were calculated from non-linear regression fit by using
GraphPad Prism.
linked to the camptothecin moiety by a carbamate link-
age, we next evaluted one- and two-ring structures con-
taining carbamate-linked side chains.
3 nM to 1.35 mM for rCE, and from 21 to 93 nM for
hCE1. Similar to results with single ring structures, data
with carbamate of b-NAP (10) exhibited inhibitory
activity for both CEs (Table 3). These data are con-
sistent with published data that carbamates are known
inhibitors of CEs.18,19
Inhibitory effect of carbamates of p-nitrophenol (NP)
and ꢀ-naphthol (NAP). None of the carbamates of
p-NP (5–8) or b-NAP (10) were metabolized by either
rCE or hCE1, but instead these compounds inhibited
the metabolism of the general CE substrate o-nitrophe-
nylacetate (NPA) by both enzymes. With the single-ring
p-NP derivatives, a 10 min pre-incubation of rCE or
hCE1 with carbamates of p-NP (5–8) resulted in a con-
centration-dependent inhibition of esterase activity as
measured by metabolism of the general esterase sub-
strate o-NPA. Table 3 shows the inhibitory potencies of
the carbamates synthesized. The 50% inhibitory con-
centrations (IC50) of these compounds ranged from
The observation that carbamates inhibited, but were not
metabolized by CEs, suggested several possibilities.
First, these compounds might have a high affinity for
the catalytic site, making them relatively irreversible
inhibitors regardless of whether the side chain is
cleaved. Second, these compounds may reversibly bind
to the active site forming a tetrahedral intermediate20
However, collapse of this transition state to cleave the
phenol could be energetically limiting. Third, the nitro
group on p-NP may interact with functional groups

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K. J. P. Yoon et al. / Bioorg. Med. Chem. 11 (2003) 3237–3244
Table 3. Inhibitory potencies of carbamates of p-nitrophenol and
b-naphthol
a
Compd
Structure
Enzyme
IC50
(mM)
Curve fitb
(R2)
5
hCE1
rCE
0.021
0.032
0.9666
0.9989
6
7
hCE1
rCE
0.089
0.200
0.9971
0.9947
hCE1
rCE
0.062
1.35
0.8778
0.9985
8
hCE1
rCE
0.093
0.003
0.9748
0.9967
10
hCE1
rCE
316.2
112.2
0.9961
0.9999
Figure 2. The effect of 4-benzyl-piperazine-1-carboxylic acid 2-nitro-
phenyl ester (9) hydrolysis by two different CEs (rCE and hCE1).
Control values for o-NP produced from
2 mM o-NPA were
171.89Æ5.56, and 46.33Æ1.67 mmoles/min/mg for rCE and hCE1,
a,bIC50 and R2 values were calculated from non-linear regression fit by
using GraphPad Prism.
respectively.
ꢀ40-fold higher than that for hCE1 (1.5Æ0.3 mmol/
min/mg). kcat/Km values were 169.2 and 68.8 min-
À1mMÀ1
for rCE and hCE1, respectively. Similar to
other ester derivatives, a lower Km value was observed
for hCE1 (46.3Æ55.7 mM) as compared to rCE
(1071Æ414.7 mM) with compound 9. The data show
that 4-benzyl-piperazine-1-carboxylic acid 2-nitro-
phenyl ester (9) demonstrated preferential hydrolysis by
rCE. Of note, the single difference between 6 (which
inhibited rCE) and 9 (which was efficiently metabolized
by rCE) is that the nitro group in phenol ring was
attached at the para- versus ortho- positions, respec-
tively, suggesting that the minor structural changes
greatly influence substrate-enzyme interaction. Collec-
tively, the above results indicate that the parent com-
pounds, linking moieties and leaving groups must be
optimized to produce compounds sufficiently selective
for a specific enzyme to be useful in VDEPT approa-
ches. Our data also indicate that it is possible to achieve
this enzyme selectivity. Specifically, the data suggest
that the design of prodrugs for use with rCE should
focus on molecules containing a carbamate-linked side
chain sufficiently large to inhibit substrate access to
human CEs compared to rCE, and that this side chain
should be in the ortho position. Further, the data sug-
gest that it may be possible to design selective inhibitors
of specific esterases by using carbamate linking moieties.
