When inhibitors do not inhibit: Critical evaluation of rational drug design targeting chorismate mutase from mycobacterium tuberculosis

Article abstract of DOI:10.1002/cbdv.201200322

Tuberculosis (TB) is a devastating disease that claims millions of lives every year. Hindered access or non-compliance to medication, especially in developing countries, led to drug resistance, further aggravating the situation. With current standard ther

Full text of DOI:10.1002/cbdv.201200322

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When Inhibitors Do Not Inhibit: Critical Evaluation of Rational Drug Design
Targeting Chorismate Mutase from Mycobacterium tuberculosis
by Steffi Munacka), Vincent Lerouxa)1)2), Kathrin Rodererb)1), Mats ꢀkvistc)d)3), Andre´ van Eerdea),
Lise-Lotte Gundersen*a), Ute Krengel*a), and Peter Kast*b)
a) University of Oslo, Department of Chemistry, P. O. Box 1033, Blindern, NO-0315 Oslo
(L.-L. G.: phone: þ47-22857019; e-mail: l.l.gundersen@kjemi.uio.no; U. K.: phone: þ47-22855461;
e-mail: ute.krengel@kjemi.uio.no)
b) ETH Zurich, Laboratory of Organic Chemistry, Wolfgang-Pauli-Strasse 10, CH-8093 Zurich
(P. K.: phone: þ41-44-6322908; e-mail: kast@org.chem.ethz.ch)
c) NorStruct, Department of Chemistry, Faculty of Science and Technology, University of Tromsø,
NO-9037 Tromsø
d) ESRF, 41 rue Jules Horowitz, F-38027 Grenoble
Dedicated to Professor D. Seebach, ETH-Zurich, on the occasion of his 75th birthday
Tuberculosis (TB) is a devastating disease that claims millions of lives every year. Hindered access or
non-compliance to medication, especially in developing countries, led to drug resistance, further
aggravating the situation. With current standard therapies in use for over 50 years and only few new
candidates in clinical trials, there is an urgent call for new TB drugs. A powerful tool for the development
of new medication is structure-guided design, combined with virtual screening or docking studies. Here,
we report the results of a drug-design project, which we based on a publication that claimed the structure-
guided discovery of several promising and highly active inhibitors targeting the secreted chorismate
mutase (*MtCM) from Mycobacterium tuberculosis. We set out to further improve on these compounds
and synthesized a series of new derivatives. Thorough evaluation of these molecules in enzymatic assays
revealed, to our dismay, that neither the claimed lead compounds, nor any of the synthesized derivatives,
show any inhibitory effects against *MtCM.
Introduction. – Tuberculosis (TB) – the ꢀwhite plagueꢁ – is threatening mankind
since millennia, and despite all advances in medicine still claims millions of lives every
year. It is caused by Mycobacterium tuberculosis (Mtb), a truly remarkable pathogen
that has a unique cell-wall structure, resulting in low permeability for many compounds.
Furthermore, the bacterium can live in equilibrium with the hostꢁs immune response.
This is possible because Mtb can survive for many years in a non-replicating state in so-
called granulomas, consisting of macrophages, dendritic cells, lymphocytes, neutrophils,
and fibroblasts [1–3]. The World Health Organization (WHO) states that currently
one third of the worldꢁs population is latently infected with the dormant bacterium,
which can reactivate when the immune response of the host is compromised [4].
Whereas citizens of developed countries hardly spare a thought for the disease, people
1
)
)
)
These authors contributed equally to this work.
Present address: University of Bordeaux, IECB, 2 rue Robert Escarpit, F-33607 Pessac.
Present address: Bionor Pharma ASA, Papirkaia 8, P.O. Box 2870, Kjørbekk, NO-3702 Skien.
2
3
ꢂ 2012 Verlag Helvetica Chimica Acta AG, Zꢃrich

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
in destitute parts of the world like Africa and Southeast Asia experience a dramatic
revival of TB. Non-access and non-compliance to medication due to insufficient
administration and monitoring possibilities has given rise to multidrug-resistant
(MDR-TB) and extensively drug-resistant (XDR-TB) strains, which require in most
cases more than two years of treatment with drugs that are less potent, more toxic, and
expensive. MDR-TB has developed resistance to the most effective first-line drugs
isoniazid, an inhibitor of mycolic acid biosynthesis [5][6], and rifampicin, which
interferes with bacterial RNA synthesis [7]. XDR-TB Strains are additionally resistant
against fluoroquinolones and second-line injectable TB drugs, such as amikacin and
kanamycin [8][9]. The molecular basis of drug resistance is in most cases associated
with mutations in the genes coding for current drug targets, including enzymes for cell
wall, nucleic acid or protein biosynthesis [10]. During suboptimal chemotherapy, such
spontaneously arising mutations are selected, leading to the emergence of resistant
strains [11]. Moreover, overexpression of efflux pump genes also appears to play an
important role, particularly for the acquired isoniazid resistance [12]. Furthermore,
there is a rising rate of coinfection with Mtb and HIV/AIDS, and the resulting
immunodeficiency enhances the burden and the mortality of TB, exacerbated by less
treatment options due to drug interference with antiretroviral therapy [13]. Unfortu-
nately, the current standard therapies, which are in use for over 50 years, are
cumbersome, demanding the administration of multidrug combinations of four first-
line TB drugs for six months, a regimen hard to comply with, especially in countries
with inadequate health-care systems.
To control TB and make medication more accessible, better treatment plans are
urgently needed, preferably addressing new targets in the bacterium to overcome the
drug-resistance problem. Several new compounds have been identified that are
currently in clinical trials. The most promising candidates are the fluoroquinolones
gatifloxacin and moxifloxacin (DNA gyrase inhibitors, now in Phase 3 clinical trials),
the diarylquinoline TCM207 (ATP synthase inhibitor), the nitroimidazoles PA-824 and
OPC-67683 (cell-wall synthesis inhibitors), as well as the oxazolidinones linezolid and
PNU-100480 (inhibitors of protein synthesis, the latter currently in Phase 2 clinical
trials) [14]. The publication of the Mtb genome in 1998 [15] has greatly facilitated the
discovery of new possible targets for TB treatment [16][17]. Currently, more than 1000
Mtb protein structures have been deposited in the Protein Data Bank (PDB), almost
half of them in complex with a ligand, including substrate analogs or first-generation
inhibitors. For example, a recent publication described several structures of the Mtb
salicylate synthase, an enzyme catalyzing the initial step in the biosynthesis of the
siderophore mycobactin, in complex with different inhibitor molecules [18]. Another
report is concerned with the structure of an enzyme of the purine salvage pathway,
purine nucleoside phosphorylase, which has been solved in complex with the
nanomolar inhibitor acyclovir [19]. While these examples represent just a small
fraction of the current structural research on Mtb proteins, they are representative of the
constant stream of new impulses to the ongoing struggle for finding better cures for TB.
In general, it is advantageous if the drug target is not present in the host in order to
minimize undesirable side effects. One pathway essential for Mtb survival [20] but not
present in humans is the shikimate pathway, which is responsible for the biosynthesis of
aromatic amino acids in bacteria, fungi, and plants [21][22]. Recently, potent inhibitors

