Cyclopentane-1,3-dione: A novel isostere for the carboxylic acid functional group. Application to the design of potent thromboxane (A2) receptor antagonists

Article abstract of DOI:10.1021/jm200980u

Cyclopentane-1,3-diones are known to exhibit pK_a values typically in the range of carboxylic acids. To explore the potential of the cyclopentane-1,3-dione unit as a carboxylic acid isostere, the physical-chemical properties of representative co

Full text of DOI:10.1021/jm200980u

ARTICLE
pubs.acs.org/jmc
Cyclopentane-1,3-dione: A Novel Isostere for the Carboxylic Acid
Functional Group. Application to the Design of Potent Thromboxane
(A2) Receptor Antagonists
Carlo Ballatore,*,†,‡ James H. Soper,‡ Francesco Piscitelli,† Michael James,‡ Longchuan Huang,† Onur Atasoylu,†
Donna M. Huryn,† John Q. Trojanowski,‡ Virginia M.-Y. Lee,‡ Kurt R. Brunden,‡ and Amos B. Smith, III†
†Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, 231 South 34th Street, Philadelphia,
Pennsylvania 19104-6323, United States
‡Center for Neurodegenerative Diseases Research, Department of Pathology and Laboratory Medicine, Institute on Aging,
University of Pennsylvania, 3600 Spruce Street, Philadelphia, Pennsylvania 19104-6323, United States
S Supporting Information
b
ABSTRACT: Cyclopentane-1,3-diones are known to exhibit pKa values
typically in the range of carboxylic acids. To explore the potential of the
cyclopentane-1,3-dione unit as a carboxylic acid isostere, the physicalꢀ
chemical properties of representative congeners were examined and compared
with similar derivatives bearing carboxylic acid or tetrazole residues. These
studies suggest that cyclopentane-1,3-diones may effectively substitute
for the carboxylic acid functional group. To demonstrate the use of the cyclo-
pentane-1,3-dione isostere in drug design, derivatives of a known thromboxane
A2 prostanoid (TP) receptor antagonist, 3-(3-(2-(4-chlorophenylsulfonami-
do)ethyl)phenyl)propanoic acid (12), were synthesized and evaluated in both
functional and radioligand-binding assays. A series of mono- and disubstituted
cyclopentane-1,3-dione derivatives (41ꢀ45) were identified that exhibit
nanomolar IC50 and Kd values similar to 12. Collectively, these studies
demonstrate that the cyclopentane-1,3-dione moiety comprises a novel isostere of the carboxylic acid functional group. Given
the combination of the relatively strong acidity, tunable lipophilicity, and versatility of the structure, the cyclopentane-1,3-dione
moiety may constitute a valuable addition to the palette of carboxylic acid isosteres.
’ INTRODUCTION
required for evaluation. Among the most commonly employed
isosteres of the carboxylic acid group, 1H-tetrazoles and 3-hydro-
xycyclobutene-1,2-diones (Figure 1A) proved to be useful in a
number of cases.2,3 Surprisingly, however, cyclopentanepolyones
have not been evaluated as carboxylic acid surrogates, although
the acidic properties of such compounds have been known for
more than 50 years.4 For example, cyclopentane-1,3-dione
(CPD) and the 2-methyl congener (1 and 2, Figure 1) have
been reported to have pKa values of 4.4 and 4.7, respectively.4
Equally important, nuclear magnetic resonance (NMR),5 infra-
red (IR) absorption,5 and X-ray crystal structure6,7 studies
demonstrate that such compounds exist almost exclusively as
fast exchanging enolꢀketone tautomers (see Figure 1B), which,
like carboxylic acids, can establish “head-to-tail” intermolecular
hydrogen bonds. In light of these interesting properties, we
reasoned that CPDs may hold considerable promise as non-
classical isosteres of the carboxylic acid moiety.
Replacement of a specific atom, or group of atoms, with
surrogates that exhibit similar physicochemical properties com-
prises a strategy known as isosteric replacement, adopted broadly
by the medicinal chemistry community to improve the properties
of a wide variety of biologically active compounds, such as
potency, selectivity, metabolic/chemical stability, and ADME-
PK properties.1,2 A typical example of such a strategy is the
replacement of a carboxylic acid moiety with a surrogate struc-
ture. Indeed, the presence of a carboxylic acid residue in the
structure of a drug or drug candidate is often responsible for
significant drawbacks, including limited permeability, toxicity,
and metabolic instability.2 At the same time, however, the acidity
of the carboxylic acid residue may be required for biological
activity; this is often the case when the carboxylic acid group is
directly involved in relatively strong ionic interactions with the
biological target (e.g., salt bridge within the active site of an
enzyme). Under such circumstances, replacement of the car-
boxylic acid moiety with a surrogate structure may be beneficial.
The outcome of any isosteric replacement, however, cannot be
readily predicted, and thus, multiple isosteres are typically
Received: July 22, 2011
Published: August 24, 2011
r
2011 American Chemical Society
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Figure 1. Selected examples of known (A) and proposed (B) isosteres of the carboxylic acid moiety.
Scheme 1a
To test this hypothesis, we designed, synthesized, and eval-
uated a focused set of model compounds to compare the physical
chemical properties of similar structures bearing a carboxylic
acid, tetrazole, or CPD residue. Electrospray ionization (ESI)
mass spectrometry (MS), 1H NMR, and computational techni-
ques were employed to evaluate the ability of the congeners to
form salts with benzamidine. To test the potential of CPD
isosteres in drug design, we also designed, synthesized, and
compared the biological properties of a series of CPD derivatives
based on a representative thromboxane A2 prostanoid (TP)
receptor antagonist (12, Figure 2)8 to the corresponding analo-
gues bearing other carboxylic acid isosteres such as the tetrazole
and amino squaric acid.
a Reagents and reaction conditions: (a) (i) lithium diisopropylamide,
ꢀ78 °C; (ii) 4-bromobenzyl bromide, ꢀ78 °C to room temp over 1 h;
(b) 2 N hydrochloric acid, acetone; (c) 4-bromobezaldehyde, Hantzsch
ester, L-proline, dichloromethane, room temp, 2 h.
alkylation at the C5 position, to obtain monoalkylated products
5 and 6 as racemic mixtures. Hydrolysis of the enolꢀether under
acidic conditions then furnished 7 and 8. For the synthesis of the
2-substituted CPD 9, we employed the reductive alkylation
protocol reported by Ramachary and Kishor;10 specifically,
4-bromobenzaldehyde was reacted with CPD in the presence
of diethyl 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate
(Hantzsch ester) to furnish 9. Construction of tetrazole 11 was
achieved as previously reported,11 while the 4-bromophenylpro-
pionic acid 10 was purchased.
Figure 2. Structure of known TP-receptor antagonist 12 (top) and
overlap of the carboxylic acid moiety of 12 with CPD derivatives
attached at either C2 or C4/C5 (bottom, A or B, respectively).
’ DESIGN AND SYNTHESIS OF MODEL COMPOUNDS
’ DESIGN AND SYNTHESIS OF CPD-CONTAINING
TP-RECEPTOR ANTAGONISTS
The CPD fragment presents two nonequivalent points of
attachment (i.e., the C2 and one of the prochiral C4/C5
positions, Figure 1B) for analogue development. Thus, to
evaluate the physicalꢀchemical properties of CPDs relative to
carboxylic acids and tetrazoles, representative model 2- and
5-monosubsituted (9 and 7, respectively, Scheme 1) and 2,5-
disubstituted (8, Scheme 1) CPD derivatives were designed and
synthesized. In each case, a 4-bromobenzyl moiety was used as a
substituent to ensure appropriate solubility in organic solvents, a
prerequisite to enable ESI-MS studies (vide infra).
Compounds 7 and 8 were prepared by reacting the appro-
priate lithiated 3-isobutoxycyclopent-2-enone (i.e., 3 or 4) with
4-bromobenzyl bromide, under the reaction conditions devel-
oped by Koreeda and co-workers9 to achieve regioselective
With respect to TP-receptor antagonist design, depending on
the point of attachment on the CPD moiety and the length of the
aliphatic spacer, two alternative structures were envisioned,
which exhibit a promising degree of overlap with the carboxylic
acid moiety of the parent compound 12 (Figure 2). We therefore
synthesized a series of both 2- and 5-substituted CPDs, as well as
different 2,5-disubstituted CPD derivatives (Scheme 2). Tetra-
zole and amino squarate derivatives of 12 (respectively 61 and
62, Table 2) were also constructed for comparison.
The synthesis of the 5-substituted CPD derivatives 41 and 2,5-
disubstituted congeners 42ꢀ45 (Scheme 2) employed similar
strategies as employed for 7 and 8. In all cases, alkylation of the
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Scheme 2a
a Reagents and reaction conditions: (a) (i-Pr)3Si-Cl, imidazole, N,N-dimethylformamide, 0 °C, 3 h; (b) NaBH4, H2O, tetrahydrofuran, 70 °C, 2 h; (c)
PPh3, I2, imidazole, Et2O, acetonitrile, 0 °C, 2 h; (d) appropriate isobutyl-protected cyclopentane-1,3-dione, lithium diisopropylamide, tetrahydrofuran,
from ꢀ78 °C to room temp; (e) tetra-n-butylammonium fluoride, tetrahydrofuran, 0 °C, 3 h; (f) NaN3, N,N-dimethylformamide, 50 °C, 45 min; (g)
PPh3, diethyl azodicarboxylate, tert-butyl (4-chlorophenyl)sulfonylcarbamate, tetrahydrofuran, room temp, 4 h; (h) (i) H2, PdꢀC, methanol, room
temp, 16 h, (ii) 4-chlorobenzenesulfonyl chloride, 2 N NaOH, 0 °C, 3 h; (i) 2 N HCl, acetone, room temp, 6ꢀ12 h; (j) 2,2,2-trifluoroacetic acid,
dichloromethane, room temp, 2ꢀ5 h.
CPD was achieved by reacting the appropriate lithiated 3-iso-
butoxycyclopent-2-enone9 (i.e., 3, 4, 17ꢀ19) with benzyl iodide
16, obtained in three steps from aldehyde 13, to furnish 20ꢀ24
(Scheme 2). Removal of the silyl-protecting group then furn-
ished alcohols 25ꢀ29. For installation of the phenylsulfonamide
moiety, we initially employed a reaction sequence consisting of
(a) conversion of alcohol 25 to the corresponding alkyl iodide
30, (b) displacement of the iodide with sodium azide (31), and
(c) reduction of the latter to the corresponding amine, followed
by in situ sulfonylation of the amine with 4-chlorobenzenesulfo-
nyl chloride to yield 32 (Scheme 2). Similar overall transforma-
tions could be achieved in comparatively fewer steps and in
higher overall yield by treating alcohols 26ꢀ29 with N-Boc-
protected phenylsulfonylamide under Mitsunobu conditions12
to furnish 33ꢀ36. Finally, treatment of 32 with 2 N hydrochloric
acid in acetone provided the desired compound 41, whereas
33ꢀ36 were subjected to stepwise deprotection of the Boc
carbamate and enolꢀether, respectively, to furnish 37ꢀ40 and
42ꢀ45 (Scheme 2).
aldehydes (14, 54) were then reacted with CPD under the
previously described reductive alkylation conditions10 to furnish
the 2-alkylated CPD derivatives 46 and 55. These intermediates
were then directly converted to alcohols 47 and 56 by treatment
with isobutanol/benzene in the presence of a catalytic amount of
p-toluenesulfonic acid at reflux. Installation of the N-Boc-pro-
tected 4-chlorophenylsulfonamide moiety via Mitsunobu
reaction12 and removal of Boc and isobutyl ether protecting
groups then furnished the desired 2-substituted CPD derivatives
50 and 59.
Syntheses of carboxylic acid 12 (Figure 2), the corresponding
alcohol 60, tetrazole 61, and amino squaric acid 62 (Table 1) are
detailed in the Supporting Information.
’ PHYSICALꢀCHEMICAL PROPERTY EVALUATION
The lipophilicity and/or acidity of compounds 1, 2, 7ꢀ11 was
determined experimentally, followed by an examination of their
ability to form salts with benzamidine, the latter investigated by
ESI-MS and NMR. Finally, computational studies were carried
out to estimate the structure and stability of the complexes of
CPD derivatives with benzamidine.
Construction of the 2-substituted CPD derivatives, 50 and 59
(Scheme 3), began with aldehydes 14 and 54, respectively. Silyl-
protected aldehyde 54 was obtained from diacid 51 via lithium
aluminun hydride reduction, followed in turn by protection as
silyl ether of one of the hydroxyl moieties (53) and pyridinium
dichlorochromate mediated oxidation of the free hydroxyl. The
Determination of pKa and log P. pKa values of compounds 1,
2, 7ꢀ11, as well as the log P and log DpH 7.4 values of compounds
7ꢀ11, were determined experimentally by Sirius Analytical
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Scheme 3a
a Reagents and reaction conditions: (a) CPD (1), Hantzsch ester, L-proline, dichloromethane, room temp, 12 h; (b) p-toluenesulfonic acid (cat.),
isobutanol/benzene, reflux, 16 h; (c) tert-butyl (4-chlorophenyl)sulfonylcarbamate, PPh3, diethyl azodicarboxylate, tetrahydrofuran, room temp, 4 h;
(d) 2,2,2-trifluoroacetic acid, dichloromethane, room temp, 2 h; (e) 2 N hydrochloric acid, acetone, room temp, 12 h; (f) LiAlH4, diethyl ether, ꢀ78 °C,
3 h; (g) (i-Pr)3Si-Cl, imidazole, N,N-dimethylformamide, 0 °C, 3 h; (h) pyridinium chlorochromate, dichloromethane, room temp, 2 h.