adjacent to the active site of CEs. This would increase
the energy of cleavage for the phenol ring from the
parent molecule. Fourth, carbamate derivatives might
be metabolized, but the parent compound or the moi-
eties generated by catalysis might bind to sites other
than the active site to alter the 3-dimensional structure
of the enzyme, thereby inhibiting the access of other
substrates to the catalytic gorge. Because CPT-11 con-
tains a carbamate-linked side chain and is metabolized
by CEs, we favor the fourth hypothesis. More specifi-
cally, as has been proposed for acetylcholinesterases,
CEs may have a ‘back door’ through which cleaved
moieties pass and bind transiently.21 In support of this
hypothesis, Bencharit et al.,21 showed that the structure
of rCE is consistent with the presence of a back door for
the 4-dipiperidino leaving group after hydrolysis of
CPT-11. After CPT-11 binds to the active site, hydro-
lysis occurs to produce SN-38 by cleaving the 4-dipi-
peridino moiety; this leaving group is then proposed to
pass through the back door to bind adjacent to the Asn
residue at position 389 in rCE. Overall, the data indicate
that it is essential to optimize both the functional and
leaving groups in designing CE prodrugs, to facilitate
rapid enzymatic turnover and therefore efficient pro-
drug activation.
Conclusion
Hydrolysis of 4-benzyl-piperazine-1-carboxylic acid
2-nitro-phenyl ester (9) by CEs. None of the synthesized
carbamates of p-NP was metabolized by rCE or hCE1.
However, the o-NP carbamate derivative, 4-benzyl-
piperazine-1-carboxylic acid 2-nitro-phenyl carbamate
(9), was preferentially hydrolyzed by rCE (Fig. 2). The
Vmax value for rCE was 54.4Æ10.4 mmol/min/mg,
The lack of tumor-specificity of anti-cancer drugs is a
significant drawback in conventional chemotherapy.
VDEPT is an approach to render anti-cancer drugs
more selective for tumor cells by expressing a prodrug-
activating enzyme specifically in tumor cells. Previously,
we reported isolation and characterization of a cDNA

K. J. P. Yoon et al. / Bioorg. Med. Chem. 11 (2003) 3237–3244
3241
encoding a rCE that efficiently converts the prodrug
CPT-11 to SN-38.3 Those studies indicated that rCE/
CPT-11 might be useful for VDEPT applications.6,11,22
mmol) and pyridine (0.1 mL, 1.25 mmol) were then
added to the mixture, and the reaction mixture was
stirred for 12 h at room temperature. Dichloromethane
was removed in vacuo and the residual reaction mixture
was extracted with ethyl acetate (3Â30 mL). The
organic layer was washed with water (2Â30 mL), and
dried over anhydrous sodium sulfate. The crude pro-
duct was purified by column chromatography using
petroleum ether/ethyl acetate (8/1) to give 5 as a white
solid (0.45 g, 37%). TLC Rf=0.6 (petroleum ether/ethyl
acetate, 8/1); mp 92–94 ꢁC; 1H NMR (300 MHz, CDCl3)
d 1.3 (q, 2H, J=12, 12 Hz, CH2CH), 1.78 (d, 2H, J=12
Hz, CH2CH), 1.8–1.9 (m, 1H, CH2CH), 2.6 (d, 2H,
J=7.5 Hz, CH2Ph), 2.83 (t, 1H, J=12 Hz, N-CH), 2.96
(t, 1H, J=12 Hz, N-CH), 4.25 (br s, 2H, N-CH2), 7.18
(d, 2H, J=7.5 Hz, Ph-H2), 7.24–7.35 (m, 3H, Ph-H3),
7.32 (d, 2H, J=9.4 Hz, ArH2), 8.25 (d, 2H, J=9.4 Hz,
ArH2); 13C NMR (125 MHz, CDCl3) 39.0, 42.6, 44.6,
122.2, 125.2, 126.2, 128.8, 129.4, 140.0, 144.8, 152.2,
156.8; MS (ESI) m/z 363.4 [M+Na]+.