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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of this pathway in Mtb have been developed, including a mechanism-based inhibitor of
the first enzyme 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHP
synthase) [23] and molecules that target the 3-dehydroquinate dehydratase [24]. In
the shikimate pathway, chorismate mutase (CM) is of central importance. CMs catalyze
the Claisen rearrangement of chorismate (1) to prephenate (2), a pericyclic reaction,
which is very rare among enzyme-catalyzed processes (Scheme 1). The most potent
inhibitor for many CMs known to date is compound 3, a transition-state analog of the
chorismate to prephenate transformation [25][26].
Scheme 1. Chorismate Mutase Reaction. Chorismate 1 is converted to prephenate 2. The oxabicyclic
dicarboxylate 3 [26][25] acts as a transition-state analog and is currently the best inhibitor of most
chorismate mutases.
Mtb features two CMs, which belong to different subclasses [27]. One is a secreted
enzyme (*MtCM, g-subclass) carrying a leader peptide sequence that directs the
protein from the cytoplasm to the surrounding medium [28][29], and the other one is
the housekeeping (cytoplasmic) CM (MtCM, d-subclass) [30]. Whereas the latter is
dedicated to the production of aromatic amino acids upon activation via complex
formation with the shikimate pathway enzyme DAHP-synthase (MtDS) [30], *MtCM
is probably connected to virulence and pathogenicity of Mtb [28][31]. Since the
shikimate pathway and thus CMs are absent in humans, these enzymes are interesting
potential drug targets. We [32] and subsequently also two other groups [29][33]
reported crystal structures of *MtCM, which provide an excellent starting point for
structure-guided drug design.
Agrawal et al. made use of this structural information for setting up virtual
screening calculations aiming at finding novel CM inhibitors tailored to specifically
interact with *MtCM [34]. They also reported experimental data suggesting that some
of their computationally derived first-generation compounds already have promising
inhibitory activity (Fig. 1). To further improve these compounds towards attaining
inhibition powers more typical for actual drugs, we set out to synthesize and test a panel
of new derivatives of the most potent of the published *MtCM inhibitors. The results

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from our enzyme inhibition studies and own modeling efforts yielded unexpected
findings regarding the validity of the previous report.
Fig. 1. *MtCM Inhibitors identified by Agrawal et al. [34]. The Ki values reported in [34] (4a: Ki ¼5.7ꢀ
1.2 mm; 5a: Ki ¼17.7ꢀ3.4 mm; 6: Ki ¼21.1ꢀ3.6 mm; 7: Ki ¼28.8ꢀ4.1 mm) could not be confirmed for 4a,
5a, and 7. Compound 6 was not reevaluated in the current work.
Results and Discussion. – Synthesis. As outlined in Scheme 2, the most promising hit
of Agrawal et al., compound 4a [34], as well as the analogs 4b–4m were obtained via
nucleophilic aromatic substitution on sulfamoyl-benzoic acid 9 using an excess of the
appropriate benzyl- or 2-phenylethylamine 8 [35] (Table 1).
Scheme 2. Synthesis of [(2-Phenylethyl)amino]- and (Benzylamino)-sulfamoylbenzoic Acids 4a–4m
(see Exper. Part for details). The presented trivial C-atom numbering was used for NMR assignments.
A Perkin-type condensation of different benzaldehydes 10 with 2-phenylacetic
acids 11 led to the compounds of the a-phenylcinnamic acid series 5a–5k (Scheme 3
and Table 2). The (E)-isomer was formed predominantly under Perkin conditions.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Table 1. Synthesis of [(2-Phenylethyl)amino]- and (Benzylamino)-sufamoylbenzoic Acids 4a–4m and
Their Activities against *MtCM
Compound
n
R
R1
R2
R3
Yield [%]
Res. Activity [%]a)
4a
4b
4c
4d
4e
4f
4g
4h
4i
2
2
2
2
2
2
2
2
1
1
1
1
1
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
MeO
H
H
H
H
H
H
H
MeO
MeO
H
MeO
H
H
Cl
F
MeO
H
MeO
MeO
H
MeO
H
H
MeO
H
H
H
MeO
H
MeO
H
MeO
H
80
85
78
87
75
60
91
94
53
36
64
61
51
100.0ꢀ4.4
101.1ꢀ5.4
114.3ꢀ5.9
102.2ꢀ6.6
100.9ꢀ5.4
95.4ꢀ6.6
99.4ꢀ6.6
106.2ꢀ7.8
88.7ꢀ15.3
100.5ꢀ6.7
88.2ꢀ28.5
96.9ꢀ5.3
106.6ꢀ7.7
4j
4k
4l
4m
H
a) Residual CM activity in the presence of 100 mm of the compound, relative to the enzymatic reaction
without added compound (i.e., negative control, defined as 100%; the standard deviation over all
measurements was ꢀ5.8%). The known inhibitor 3, used as a positive control, reduced the CM activity to
29.3ꢀ0.7% already at a concentration of 11.1 mm. Details of the applied stop assay for CM activity are
described in the Exper. Part.
Scheme 3. Synthesis of Phenyl-cinnamic Acids 5a–5k. The presented C-atom numbering was used for
NMR assignments.
a) Ac2O/Et3N 2 :1, reflux 24 h.
However, the (Z)-isomer, which was reported as an active inhibitor of *MtCM [34],
was generally obtained in moderate yields by prolonged reaction times at high
temperatures, probably promoting the thermal isomerization of the formed (E)-isomer
[36][37]. The isomers of compounds 5e–5h could not be separated by column
chromatography, and the (Z)-isomers were, therefore, not further characterized. Pure
(E)-isomers of 5e–5h were obtained by short reaction times. Selective NOESY