(UK) via UV-metric or potentiometric (pH-metric) methods.
The results are summarized in Table 1 (see Supporting Informa-
tion for full reports).
the mass of the [CPD benzamidine] complex. As illustrated in
3
Figure 3, in all cases the test compound benzamidine ion+ was
3
readily detectable by ESI-MS.
Competition-binding studies were conducted by comparing
Evaluation of the Ability of Model CPD Derivatives to
Form Salts with Benzamidine. The structure and stability of the
amidine complexes with carboxylate and tetrazolate anions have
been investigated with a combination of ESI-MS and NMR
techniques,13,14 suggesting that this experimental model could be
useful to evaluate and compare carboxylic acid isosteres. Thus,
we employed similar strategies to evaluate the ability of CPDs
7ꢀ9 to form salts with amidines. For ESI-MS studies, a 10 μM
solution of each test compound in acetonitrile was mixed with an
equal volume of a 10 μM solution of benzamidine in acetonitrile.
The solubility of 7ꢀ11 in acetonitrile had been predetermined,
and each of the compounds was found to be fully soluble at
10 μM (see Supporting Information). After a 5 min incubation
period, the resulting mixtures were analyzed by ESI-MS to detect
the intensity of the signal corresponding to the [8 benzamidine]
3
complex with and without coaddition of a competing acid/acid
surrogate (i.e., 7, 9ꢀ11; see Figure 4). In these experiments,
benzamidine, compound 8, and the competing acid/acid surro-
gate were mixed in a 1:1:1 ratio in acetonitrile and after a 5 min
incubation time, the mixtures were analyzed by ESI-MS to
monitor for the presence of the [8 benzamidine] complex.
3
The relative intensity of the [8 benzamidine] complex in the
3
presence or absence of competing acid/acid surrogate provided
qualitative information on the binding affinity of CPD derivatives
for amidines, compared to acid 10 or tetrazole 11 (Figure 4). As
shown in Figure 4, co-incubation of equimolar amounts of
benzamidine, 8, and 9 resulted in a ∼50% reduction in the peak
intensity assigned to the [8 benzamidine] complex, while ana-
3
a
logous co-incubations of benzamidine and 8 with 7, 10, or 11
Table 1. pKa, log P, and log DpH 7.4
resulted in a comparatively smaller reduction (i.e., <50%) of the
compd
pKa
log P
NDc
NDc
log DpH 7.4
[8 benzamidine] signal. Thus, under the experimental condi-
3
tions employed in the competition studies, all CPD derivatives
appeared to have greater affinity for benzamidine than either the
corresponding carboxylic acid (10) or tetrazole (11) compound
(Figure 4).
1
4.20 ( 0.01 (4.4)b
4.47 ( 0.01 (4.7)b
3.96 ( 0.01
NDc
NDc
2
7
3.02 ( 0.01
3.28 ( 0.01
3.01 ( 0.01
3.00 ( 0.01
2.21 ( 0.01
ꢀ0.42
0.11
8
4.24 ( 0.01
The interaction of the CPDꢀbenzamidine complex was also
1
9
4.20 ( 0.02
ꢀ0.19
0.01
investigated by H NMR. The observed upfield shift of the
10
4.33 ( 0.01
signal corresponding to the proton in position 2 of CPD 1 at
different molar ratios with benzamidine was used to conduct Job
plot analysis (see Supporting Information).15 On the basis of
such an analysis, the minimum of the plot corresponds to the
11
4.62 ( 0.01
ꢀ0.42
a All determinations were carried out via UV-metric or potentiometric
(pH-metric) method. b Literature value.5 c Not determined.
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Figure 5. 1H NMR Job plot analysis of [1 benzamidine] complex showing
3
1:1 stoichiometry. The plot is derived from the chemical shift of the C2-H of
1 at different molar ratios with benzamidine. The total concentration is kept
constant (i.e., 0.125 M) in each experiment. The mole fraction x is defined as
[1]/([1] + [benzamidine]); Δ∂ = ∂complex ꢀ ∂free
.
Initially, quantum mechanical (QM) calculations were per-
formed both to explore the ground state geometries and to
define the energy differences of the two tautomeric forms of a
representative CPD (i.e., 7 and 8). These studies revealed that
regardless of solvent medium (i.e., no solvent/vacuum, water, or
chloroform), the enolꢀketone tautomer is comparatively more
stable than the diketone tautomer (e.g., for 8 the energy
difference is 0.91 kcal/mol, B3LYP 6-31G+(d,p) in the gas
phase, and on average 0.74 kcal/mol in water and chloroform;
see Supporting Information). Next, the geometry and stability of
Figure 3. [CPD benzamidine] complexes detected by ESI-MS.
3
[8 benzamidine] complexes were investigated. Optimization of
3
possible salt geometries revealed two possible scenarios (A and B),
originating from the initial tautomers (i.e., diketone and enolꢀ
ketone): (a) a bicoordinated “stacked” geometry (Figure 6A)
characterized by two intermolecular interactions between the
diketone tautomer and the amidine moiety; (b) a monocoordi-
nated “linear” geometry, wherein a single interaction between the
amidine nitrogen and the enolate oxygen is observed (Figure 6B).
Quantum mechanical calculations (gas phase) and with a water
solvation model suggest that the latter salt bridge geometry
(Figure 6B) is more stable than the former. Disruption of the π
system in the geometry in Figure 6A is believed to be responsible
for the energy difference. Calculated energies for the monocoor-
dinated CPD salt (Figure 6B) were found to be similar to those
corresponding to the bicoordinated salt bridges of carboxylic acids
with amidine bases (see Supporting Information).
’ BIOLOGICAL EVALUATION
To evaluate whether CPD isosteres could mimic the activity of
biologically active compounds, CPD analogues 41ꢀ45 and 59
were evaluated for their ability to act as antagonists of the human
and mouse TP-receptor by a functional inositol monophosphate
(IP1) assay that measures receptor activation. The activity of test
compounds was compared with known antagonist 12,8 as well as
with tetrazole 61 and amino squaric acid 62. Analogues 37ꢀ39
and 50 were also examined for comparison. Briefly, the IP1 assay
depends on a homogeneous time-resolved fluorescence method
that permits the measurement of IP1, which is a stable metabolite
of the interacellular signal transduction molecule, inositol triphosphate
Figure 4. Relative intensity of the [8 benzamidine]+ signal in a 1:1
mixture of 8 and benzamidine (100%) or in 1:1:1 mixtures of 8,
benzamidine, and competing acid/acid surrogate (i.e., 7, 9ꢀ11).
3
stoichiometry of the CPDꢀbenzamidine interaction. These
studies confirmed that CPD forms a 1:1 complex with benzami-
dine (Figure 5).
Computational Studies. CPDs possess two tautomeric forms
that can potentially generate different salt bridge geometries.
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Figure 6. Calculated bicoordinated “stacked” (A) and monocoordinated “linear” (B) geometries of the [8 benzamidine] complex in gas phase.
3
(IP3). Activation of the TP-receptorisknown to result inincreased
release of IP3;16 thus, an antagonist to this receptor will inhibit
agonist-induced increase of IP1 in QBI293 cells that have been
stably transfected with the human or mouse TP receptor. As
illustrated in Table 2, a range of CPD analogues were evaluated
and derivatives 42ꢀ45 exhibited activity comparable to that of the
known antagonist 12 as well as those of the tetrazole and amino
squarate derivatives (61 and 62, respectively).
Finally, the binding affinity of a representative subset of active
and inactive compounds against the human and mouse TP-
receptor was evaluated via radioligand scintillation proximity
assay. In this assay, membrane preparations from cells expressing
the human or mouse TP receptor associate with scintillant beads
that emit measurable photons upon binding of the radiolabeled
TP antagonist, [3H]SQ 29548, to receptors within the mem-
brane. Thus, a ligand that competes for binding of the radiolabeled
compound to the TP-receptor will cause a concentration-dependent
decrease in signal that can be quantified to yield a binding constant
for the competitive molecule.17 Consistent with the functional assay
results, these radioligand binding studies reveal that CPD derivatives
41 and 42 exhibit binding affinity values that are comparable to that
of the known antagonist 12, aswellasthoseofthetetrazole(61) and
the amino squarate (62) derivatives (Table 3).
intracellular hydrophobic segment between the III, V, and VI
helices, with a favorable hydrogen bond to the Ser-201 residue
(Figure 7A). Next, the binding modes of compound 12, and of a
representative CPD derivative (42), were evaluated and com-
pared (Figure 7B and Figure 7C, respectively). As shown in
Figure 7B,C and in Figure 8, compounds 12 and 42 were found
to share similar hydrophobic interactions between the central
phenyl ring and the pocket formed by Leu-261, Leu-294, Ala-297,
and Met-112, as well as between the phenylsulfonamide moiety
and the pocket between helices III and IV (Trp-258 and Gly-116,
see Figure 8). Interestingly, while the carboxylic acid moiety of
12 interacted with Arg-295 via the expected bicoordinated salt
bridge, the corresponding CPD isostere in 42 was found to
establish a monocoordinated salt bridge with the guanidinium
moiety of Arg-295 as well as a hydrogen bond with the backbone
nitrogen of Thr-298 (Figure 8). Equally interesting, the 2-position
of the CPD fragment appears to be directed through the groove of
helix VII, suggesting that an increase in steric hindrance at this
position may be well tolerated. This observation is consistent with
functional and receptor binding data (cf., Tables 2 and 3).
’ DISCUSSION
Although CPDs share important similarities with the car-
boxylic acid moiety, including comparable pKa values and the
ability to establish H-bonds, there are no reports of this isostere
employed as a surrogate of the carboxylic acid moiety in drug
design. To evaluate CPDs as potential carboxylic acid isosteres,
we compared the properties of representative mono- and disub-
stituted CPD analogues with similar compounds bearing car-
boxylic acid or tetrazole moieties. These studies confirmed the
intrinsic acidity of the CPD fragment and revealed that com-
pounds 7ꢀ9, depending on the substitution pattern, can be
somewhat more acidic than the corresponding carboxylic acid
and tetrazole counterparts. Furthermore, the lipophilicity of
these compounds is also either equal or higher than acid 10
and tetrazole 11 (Table 1). Importantly, the ability of CPD
’ DOCKING STUDIES
A TP-receptor model was developed via homology modeling
as previously described.18 Docking studies were then carried out
using the Autodock software package, initially employing the
natural ligand thromboxane A2 (TXA2). Since previous reports
suggest that Arg-295 and Ser-201 residues in the intracellular
region are important constituents of the binding pocket within
the TP-receptor,19 docking of TXA2 was conducted by setting a
salt bridge between the carboxylic acid residue of TXA2 and the
side chain of Arg-295 as a constraint. In agreement with previous
docking studies by Yamamoto and co-workers,20 our studies
indicate that the polycarbon side chain of TXA2 extends into the
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Table 2. TP-Receptor Antagonist Activity of Test
Compounds
Table 3. TP-Receptor Binding Affinity of Selected
Compounds, Determined by Radioligand-Binding Assay
calculated salt bridge formation energies; however, the lack of cor-
relation observed may be due to the experimental conditions
employed in the ESI-MS studies, particularly the fact that these
experiments are performed in organic solvent. Taken together,
these results strongly suggested that the CPD fragment holds great
promise as an effective surrogate for the carboxylic acid moiety.
To verify this hypothesis in the context of a biologically active
system, analogues of the known TP-receptor antagonist 128 were
designed by replacing the carboxylic acid moiety of this com-
pound with a CPD fragment linked at either the C2 or C5
position (Figure 2). The TP-receptor is an important G-protein-
coupled receptor (GPCR) implicated in several pathophysiolo-
gical processes, such as vasoconstriction, aggregation of
platelets,21 and more recently, Alzheimer’s disease.22 A number
of TP-receptor antagonists have been reported, and many of
these, like 12, comprise an aromatic ring connected to both a
phenylsulfonamide and a carboxylic acid moiety via intervening
aliphatic spacers, typically of two to three carbons in length.21
Previous studies suggest that the carboxylic acid moiety of TP-
receptor antagonists are involved in the formation of a salt bridge
with Arg-295 of the receptor.20 Thus, the presence of a carboxylic
acid moiety may be a general prerequisite for receptor binding
and activity. This requirement is clearly the case for compound
12, as highlighted by the dramatic loss in activity in both the
functional and radioligand-binding assays observed when the
corresponding alcohol derivative 60 is employed (Tables 2 and
3). Conversely, replacement of the carboxylic acid moiety with
either tetrazole (61) or squaric acid (62) led to derivatives with
activity and binding affinity generally comparable to 12. Among
the CPD derivatives, interesting differences emerged depending
on where the CPD moiety is attached (i.e., C2 or C5). While C2
substituted derivatives 50 and 59 were essentially devoid of any
activity in the functional assay, all C5 substituted compounds
(41ꢀ45) exhibited IC50 values in the same range of carboxylic
acid 12 (Table 2). These observations were confirmed by
radioligand-binding data (Table 3), demonstrating that represen-
tative examples of C2 and C5 linked CPD derivatives exhibited
derivatives 7ꢀ9 to form complexes with benzamidine was
demonstrated by ESI-MS and NMR. Of particular note, our
studies suggest that CPDs can reversibly form 1:1 complexes
with benzamidine regardless of the substitution pattern (cf.,
Figures 3 and 5). Computational studies indicated that the most
likely salt bridge geometry involves monocoordinated structures
between the amidine nitrogen and the enolate oxygen of
the CPD. Although our studies focused exclusively on the
CPDꢀbenzamidine complexes, similar salt geometries may also
exist between CPDs and other amines, including the amino
groups on lysine and histidine side chains. Interestingly, results
from competition-binding studies indicate that, under the ex-
perimental conditions, the CPDꢀbenzamidine complex may be
more stable than the corresponding tetrazolateꢀbenzamidine or
carboxylateꢀbenzamidine complexes (Figure 4). These results
do not appear to correlate exactly with either the pKa values or
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Figure 8. Schematic representation of key interactions of 12 and 42
with TP-receptor.