In the current study, we synthesized additional potential
substrates for mammalian CEs and investigated their
specificity for catalytic hydrolysis by rCE. Since both
the functional and linking groups of the substrate
structures influence cleavage activity of mammalian
CEs, we modified these two features to identify com-
pounds selective for rCE. Our study showed that ester-
linked substrates showed little difference in hydrolysis
by rCE and hCE1, regardless of substrate size. Further,
all carbamates of p-NP inhibited both enzymes. How-
ever, a carbamate derivative of o-NP (9) was hydrolyzed
preferably by rCE. This information is being used to
design prodrugs that, when activated, have mechanisms
of cytotoxicity complementary to that of CPT-11, for
future VDEPT applications.
The following compounds (6–10) were prepared in a
similar manner as described above for compound 5.
Experimental
Synthesis
4-Benzyl piperazine-1-carboxylic acid 4-nitrophenyl ester
(6). The reaction of p-nitrophenol (0.8 g, 5.8 mmol),
pyridine (2.3 mL, 28.5 mmol) in dichloromethane (20
mL), triphosgene (2 g, 6.9 mmol) and 1-benzyl piper-
azine (2.5 g, 14.4 mmol) gave a crude product. This
crude mixture was purified by column chromatography
using petroleum ether/ethyl acetate (9/1) to give 6 as a
white solid (0.27 g, 14%). TLC Rf=0.4 (petroleum
ether/ethyl acetate, 9/1); mp 238–239 ꢁC; 1H NMR
(500 MHz, CDCl3) d 2.8 (br s, 2H, N-CH2), 3.5 (br s,
2H, CH2Ph), 3.95 (br s, 1H, N-CH), 4.18–4.38 (m, 5H,
2Â N-CH2, N-CH), 7.3 (d, 2H, J=8.3 Hz, ArH2),
7.48–7.52 (m, 3H, Ph-H3), 7.63–7.65 (m, 2H, Ph-H2),
8.3 (d, 2H, J=8.3 Hz, ArH2); 13C NMR (75 MHz,
CDCl3) d 49.87, 59.08, 122.17, 122.37, 124.70, 125.10,
128.57, 128.83, 129.36, 131.28, 144.48, 151.26, 155.59;
MS (ESI) m/z 342.3 [M+H]+.
Materials. All the anhydrous solvents and starting
materials were purchased from Aldrich Chemical Com-
pany (Wilwaukee, WI). All reagent grade solvents used
for chromatography were purchased from Fisher Scien-
tific (Suwanee, GA) and Flash column chromatography
silica cartridges (MetaFlash) were obtained from Ansys
Technologies Inc. (Lake Forest, CA).
Chemistry general
Triphosgene reaction was performed under a dry argon
atmosphere. The reactions were monitored by thin layer
chromatography (TLC) on pre-coated Merck 60 F254
silica gel plates and visualized using UV light (254 nm).
Biotage FLASH 25+ column chromatography system
was used to purify mixtures. Melting points were deter-
mined on a Thomas Hoover Capillary melting point
apparatus (Thomas Scientific, Swedesboro, NJ) and are
4-(Furan-2-carbonyl)-piperazine-1-carboxylic acid 4-
nitro-phenyl ester (7). The reaction of p-nitrophenol
(0.8 g, 5.8 mmol), pyridine (2.3 mL, 28.5 mmol) in di-
chloromethane (20 mL), triphosgene (2 g, 6.9 mmol)
and 1-(2-furosyl)-piperazine (2.5 g, 14.4 mmol) gave a
crude product. This crude product was recrystalized
with methanol to give 7 as off-white solid (0.98 g, 49%).
TLC Rf=0.25 (petroleum ether/ethyl acetate, 9/1); mp
1
uncorrected. All H and 13C NMR spectra were recor-
ded on a Bruker ARX-300 (300 and 75 MHz for 1H and
13C NMR, respectively) or Varian INOVA-500 (500
1
and 125 MHz for H and 13C NMR, respectively) spec-
trometers. Chemical shifts are reported in ppm (d) rela-
tive to residual solvent peak or internal standard
(tetramethylsilane) and coupling constants (J) are
reported in hertz (Hz). Mass spectra were recorded on a
Bruker Esquire LCMS using ESI.