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Table 2. Synthesis of Phenyl-cinnamic Acids 5a–5k and Their Activities against *MtCM
Compound
R
R1
R2
R3
Yield [%]
Res. Activity [%]b)
(E) (Z)
96.2ꢀ5.7 90.3ꢀ3.1
(E)
(Z)
5a
5b
5c
5d
Cl
H
Me
MeO
Cl
Cl
Cl
Cl
Cl
NO2
NO2
NO2
NO2
H
H
H
H
MeO
MeO
MeO
MeO
H
MeO
H
MeO
H
MeO
MeO
MeO
MeO
H
51
69
42
54
45
20
43
47
55
31
50
38
27
31
20
–c)
–c)
–c)
–c)
35
9
96.3ꢀ7.7
93.0ꢀ6.5
96.0ꢀ5.0
88.6ꢀ4.8
103.1ꢀ6.7
97.4ꢀ8.7
88.2ꢀ18.5
94.5ꢀ6.2
93.3ꢀ5.1
93.2ꢀ6.8
87.3ꢀ7.2
97.3ꢀ6.4
90.9ꢀ5.3
5ea)
5fa)
5ga)
5ha)
5i
–
–
–
–
H
MeO
MeO
H
H
MeO
NO2
NO2
NO2
105.0ꢀ6.7
103.7ꢀ4.6
108.7ꢀ4.8
5j
5k
Cl
Cl
MeO
H
31
a) The (E)- and (Z)-isomers of compounds 5e–5h could not be separated by column chromatography.
Pure (E)-isomers were obtained by short reaction times, whereas the (Z)-isomers could not be obtained
in pure form (see Exper. Part for details). b) Residual CM activity relative to the enzymatic reaction
without added compounds; see the footnote of Table 1. c) Not isolated.
experiments for the major isomer of 5a revealed that the compound is the (E)-isomer
based on the correlation that was observed between HꢁC(6’/2’) and HꢁC(6’’)
(Scheme 3). The signal for the C¼C bond H-atom (HꢁC(3)) was shifted to significantly
higher ppm values in the (E)-isomers of the entire a-phenylcinnamic acid series [38]. If
available, the pure (E)- and (Z)-stereoisomers were separately subjected to enzymatic
assays (Table 2).
Finally, another compound identified as being active by Agrawal et al. [34] was
synthesized as shown in Scheme 4, following literature procedures. Installation of a
methyl ester in 12 [39] and subsequent nucleophilic aromatic substitution led to the
methylamino derivative 7 that could be obtained with an overall yield of 88% [40].
Scheme 4. Synthesis of Methyl 4-(Methylamino)-3-nitrobenzoate (7)
a) 3.1 equiv. SOCl2, MeOH, reflux, 4 h; 93%. b) 13 equiv. MeNH2 (40% soln. in H2O), 2 equiv. K2CO3,
r.t., 15 h; 95%.
Structure elucidation of all isolated compounds by using multiple 1D and 2D-NMR
experiments, and MS methods as well as elemental analysis of representative molecules
of each series left no doubt that we had exactly the hit compounds at hand that were
reported by Agrawal et al. [34].
Molecular Modeling. In parallel to the classical medicinal-chemistry approach, we
set out to explore the chemical space around the reference structures 4a, 5a, 6, and 7

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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(Fig. 1) by virtual screening investigations similar to those already reported by Agrawal
et al. [34]. Starting from the same apo *MtCM structure (PDB ID: 2F6L, [29])4) and
using the extended binding site definition as described in the Exper. Part, we docked the
four reportedly active (Fig. 1) and eleven inactive compounds of Agrawal et al. [34].
While compound 4a indeed emerged as the top performer regarding its scoring function
value, none of the tested compounds bound in a manner characteristic of a well-binding
ligand. For instance, some highly polar chemical groups are unmatched on the receptor.
Such binding modes are not commonly observed for good inhibitors, such as 3 in
complex with *MtCM (PDB: 2FP2, [32]). This being said, our docking models are at
least more realistic than those illustrated by Agrawal et al. [34], which are definitely not
meaningful. For example, in Figure 8 of [34], compound 4a features a carboxylate
group juxtaposed to the carboxylate of Glu109, and, in the second illustrated example,
compound 5a appears to ignore a large solvated area of the binding site, including
residues Glu106 (mislabeled as Glu105) and Glu109, which are crucial for ligand
binding in the active site of *MtCM [32]. Our own docking calculations, exemplified
with 4a in Fig. 2, predicted more extensive Van der Waals contacts to the *MtCM
surface. In addition, the electrostatic interactions were improved, with the sulfonamide
group of 4a next to Glu106, and the carboxylate forming a salt bridge with Arg49.
Nevertheless, while there were no obvious violations of basic bimolecular association
rules, these interactions are still suboptimal. In summary, none of the compounds
reported in the present publication and in the past Agrawal et al. report [34] exhibited a
particularly attractive binding mode to the apo *MtCM target conformation.
Protein Crystallography. The newly synthesized compound 4a and analogs were
subjected to soaking experiments with the target enzyme to structurally investigate
possible binding sites. The potential inhibitor molecules were added as solids to the
drop containing the *MtCM protein crystals and equilibrated overnight or soaked in
the cryoprotectant solution containing the potential inhibitors at 5-mm concentration
(1:5 molar ratio of *MtCM to inhibitor). Data sets were collected for different crystals
and the *MtCM structures were solved for soaks with 4a (1.45 ꢄ), 4l (1.45 ꢄ), 4m
(1.70 ꢄ), and 4k (1.50 ꢄ). However, even though we applied similar soaking conditions
as for the *MtCM complex structure with the transition-state analog 3 [32][41], and the
crystals diffracted to high resolution, the refined structures showed no electron density
for any of the new inhibitors. Acetate from the buffer solution was the only compound
found to be present in the obtained *MtCM crystal structures.
Enzyme Activity Assays. To assess the inhibitory effects of the synthesized
sulfamoyl-benzoic acids 4a–4m, the a-phenylcinnamic acids 5a–5k, and the compound
7, a discontinuous (stop) assay for chorismate mutase activity was implemented to
rapidly evaluate the potential of the synthesized compounds. The initial rate of the
*MtCM-catalyzed conversion of chorismate to prephenate in the absence and presence
of the potential inhibitors was determined. The compounds were dissolved in DMSO to
10 mm and investigated at a final concentration of 100 mm in the enzyme assay (75 mm
chorismate, 2 nm *MtCM). Product formation was followed by quantitative acidic
4
)
We used the apo *MtCM structure solved by Kim et al. [29] rather than our own structure (PDB ID:
2FP1) [32], even though the former was solved at slightly lower resolution (1.70 vs. 1.55 ꢄ), in order
to stay as close as possible to the protocol used by Agrawal et al. [34].

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Fig. 2. Docking model for the previously claimed lead compound 4a. A model of *MtCM interacting with
ligand 4a was generated by GOLD v3.2 as described in the Exper. Part. It was not further refined to
account for ligand-induced changes; thus, *MtCM atom coordinates still correspond to the input crystal
structure (PDB ID: 2F6L). a) 3D Snapshot prepared with VMD [60]. The protein backbone in cartoon
representation (black) shows the secondary structure of *MtCM (N and C termini labeled), whereas a
surface representation was chosen to emphasize the residues that were explicitly used to define the
receptor binding site of *MtCM for GOLD (different colors indicate different contiguous sequence
stretches). b) 2D Schematic representation generated with PoseView [61] depicting the major predicted
proteinꢁligand interactions. H-Bonding distances in ꢄ. The indicated scoring function value is related to
GOLD modeling approximations upon exploring the conformational landscape of 4a in the *MtCM
receptor environment; it is strongly target-dependent and should never be correlated to Ki values.
Independent docking calculations favored the same binding mode shown here for 4a also for analogs 4b–
4m, with comparable scoring function (Goldscore) values. Note that a docking approach can only predict
a most-probable binding mode for a given receptorꢁligand pair, assuming binding actually occurs.
However, it possesses very limited predictive power regarding the experimental potency of ligand
binding to the chosen target.
conversion of prephenate into phenylpyruvic acid and determining the absorbance of
the latter at 320 nm under basic conditions. For comparison, the reaction was also
carried out in the presence of the oxabicyclic dicarboxylic acid 3, which is currently the
most potent inhibitor of *MtCM (Ki 3.7 mm) [28]. When 3 was provided at a final
concentration of 11.1 mm, the catalytic activity of *MtCM was lowered to 29ꢀ1%
(activity in the absence of 3 corresponds to 100ꢀ1%). The activities measured in the