2-position of the CPD can be adjacent to the groove of helix VII
of the TP-receptor, indicating that steric hindrance at the C2
would be relatively well tolerated. This characteristic may be
exploited to design analogues with increased complementarity
with the receptor and/or to modify the physicalꢀchemical proper-
ties (e.g., lipophilicity) of the compound.
’ CONCLUSIONS
Collectively, the studies presented here demonstrate that the
CPD fragment comprises a viable new surrogate for the car-
boxylic acid moiety with potential applications in drug design. In
addition to the significant intrinsic acidity and the ability to form
salt bridges, our results demonstrate that the CPD moiety may
permit substantial structural differentiation of the acidic residue.
This characteristic may be particularly desirable when attempting
to modulate drugꢀtarget/off-target interactionsand/or physicalꢀ
chemical properties of biologically active compounds. As a result,
the CPD would appear to be a valuable addition to the existing
palette of carboxylic acid isosteres.
’ EXPERIMENTAL SECTION
Materials and Methods. All solvents were reagent grade. All
reagents were purchased from Aldrich or Acros and used as received.
Thin layer chromatography (TLC) was performed with 0.25 mm E.
Merck precoated silica gel plates. Flash chromatography was performed
with silica gel 60 (particle size 0.040ꢀ0.062 mm) supplied by Silicycle
and Sorbent Technologies. Spots were detected by viewing under a UV
light. Yields refer to chromatographically and spectroscopically pure
compounds. Infrared spectra were recorded on a Jasco Model FT/IR-
480 Plus spectrometer. All melting points were obtained on a Thomas-
Hoover apparatus. Proton (1H) and carbon (13C) NMR spectra were
recorded on a Bruker AMX-500 spectrometer. Chemical shifts were
reported relative to solvents (CDCl3 7.27 ppm; CD3OD 3.35 ppm;
DMSO-d6 2.5 ppm, acetone-d6 2.05 ppm). As previously observed,10
CPDs are mixtures of rapidly interconverting tautomers that can complicate
the interpretation of 13C NMR spectra. High-resolution mass spectra were
measured at the University of Pennsylvania Mass Spectrometry Service
on either a VG Micromass 70/70H or VG ZAB-E spectrometer. Single-
crystal X-ray structure determinations were performed at the University
of Pennsylvania with an Enraf Nonius CAD-4 automated diffractometer.
Analytical reversed-phased (Sunfire C18; 4.6 mm ꢁ 50 mm, 5 mL) high-
performance liquid chromatography (HPLC) was performed with a
Figure 7. Docking of thromboxane A2 (A), 12 (B), and 42 (C) to the
TP-receptor.
drastically different binding affinities. Given that ESI-MS studies
with model compounds 7ꢀ9 did not reveal significant differences
between the C2 and C5 linked CPD congeners in their ability to
generate complexes with benzamidine, the results from the func-
tional assay suggest that the position and/or orientation of the
enolꢀketone moiety may prove critical in the relatively confined
environment of the receptor. Also of note, the steric hindrance of
the C2 substituents of 41ꢀ45 did not appear to impact the activity
of the analogues, suggesting that effective electrostatic interactions
with the TP-receptor may take place if the CPD moiety is linked at
C5, even in the presence of relatively large substituents linked at
C2. Consistent with these results, docking studies revealed that the
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Water binary gradient module 2525 equipped with Waters 2996 PDA
and Water micromass ZQ. All samples were analyzed employing a linear
gradient from 10% to 90% of acetonitrile in water over 8 min and flow
rate of 1 mL/min, and unless otherwise stated, the purity level was >95%.
Preparative reverse phase HPLC purification was performed employing
Waters SunFire prep C18 OBD columns (5 μm, 19 mm ꢁ 50 mm, or
19 mm ꢁ 100 mm). All samples were purified employing a linear
gradient from 10% to 90% of acetonitrile in water over 15 min and flow
rate of 20 mL/min. The preparative HPLC system was equipped with
Gilson 333 pumps, a 215 liquid handler, 845Z injection module, and UV
detector. Unless otherwise stated, all final compounds were found to be
>95% pure as determined by HPLC/MS and NMR.
2-(4-Bromobenzyl)-3-hydroxycyclopent-2-enone (9). To a
solution of cyclopentane-1,3-dione (0.200 g, 2.0 mmol), diethyl 2,6-dimethyl-
1,4-dihydropyridine-3,5-dicarboxylate (0.520 g, 2.0 mmol), and L-proline
(0.012 g) in dichloromethane (6.6 mL) was added 4-bromobenzaldehyde
(1.130 g; 6.0 mmol), and the resulting mixture was allowed to stir at room
temperature for 30 min. Purification by silica gel column chromatography
using a gradient of ethyl acetate in hexanes as eluant provided 9 (75% yield).
Mp: 199ꢀ201 °C (from methanol). 1H NMR (CD3OD): δ 2.51 (s, 4H),
3.38 (s, 2H), 7.12 (d, J= 8.2 Hz, 2H), 7.33 (d, J= 8.3 Hz, 2H) ppm. 13CNMR
(MeOD): δ 27.2, 31.5, 117.6, 120.5, 131.5, 132.3, 141.0 ppm. IR (film):
ν 2911, 2532, 1565 cmꢀ1. HRMS (ESIꢀ): calculated for C12H10BrO2
ꢀ
264.9864; found 264.9872.
5-(4-Bromobenzyl)-3-isobutoxycyclopent-2-enone (5).
3-Isobutoxycyclopent-2-enone (3, 0.130 g, 0.84 mmol) in anhydrous
tetrahydrofuran (2.0 mL) was cooled to ꢀ78 °C, and a freshly prepared 1 M
solution of lithium diisopropylamide (1.0 mL, 1.0 mmol) was added
dropwise. The mixture was stirred at ꢀ78 °C for 45 min, and a solution
of 1-bromo-4-(bromomethyl)benzene (0.210 g, 0.84 mmol) in anhydrous
tetrahydrofuran (3.0 mL) was added dropwise. The resulting mixture was
stirred for 1 h, allowing the temperature to rise to room temperature.
The reaction was quenched with a saturated aqueous solution of
ammonium chloride (3.0 mL). The aqueous phase was extracted
with ethyl acetate (2 ꢁ 10 mL), and combined organic layers were
washed with brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. Purification by silica gel column chromatog-
raphy using a gradient of ethyl acetate in hexanes as eluant provided
3-(2-Hydroxyethyl)benzaldehyde (13). A solution 2-(3-bro-
mophenyl)ethanol (1.00 g, 0.67 mL, 4.9 mmol) and N,N,N0,N0-tetra-
methylethylenediamine (1.5 mL) in anhydrous diethyl ether (10 mL)
was cooled to ꢀ78 °C in dry iceꢀacetone bath. n-Butyllithium (2.4 M
solution in hexanes, 4.0 mL, 9.8 mmol) was added dropwise, and the
resulting mixture was allowed to warm to ꢀ20 °C (over 1 h) and then
cooled again to ꢀ78 °C. Anhydrous N,N-dimethylformamide (5 mL)
was added, and the resulting mixture was warmed to room temperature
and stirred for an additional 1 h. The reaction was quenched with a
saturated solution of ammonium chloride, and the aqueous portion was
extracted with ethyl acetate. The combined extracts were washed
with brine, dried over magnesium sulfate, filtered, and concentrated in
vacuo. Purification by silica gel column chromatography using ethyl
acetateꢀhexanes, 2:3, as eluant furnished 13 as a colorless oil (88%
yield). 1H NMR (CDCl3): δ 1.69 (broad s, 1H), 2.96 (t, J = 6.5 Hz, 2H),
3.91 (t, J = 6.5 Hz, 2H), 7.47ꢀ7.53 (m. 2H), 7.74ꢀ7.76 (m, 2H), 10.00
(s, 1H) ppm. 13C NMR (CDCl3): δ 39.0, 63.4, 128.4, 129.4, 130.1,
1
5 as a white solid (40% yield). H NMR (CDCl3): δ 0.94 (d, J =
6.7 Hz, 6H), 2.00 (m, 1H), 2.27 (dd, J = 17.7, 2.2 Hz, 1H), 2.55ꢀ2.61
(m, 2H), 2.71ꢀ2.74 (m, 1H), 3.12 (dd, J = 14.0, 4.2 Hz, 1H), 3.67
(d, J = 6.4 Hz, 2H), 5.20 (s, 1H), 7.04 (d, J = 8.3 Hz, 2H), 7.35ꢀ7.36
(m, 2H) ppm. 13C NMR (CDCl3): δ 19.05, 19.07, 27.9, 34.1, 36.4,
46.4, 78.1, 104.0, 120.3, 130.8, 131.6, 138.5, 189.2, 206.7 ppm. IR
135.5, 136.9, 140.1, 192.6 ppm. IR (film): ν 3381, 1696 cmꢀ1
.
3-(2-((Triisopropylsilyl)oxy)ethyl)benzaldehyde (14). Chloro-
triisopropylsilane (0.640 g, 0.71 mL, 3.3 mmol) was added dropwise to a
solution of 13 (0.450 g, 3.0 mmol) and 1H-imidazole (0.410 g, 6.0 mmol)
in anhydrous N,N-dimethylformamide (10 mL) at 0 °C, and the resulting
mixture was stirred at 0 °C for 3 h. The reaction mixture was then diluted
with water, and the aqueous portion was extracted with diethyl ether. The
combined extracts were washed with brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. Purification by silica gel column
chromatography using ethyl acetateꢀhexanes, 1:9, as eluant furnished 14
as colorless oil (96% yield). 1H NMR (CDCl3): δ 1.00ꢀ1.10 (m, 21H),
2.93 (t, J = 6.6 Hz, 2H), 3.93 (t, J = 6.6 Hz, 2H), 7.45 (t, J = 7.5 Hz, 1H),
7.52 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.76 (s, 1H), 10.00
(s, 1H) ppm. 13C NMR (CDCl3): δ 12.1, 18.1, 39.5, 64.4, 127.9, 129.0,
130.6, 135.7, 136.7, 140.9, 192.7 ppm. IR (film): ν 1703 cmꢀ1. MS [ESI]+:
calculated for C18H31O2Si+ 307.20; found 307.20.
(film): ν 2962, 2931, 2876, 1692, 1593 cmꢀ1
.
5-(4-Bromobenzyl)-3-isobutoxy-2-methylcyclopent-2-enone
(6). 6 was prepared as 5 from 4. Yield 20%. 1H NMR (CDCl3): δ 0.97
(d, J = 6.5 Hz, 6H), 1.65 (s, 3H), 1.65ꢀ2.00 (m, 1H), 2.21ꢀ2.25
(m, 1H), 2.52ꢀ2.57 (m, 1H), 2.60ꢀ2.65 (m, 1H), 2.73ꢀ2.76 (m, 1H),
3.22 (dd, J = 16.5, 4.5 Hz, 1H), 3.83 (dd, J = 1.5, 0.5 Hz, 2H), 7.08 (d, J =
8.5 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 6.2,
18.9, 28.8, 31.0, 36.7, 45.8, 75.7, 115.4, 120.2, 130.7, 131.6, 138.6, 183.2,
206.1 ppm. IR (film): ν 3384, 3283, 1725 cmꢀ1. MS (ESI+): calculated
for C17H22BrO2+ 337.08; found 337.01.
5-(4-Bromobenzyl)-3-hydroxycyclopent-2-enone (7). To a
mixture of 5 (0.110 g, 0.34 mmol) in acetone (4.2 mL), 2 N hydrochloric
acid (1.7 mL) was added at room temperature. The mixture was stirred
for 16 h. The reaction mixture was then concentrated under reduced
pressure and the residue is purified by preparative reverse phase HPLC
providing 7 as a white solid (46% yield). Mp: 206ꢀ208 °C (from
methanol). 1H NMR (MeOD): δ 2.20 (dd, J = 18.1, 2.5 Hz, 1H), 2.52
(dd, J = 18.1, 6.9 Hz, 1H), 2.63 (dd, J = 13.8, 9.3 Hz, 1H), 2.93 (m, 1H),
3.08 (dd, J = 13.8, 4.3 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.4
Hz, 2H) ppm. 13C NMR (MeOD): δ 37.47, 37.61, 46.0, 105.9, 121.3,
132.2, 132.6, 139.7, 200.1, 204.9 ppm. IR (film): ν 2920, 2680, 2562,
1713, 1555 cmꢀ1. HRMS [ESI]ꢀ: calculated for C12H10O2Brꢀ 264.9864;
found 264.9869.