157–159 ꢁC; H NMR (300 MHz, CDCl3) d 3.7 (d, 4H,
1
J=4 Hz, 2ÂN-CH2 ), 3.9 (s, 4H, 2ÂN-CH2), 6.54 (d,
1H, J=3.2 Hz, furosyl H), 7.12 (d, 1H, J=3.2 Hz, fur-
osyl H), 7.34 (d, 2H, J=8.3 Hz, ArH2), 7.5 (s, 1H, fur-
osyl H), 8.3 (d, 2H, J=8.3 Hz, ArH2); 13C NMR
(75 MHz, CDCl3) d 43.60, 44.17, 46.32, 111.01, 116.71,
116.93, 121.72, 124.54, 143.51, 144.46, 147.04, 151.62,
155.43, 158.64; MS (ESI) m/z 368.3 [M+Na]+.
General procedure for carbamates of nitrophenol and
naphthol
4-Benzyl-piperidine-1-carboxylic acid 4-nitrophenyl ester
(5). To a stirred solution of p-nitrophenol (0.5 g, 3.6
mmol), pyridine (1.45 mL, 18 mmol) in dichloro-
methane (20 mL), triphosgene (1.28 g, 4.3 mmol) was
added under argon atmosphere. After 1 h stirring at
room temperature, TLC was used to confirm that reac-
tions were complete. 4-benzyl piperidine (1.58 mL, 9
4-[(4-Chloro-phenyl)-phenyl-methyl]-piperazine-1-car-
boxylic acid 4-nitro-phenyl ester (8). The reaction of
p-nitrophenol (0.35 g, 2.5 mmol), pyridine (0.9 mL, 11.2
mmol) in dichloromethane (20 mL), triphosgene (0.8 g,

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K. J. P. Yoon et al. / Bioorg. Med. Chem. 11 (2003) 3237–3244
2.7 mmol) and 4-(chloro-benzhydryl)-piperazine (1.5 g,
5.2 mmol) gave a crude product. This crude product
was purified by column chromatography using petro-
leum ether/ethyl acetate (9/1) to give 8 as a white solid
(0.38 g, 33%). TLC Rf=0.6 (petroleum ether/ethyl ace-
tate, 9/1); mp 65–66 C; H NMR (300 MHz, CDCl3) d
2.5 (t, 4H, J=4 Hz, 2ÂN-CH2), 3.6 (br s, 2H, N-CH2),
3.7 (br s, 2H, N-CH2), 4.3 (s, 1H, CHAr2), 7.25–7.35
(m, 7H, ArH7), 7.36–7.42 (m, 2H, ArH2), 7.4 (d, 2H,
J=8.8 Hz, ArH2), 8.35 (d, 2H, J=8.8 Hz, ArH2); 13C
NMR (75 MHz, CD3OD) d 50.53, 50.72, 74.36, 121.72,
124.07, 125.10, 126.56, 127.06, 127.79, 127.85, 128.60,
131.96, 144.49, 152.00, 155.71; MS (ESI) m/z 474.3
[M+Na]+.
in vacuo. Excess dichloromethane was added, and the
mixture was washed with water (2Â30 mL) and dried
over sodium sulfate. After the solvent was removed in
vacuo, the mixture was subjected to column chromato-
graphy with petroleum ether/ethyl acetate (4/1) to give
11 as a white solid (0.3 g, 17%). TLC Rf=0.8 (petro-
leum ether/ethyl acetate, 4/1); mp 132–133 ꢁC; 1H NMR
(500 MHz, CDCl3) d 7.42 (d, 2H, J=9.1 Hz, PhH2),
7.55 (t, 2H, J=7.6 Hz, PhH2), 7.7 (t, 1H, J=7.6 Hz,
PhH), 8.2 (d, 2H, J=7.6 Hz, ArH2), 8.34 (d, 2H, J=7.5
Hz, ArH2); 13C NMR (75 MHz, CDCl3) d 122.09,
124.72, 128.05, 128.27, 129.79, 133.71, 144.89, 155.23,
163.68; MS (ESI) m/z 266.0 [M+Na]+.