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2515
presence of the synthesized compounds tested ranged from 87ꢀ7% to 114ꢀ6%
(Tables 1 and 2). For the reported lead compounds, the remaining catalytic activity of
*MtCM was 100ꢀ4% for 4a, 90ꢀ3% for 5a, and 103ꢀ5% for 7. Thus, contrary to the
published results [34], none of the claimed lead compounds showed any inhibitory
effect on *MtCM, and neither did any of the various analogs.
For an independent confirmation of the results from the stop assay, the newly
synthesized compound 4a was tested in a direct and continuous kinetic chorismate
mutase assay, whereby the consumption of the substrate chorismate by *MtCM is
observed spectrophotometrically. The assay setup and conditions were similar to those
applied by Agrawal et al. [34], except that these authors used a surprisingly high
*MtCM concentration (100 nm, and thus 50-fold higher than the concentration we
used), which, according to our experiments, would make it difficult to observe
meaningful initial velocities of chorismate consumption. Kinetic parameters from the
conversion of chorismate to prephenate were determined in the presence of 4a at
different concentrations (Fig. 3,a). We found that the kcat and Km values were not
Fig. 3. Determination of the inhibitory effect of compound 4a on *MtCM activity. a) Curve fits using the
MichaelisꢁMenten equation to correlate initial velocities of chorismate consumption by the secreted
chorismate mutase from M. tuberculosis (*MtCM) with the chorismate concentration. Initial velocities
(v0) were normalized by the *MtCM concentration in the assay. Individual curves (color coded as
indicated below) were obtained in the presence of different concentrations of 4a, ranging from 0 mm
(open red circles), 1 mm (open blue squares), 5 mm (open green diamonds), 15 mm (black crosses), 25 mm
(pink plus signs) to 50 mm (open cyan triangles). The inset of panel a) shows a simulation of expected
curves (broken lines) for theoretical Ki values of 5.7 mm (filled black squares) and 30 mm (filled gray
diamonds) at a concentration of 50 mm of a competitive inhibitor. Filled red circles show the simulated
curve if no inhibitor is present. b) LineweaverꢁBurk plot of the experimental data and the simulations
from a), using symbols and colors as defined above. For the inset in b, Km,app derived from the linear
regression analysis of all experimental values was plotted against the inhibitor concentration (filled red
circles). The linear fit through these points (solid red line) does not yield a meaningful Ki value for 4a.
For comparison, the simulated curves (broken lines) for theoretical Ki values of 5.6 and 30 mm are shown
with filled black squares and gray diamonds, respectively.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
significantly affected by the addition of 4a. Irrespective of the presence or absence of
4a, we observed for kcat 32.3ꢀ1.1 sꢁ1 and for Km 20.3ꢀ1.5 mm, which is within a factor of
two of previously published values for a comparable *MtCM variant upon interpo-
lation to pH 6.5 [28]. Indeed, a LineweaverꢁBurk analysis of the experimentally
obtained data and plotting Km,app vs. the concentration of 4a did not yield a meaningful
Ki value (Fig. 3,b). This is in stark contrast to the previously published Ki value of
5.7 mm for this compound [34].
Conclusions. – In a previous publication [34], the discovery of four active
compounds against the secreted enzyme *MtCM from the pathogen Mtb was reported.
As one of few successes of an in silico drug-finding strategy against TB, this article did
not go unnoticed [42–44]. The initial goal of the work described here was to use those
prior results as a starting point and an inspiration for the discovery of additional active
compounds. We chose a traditional medicinal-chemistry approach supported by
molecular docking in order to determine precise structureꢁactivity relationships,
aiming at ultimately discovering novel lead compounds with a better pharmacokinetic
(more drug-like) profile than 4a, 5a, 6, and 7.
The final results of our in silico studies drastically differed from those reported by
Agrawal et al. [34], despite comparable input data and a similar docking strategy. We
conclude from a critical analysis of both our own data and the report of Agrawal et al.
that the structural models from the latter study are not reliable. We first assumed that
the discovery of active compounds guided by such flawed modeling could have been the
result of chance. However, our subsequent rigorous activity testing of three of the four
compounds identified by Agrawal et al., i.e., 4a, 5a, and 7, revealed that they are actually
inactive. Given all the shortcomings identified, one may start wondering whether the
compounds assayed in the previous study were really the ones they showed, given that
the characterization of those compounds is not documented.
Independent of our own investigation, another group pursued a similar computer-
aided medicinal-chemistry investigation for *MtCM inhibitors [45] based on the claims
by Agrawal et al. [34]. Using repeated cycles of fully-flexible docking combined with
molecular-dynamics simulations, Sikdar [45] identified *MtCM conformations that
provided qualitative agreement with the reported affinity measurements. This more
complex modeling approach was obviously biased for discovering analogs of 4a, 5a, 6,
and 7, and its predictive capability critically depends on the actual experimental
potency of those compounds. Like us, Sikdar used modeling to guide the synthesis of
further inhibitor candidates; however, the newly synthesized compounds were
subjected to biological in vivo assays rather than to enzyme inhibition studies in vitro.
In any case, those experimental results turned out to be equally disappointing [45].
While totally unrealistic ligand binding modes can sometimes be found in published
proteinꢁligand X-ray structures [46–48], this problem is seen even more frequently in
computer-generated models of proteinꢁligand complexes. In fact, such illustrations,
which are often encountered in medicinal-chemistry journals, appear to be more like
the graphical equivalent of the astronomerꢁs ꢀartist renderingsꢁ than chemically
meaningful representations. We assume these cases to be mostly the result of a
combination of good intent (providing a structural interpretation of experimental
data), lack of expertise, the desire to highlight rational design over serendipity, and