(3-(2-((Triisopropylsilyl)oxy)ethyl)phenyl)methanol (15).
To a solution of 14 (0.500 g, 1.63 mmol) in tetrahydrofuran (4 mL)
and water (0.25 mL), sodium borohydride (0.062 g, 1.63 mmol) was
added. The reaction mixture was heated to reflux for 2 h. After the
mixture was cooled, water was added. The aqueous portion was
extracted with ethyl acetate. The combined extracts were washed with
brine, dried over magnesium sulfate, filtered, and concentrated in vacuo.
The residue was dried under high vacuum to afford 15 as colorless oil
(99% yield). 1H NMR (CDCl3): δ 0.91ꢀ1.17 (m, 21H), 1.62 (broad t,
J = 5.9 Hz, 1H), 2.88 (t, J = 7.1 Hz, 2H), 3.89 (d, J = 7.1 Hz, 2H), 4.68
(d, J = 5.7 Hz, 2H), 7.17 (d, J = 7.4 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H), 7.24
(s, 1H), 7.28 (t, J = 7.5 Hz, 1H) ppm. 13C NMR (CDCl3): δ 12.2, 18.2,
39.9, 64.9, 65.7, 125.0, 128.1, 128.7, 128.8, 139.9, 141.0 ppm. IR (film):
5-(4-Bromobenzyl)-3-hydroxy-2-methylcyclopent-2-enone
(8). 8 was prepared as 7 from 6. Yield 84%. Mp: 180ꢀ181 °C (from
methanol). 1H NMR (CD3OD): δ 1.55 (s, 3H), 2.16 (d, J = 17.5 Hz,
1H), 2.49 (d, J = 17.5 Hz, 1H), 2.58 (dd, J = 13.5, 8.5 Hz, 1H),
2.84ꢀ2.88 (m, 1H), 3.11 (dd, J = 14.0, 4.0 Hz, 1H), 7.13 (d, J = 8.5 Hz,
2H), 7.40 (d, J = 8.5 Hz, 2H) ppm. 13C NMR (CD3OD): δ 5.7, 36.0,
37.8, 45.2, 114.1, 121.3, 132.2, 132.6, 139.8 ppm. IR (film): ν 2922,
ν 3325 cmꢀ1
.
(3-(Iodomethyl)phenethoxy)triisopropylsilane (16). A mixture
of 15 (0.500 g, 1.63 mmol), triphenylphosphine (1.280 g, 4.89 mmol),
imidazole (0.360 g, 5.22 mmol), and iodine (1.320 g, 5.22 mmol) in diethyl
ether (20 mL) and acetonitrile (6.0 mL) was stirred at 0 °C for 2 h. The
reaction mixture was diluted with ether and washed with water and a 20 wt %
2639, 1570 cmꢀ1. MS (ESI+): calculated for C13H14BrO2 281.02;
+
found 281.13.
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Journal of Medicinal Chemistry
ARTICLE
solution of thiosulfate in water. The organic layer was dried over magnesium
sulfate, filtered, and concentrated in vacuo. Purification by silica gel column
chromatography using ethyl acetateꢀhexanes, 1:9, as eluant provided 16 as
colorless oil (64% yield). 1H NMR (CDCl3): δ 1.00ꢀ1.11 (m, 21H), 2.83
(t, J = 6.9 Hz, 2H), 3.89 (t, J = 6.9 Hz, 2H), 4.4 (s, 2H), 7.11 (d, J = 6.9 Hz,
1H), 7.20ꢀ7.27 (m, 3H) ppm. 13C NMR (CDCl3): δ 6.1, 12.2, 18.2, 39.7,
64.7, 126.8, 128.9, 129.0, 129.8, 139.2, 140.3 ppm. MS [ESI]+: calculated for
C18H32IOSi+ 419.12; found 419.10.
2-(Cyclopropylmethyl)-3-isobutoxy-5-(3-(2-((triisopropylsilyl)-
oxy)ethyl)benzyl)cyclopent-2-enone (24). 24 was prepared
1
as 20 from 16 and 19. Yield: 44%. H NMR (CDCl3): δ 0.10ꢀ0.13
(m, 2H), 0.33ꢀ0.36 (m, 2H), 0.87 (m, 1H), 0.95 (dd, J = 6.7, 0.6 Hz,
6H), 1.02ꢀ1.07 (m, 21H), 1.97 (m, 1H), 2.06 (d, J = 6.8 Hz, 2H),
2.27ꢀ2.31 (m, 1H), 2.49 (dd, J = 14.1, 10.6 Hz, 1H), 2.63 (dd, J = 17.4,
6.9 Hz, 1H), 2.78 (m, 1H), 2.82 (t, J = 7.1 Hz, 2H), 3.28 (dd, J = 14.1,
4.0 Hz, 1H), 3.80 (d, J = 6.5 Hz, 2H), 3.86 (t, J = 7.2 Hz, 2H), 7.05
(m, 3H), 7.18 (t, J = 7.9 Hz, 1H) ppm. 13C NMR (CDCl3): δ 4.6, 10.0,
12.1, 18.1, 19.0, 26.0, 28.8, 31.0, 37.4, 39.9, 46.2, 64.9, 75.5, 119.6, 126.7,
127.3, 128.4, 129.8, 139.6, 139.7, 183.6, 206.1 ppm.
3-Isobutoxy-5-(3-(2-((triisopropylsilyl)oxy)ethyl)benzyl)-
cyclopent-2-enone (20). A solution of diisopropylamine (0.040 g,
0.05 mL, 0.38 mmol) in anhydrous tetrahydrofuran (0.3 mL) was cooled
to ꢀ78 °C, and n-butyllithium (2.3 M solution in hexanes, 0.15 mL,
0.35 mmol) was added dropwise. After being stirred at ꢀ78 °C for 1 h, a
solution of 39 (0.050 g, 0.32 mmol) in anhydrous tetrahydrofuran
(2 mL) was added dropwise, and the reaction mixture was stirred at
ꢀ78 °C for 45 min. A solution of 16 (0.150 g, 0.35 mmol) in anhydrous
tetrahydrofuran (1.5 mL) was then added dropwise, and the reaction
mixture was allowed to warm to room temperature. The reaction was
quenched with a saturated solution of ammonium chloride, and the
aqueous phase was extracted with ethyl acetate. The combined organic
extracts were washed with brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. Purification by silica gel column chroma-
tography using ethyl acetateꢀhexanes, 1:3, as eluant furnished 20 as
colorless oil (49% yield). 1H NMR (CDCl3): δ 0.95 (d, J = 6.5 Hz, 6H),
1.00ꢀ1.10 (m, 21H), 1.20ꢀ2.10 (m, 1H), 2.32ꢀ2.37 (m, 1H),
2.49ꢀ2.61 (m, 2H), 2.76ꢀ2.80 (m, 1H), 2.82 (t, J = 7.1 Hz, 2H), 3.26
(dd, J = 13.9, 4.0 Hz, 1H), 3.71 (d, J = 7.1 Hz, 2H), 3.87 (t, J = 7.1 Hz, 2H),
5.25(s, 1H), 7.03ꢀ7.07 (m, 3H), 7.19 (t, J = 8.1 Hz, 1H) ppm. 13C NMR
(CDCl3): δ 12.1, 18.1, 19.1, 28.0, 34.5, 37.3, 39.9, 46.9, 65.0, 78.1, 103.8,
126.7, 127.3, 128.5, 129.9, 139.6, 139.7, 189.4, 207.3 ppm. IR (film):
ν 1696, 1595 cmꢀ1. MS [ESI]+: calculated for C27H44O3NaSi+ 467.30;
found 467.30.
5-(3-(2-Hydroxyethyl)benzyl)-3-isobutoxycyclopent-2-enone
(25). A solution of tetrabutylammonium fluoride (1 M in tetrahydro-
furan, 0.47 mL, 0.47 mmol) was added dropwise to a solution of 20
(0.071 g, 0.16 mmol) in anhydrous tetrahydrofuran (1 mL) at 0 °C. The
reaction mixture was stirred at 0 °C for 3 h and then quenched with a
saturated solution of ammonium chloride. The aqueous layer was
extracted with diethyl ether, and the combined organic layers were
washed with brine, dried over magnesium sulfate, filtered, and concen-
trated in vacuo. Purification by silica gel column chromatography using
dichloromethaneꢀmethanol, 95:5, as eluant provided 25 as colorless oil
(82% yield). 1H NMR (CDCl3): δ 0.96 (d, J = 6.7 Hz, 6H), 1.98ꢀ2.16
(m, 1H), 2.34 (dd, J = 17.7, 2.3 Hz, 1H), 2.54ꢀ2.63 (m, 2H), 2.75ꢀ2.80
(m, 1H), 2.84 (t, J = 6.7 Hz, 2H), 3.22 (dd, J = 13.9, 4.1 Hz, 2H), 3.70
(d, J = 5.8 Hz, 2H), 3.83 (t, J = 6.6 Hz, 2H), 5.24 (s, 1H), 7.05ꢀ7.08
(m, 3H), 7.21 (t, J = 8.1 Hz, 1H) ppm. 13C NMR (CDCl3): δ 19.1, 28.0,
34.4, 37.2, 39.3, 46.7, 63.7, 78.2, 103.9, 127.1, 127.2, 128.8, 129.8, 139.0,
139.9, 189.5, 207.5 ppm. IR (film): ν 3399, 1685, 1590 cmꢀ1. MS
[ESI]+: calculated for C18H25O3+ 289.18; found 289.21.
5-(3-(2-Hydroxyethyl)benzyl)-3-isobutoxy-2-methylcyclopent-
2-enone (26). 26 was prepared as 25 from 21. Yield: 78%. 1H NMR
(CDCl3): δ 0.95ꢀ0.96 (m, 6H), 1.64 (s, 3H), 1.92ꢀ2.00 (m, 2H), 2.28
(d, J = 17.4 Hz, 1H), 2.52 (dd, J = 14 and 10.4 Hz, 1H), 2.60ꢀ2.65 (m,
1H), 2.75ꢀ2.80 (m, 1H), 2.85 (t, J = 7.5 Hz, 2H), 3.26 (dd, J = 14.0 and
4.0 Hz, 1H), 3.80ꢀ3.86 (m, 4H), 7.05ꢀ7.09 (m, 3H), 7.23 (t, J = 7.8 Hz,
1H) ppm. 13C NMR (CDCl3): δ 6.2, 19.0, 28.9, 31.3, 37.5, 39.3, 46.1,
63.8, 75.7, 115.4, 127.1, 127.2, 128.8, 129.8, 139.0, 140.1, 183.3, 206.7
ppm. IR (film): ν 3404, 1680, 1620 cmꢀ1. MS [ESI]+: calculated for
C19H27O3+ 303.19; found 303.20.
2-Ethyl-5-(3-(2-hydroxyethyl)benzyl)-3-isobutoxycyclopent-
2-enone (27). 27 was prepared as 25 from 22. Yield: 86%. 1H NMR
(acetone-d6): δ 0.93ꢀ0.96 (m, 9H), 1.94 (dt, J = 13.3, 6.7 Hz, 1H), 2.08
(q, J = 7.6 Hz, 2H), 2.42 (dt, J = 17.3, 1.1 Hz, 1H), 2.54 (dd, J = 13.7,
9.7 Hz, 1H), 2.67 (ddt, J = 9.6, 4.4, 2.3 Hz, 1H), 2.72ꢀ2.79 (m, 3H), 3.10
(dd, J = 13.7, 4.0 Hz, 1H), 3.70 (bs, 1H), 3.73 (t, J = 7.1 Hz, 2H), 3.92
(dd, J = 6.5, 3.1 Hz, 2H), 7.07 (t, J = 6.4 Hz, 2H), 7.12 (s, 1H), 7.17 (t, J =
7.5 Hz, 1H) ppm. 13C NMR (acetone-d6): δ 13.5, 15.7, 19.5, 29.9, 31.6,
38.1, 40.8, 47.2, 64.4, 76.3, 121.4, 127.9, 128.1, 129.5, 131.0, 140.96,
140.98, 184.4, 205.8 ppm.
5-(3-(2-Hydroxyethyl)benzyl)-3-isobutoxy-2-isopropylcy-
clopent-2-enone (28). 28 was prepared as 25 from 23. Yield: 90%.
1H NMR (acetone-d6): δ 1.07 (d, J = 6.7 Hz, 6H), 1.22 (t, J = 7.6 Hz,
6H), 2.02ꢀ2.09 (m, 1H), 2.43ꢀ2.48 (m, 1H), 2.69ꢀ2.84 (m, 4H), 2.89
(t, J = 7.0 Hz, 2H), 3.00 (s, 1H), 3.18 (dd, J = 13.1, 3.1 Hz, 1H), 3.83
(t, J = 7.0 Hz, 2H), 3.97 (dd, J = 6.4, 1.1 Hz, 2H), 7.17ꢀ7.21 (m, 3H),
7.33 (t, J = 7.5 Hz, 1H) ppm. 13C NMR (acetone-d6): δ 19.7, 21.3, 21.3,
24.4, 30.1, 31.6, 38.3, 40.7, 47.1, 64.5, 76.8, 118.9, 125.2, 128.4, 128.5,
129.8, 131.5, 141.1, 185.2, 206.7 ppm.