ꢁ
1
The following compounds (12–15) were prepared in a
similar manner as described above for compopund 11.
4-Benzyl-piperazine-1-carboxylic acid 2-nitro-phenyl es-
ter (9). The reaction of o-nitrophenol (0.8 g, 5.8 mmol),
pyridine (2.3 mL, 28.5 mmol) in dichloromethane (20
mL), triphosgene (2 g, 6.9 mmol) and 1-benzyl piper-
azine (2.5 g, 14.4 mmol) gave a crude product. This
crude product was purified by column chromatography
using petroleum ether/ethyl acetate (9/1) to give 9 as a
white solid (0.29 g, 15%). TLC Rf=0.3 (petroleum
ether/ethyl acetate, 9/1); mp 206–208 ꢁC; 1H NMR
(500 MHz, CDCl3) d 2.9 (q, 1H, J=11.3, 11.3 Hz,
N-CH), 3.05 (q, 1H, J=11.3, 11.3 Hz, N-CH), 3.4 (t,
2H, J=11.3 Hz, CH2Ph), 3.9 (t, 1H, J=12.5 Hz,
N-CH), 4.15 (t, 1H, J=12.5 Hz, N-CH), 4.15–4.4 (m,
4H, 2ÂN-CH2), 7.31, 7.54 (dd, 1H, J=2.2, 8.7 Hz,
ArH), 7.43, 7.5 (td, 1H, J=2.2, 6.5 Hz, ArH), 7.46–7.5
(m, 3H, PhH3), 7.7, 7.75 (td, 1H, J=2.2, 6.5 Hz,
ArH), 7.69–7.73 (m, 2H, PhH2), 8.14, 8.18 (dd, 1H,
J=2.2, 8.7 Hz, ArH); 13C NMR (75 MHz, CDCl3) d
50.46, 60.03, 60.45, 124.31, 124.86, 125.33, 125.50,
126.30, 127.00, 127.26, 128.83, 129.87, 130.83, 134.80,
134.97, 140.77, 143.22, 143.76, 151.45; MS (ESI) m/z
342.3 [M+H]+.
3,4,5-Trimethoxy-benzoic acid 4-nitro-phenyl ester (12).
The reaction of p-nitrophenol (1 g, 7.2 mmol), triethyl-
amine (1 mL, 7.2 mmol) in dichloromethane (30 mL)
and 3,4,5-trimethoxy benzoyl chloride (5 g, 21.6 mmol)
gave a crude product. This crude product was purified
by column chromatography using petroleum ether/ethyl
acetate (9/1) to give 12 as a white solid (0.31 g, 13%).
TLC Rf=0.4 (petroleum ether/ethyl acetate, 9/1); mp
ꢁ
1
164–165 C; H NMR (500 MHz, CDCl3) d 4.0 (s, 9H,
3ÂOCH3), 7.42 (dd, 2H, J=2.6, 6.6 Hz, ArH2), 7.46 (s,
2H, ArH2), 8.36 (dd, 2H, J=2.6, 6.6 Hz, ArH2); 13C
NMR (75 MHz, CDCl3) d55.85, 60.20, 107.19, 122.51,
122.82, 124.71, 142.77, 144.82, 152.63, 155.34, 163.17;
MS (ESI) m/z 356.1 [M+Na]+.