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2517
confirmation bias. The latter is certainly one of molecular modelerꢁs worst bad habits
[49].
Our discovery that none of the three resynthesized compounds previously touted to
be good inhibitors of *MtCM showed any inhibitory activity, when subjected to
thorough enzymatic characterization, stands in glaring contrast to the previously
published results. Our findings, which uncovered obvious flaws in computer modeling
and experimental protocols, once more caution against trusting too uncritically the
shoulders on which we choose to stand.
We thank Dr. Rosalino Pulido for the synthesis of inhibitor 3 and the ESRF, Grenoble, for beamtime
allocation. Dirk Petersen from the NMR center, University of Oslo, performed selective NOESY
experiments. We are further grateful to the Theoretical and Computational Biophysics group at the
University of Illinois, Urbana-Champaign for providing free access to the VMD software (http://
www.ks.uiuc.edu/Research/vmd/) and the ZBH Center for Bioinformatics at the University of Hamburg,
Germany, for the availability of the PoseViewWeb server (http://poseview.zbh.uni-hamburg.de/
poseview/wizard). This study was supported by funds from the University of Oslo, the Swiss National
Science Foundation (grant no. 3100A0-116475), and the ETH Zurich.
Experimental Part
General. Chorismic acid was produced and isolated as described in [50]. The transition-state analog 3
was synthesized by R. Pulido according to a published procedure [51]. Abbreviations: BSA, bovine serum
albumin; PEG, polyethylene glycol. Reagents that were used in the synthesis were purchased from Sigma
Aldrich. Solvents were dried and distilled according to literature procedures before use. Dry CH2Cl2 and
DMF were obtained from an MBraun SPS-800 Compact solvent purification system. 2-Nitro-3,4-
dimethoxybenzaldehyde [52], 2-nitro-5-methoxybenzaldehyde [53], 2-nitro-4-methoxybenzaldehyde
[54], 2-(3,4-dimethoxyphenyl)-N-methylethanamine [55], and methyl 4-fluoro-3-nitrobenzoate (13) [39]
were synthesized according to literature procedures. NMR Spectra of those compounds were in
agreement with the previously published ones. Flash chromatography (FC): silica gel 60 (0.040–
0.063 mm mesh; Merck). TLC: silica gel 60 F254 on aluminum (Merck). M.p.: Bꢀchi Melting Point B-545,
uncorrected. NMR Spectra5): Bruker DPX 300 and AVII 400 spectrometer; chemical shifts, d, in ppm for
1H and 13C, coupling constants (J) in Hz. MS-EI: Waters (Micromass) Prospec Q instrument, 70 eV
ionizing voltage, in m/z (%). MS-ESI: Bruker Apex 47e FT-ICR mass spectrometer. Elemental analyses
were performed by Lianne Hill, School of Chemistry, University of Birmingham.
Computer Docking Experiments. Docking calculations [56][57] were performed using GOLD
version 3.2 [58]. GOLD is a semi-flexible docking program: the conformational space of the docked
compounds is fully explored by a genetic algorithm, while the target active site is kept rigid (except that
targeted terminal NꢁH bonds are allowed to rotate as to provide H-bonds). The Goldscore scoring
function was used with the ꢀrelative ligand energyꢁ option turned on. The genetic algorithm parameters
are automatically adjusted as a function of each input moleculeꢁs number of rotatable bonds (autoscale,
1.0). A maximum of 50 docking runs were performed for each candidate compound, with calculations
being terminated as soon as the current top 5 solutions (according to the Goldscore value) converged to
an equivalent conformational state (defined as root-mean-square deviation<1 ꢄ). All other GOLD
parameters were kept to the default values. The enzyme active site was defined as consisting of the
following *MtCM residues: 48–50, 55–57, 59–61, 65–77, 101–110, and 133–135. The binding region for
docking was thereby significantly larger compared to both the transition-state analog (3)-bound catalytic
site structures [32] and previously reported protocols [34], allowing i) to accommodate the conforma-
tional relaxation of the catalytic site in the absence of the transition-state analog 3, and ii) to catch
possible alternative binding modes involving residues that are not part of the catalytic site, but accessible
5
)
For assignments, the C-atom numberings indicated in Schemes 2 and 3 were used.

2518
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
in this relaxed unbound state. No automated binding site definition algorithm operating from a central
coordinate was used, as those are particularly inaccurate except for well-defined, closed cavities.
Modeling of the Docking Target *MtCM Structure. As a target structure, we selected the apo *MtCM
structure with the PDB-ID 2F6L [29], matching the Agrawal et al. protocol [34] that was initially
considered as a successful reference. There are other Mtb CM crystal structures available in the PDB; the
preparation of these for structure-based computational investigations would be similar. The H-atoms
(not resolved at the resolution reported for the published structures) were added to their most probable
positions at pH 7. The protonation state of histidines was set arbitrarily, which is acceptable considering
that no His is present in the active sites, thus having no effect on ligand-docking calculations. Water
molecules present in the X-ray crystal structures were removed.
Generation of Ligand Candidates. For each candidate compound, a low-energy conformational state
suitable as input for semi-flexible docking was generated using CORINA [59]. Stereoisomers and
compounds having multiple possible ring and double-bond configurations led to separate entries. In
addition to the corresponding CORINA option, an in-house program was used to ensure that the atom
and bond types in the MOL2 file format were conforming to some non-standard GOLD-specific
requirements, as described extensively in the GOLD manual.
Analysis of Docking Results. Docking poses were investigated using VMD [60] and PoseView [61].
The former program was used to manipulate the 3D proteinꢁligand models, with the help of 2D
schematic representations generated by the latter.
Protein Production and Purification. The C-terminally (His)6-tagged *MtCM was overproduced in
Escherichia coli strain KA29 carrying plasmid pKTU3-HCT. The protein was subsequently purified by
Ni-NTA affinity chromatography as described in [28]. As far as we can tell, the *MtCM used in this study
has the same format (i.e., including the C-terminal (His)6 tag) as the one used by Agrawal et al. [34].
Crystallization and Structure Determination. *MtCM Crystals were obtained as described in [32]
using PEG 4000 as precipitant (10% (w/v)) in 0.2m AcONa and 0.1m Na-cacodylate buffer, pH 6.0. The
crystals were either soaked in 5 mm solns. of the synthesized compounds in the cryoprotectant (30% (v/v)
PEG 400), or the compounds were added to the drop containing *MtCM crystals and equilibrated
overnight. Cryoprotected crystals were flash-frozen in liquid N2. Data sets could be obtained for *MtCM
soaked with 4a (1.45 ꢄ), 4l (1.45 ꢄ), 4m (1.7 ꢄ), and 4k (1.5 ꢄ) added as solids. Crystallographic data
were collected on beamline ID23-2 at the ESRF, Grenoble [62], processed with iMosflm [63] and scaled
with Scala [64]. The structures were found to be isomorphous to the published apo structure [PDB ID:
2FP1][32], which was used as a starting model. Rigid-body and restrained refinement were carried out
with Refmac5 [65] and model building with Coot [66]. Fo ꢁFo maps were calculated after first scaling the
observed intensities to those from the published apo structure and using the calculated phases from the
latter. This was carried out using FHSCALE and FFT of the CCP4 program suite [67].
Discontinuous Chorismate Mutase Assay (Stop Assay). Initial rates of the enzyme-catalyzed
conversion of chorismate to prephenate were determined. Reactions were carried out in a volume of
500 ml in 50 mm potassium phosphate, pH 7.5, 75 mm chorismate, 2 nm *MtCM, 0.1 mg/ml BSA, and
0.005% Tween 20. The synthesized inhibitor candidates were dissolved in DMSO to 10 mm, added to the
assay solns. (100 mm final concentration in the assay), and the mixture was incubated at 308 in a
thermomixer at 550 rpm. Individual reactions were stopped after 3.5, 6.5, or 10 min by addition of 50 ml
5m HCl, and incubation was continued for 10 min at 308 to ensure quantitative conversion of prephenate
to phenylpyruvic acid. Thereafter, 50 ml 10m NaOH were added to deprotonate the phenylpyruvic acid to
phenylpyruvate. The amount of prephenate formed from chorismate could thus be monitored by
measuring the absorption of phenylpyruvate at 320 nm (e320 ¼17,500 mꢁ1 cmꢁ1). Samples quenched at
0 min incubation yielded blank absorbance values to be subtracted from the absorbance data obtained at
the different incubation time points. Initial reaction rates were derived from the slope of the resulting
graph (A320nm vs. time) and corrected by subtracting the slope of the uncatalyzed rearrangement reaction
occurring at 308. The slope and standard deviation of the resulting curve were determined. The initial
catalytic velocities (v0) were independently determined at least in duplicates (up to ten measurements for
selected compounds) for each of the inhibitor candidates. For each kinetic run with a given compound, an
assay without the inhibitor was carried out as well. A direct comparison of the corresponding v0 values
yielded the percentage of residual activity (the activity of the control measurement without compounds