3-Isobutoxy-2-methyl-5-(3-(2-((triisopropylsilyl)oxy)ethyl)-
benzyl)cyclopent-2-enone (21). 21 was prepared as 20 from 16
1
and 4. Yield: 56%. H NMR (CDCl3): δ 0.95ꢀ1.08 (m, 27H), 1.65
(s, 3H), 1.93ꢀ1.99 (m, 1H), 2.25ꢀ2.30 (m, 1H), 2.45 (dd, J = 14.0, 10.8
Hz, 1H), 2.58ꢀ2.63 (m, 1H), 2.76ꢀ2.78 (m, 1H), 2.83 (t, J = 7.1 Hz,
2H), 3.30 (dd, J = 14.1, 3.9 Hz, 1H), 3.82 (d, J = 6.6 Hz, 2H), 3.87 (t, J =
7.3 Hz, 2H), 7.03ꢀ7.08 (m, 3H), 7.20 (t, J = 7.5 Hz, 1H) ppm. 13C NMR
(CDCl3): δ 6.2, 12.1, 18.1, 18.9, 28.8, 31.3, 37.5, 39.8, 46.2, 64.9, 75.6,
115.3, 126.6, 127.3, 128.4, 129.8, 139.6, 139.8, 183.3, 206.6 ppm. IR
(film): ν 1736, 1652, 1634 cmꢀ1. MS [ESI]+: calculated for C28H47O3Si+
459.32; found 459.28.
2-Ethyl-3-isobutoxy-5-(3-(2-((triisopropylsilyl)oxy)ethyl)-
benzyl)cyclopent-2-enone (22). 22 was prepared as 20 from 16
and 17. Yield: 48%. 1H NMR (CDCl3): δ 0.96 (dd, J = 6.7, 1.6 Hz, 6H),
0.99ꢀ1.07 (m, 24H), 1.97 (dt, J = 13.3, 6.7 Hz, 1H), 2.16 (q, J = 7.6 Hz,
2H), 2.27 (dt, J = 17.5, 1.0 Hz, 1H), 2.48 (dd, J = 14.0, 10.7 Hz, 1H), 2.60
(dd, J = 17.4, 6.8 Hz, 1H), 2.74ꢀ2.77 (m, 1H), 2.83 (t, J = 7.2 Hz, 2H),
3.29 (dd, J = 14.1, 4.0 Hz, 1H), 3.80 (d, J = 6.5 Hz, 2H), 3.86ꢀ3.89 (m,
2H), 7.06 (dt, J = 12.4, 6.7 Hz, 3H), 7.20 (t, J = 7.5 Hz, 1H) ppm. 13C NMR
(CDCl3):δ12.2, 12.8, 14.8, 18.2, 19.0, 28.9, 31.0, 37.5, 39.9, 46.2, 65.0, 75.5,
121.4, 126.8, 127.3, 128.5, 129.9, 139.65, 139.80, 183.3, 206.2 ppm.
3-Isobutoxy-2-isopropyl-5-(3-(2-((triisopropylsilyl)oxy)-
ethyl)benzyl)cyclopent-2-enone (23). 23 was prepared as 20
from 16 and 18. Yield: 68%. 1H NMR (CDCl3): δ 0.96 (dd, J = 6.7, 0.9
Hz, 6H), 1.02ꢀ1.08 (m, 21H), 1.09ꢀ1.14 (m, 6H), 1.96 (dd, J = 13.3,
6.6 Hz, 1H), 2.25 (dd, J = 17.4, 2.3 Hz, 1H), 2.49 (dd, J = 14.0, 10.5 Hz,
1H), 2.58 (dd, J = 17.4, 6.9 Hz, 1H), 2.72 (td, J = 7.3, 3.3 Hz, 1H), 2.77
(t, J = 7.1 Hz, 1H), 2.83 (t, J = 7.1 Hz, 2H), 3.25 (dd, J = 14.0, 4.0 Hz,
1H), 3.78 (d, J = 6.0 Hz, 2H), 3.87 (t, J = 7.2 Hz, 2H), 7.05 (dt, J = 13.4,
7.0 Hz, 3H), 7.19 (t, J = 7.5 Hz, 1H) ppm. 13C NMR (CDCl3): δ 12.2,
18.2, 19.1, 20.5, 23.0, 28.9, 30.7, 37.5, 39.9, 46.0, 65.0, 75.5, 124.8, 126.8,
127.3, 128.4, 129.9, 139.59, 139.72, 183.0, 205.7 ppm.
2-(Cyclopropylmethyl)-5-(3-(2-hydroxyethyl)benzyl)-3-
isobutoxycyclopent-2-enone (29). 29 was prepared as 25 from
24. Yield: 99%. 1H NMR (CDCl3): δ 0.09ꢀ0.12 (m, 2H), 0.33ꢀ0.37
(m, 2H), 0.87ꢀ0.91 (m, 1H), 0.96 (d, J = 6.7 Hz, 6H), 1.80 (bs, 1H),
1.97 (dt, J = 13.3, 6.7 Hz, 1H), 2.06 (d, J = 6.8 Hz, 2H), 2.28ꢀ2.32
(m, 1H), 2.55 (dd, J = 14.1, 10.3 Hz, 1H), 2.66 (dd, J = 17.4, 6.8 Hz, 1H),
6978
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Journal of Medicinal Chemistry
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2.77ꢀ2.81 (m, 1H), 2.85 (t, J = 6.6 Hz, 2H), 3.26 (dd, J = 14.1, 4.2 Hz,
1H), 3.81 (d, J = 6.4 Hz, 2H), 3.85 (t, J = 6.6 Hz, 2H), 7.07ꢀ7.09
(m, 3H), 7.23 (t, J = 7.7 Hz, 1H) ppm. 13C NMR (CDCl3): δ 4.6, 10.0,
19.0, 26.0, 28.9, 31.1, 37.4, 39.4, 46.1, 63.8, 75.6, 119.7, 127.2, 128.8, 129.8,
1H), 2.26ꢀ2.28 (m, 1H), 3.03 (t, J = 7.4 Hz, 2H), 3.31 (dd, J = 14.0, 3.4 Hz,
1H), 3.79ꢀ3.84 (m, 2H), 4.04 (t, J = 5.85 Hz, 2H), 7.09ꢀ7.12 (m, 3H),
7.24 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 8.3 Hz, 2H)
ppm. 13C NMR (CDCl3): δ 6.2, 19.0, 28.1, 28.9, 31.4, 36.6, 37.5, 46.2, 48.6,
75.7, 84.8, 115.3, 127.4, 129.0, 129.1, 129.6, 129.9, 138.4, 138.9, 139.9, 140.5,
150.8, 183.5, 206.5 ppm. IR (film): ν 1729, 1688, 1631 cmꢀ1. MS [ESI]+:
calculated for C30H39ClNO6S+ 576.22; found 576.06.
+
139.0, 140.1, 183.9, 206.3 ppm. HRMS (ESI+): calculated for C22H31O3
343.2267; found 343.2268.
5-(3-(2-Iodoethyl)benzyl)-3-isobutoxycyclopent-2-enone
(30). 30 was prepared as 16 from 25. Yield: 88%. 1H NMR (CDCl3):
δ 0.96 (d, J = 6.7 Hz, 6H), 1.99ꢀ2.07 (m, 1H), 2.35 (dd, J = 17.8, 2.3 Hz,
1H), 2.59ꢀ2.63 (m, 2H), 2.77ꢀ2.81 (m, 1H), 3.14 (t, J = 7.7 Hz, 2H),
3.23 (dd, J = 13.9, 4.1 Hz, 1H), 3.35 (t, J = 7.3 Hz, 2H), 3.70 (d, J =
6.5 Hz, 2H), 5.25 (s, 1H), 7.02ꢀ7.04 (m, 2H), 7.10 (d, J = 7.6 Hz, 1H),
7.22 (t, J = 7.7 Hz, 1H) ppm. 13C NMR (CDCl3): δ 6.0, 19.1, 28.0, 34.3,
37.1, 40.3, 46.7, 78.1, 104.0, 126.5, 127.6, 128.9, 129.2, 140.0, 140.9, 189.4,
tert-Butyl (4-Chlorophenyl)sulfonyl(3-((3-ethyl-4-isobutoxy-
2-oxocyclopent-3-en-1-yl)methyl)phenethyl)carbamate (34).
1
34 was prepared as 33 from 27. Yield: 100%. H NMR (acetone-d6):
δ 1.00 (dd, J = 6.7, 1.3 Hz, 6H), 1.04 (t, J = 7.5 Hz, 3H), 1.39 (s, 9H), 1.99
(dt, J = 13.4, 6.7 Hz, 1H), 2.16 (q, J = 7.5 Hz, 2H), 2.43ꢀ2.39 (m, 1H),
2.63 (dd, J = 13.8, 9.7 Hz, 1H), 2.72 (dd, J = 17.3, 6.8 Hz, 1H), 2.77
(m, 1H), 3.07 (t, J = 7.3 Hz, 2H), 3.18 (dd, J = 13.8, 4.0 Hz, 1H), 3.93 (d,
J = 6.6 Hz, 2H), 4.12 (t, J = 7.3 Hz, 2H), 7.19 (m, 3H), 7.32 (t, J = 7.5 Hz,
1H), 7.63 (d, J = 8.6 Hz, 2H), 7.82 (d, J = 8.6 Hz, 2H) ppm.
207.2 ppm. IR: ν 1691, 1592cmꢀ1. MS[ESI]+: calculatedfor C18H24IO2
+
398.08; found 398.07.
tert-Butyl (4-Chlorophenyl)sulfonyl(3-((4-isobutoxy-3-
isopropyl-2-oxocyclopent-3-en-1-yl)methyl)phenethyl)-
carbamate (35). 35 was prepared as 33 from 28. Yield: 80%. 1H NMR
(CD3OD): δ 0.98 (d, J = 6.7 Hz, 6H), 1.15 (dd, J = 10.3, 7.1 Hz, 6H),
1.30ꢀ1.42 (m, 9H), 1.96 (dt, J = 13.3, 6.6 Hz, 1H), 2.42 (d, J = 15.9 Hz,
1H), 2.63 (dd, J = 13.7, 9.1 Hz, 1H), 2.71ꢀ2.78 (m, 3H), 3.03 (t, J =
7.1 Hz, 2H), 3.16 (dd, J = 13.7, 3.8 Hz, 1H), 3.92 (d, J = 6.3 Hz, 2H), 4.12
(t, J = 7.1 Hz, 2H), 7.14 (m, 3H), 7.26 (d, J = 7.4 Hz, 1H), 7.59 (d, J =
8.7 Hz, 2H), 7.78 (d, J = 8.7 Hz, 2H) ppm. 13C NMR (CD3OD): δ 19.3,
20.85, 20.88, 24.1, 28.3, 30.0, 31.7, 37.4, 38.0, 47.0, 77.1, 85.7, 125.3,
128.63, 128.67, 129.9, 130.3, 131.0, 131.3, 139.8, 140.4, 140.85, 140.94,
152.2, 187.4, 208.8 ppm.
tert-Butyl(4-Chlorophenyl)sulfonyl(3-((3-(cyclopropylmethyl)-
4-isobutoxy-2-oxocyclopent-3-en-1-yl)methyl)phenethyl)-
carbamate (36). 36 was prepared as 33 from 29. Yield: 78%. 1H NMR
(CDCl3): δ 0.09ꢀ0.12 (m, 2H), 0.33ꢀ0.36 (m, 2H), 0.87ꢀ0.90 (m, 1H),
0.93 (dd, J = 6.7, 1.8 Hz, 6H), 1.33 (s, 9H), 1.95 (dt, J = 13.3, 6.7 Hz,
1H), 2.06 (d, J = 6.8 Hz, 2H), 2.31 (dd, J = 17.6, 1.9 Hz, 1H), 2.51 (dd,
J = 14.1, 10.7 Hz, 1H), 2.63 (dd, J = 17.6, 6.8 Hz, 1H), 2.78ꢀ2.82
(m, 1H), 3.02 (t, J = 7.5 Hz, 2H), 3.30 (dd, J = 14.1, 4.0 Hz, 1H), 3.80
(dd, J = 6.5, 3.3 Hz, 2H), 4.03 (t, J = 7.5 Hz, 2H), 7.10 (q, J = 8.8 Hz, 3H),
7.23 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 8.7 Hz,
2H) ppm.