Benzoic acid 2-nitro-phenyl ester (13). The reaction of o-
nitrophenol (1 g, 7.2 mmol), triethylamine (1 mL, 7.2
mmol) in dichloromethane (30 mL) and benzoyl chlo-
ride (3 g, 21.3 mmol) gave a crude product. This crude
product was purified by column chromatography using
petroleum ether/ethyl acetate (9/1) to give 13 as a off-
white solid (0.19 g, 11%). TLC Rf=0.4 (petroleum
ether/ethyl acetate, 4/1); mp 54–56 ꢁC; 1H NMR
(500 MHz, CDCl3) d 7.44–7.62 (m, 5H, ArH5), 7.72–
7.82 (m, 2H, ArH2), 8.18–8.32 (m, 2H, ArH2); 13C
NMR (75 MHz, CDCl3) d 124.86, 125.29, 126.08,
127.95, 128.17, 128.76, 129.67, 129.99, 133.25, 133.59,
134.08, 143.84, 163.83, 171.26; MS (ESI) m/z 266.0
[M+Na]+.
4-Benzyl-piperazine-1-carboxylic acid naphthalene-2-yl
ester (10). The reaction of b-naphthol (0.15 g, 1.0
mmol), pyridine (0.5 mL, 6.2 mmol) in dichloromethane
(20 mL), triphosgene (0.62 g, 2 mmol) and 1-benzyl
piperazine (0.47 g, 2.6 mmol) gave a crude product. This
crude mixture was purified by column chromatography
using petroleum ether/ethyl acetate (4/1) to give 10 as a
white powder (0.065 g, 18%). TLC Rf=0.25 (petroleum
1
ether/ethyl acetate, 4/1); H NMR (500 MHz, CDCl3) d
2.5 (t, 4H, J=4.2 Hz, 2ÂN-CH2), 3.6 (s, 2H, CH2Ph),
3.62 (s, 2H, N-CH2), 3.72 (s, 2H, N-CH2), 7.25–7.36 (m,
5H, ArH5), 7.42–7.52 (m, 2H, ArH2) 7.58 (d, 1H, J=2.7
Hz, ArH), 7.78–7.88 (m, 4H, ArH4); MS (ESI) m/z
347.3 [M+H]+.
Benzoic acid naphthalen-2-yl-ester (14). The reaction of
b-naphthol (1 g, 6.9 mmol), triethylamine (1 mL, 7.2
mmol) in dichloromethane (30 mL) and benzoyl chlo-
ride (3 g, 21.3 mmol) gave a crude product. This crude
product was purified by column chromatography using
petroleum ether/ethyl acetate (4/1) to give 14 as a white
solid (0.8 g, 47%). TLC Rf=0.6 (petroleum ether/ethyl
acetate, 4/1); mp 95–96 ꢁC; 1H NMR (500 MHz, CDCl3)
d 7.43 (dd, 1H, J=2.3, 8 Hz, ArH), 7.54–7.63 (m, 4H,
ArH4), 7.72 (d, 1H, J=6.8 Hz, ArH), 7.76 (d, 1H, J=8
Hz, ArH), 7.9 (d, 1H, J=6.8 Hz, ArH), 7.94 (d, 1H,
J=6.8 Hz, ArH), 7.98 (d, 1H, J=8 Hz, ArH), 8.32 (d,
2H, J=6.8 Hz, ArH2); 13C NMR (75 MHz, DMSO-d6)
d 118.62, 121.54, 125.80, 126.64, 127.46, 127.66, 128.90,
129.33, 129.76, 130.27, 131.05, 133.33, 133.98, 134.99,
148.28, 164.74; MS (ESI) m/z 271.1 [M+Na]+.
General procedure for carboxylic esters of nitrophenol
and naphthol
Benzoic acid 4-nitro-phenyl ester (11). Triethylamine (1
mL, 7.2 mmol) was added to a solution of p-nitrophenol
(1 g, 7.2 mmol) in dichloromethane (30 mL). Benzoyl
chloride (3 g, 21.3 mmol) was added slowly to the mix-
ture in an ice bath. The ice bath was then removed, and
the reaction mixture was stirred overnight at room
temperature. The reaction solvent was then evaporated

K. J. P. Yoon et al. / Bioorg. Med. Chem. 11 (2003) 3237–3244
3243
3,4,5-Trimethoxy-benzoic acid naphthalen-2-yl-ester
(15). The reaction of b-naphthol (1 g, 6.9 mmol), trie-
thylamine (1 mL, 7.2 mmol) in dichloromethane (30
mL) and 3,4,5-trimethoxy benzoyl chloride (4.5 g, 19.5
mmol) gave a crude product. This crude product was
column chromatographed with petroleum ether/ethyl
acetate (9/1) to give 15 as a white solid (1.1 g, 46%).