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2519
added was set to 100%). For the duplicate measurements, the error was obtained as the square root of the
pooled variances of the curve fits from the linear regression analysis of each measurement
ꢀ
ꢁ
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
s ¼ ðS s2Þ=n . When additional kinetic runs were performed, the error was obtained as the
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
n
i
2
ꢀ
standard deviation of the mean, according to s ¼ Snðxi ꢁ xÞ =n ꢁ 1. As a positive control, the reaction
was also carried out in the presence of the well-established transition state analog inhibitor 3 at a final
concentration of 11.1 mm [26][28].
Continuous Chorismate Mutase (CM) Assay. Compound 4a was dissolved in 50 mm BTP (¼1,3-
bis[tris(hydroxymethyl)methylamino]propane), pH 7.5, to yield a stock soln. with a concentration of
0.8 mm. Continuous CM assays were performed essentially as described in [28]. Specifically, we recorded
the disappearance of chorismate at 274 nm (e274 ¼2630 mꢁ1 cmꢁ1) at 308 in 50 mm potassium phosphate
buffer, pH 6.5, in the presence of 2 nm *MtCM. Initial velocities were always corrected for the
spontaneous background reaction at 308 (kuncat ¼1.2 · 10ꢁ5 sꢁ1). The concentration of the presumed
inhibitor 4a was varied from 0 to 50 mm. The data were fitted to the MichaelisꢁMenten equation (v0 ¼kcat
·
[E]0 ·[S]/(Km þ[S])) with the program KaleidaGraph (Synergy Software). Km,app. was determined from
the x-axis intercept of the 1/v0 vs. 1/[S] LineweaverꢁBurk plot. By replotting Km,app. against the inhibitor
concentration, ꢁKi can be read from the (negative) x-axis intercept [68].
Syntheses. General Procedure for the Syntheses of [(2-Phenylethyl)amino]-sulfamoylbenzoic Acids
4a–4f. A mixture of 4-chloro-3-nitro-5-sulfamoylbenzoic acid (9; 1.40 g, 5.00 mmol) and the appropriate
amine 8 (15.0 mmol, 3.00 equiv.) in H2O (15 ml) was refluxed for 2.5 h. After cooling to r.t., the mixture
was strongly alkalinized through addition of 2m NaOH (pH ca. 11), and the aq. layer was extracted with
Et2O (3ꢂ50 ml). Subsequently, the crude product was precipitated via addition of 2m HCl (pH ca. 2).
The resulting solids were collected and purified through recrystallization from EtOH.
4-{[2-(3,4-Dimethoxyphenyl)ethyl]amino}-3-nitro-5-sulfamoylbenzoic Acid (4a). Yield: 1.71 g
(4.02ꢂ10ꢁ3 mol, 80%). Dark yellow fibroid solid. M.p. 234.8–235.48. 1H-NMR (300 MHz, (D6)DMSO):
2.81 (t, J¼7.0, CH2(8)); 3.13 (dt, J¼7.0, 5.5, CH2(7)); 3.69 (s, MeO); 3.72 (s, MeO); 6.73 (dd, J¼8.1, 1.7,
HꢁC(6’)); 6.83 (d, J¼8.1, HꢁC(5’)); 6.84 (d, J¼1.7, HꢁC(2’)); 7.08 (t, J¼5.5, NH); 7.94 (s, H2NSO2); 8.30
(d, J¼2.1, HꢁC(2) or HꢁC(6)); 8.44 (d, J¼2.1, HꢁC(2) or HꢁC(6)); 13.23 (br. s, COOH). 13C-NMR
(75 MHz, (D6)DMSO): 164.98 (COOH); 148.66 (C(3’)); 147.50 (C(4’)); 142.35 (C(4)); 136.64 (C(3)/
C(5)); 133.11 (C(6)/C(2)); 131.29 (C(6)/C(2)); 130.43 (C(3)/C(5)); 130.32 (C(1’)); 120.61 (C(6’)); 116.80
(C(1)); 112.65 (C(2’)); 112.04 (C(5’)); 55.48 (MeO); 55.27 (MeO); 48.23 (C(7)); 34.92 (C(8)). EI-MS:
425 (9, Mþ ), 274 (5), 257 (5), 245 (6), 152 (32), 151 (100), 107 (6). HR-EI-MS: 425.0892 (Mþ,
C17H19N3O8Sþ ; calc. 425.0893).
3-Nitro-4-[(2-phenylethyl)amino]-5-sulfamoylbenzoic Acid (4b). Yield: 1.54 g (4.23ꢂ10ꢁ3 mol,
85%). Yellow fibroid solid. M.p. 207.6–208.18 ([35]: 208.0–208.58). 1H-NMR (300 MHz, (D6)DMSO):
2.88 (t, J¼6.8, CH2(8)); 3.16 (dt, J¼7.3, 6.8, CH2(7)); 7.11 (t, J¼4.9, NH); 7.18–7.31 (m, 5 arom. H); 7.94
(s, H2NSO2); 8.32 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.46 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 13.24 (br. s, COOH).
13C-NMR (75 MHz, (D6)DMSO): 165.02 (COOH); 142.26 (C(4)); 138.05 (C(1’)); 136.76 (C(5)/C(3));
133.20 (C(6)/C(2)); 131.34 (C(6)/C(2)); 130.41 (C(5)/C(3)); 128.76 (2 Ph-C); 128.56 (2 Ph-C); 126.60
(C(4’)); 116.97 (C(1), 47.93 (C(7)); 35.29 (C(8)). EI-MS: 365 (5, Mþ ), 275 (16), 274 (100), 257 (50), 245
(56), 181 (13), 165 (12), 153 (11), 92 (13), 91 (80), 77 (14), 65 (13). HR-EI-MS: 365.0674 (Mþ,
C15H15N3O6Sþ ; calc. 365.0682).
4-{[2-(3-Methoxyphenyl)ethyl]amino}-3-nitro-5-sulfamoylbenzoic Acid (4c). Yield: 1.54 g (3.90ꢂ
10ꢁ3 mol, 78%). Yellow fibroid solid. M.p. 199.3–200.28 ([45]: 195.1–199.88). 1H-NMR (300 MHz,
(D6)DMSO) 2.85 (t, J¼7.5, CH2(8)); 3.15 (dt, J¼7.5, 5.5, CH2(7)); 3.72 (s, MeO); 6.76–6.81 (m,
HꢁC(2’), HꢁC(4’), HꢁC(6’)); 7.08 (t, J¼5.1, NH); 7.19 (t, J¼8.1, HꢁC(5’)); 7.93 (s, H2NSO2); 8.31 (d, J¼
2.1, HꢁC(2)/HꢁC(6)); 8.44 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 13.23 (br. s, COOH). 13C-NMR (75 MHz,
(D6)DMSO): 164.93 (COOH); 159.31 (C(3’)); 142.22 (C(4)); 139.57 (C(1’)); 136.67 (C(5)/C(3)); 133.09
(C(6)/C(2)); 131.25 (C(6)/C(2)); 130.36 (C(5)/C(3)); 129.53 (C(5’)); 120.84 (C(6’)); 116.86 (C(1)); 114.26
(C(2’)); 112.17 (C(4’)); 54.85 (MeO); 47.86 (C(7)); 35.30 (C(8)). EI-MS: 395 (17, Mþ ), 275 (17), 274
(100), 257 (58), 245 (70), 197 (10), 181 (17), 165 (11), 153 (13), 135 (10), 122 (62), 121 (100), 92 (10), 91
(38), 78 (18), 77 (20), 65 (15). HR-EI-MS: 395.0780 (Mþ, C16H17N3O7Sþ ; calc. 395.0787). Anal calc. for
C16H17N3O7S (395.39): C 48.60, H 4.33, N 10.63; found: C 48.72, H 3.99, N 10.77.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