5-(3-(2-Azidoethyl)benzyl)-3-isobutoxycyclopent-2-enone
(31). A mixture of 30 (0.044 g, 0.11 mmol) and sodium azide (0.007 g,
0.11 mmol) in dry N,N-dimethylformamide (0.5 mL) was heated at
50 °C for 45 min. The reaction mixture was then diluted with water, and
the aqueous portion was then extracted with ethyl acetate. The organic
extracts were washed with brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The residue so obtained was dried under
high vacuum to furnish 31 (76% yield). 1H NMR (CDCl3): δ 0.97 (d, J =
6.7 Hz, 6H), 2.00ꢀ2.08 (m, 1H), 2.34 (dd, J = 17.8, 2.2 Hz, 1H),
2.57ꢀ2.63 (m, 2H), 2.77ꢀ2.81 (m, 1H), 2.87 (t, J = 7.2 Hz, 2H), 3.24
(dd, J = 13.9, 4.1 Hz, 1H), 3.49 (t, J = 7.2 Hz, 2H), 3.70 (d, J =
6.5 Hz, 2H), 5.25 (s, 1H), 7.06ꢀ7.09 (m, 3H), 7.23 (t, J = 7.9 Hz, 1H)
ppm. 13C NMR (CDCl3): δ 19.1, 27.9, 34.3, 35.4, 37.1, 46.7, 52.6, 78.1,
103.9, 126.9, 127.5, 128.9, 129.5, 138.4, 140.1, 189.4, 207.2 ppm. IR
+
(film):ν 2097, 1692, 1592cmꢀ1. MS[ESI]+: calculatedfor C18H24N3O2
314.18; found 314.07.
4-Chloro-N-(3-((4-isobutoxy-2-oxocyclopent-3-en-1-yl)-
methyl)phenethyl)benzenesulfonamide (32). A mixture of 31
(0.059 g, 0.19 mmol) and Pd on C (10 wt %, 0.006 g) in methanol (3 mL)
was stirred at room temperature for 1 h under 1 atm of hydrogen. The
reaction mixture was filtered to remove the catalyst. Water (0.5 mL) and
2 N sodium hydroxide (0.27 mL) were added, and the resultant mixture was
cooled to 0 °C. 4-Chlorophenylsulfonyl chloride (0.082 g, 0.39 mmol) was
added. The reaction mixture was stirred at 0 °C for 4 h and then diluted with
ethyl acetate. The organic layers were collected, washed with brine, dried
over magnesium sulfate, filtered, and concentrated in vacuo. Purification by
silica gel column chromatography, using ethyl acetateꢀhexanes, 1:1, as
4-Chloro-N-(3-((2-hydroxy-4-oxocyclopent-2-en-1-yl)methyl)-
phenethyl)benzenesulfonamide (41). A mixture of 32 (0.018 g,
0.04 mmol) in acetone (0.5 mL) and 2 N hydrochloric acid (0.2 mL) was
stirred at room temperature for 6 h. The reaction mixture was diluted
with water, and the acetone was evaporated under reduced pressure. The
aqueous phase was extracted with ethyl acetate, and the combined
organic layers were washed with brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. Purification by reverse phase
1
eluant, provided 32 (22% yield). H NMR (CDCl3): δ 0.98 (d, J =
6.5 Hz, 6H), 1.99ꢀ2.10 (m, 1H), 2.32 (dd, J= 17.5, 2.7 Hz, 1H), 2.61ꢀ2.65
(m, 2H), 2.75 (t, J= 6.6 Hz, 2H), 3.15 (dd, J = 13.9, 4.5 Hz, 1H), 3.22ꢀ3.26
(m, 2H), 3.71ꢀ3.73 (m, 3H), 4.47 (broad t, J = 6.2 Hz, 1H), 5.26 (s, 1H),
6.92ꢀ6.94 (m, 2H), 7.08 (d, J = 7.6 Hz, 1H), 7.21 (t, J = 7.8 Hz, 1H), 7.47
(d, J = 8.6 Hz, 2H), 7.75 (d, J = 8.6 Hz, 2H) ppm. MS [ESI]+: calculated for
C24H29ClNO4S+ 462.15; found 462.21.
tert-Butyl (4-Chlorophenyl)sulfonyl(3-((2-isobutoxy-3-methyl-
4-oxocyclopent-2-en-1-yl)methyl)-phenethyl)carbamate (33).
Diethyl diazodicarboxylate (40 wt % in toluene, 0.08 mL, 0.28 mmol) was
added dropwise to a solution of 26 (0.021 g, 0.07 mmol), N-boc-4-
chlorobenzenesulfonamide (0.032 g, 0.11 mmol), and triphenylphosphine
(0.058 g, 0.22 mmol) in anhydrous tetrahydrofuran (3 mL) at 0 °CunderAr
atmosphere, and the mixture was stirred at room temperature for 4 h. The
reaction was quenched with water, and the resulting aqueous portion was
extracted with diethyl ether. The combined organic layers were washed with
brine, dried over magnesium sulfate, filtered, and concentrated in vacuo.
Purification by silica gel column chromatography using ethyl aceta-
teꢀhexanes, 1:3, as eluant furnished 33 (96% yield). 1H NMR (CDCl3):
δ 0.94 (d, J = 6.8 Hz, 6H), 1.34 (s, 9H), 1.65 (s, 3H), 1.91ꢀ1.99 (m, 1H),
2.29 (d, J = 17.5 Hz, 1H), 2.45ꢀ2.49 (m, 1H), 2.60 (dd, J = 17.5, 6.7 Hz,
1
preparative HPLC provided 41 as white solid. Yield: 88%. H NMR
(DMSO-d6): δ 2.05ꢀ2.09 (m, 1H), 2.37ꢀ2.39 (m, 1H), 2.46 (dd, J =
13.7, 10.2 Hz, 1H), 2.64 (t, J = 7.3 Hz, 2H), 2.74ꢀ2.78 (m, 1H),
2.95ꢀ3.01 (m, 3H), 5.06 (s, 1H), 6.97 (d, J = 7.6 Hz, 1H), 6.99 (s, 1H),
7.04 (d, J = 7.6 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.64 (d, J = 8.6 Hz, 2H),
7.76 (d, J = 8.6 Hz, 2H), 7.82 (t, J = 5.7 Hz, 1H) ppm. 13C NMR
(DMSO-d6): δ 22.1, 29.0, 35.2, 36.7, 44.0, 104.5, 126.5, 126.8, 128.3,
128.4, 129.1, 129.6, 137.2, 138.5, 139.4, 139.6 ppm. IR (film): ν 3402, 1730,
1647, 1580 cmꢀ1. HRMS [ESI]+: calculated for C20H20ClNNaO4S+
428.0699; found 428.0714.
4-Chloro-N-(3-((4-isobutoxy-3-methyl-2-oxocyclopent-3-
en-1-yl)methyl)phenethyl)benzenesulfonamide (37). To a
solution of 33 (0.100 g, 0.173 mmol) in dichloromethane (5 mL) was
added 2,2,2-trifluoroacetic acid (1.5 mL). The resultant mixture was
stirred at room temperature for 1 h. The reaction mixture was
concentrated in vacuo and purified by preparative reverse phase HPLC
to obtain 37 (73% yield). 1H NMR (CDCl3): δ 0.97 (dd, J = 6.7, 1.3 Hz,
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Journal of Medicinal Chemistry
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6H), 1.65 (t, J = 1.6 Hz, 3H), 1.98 (dt, J = 13.4, 6.7 Hz, 1H), 2.28 (dt, J =
17.4, 1.9 Hz, 1H), 2.53 (dd, J = 14.0, 10.2 Hz, 1H), 2.65 (ddd, J = 17.4,
6.8, 1.6 Hz, 1H), 2.75ꢀ2.79 (m, 3H), 3.21ꢀ3.26 (m, 3H), 3.85 (d, J =
6.6 Hz, 2H), 4.45 (t, J = 6.2 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 7.09 (d, J =
7.6 Hz, 1H), 7.22 (t, J = 7.4 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.75 (d, J =
8.7 Hz, 2H) ppm. 13C NMR (CDCl3): δ 5.3, 18.3, 28.4, 30.9, 35.7, 36.9,
44.1, 45.7, 75.7, 114.9, 126.65, 126.85, 128.04, 128.13, 128.5, 129.09,
138.3, 138.59, 138.72, 139.2, 185.5, 208.1 ppm. IR (film): ν 2926, 1679,
1618 cmꢀ1. HRMS [ESI]+: calculated for C25H31ClNO4S+ 476.1662;
found 476.1656.
of 38 (0.05 g, 0.11 mmol) in acetone (5 mL) was added 2 N hydrochloric
acid (5 mL). The reaction mixture was stirred at room temperature for
13 h and then concentrated in vacuo. The aqueous portion was extracted
with ethyl acetate (10 mL ꢁ 2), and the organic portion was washed with
brine (5 mL ꢁ 2). The organic layer was dried with anhydrous sodium
sulfate, filtered, and concentrated in vacuo. Purification by reverse phase
1
preparative HPLC provided the desired compound (75% yield). H
NMR (CD3OD): δ 0.93ꢀ0.96 (m, 3H), 2.05ꢀ2.17 (m, 3H), 2.45 (dd,
J = 17.7, 6.8 Hz, 1H), 2.59 (dd, J = 13.7, 9.2 Hz, 1H), 2.69ꢀ2.74
(m, 2H), 2.82 (m, 1H), 3.11 (m, 3H), 6.99 (t, J = 2.0 Hz, 2H), 7.07
(s, 1H), 7.16 (d, J = 7.8 Hz, 1H), 7.57ꢀ7.58 (m, 2H), 7.79ꢀ7.81
(m, 2H) ppm. 13C NMR (CDCl3): δ 12.8, 14.5, 29.4, 29.9, 32.0, 35.9,
37.8, 44.4, 53.9, 120.1, 127.0, 127.8, 128.7, 129.1, 129.7, 130.1, 137.9,
4-Chloro-N-(3-((3-ethyl-4-isobutoxy-2-oxocyclopent-3-
en-1-yl)methyl)phenethyl)benzenesulfonamide (38). A solu-
tion of 34 (0.122 g, 0.207 mmol) in dichloromethane (5 mL) and 2,2,2-
trifluoroacetic acid (3 mL) was stirred at room temperature for 5 h. The
reaction mixture was concentrated in vacuo and purified by reverse
phase preparative HPLC to provide 38 as a colorless oil (80% yield).
1H NMR (CDCl3): δ 0.95 (d, J = 6.7 Hz, 6H), 0.98 (t, J = 7.5 Hz, 3H),
1.96 (dt, J = 13.3, 6.7 Hz, 1H), 2.14 (q, J = 7.5 Hz, 2H), 2.26 (dd, J = 17.4,
0.9 Hz, 1H), 2.53 (dd, J = 14.0, 10.0 Hz, 1H), 2.64 (dd, J = 17.4, 6.8 Hz,
1H), 2.71ꢀ2.78 (m, 3H), 3.17ꢀ3.24 (m, 3H), 3.82 (d, J = 6.5 Hz, 2H),
4.87 (t, J = 6.1 Hz, 1H), 6.94ꢀ6.96 (m, 2H), 7.06 (d, J = 7.5 Hz, 1H),
7.18 (d, J = 7.5 Hz, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H)
ppm. 13C NMR (CDCl3): δ 12.8, 14.8, 19.0, 28.9, 31.1, 36.0, 37.4, 44.4,
46.0, 75.7, 121.4, 126.9, 127.6, 128.7, 129.0, 129.56, 129.62, 138.0, 138.8,
138.5, 139.5, 139.6 ppm. IR (film): ν 3388, 2922, 2354, 1608 cmꢀ1
.
HRMS (ESIꢀ): calculated for C22H23NO4SClꢀ 432.1036; found 432.1036.
4-Chloro-N-(3-((4-hydroxy-3-isopropyl-2-oxocyclopent-
3-en-1-yl)methyl)phenethyl)benzenesulfonamide (44). 44
1
was prepared as 43 from 39. Yield: 50%. H NMR (CDCl3): δ 1.10
(d, J = 6.9 Hz, 6H), 2.17 (bs, 1H), 2.45 (dd, J = 17.4, 5.3 Hz, 1H),
2.59ꢀ2.78 (m, 5H), 3.04ꢀ3.11 (m, 3H), 6.97 (d, J = 9.7 Hz, 2H), 7.03
(d, J = 5.9 Hz, 1H), 7.15 (s, 1H), 7.55 (d, J = 7.3 Hz, 2H), 7.78 (d, J = 7.7
Hz, 2H) ppm. 13C NMR (CDCl3): δ 20.70, 20.72, 24.1, 35.84,
37.2, 38.3, 45.3, 45.7, 123.6, 128.0, 128.5, 129.64, 129.78, 130.5, 130.8,
139.8, 140.1, 140.5, 141.0 ppm. IR (film): ν 3268, 2962, 2930, 2873,
1672, 1609 cmꢀ1. HRMS (ESI+): calculated for C23H26ClNO4SNa+
470.1169; found 470.1165.
139.2, 140.4, 183.5, 206.1 ppm. IR (film): ν 2960, 2920, 2852, 1613 cmꢀ1
.
HRMS[ESI]+:calculatedforC26H32NO4NaSCl+ 512.1638; found 512.1627.