TLC Rf=0.4 (petroleum ether/ethyl acetate, 9/1); mp
amount of b-NAP produced was determined from a
b-NAP standard curve (r2=0.975). Enzyme kinetics
were determined using methods modified from those of
Huang et al.26 Initial velocities were recorded as OD/
min/mg purified enzyme, and corrected for background
(nonenzymatic, time-dependent) hydrolysis. R2 values
were obtained from these curve fits.
114–116 ꢁC; H NMR (500 MHz, CDCl3) d 4.0 (s, 9H,
Enzymatic inhibition of CEs, by derivatives of p-nitro-
phenol or ꢀ-naphthol. Reaction mixtures (300 mL) con-
tained CEs (rCE or hCE1), 2 mM o-NPA, and a range
(3.1–1000 nM and 78–2500 mM for p-NP and b-NAP
derivatives, respectively) of concentrations of synthe-
sized compounds in 50 mM HEPES buffer at pH 7.4,
1
3ÂOCH3), 7.38 (dd, 1H, J=2.3, 8.6 Hz, ArH), 7.5–7.58
(m, 4H, ArH4), 7.7 (d, 1H, J=2.3 Hz, ArH), 7.86 (d,
1H, J=8.6 Hz, ArH), 7.91 (d, 1H, J=8.6 Hz, ArH ),
7.94 (d, 1H, J=8.6 Hz, ArH); 13C NMR (75 MHz,
CDCl3) d 55.82, 60.45, 107.03, 118.21, 120.76, 123.91,
125.24, 126.09, 127.14, 127.30, 128.95, 131.02, 133.33,
142.45, 148.15, 152.60, 164.47; MS (ESI) m/z 361.2
[M+Na]+.
25 ꢁC.
Absorbance
was
monitored
spectro-
photometrially for 0.5–10 min in 96-well flat bottom
clear polystyrene cell culture plates (Corning Costar) at
420 nm using a spectrophotometer (Dynex Technolo-
gies, VA), as described above. Enzyme activity was
expressed as percent of controls containing no ‘inhi-
bitor’. IC50 values (inhibition concentration at 50%)
were obtained by nonlinear regression fit analysis using
GraphPad Prism software. R2 values were obtained
from these curve fits.
Enzymes and assays
Purified carboxylesterases. Pure rCE and hCE1 were
prepared from baculovirus-infected cell culture media as
described previously.23
Hydrolysis of esters of nitrophenol (NP) by CEs. The
hydrolysis of esters of NP was determined spectro-
photometrically in 96-well plates (Dynex Technologies,
Chantilly, VA) as described previously.24 The incu-
bation mixture contained various concentrations (0.05–
2 mM) of substrate and 3 mg/mL of CE in 220 mL of 50
mM HEPES buffer, pH 7.4. The time-dependent libera-
tion of p- or o-NP was monitored for 0.5 to 10 min by
the change in A420. Data were transferred to a compu-
ter, and the CE activity was calculated using a Micro-
soft Excel spreadsheet.
Acknowledgements
This work was supported in part by NIH grant
CA76202, CA79763, P30 CA21765, and by the Amer-
ican Lebanese Syrian Associated Charities (PMP,
MKD, CLM, KJPY) and the College of Pharmacy,
University of Tennessee Health Science Center (REL
and KJPY).
Km, Vmax, and kcat values were determined using sub-
strate concentrations of 0.005, 0.05, 0.5, 5, 50, 250, 500,
and 1,000 mM. Triplicate experiments were done, and in
each experiment all data points were performed in tri-
plicate on the same plate with a matching set of blank
CE-free controls and also positive controls (o-NPA)
with/without CEs. Values were determined from hyper-
bolic plots of reaction velocities versus substrate con-
centrations using the Prism software (GraphPad, San
Diego, CA).7 R2 values were obtained from these curve
fits.
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