4-{[2-(4-Methoxyphenyl)ethyl]amino}-3-nitro-5-sulfamoylbenzoic Acid (4d). Yield: 1.71 g (4.32ꢂ
10ꢁ3 mol, 87%). Yellow fibroid solid. M.p. 234.4–234.98. 1H-NMR (300 MHz, (D6)DMSO) 2.81 (t, J¼
7.2, CH2(8)); 3.10 (dt, J¼7.2, 5.5,CH2(7)); 3.70 (s, MeO); 6.83–6.86 (m, HꢁC(2’), HꢁC(6’)); 7.08 (t, J¼
5.5, NH); 7.13–7.16 (m, HꢁC(3’), HꢁC(5’)); 7.93 (s, H2NSO2); 8.31 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.44 (d,
J¼2.1, HꢁC(2)/HꢁC(6)); 13.23 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 164.96 (COOH);
158.00 (C(4’)); 142.22 (C(4)); 136.66 (C(5)/C(3)); 133.11 (C(6)/C(C2)); 131.27 (C(6)/C(C2)); 130.29
(C(5)/C(3)); 129.80 (C(1’)); 129.73 (C(2’), C(6’)); 116.81 (C(1)); 113.95 (C(3’), C(5’)); 54.97 (MeO);
48.11 (C(7)); 34.37 (C(8)). EI-MS: 395 (3, Mþ ), 274 (11), 257 (8), 245 (10), 122 (37), 121 (100), 91 (7),
78 (6), 77 (8). HR-EI-MS: 395.0779 (Mþ, C16H17N3O7Sþ ; calc. 395.0787).
4-{[2-(2-Methoxyphenyl)ethyl]amino}-3-nitro-5-sulfamoylbenzoic Acid (4e). Yield: 1.49 g (3.77ꢂ
10ꢁ3 mol, 75%). Yellow crystalline powder. M.p. 221.9–222.48. 1H-NMR (300 MHz, (D6)DMSO): 2.86
(t, J¼7.7, CH2(8)); 3.12 (dt, J¼7.7, 5.3, CH2(7)); 3.76 (s, MeO); 6.84 (dt, J¼7.3, 1.1, HꢁC(5’)); 6.94 (d, J¼
7.7, HꢁC(3’)); 7.07 (t, J¼5.1, NH); 7.14 (dd, J¼7.3, 1.7, HꢁC(6’)); 7.20 (dt, J¼7.3, 1.7, HꢁC(4’)); 7.94 (s,
H2NSO2); 8.30 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.44 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 13.22 (br. s, COOH).
13C-NMR (75 MHz, (D6)DMSO): 164.98 (COOH); 157.15 (C(2’)); 142.34 (C(4)); 136.67 (C(3)/C(5));
133.11 (C(6)/C(2)); 131.30 (C(6)/C(2)); 130.32 (C(6’)); 128.14 (C(4’)); 125.58 (C(1’)); 120.37 (C(5’));
116.78 (C(1)); 110.73 (C(3’)); 55.22 (MeO); 46.19 (C(7)); 30.29 (C(8)). ESI-MS: 396 (100, [MþH]þ ),
418 (8, [MþNa]þ ). HR-EI-MS: 395.0780 (Mþ, C16H17N3O7Sþ ; calc. 395.0787). Anal calc. for
C16H17N3O7S (395.39): C 48.60, H 4.33, N 10.63; found: C 48.91, H 4.06, N 10.83.
4-{[2-(3-Chlorophenyl)ethyl]amino}-3-nitro-5-sulfamoylbenzoic Acid (4f). Yield: 1.19 g (2.99ꢂ
10ꢁ3 mol, 60%). Yellow crystalline powder. M.p. 205.5–206.18. 1H-NMR (300 MHz, (D6)DMSO): 2.89
(t, J¼7.5, CH2(8)); 3.17 (dt, J¼7.5, 5.5, CH2(7)); 7.06 (t, J¼5.3, NH); 7.17–7.33 (m, HꢁC(2’), HꢁC(4’),
HꢁC(5’), HꢁC(6’)); 7.95 (s, H2NSO2); 8.31 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.45 (d, J¼2.1, HꢁC(2)/
HꢁC(6)); 13.24 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 164.93 (COOH); 142.24 (C(4));
140.59 (C(1’)); 136.72 (C(5)/C(3)); 133.13 (C(3’)); 133.07 (C(6)/C(2)) 131.26 (C(6)/C(2)); 130.45 (C(5)/
C(3)); 130.30 (C(5’)/C(4’)); 128.67 (C(6’)/C(2’)); 127.52 (C(5’)/C(4’)); 126.57 (C(6’)/C(2’)); 117.03 (C(1));
47.57 (C(7)); 34.82 (C(8)). ESI-MS: 400 (100, [MþH]þ ), 422 (4, [MþNa]þ ). HR-EI-MS: 399.02931
(Mþ, C15H14ClN3O6Sþ ; calc. 399.02918). Anal calc. for C15H14ClN3O6S (399.81): C 45.06, H 3.53, N 10.51;
found: C 45.34, H 3.21, N 10.71.
4-{[2-(3-Fluorophenyl)ethyl]amino}-3-nitro-5-sulfamoylbenzoic Acid (4g). 4-Chloro-3-nitro-5-sulfa-
moylbenzoic acid (9; 500 mg, 1.78 mmol) and 3-fluorophenethylamine (¼ N-ethyl-3-fluoroaniline;
5.34 mmol, 0.70 ml, 3.00 equiv.) were refluxed in H2O (5 ml) for 2.5 h, and workup was performed as
described in the General Procedure. The crude product was purified through recrystallization from
1
CH2Cl2. Yield: 628 mg (1.64ꢂ10ꢁ3 mol, 91%). Yellow solid. M.p. 181.2–182.38. H-NMR (300 MHz,
(D6)DMSO): 2.91 (t, J¼7.5, CH2(8)); 3.18 (dt, J¼7.5, 5.7, CH2(7)); 6.09–7.09 (m, HꢁC(2’), HꢁC(4’),
HꢁC(6’), NH); 7.32 (dt, J¼7.9, 1.7, HꢁC(5’)); 7.96 (s, H2NSO2); 8.32 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.45 (d,
J¼2.1, HꢁC(6)/HꢁC(2)); 13.24 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 164.93 (COOH);
163.82 (C(3’)); 142.2 (C(4)); 140.88 (C(1’)); 136.73 (C(5)/C(3)); 133.15 (C(6)/C(2)); 131.29 (C(6)/C(2));
130.45 (C(5’)); 124.92 (C(6’)); 117.01 (C(1)); 115.42 (C(2’)); 113.25 (C(4’)); 47.58 (C(7)); 34.90 (C(8)).
ESI-MS: 384 (100, [MþH]þ ), 406 (6, [MþNa]þ ). HR-EI-MS: 383.05893 (Mþ, C15H14FN3O6Sþ ; calc.
383.05873). Anal. calc. for C15H14FN3O6S (383.35): C 47.00, H 3.68, N 10.96; found: C 47.17, H 3.64, N
10.76.
4-{[2-(3,4-Dimethoxyphenyl)ethyl](methyl)amino}-3-nitro-5-sulfamoylbenzoic Acid (4h). Com-
pound
9 (157 mg, 0.560 mmol, 1.00 equiv.) and 2-(3,4-dimethoxyphenyl)-N-methylethanamine
(328 mg, 1.68 mmol, 3.00 equiv.) were refluxed for 11 h in H2O (5 ml). The product was obtained
through extraction of the acidified aq. layer (pH ca. 2) with AcOEt (3ꢂ10 ml) and purified via
recrystallization from CH2Cl2. Yield: 230 mg (5.24ꢂ10ꢁ3 mol, 94%). Orange solid. M.p. 180.3–181.28.
1H-NMR (400 MHz, (D6)DMSO): 2.68–2.72 (m, CH2(8)); 2.90 (s, MeN); 3.15–3.19 (m, CH2(7)); 3.70
(s, MeO(4’)); 3.72 (s, MeO(3’)); 6.67 (dd, J¼8.1, 2.0, HꢁC(6’)); 6.78 (d, J¼2.1, HꢁC(2’)); 6.85 (d, J¼8.0,
HꢁC(5’)); 7.60 (s, H2NSO2); 8.40 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.70 (d, J¼2.1 HꢁC(2)/HꢁC(6)); 13.74
(COOH). 13C-NMR (101 MHz, DMSO): 164.55 (COOH); 149.51 (C(3)/C(5)); 148.65 (C(3’’)); 147.26
(C(4’’)); 145.91 (C(4)); 144.01 (C(3)/C(5)); 132.84 (C(2)/C(6)); 131.52 (C(1’)); 128.96 (C(2)/C(6));
127.08 (C(5)); 120.42 (C(6’’)); 112.50 (C(2’)); 112.00 (C(5’)); 57.39 (C(7)); 55.51 (MeO); 55.34 (MeO);