4-Chloro-N-(3-((4-isobutoxy-3-isopropyl-2-oxocyclopent-
3-en-1-yl)methyl)phenethyl)benzenesulfonamide (39). 39
4-Chloro-N-(3-((3-(cyclopropylmethyl)-4-hydroxy-2-oxo-
cyclopent-3-en-1-yl)methyl)phenethyl)benzenesulfonamide
(45). 45 was prepared as 43 from 40. Yield 56%. 1H NMR (CD3OD):
δ 0.04ꢀ0.05 (m, 2H), 0.27ꢀ0.30 (m, 2H), 0.81ꢀ0.85 (m, 1H), 2.00
(d, J = 6.7 Hz, 2H), 2.17 (dd, J = 17.8, 1.8 Hz, 1H), 2.46 (d, J = 6.8 Hz,
1H), 2.58 (dd, J = 13.8, 9.1 Hz, 1H), 2.68 (t, J = 7.4 Hz, 2H), 2.80ꢀ2.88
(m, 1H), 3.07 (m, 3H), 6.95ꢀ6.96 (m, 2H), 7.02 (d, J = 7.7 Hz, 1H),
7.14 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 8.7 Hz, 2H)
ppm. 13C NMR (CD3OD): δ 4.85, 4.87, 10.9, 26.2, 30.9, 37.2, 38.3, 45.8,
118.4, 128.0, 128.5, 129.72, 129.81, 130.6, 130.8, 139.8, 140.1, 140.6,
141.0 ppm. IR (film): ν 3279, 2921, 2852, 1729, 1584 cmꢀ1. HRMS
(ESI+): calculated for C24H25ClNO4S+ 458.1193; found 458.1190.
3-Hydroxy-2-(3-(2-((triisopropylsilyl)oxy)ethyl)benzyl)-
cyclopent-2-enone (46). To a solution of 14 (0.920 g, 3.0 mmol),
cyclopentane-1,3-dione (0.098 g, 1.0 mmol), and L-proline (0.005 g)
in dichloromethane (5 mL) was added diethyl 2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (0.253 g, 1.0 mmol). The result-
ing mixture was allowed to stir at room temperature for 12 h.
Purification by silica gel column chromatography using a gradient
of ethyl acetate in hexanes as eluant provided 46 (76% yield).
1H NMR (CD3OD): δ 1.01ꢀ1.04 (m, 21H), 2.47 (s, 4H), 2.75 (t, J =
6.8 Hz, 2H), 3.40 (s, 2H), 3.85 (t, J = 6.8 Hz, 2H), 6.95ꢀ6.97 (m, 1H),
7.03 (d, J = 7.6 Hz, 1H), 7.08 (t, J = 7.4 Hz, 2H) ppm. 13C NMR
(CD3OD): δ 13.3, 18.6, 27.8, 31.5, 40.9, 66.2, 118.4, 127.4, 127.8,
129.1, 130.4, 140.4, 141.5 ppm.
2-(3-(2-Hydroxyethyl)benzyl)-3-isobutoxycyclopent-2-enone
(47). A mixture containing 46 (0.294 g, 0.757 mmol), p-toluenesulfonic
acid (0.005 g), isobutanol (1.0 mL), and benzene (4.0 mL) was heated to
reflux for 12 h. The reaction mixture was then concentrated in vacuo and
the residue was purified by silica gel column chromatography using 5%
of methanol in dichloromethane as eluant, providing 47 (81% yield).
1H NMR (CDCl3): δ 0.96 (d, J = 6.7 Hz, 6H), 2.00 (m, 1H), 2.39 (dt, J =
5.0, 2.4 Hz, 2H), 2.61 (t, J = 4.6 Hz, 3H), 2.77 (t, J = 6.9 Hz, 2H), 3.41
(s, 2H), 3.76 (t, J = 6.9 Hz, 2H), 3.88 (d, J = 6.4 Hz, 2H), 6.97 (d, J =
7.3 Hz, 1H), 7.11 (m, 3H) ppm. 13C NMR (CDCl3): 19.0, 25.2, 27.4,
28.8, 33.6, 39.4, 63.7, 75.7, 119.9, 126.6, 126.7, 128.4, 129.4, 138.7, 140.6,
185.3, 204.5 ppm. IR (film): ν 3399, 1685, 1590 cmꢀ1. MS (ESI+):
calculated for C18H25O3+ 289.18; found 289.16.
1
was prepared as 38 from 35. Yield: 99%. H NMR (CDCl3): δ 0.87
(d, J = 6.7 Hz, 6H), 1.02 (dd, J = 15.4, 7.0 Hz, 6H), 1.85 (m, 1H),
2.29ꢀ2.33 (m, 1H), 2.55ꢀ2.69 (m, 6H), 2.98 (dd, J = 13.4, 3.2 Hz, 1H),
3.05 (t, J = 7.2 Hz, 2H), 3.82 (d, J = 6.3 Hz, 2H), 6.90ꢀ6.93 (m, 2H),
6.97 (d, J = 7.6 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 7.46 (d, J = 8.5 Hz, 2H),
7.71 (d, J = 8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 19.3, 20.78, 20.81,
24.1, 29.9, 31.4, 37.0, 37.9, 45.6, 46.9, 77.0, 125.4, 128.0, 128.4, 129.64,
129.71, 130.5, 130.8, 139.7, 140.0, 140.4, 140.9, 187.5, 208.9 ppm. IR
(film): ν 3268, 2963, 2930, 2874, 1671, 1608, 1470, 1383, 1335 cmꢀ1
.
HRMS (ESIꢀ): calculated for C27H33ClNO4Sꢀ 502.1819; found 502.1802.
4-Chloro-N-(3-((3-(cyclopropylmethyl)-4-isobutoxy-2-
oxocyclopent-3-en-1-yl)methyl)phenethyl)benzenesulfonamide
(40). 40 was prepared as 37 from 36. Yield: 29%. 1H NMR (CD3OD):
δ 0.05ꢀ0.06 (m, 2H), 0.30ꢀ0.32 (m, 2H), 0.84 (m, 1H), 0.98 (d, J = 7.0
Hz, 6H), 1.95 (m, 1H), 2.03 (d, J = 6.8 Hz, 2H), 2.45 (m, 1H),
2.65ꢀ2.69 (m, 1H), 2.74 (t, J = 7.2 Hz, 2H), 2.80ꢀ2.83 (m, 2H),
3.09ꢀ3.15 (m, 3H), 3.95 (dd, J = 6.4, 0.7 Hz, 2H), 7.00ꢀ7.04 (m, 2H),
7.08 (d, J = 7.6 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.57 (d, J = 8.7 Hz, 2H),
7.78ꢀ7.80 (m, 2H) ppm. 13C NMR (CD3OD): δ 5.0, 10.8, 19.2, 26.7,
30.0, 31.7, 37.0, 37.9, 45.6, 47.2, 77.2, 120.3, 128.1, 128.4, 129.73, 129.74,
130.5, 130.8, 139.8, 140.2, 140.5, 141.0, 188.4, 209.7 ppm. HRMS
(ESIꢀ): calculated for C28H33ClNO4Sꢀ 514.1819; found 514.1809.
4-Chloro-N-(3-((2-hydroxy-3-methyl-4-oxocyclopent-2-en-
1-yl)methyl)phenethyl)benzenesulfonamide (42). 42 was pre-
pared as 43 from 37. Yield 54%. 1H NMR (DMSO-d6): δ 1.43 (s, 3H),
1.98 (d, J = 17.4 Hz, 1H), 2.30 (dd, J = 17.3, 7.0 Hz, 1H), 2.37 (dd,
J = 13.5, 10.2 Hz, 1H), 2.62ꢀ2.65 (m, 3H), 2.94ꢀ2.98 (m, 2H), 3.03
(dd, J = 13.5, 3.6 Hz, 1H), 6.94ꢀ6.97 (m, 2H), 7.02 (d, J = 7.6 Hz, 1H),
7.14 (t, J = 7.5 Hz, 1H), 7.64 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 8.6 Hz, 2H),
7.84 (broad t, J = 5.6 Hz, 1H) ppm. 13C NMR (DMSO-d6): δ 5.9, 22.0,
35.3, 37.0, 43.6, 44.0, 110.8, 126.4, 126.8, 128.2, 128.4, 129.2, 129.4,
137.2, 138.4, 139.3, 139.9, 212.2, 214.7 ppm. IR (film): ν 3279, 2925,
1608, cmꢀ1. HRMS [ESI]+: calculated for C21H22ClNNaO4S+
442.0856; found 442.0847.
4-Chloro-N-(3-((3-ethyl-4-hydroxy-2-oxocyclopent-3-en-1-
yl)methyl)phenethyl)benzenesulfonamide (43). To a mixture
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tert-Butyl (4-Chlorophenyl)sulfonyl(3-((2-isobutoxy-5-
oxocyclopent-1-en-1-yl)methyl)phenethyl)carbamate (48).
48 was prepared as 33 from 47. Yield: 92%. 1H NMR (CDCl3): δ 0.99
(d, J = 6.7 Hz, 6H), 1.37 (s, 9H), 2.04 (td, J = 13.1, 6.4 Hz, 1H), 2.44 (m,
2H), 2.65 (m, 2H), 2.98 (t, J = 7.8 Hz, 2H), 3.46 (s, 2H), 3.91 (d, J =
6.5 Hz, 2H), 4.00 (dd, J = 8.6, 7.0 Hz, 2H), 7.02 (m, 1H), 7.17ꢀ7.18 (m,
3H), 7.44 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.7 Hz, 2H) ppm. 13C NMR
(CDCl3): δ 19.1, 25.3, 27.5, 28.1, 28.9, 33.7, 36.6, 48.6, 75.8, 84.7, 119.9,
126.8, 127.2, 128.7, 129.1, 129.53, 129.63, 138.1, 138.9, 139.8, 140.9,
150.8, 185.2, 204.4 ppm. MS (ESI+): calculated for C29H37ClNO6S+
562.20; found 561.83.
(CDCl3): δ 12.2, 18.1, 39.4, 39.9, 63.8, 65.0, 126.9, 127.5, 128.6, 130.1,
138.5, 139.8 ppm.
2-(3-(2-((Triisopropylsilyl)oxy)ethyl)phenyl)acetaldehyde
(54). To a solution of 53 (0.170 g, 0.527 mmol) in dichloromethane
(4 mL) was added DessꢀMartin periodinane (0.270 g, 0.632 mmol).
The mixture was stirred at room temperature for 4 h. The reaction
mixture was filtered through a pad of Celite and concentrated in vacuo.
Purification by silica gel column chromatography, using ethyl acetate
1
in hexanes as eluant, provided 54 (77% yield). H NMR (CDCl3):
δ 1.01ꢀ1.11 (m, 21H), 2.87 (t, J = 7.0 Hz, 2H), 3.65 (d, J = 2.5 Hz, 2H),
3.92 (t, J = 7.0 Hz, 2H), 7.08 (d, J = 7.5 Hz, 1H), 7.12 (s, 1H), 7.19
(d, J = 7.5 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 9.74 (t, J = 2.5 Hz, 1H) ppm.
13C NMR (CDCl3): δ 12.1, 18.1, 39.7, 50.7, 64.8, 127.5, 128.4, 129.0,
130.7, 131.8, 140.5, 199.5 ppm.
3-Hydroxy-2-(3-(2-((triisopropylsilyl)oxy)ethyl)phenethyl)-
cyclopent-2-enone (55). 55 was prepared as 46 from 54. Yield:
74%. 1H NMR (CD3OD): δ 0.98 (m, 21H), 2.33 (q, J = 7.7 Hz, 2H),
2.40 (d, J = 7.4 Hz, 4H), 2.60 (q, J = 7.5 Hz, 2H), 2.73 (q, J = 6.9 Hz, 2H),
3.82 (q, J = 7.0 Hz, 2H), 6.97 (m, 3H), 7.07 (t, J = 7.4 Hz, 1H) ppm. 13C
NMR (CD3OD): δ 13.3, 18.6, 24.2, 31.4, 34.9, 40.9, 66.2, 118.2, 127.3,
127.8, 129.2, 130.5, 140.44, 140.45, 143.3 ppm.
2-(3-(2-Hydroxyethyl)phenethyl)-3-isobutoxycyclopent-
2-enone (56). 56 was prepared as 47 from 55. Yield: 66%. 1H NMR
(CDCl3): δ 0.98 (d, J = 6.5 Hz, 6H), 1.98ꢀ2.03 (m, 1H), 2.41ꢀ2.47
(m, 4H), 2.60ꢀ2.62 (m, 2H), 2.73 (t, J = 7.5 Hz, 2H), 2.84 (t, J = 6.5 Hz,
2H), 3.84ꢀ3.87 (m, 4H), 7.02ꢀ7.08 (m, 3H), 7.20 (t, J = 7.5 Hz,
1H) ppm.
4-Chloro-N-(3-((2-isobutoxy-5-oxocyclopent-1-en-1-yl)-
methyl)phenethyl)benzenesulfonamide (49). To a solution of
48 (0.120 g, 0.214 mmol) in dichloromethane (5.0 mL) was added 2,2,2-
trifluoroacetic acid (1.5 mL). The resulting mixture was stirred at room
temperature for 0.5 h. The reaction mixture was concentrated in vacuo
and purified by reverse phase preparative HPLC to furnish 49 as white
1
solid (51% yield). H NMR (CDCl3): δ 0.99 (d, J = 6.7 Hz, 6H),
2.00ꢀ2.06 (m, 1H), 2.45 (m, 2H), 2.66 (m, 2H), 2.71 (t, J = 6.8 Hz, 2H),
3.19 (q, J = 6.6 Hz, 2H), 3.42 (s, 2H), 3.92 (d, J = 6.5 Hz, 2H), 4.71 (t, J =
6.1 Hz, 1H), 6.84ꢀ6.86 (m, 1H), 7.02 (s, 1H), 7.13 (m, 2H), 7.45 (d, J =
8.6 Hz, 2H), 7.71 (d, J = 8.6 Hz, 2H) ppm. 13C NMR (CDCl3): δ 19.1,
25.3, 27.5, 28.9, 33.7, 35.9, 44.4, 75.8, 119.9, 126.3, 127.4, 128.71, 128.89,
129.3, 129.5, 137.6, 138.8, 139.2, 141.1, 185.2, 204.5 ppm. IR (film): ν
3270, 3202, 2962, 2929, 2875, 1678, 1616 cmꢀ1. HRMS (ESIꢀ):
calculated for C24H27ClNO4Sꢀ 460.1349; found 460.1348.
4-Chloro-N-(3-((2-hydroxy-5-oxocyclopent-1-en-1-yl)methyl)-
phenethyl)benzenesulfonamide (50). 50 was prepared as 43
from 49. Mp: 176ꢀ178 °C (from ethyl acetate). 1H NMR (DMSO-d6):
δ 2.37 (s, 4H), 2.60 (t, J = 7.5 Hz, 2H), 2.91ꢀ2.95 (m, 2H), 3.27 (s, 2H),
6.89 (d, J = 7.5 Hz, 1H), 6.94ꢀ7.01 (m, 2H), 7.09 (t, J = 7.5 Hz, 1H),
7.65 (d, J = 7.8 Hz, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.82 (broad t, J =
5.7 Hz, 1H), 11.9 (broad s, 1H) ppm. 13C NMR (DMSO-d6): δ 26.4,
35.3, 44.0, 115.2, 125.7, 126.1, 128.0, 128.4, 128.5, 129.3, 137.1, 138.1,
139.3, 140.7 ppm. IR (film): ν 3090, 1610 cmꢀ1. HRMS [ESI]+:
calculated for C20H20ClNNaO4S+ 428.0699; found 428.0713.
2,20-(1,3-Phenylene)diethanol (52). To a solution of 2,20-(1,3-
phenylene)diacetic acid (1.000 g, 5.15 mmol) in anhydrous tetrahydrofuran
(15 mL) was added lithium aluminum hydride (0.5860 g, 15.45 mmol) at
0 °C. The reaction mixture was stirred at 0 °C for 3 h and finally
quenched by addition of hydrochloric acid (1 N) until pH 7 was obtained.
The solution was filtered through filter paper, and the aqueous portion
was extracted with ethyl acetate (15 mL ꢁ 2). The organic layer was then
washed with brine (15 mL ꢁ 2), dried over sodium sulfate, filtered, and
concentrated in vacuo. Purification by silica gel column chromatography,
eluting with a gradient of ethyl acetate in hexanes (50%ꢀ80%), provided
52 (61% yield). 1H NMR (MeOD): δ 2.79 (t, J = 7.1 Hz, 4H), 3.74 (t, J =
7.1 Hz, 4H), 7.05 (dd, J = 7.5, 1.5 Hz, 2H), 7.09 (s, 1H), 7.19 (t, J = 7.5
Hz, 1H) ppm. 13C NMR (MeOD): δ 40.3, 64.3, 127.9, 129.5, 130.8,
140.4 ppm.
2-(3-(2-((Triisopropylsilyl)oxy)ethyl)phenyl)ethanol (53).
To a solution of 2,20-(1,3-phenylene)diethanol (0.430 g, 2.59 mmol)
and imidazole (0.176 g, 2.59 mmol) in anhydrous N,N-dimethylforma-
mide (15 mL) was slowly added chlorotriisopropylsilane (0.22 mL,
1.03 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 3 h. The
reaction was quenched by addition of water (5 mL). The aqueous
portion was extracted with ethyl acetate (15 mL ꢁ 2), and the organic
portion was washed with brine (10 mL ꢁ 2). The organic layer was dried
over anhydrous sodium sulfate, filtered, and concentrated in vacuo.
Purification by column chromatography using a mixture of 1:10 ethyl
acetate/hexanes as eluant provided 53 as colorless oil (35% yield).
1H NMR (CDCl3): δ 1.09ꢀ1.05 (m, 21H), 1.85 (s, 1H), 2.86 (q, J =
6.7 Hz, 4H), 3.85 (t, J = 6.7 Hz, 2H), 3.91 (t, J = 7.1 Hz, 2H), 7.09 (d, J =
7.6 Hz, 1H), 7.11ꢀ7.12 (m, 2H), 7.24 (t, J = 7.8 Hz, 1H) ppm. 13C NMR
tert-Butyl (4-Chlorophenyl)sulfonyl(3-(2-(2-isobutoxy-5-
oxocyclopent-1-en-1-yl)ethyl)phenethyl)carbamate (57). 57
1
was prepared as 33 from 56. Yield: 43%. H NMR (CDCl3): δ 0.98
(d, J = 6.5Hz, 6H), 1.59 (s, 9H), 1.98ꢀ2.03 (m, 1H), 2.43ꢀ2.45 (m, 4H),
2.62 (t, J = 4.5 Hz, 2H), 2.73 (t, J = 8.0 Hz, 2H), 2.99ꢀ3.03 (m, 2H),
3.83ꢀ3.86 (m, 2H), 3.98ꢀ4.01 (m, 1H), 4.19ꢀ4.24 (m, 1H), 7.05ꢀ7.11
(m, 3H), 7.21 (t, J = 7.5 Hz, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.77 (d, J =
8.5 Hz, 2H) ppm. 13C NMR (CDCl3): δ 19.1, 23.5, 25.2, 28.1, 28.9, 33.8,
33.9, 36.7, 48.8, 75.6, 84.8, 119.9, 126.7, 127.0, 128.7, 129.1, 129.4, 129.7,
138.0, 139.0, 139.9, 142.9, 150.9, 184.9, 204.9 ppm.
4-Chloro-N-(3-(2-(2-isobutoxy-5-oxocyclopent-1-en-1-yl)-
ethyl)phenethyl)benzenesulfonamide (58). 58 was prepared as
42 from 57. Yield 28%. 1H NMR (CDCl3): δ 1.00 (d, J = 6.7 Hz, 6H),
2.06ꢀ1.98 (m, 1H), 2.45ꢀ2.39 (m, 4H), 2.61 (m, 2H), 2.69 (t, J =
7.7 Hz, 2H), 2.73 (t, J = 6.7 Hz, 2H), 3.22 (q, J = 6.3 Hz, 2H), 3.88 (d, J =
6.5 Hz, 2H), 5.03ꢀ5.04 (bs, 1H), 6.86 (d, J = 7.5 Hz, 1H), 6.95 (s, 1H),
6.99 (d, J = 7.6 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 7.44 (d, J = 8.6 Hz, 2H),
7.75 (d, J = 8.6 Hz, 2H) ppm. 13C NMR (CDCl3): δ 19.1, 23.2, 25.2,
28.9, 33.6, 33.8, 36.0, 44.5, 75.8, 119.8, 126.4, 127.2, 128.67, 128.72,
129.2, 129.5, 137.7, 139.00, 139.08, 142.6, 185.9, 205.5 ppm. HRMS
[ESI]+: calculated for C25H30ClNO4SNa+ 498.1482; found 498.1476.
4-Chloro-N-(3-(2-(2-hydroxy-5-oxocyclopent-1-en-1-yl)-
ethyl)phenethyl)benzenesulfonamide (59). 59 was prepared as
1
43 from 58. Yield 84%. H NMR (DMSO-d6): δ 2.26 (dd, J = 9.5,
6.8 Hz, 2H), 2.34 (bs, 4H), 2.57 (dd, J = 9.4, 6.7 Hz, 2H), 2.66 (t, J =
7.5 Hz, 2H), 2.98 (q, J = 6.7 Hz, 2H), 6.95 (d, J = 6.6 Hz, 2H), 7.02 (d, J =
7.6 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.77 (d, J =
8.6 Hz, 2H), 7.83 (t, J = 5.7 Hz, 1H), 11.55 (s, 1H) ppm. 13C NMR
(DMSO-d6): δ 22.8, 33.3, 35.3, 44.1, 115.5, 125.98, 126.09, 128.2,
128.41, 128.51, 129.3, 137.1, 138.3, 139.4, 142.0 ppm. IR: ν 3253, 2924,
1709, 1587 cmꢀ1. HRMS [ESI]ꢀ: calculated for C21H21ClNO4Sꢀ
418.0880; found 418.0878.
CPDꢀBenzamidine Salt Detection with ESI-MS. CPDꢀ
benzamidine salts were detected using ESI-MS with an Acquity TQMS
controlled by MassLynx software (Waters Corporation, Milford, MA, U.S.).
Source gas flow rates, temperatures, and voltages were optimized for the
detection of intact salt ions. CPD/benzamidine mixtures (10 μM/10 μM)
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Journal of Medicinal Chemistry
ARTICLE
in acetonitrile were vortexed at room temperature for 5 min and then
infused using the detector’s syringe pump at 30 μL/min. Mass spectra
were acquired in positive ion mode with a 0.5 s scan rate over 30 s. Scans
1ꢀ50 were combined and analyzed.
analytical reports for pKa determinations for compounds 1 and 2;
and pKa/log P determinations for compounds 7ꢀ9. This material
is available free of charge via the Internet at http://pubs.acs.org.
CPDꢀBenzamidine Relative Affinity by Competition.
Compound 8 and benzamidine were mixed with a competing acid at
equimolar amounts (10 μM/10 μM/10 μM) in acetonitrile. Mixtures
were vortexed at room temperature for 10 min and then infused into the
mass spectrometer at 30 μL/min. After stabilization of the infusion flow,
mass spectra were acquired in positive ion mode with a 0.2 s scan rate
over 30 s. Scans 1ꢀ100 were combined, and the intensity of the
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (215) 898-4891. E-mail: bcarlo@sas.upenn.edu. Ad-
dress: Department of Chemistry, School of Arts and Sciences,
University of Pennsylvania, 231 South 34th Street, Philadelphia,
PA 19104-6323.
8ꢀbenzamidine salt ion, [8(79Br) benzamidine + H]+ (m/z 401), was
3
recorded. Affinities between acids and the benzamidine base were deter-
mined relative to 8 as the average (N = 3) reduction of intensity of the
8ꢀbenzamidine salt ion in the presence of a competing acid.
’ ACKNOWLEDGMENT
A.B.S. dedicates this article to William C. Agosta (Professor
Emeritus, Rockefeller University, New York, NY), outstanding
scientist/scholar, author, and mentor, who first brought to our
attention the remarkable acidity of the cyclopentane-1,3-dione
class of molecules. Financial support for this work has been
provided by the NIH/NIA (Grant AG034140) and the NSF
(Grant CHE-0840438, X-ray facility).
IP1 Functional Assay. The activity of the TP-receptor was
measured by quantifying cellular levels of the IP3 metabolite, IP1, using
a homogeneous time-resolved fluorescence (HTRF) assay kit (IP-One
Tb, Cisbio, Bedford, MA, U.S.). QBI-HEK 293A cells that were stably
transfected with human or mouse TP receptor cDNA were plated into
384-well plates at 10 000 cells/well in DMEM containing 4.5 g/L
glucose, 10% fetal bovine serum, L-glutamine, and penicillin/strepto-
mycin. The cells were incubated for 16 h at 37 °C with 5% CO2, after
which culture medium was removed and the cells were then incubated
for 15 min at 37 °C with 5% CO2 in 10 mM Hepes, 1 mM CaCl2, 0.4 mM
MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose, 50 mM LiCl, pH
7.4, containing varying concentrations of test antagonist. The TP
receptor agonist, I-BOP ([15-(1α,2β(5Z),3α-(1E,3S),4α)]-7-[3-hydroxy-
4-(p-iodophenoxy)-1-butenyl-7-oxabicycloheptenoic acid), was added
at 1.6 nM and incubated for 1 h at 37 °C with 5% CO2. Tb-labeled anti-
IP1 cryptate and D2-labeled IP1 were subsequently added in lysis buffer
and incubated for 1 h at 25 °C according to the manufacturer’s
instructions. Plates were read on a Spectramax M5 microplate reader,
with data expressed as the ratio of 665 nm/620 nm fluorescence.
Scintillation Proximity Binding Assay. QBI-HEK 293A cells
expressing hTP or mTP receptor were grown as described above and
harvested in phosphate-buffered saline with 1 mM EDTA. The cell pellet
underwent homogenization in 20 mM Hepes, 1 mM EGTA, and 0.5 mM
DTT with protease inhibitor cocktail, followed by centrifugation at
1000g for 10 min at 8 °C to remove cell debris. The resulting supernatant
was centrifuged at 21 000 rpm for 30 min at 4 °C, with the pellet
resuspended in 20 mM Hepes, 1 mM EGTA, 100 mM NaCl. Membrane
preparations were normalized to protein level as determined with a
bicinchoninic acid assay and stored at ꢀ80 °C. Test antagonists were
incubated at 10 different concentrations with 100 μg of PVT-WGA SPA
beads (PerkinElmer, Waltham, MA, U.S.), 62.5 μg of membrane, and
20 nM 3H-SQ29,548 (PerkinElmer, Waltham, MA, U.S.) in 50 mM Tris,
4 mM CaCl2, 0.1% ascorbic acid, pH 7.5, for 2 h at 25 °C in 384-well
polystyrene plates. Plates were sealed and read on a scintillation counter.
The percent total binding was determined, with total binding calculated
’ ABBREVIATIONS USED
CPD, cyclopentane-1,3-dione; TP, thromboxane A2 prostanoid;
IP1, inositol monophosphate
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures for
b
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