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2521
41.73 (MeN); 33.77 (C(8)). MS-EI: 439 (3, Mþ ), 290 (6), 289 (13), 288 (100), 151 (25). HR-EI-MS:
439.1058 (Mþ, C18H21N3O8Sþ ; calc. 439.1049).
4-(Benzylamino)-3-nitro-5-sulfamoylbenzoic Acid (4i). A mixture of 9 (1.00 g, 3.56 mmol) and
PhCH2NH2 (1.53 g, 14.3 mmol, 4.00 equiv.) in H2O (5 ml) were stirred for 3 h at r.t. After standing for
30 min, the H2O phase was decanted from the formed solids, which were subsequently dissolved in large
amounts of hot H2O. The resulting soln. was treated with 4m HCl to precipitate the crude product, which
was purified through recrystallization from MeOH. Yield: 663 mg (1.89ꢂ10ꢁ3 mol, 53%). Yellow solid.
M.p. 181.1–183.08 ([35]: 1888 (dec)). 1H-NMR (300 MHz, (D6)DMSO): 4.32 (d, J¼5.5, CH2(7)); 7.22–
7.33 (m, 5 arom. H); 7.43 (t, J¼5.5, NH); 8.06 (s, H2NSO2); 8.27 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.49 (d, J¼
2.1 HꢁC(2)/HꢁC(6)); 13.26 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 165.02 (COOH); 142.00
(C(5)); 136.94 (C(5)/C(3)); 136.72 (C(1’)); 133.18 (C(6’)/C(2’)); 131.35 (C(6’)/C(2’)); 130.95 (C(3)/
C(5)); 128.59 (2 Ph-C); 127.85 (2 Ph-C); 127.80 (C(4’)); 117.19 (C(1)); 49.62 (C(7)). ESI-MS: 350.1 (100,
[MꢁH]þ ). HR-EI-MS: 351.0527 (Mþ, C14H13N3O6Sþ ; calc. 351.0525).
4-[(3,4-Dimethoxybenzyl)amino]-3-nitro-5-sulfamoylbenzoic Acid (4j). Compound 9 (300 mg,
1.07 mmol) and 3,4-dimethoxybenzylamine (0.65 ml, 4.3 mmol, 4.0 equiv.) in H2O (2.5 ml) were stirred
overnight (ca. 15 h) at r.t. The formed solids were dissolved in large amounts of hot H2O and treated with
4m HCl, resp. The product was obtained through extraction with AcOEt (3ꢂ10 ml). Yield: 158 mg
(3.84ꢂ10ꢁ3 mol, 36%). Yellow solid. M.p. 180.4–181.38. 1H-NMR (300 MHz, (D6)DMSO): 3.65 (s,
MeO); 3.70 (s, MeO); 4.20 (d, J¼5.1, CH2(7)); 6.77 (dd, J¼8.1, 1.7, HꢁC(6’)); 6.85 (d, J¼8.1, HꢁC(5’));
6.88 (d, J¼1.7, HꢁC(2’)); 7.35 (t, J¼5.1, NH); 8.10 (s, H2NSO2); 8.28 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.48
(d, J¼2.1, HꢁC(2)/HꢁC(6)); 13.43 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 164.98 (COOH);
148.66 (C(4’)/C(3’)); 148.36 (C(4’)/C(3’)); 142.04 (C(4)); 136.92 (C(5)/C(3)); 133.07 (C(6)/C(2)); 131.36
(C(6)/C(2)); 131.02 (C(5)/C(3)); 128.80 (C(1’)); 120.39 (C(6’)); 117.12 (C(1)); 111.69 (C(6’)/C(2’));
111.52 (C(6’)/C(2’)); 55.42 (MeO); 55.33 (MeO); 49.48 (C(7)). EI-MS: 411 (24, Mþ ), 261 (30), 244 (13),
166 (16), 151 (100), 135 (6), 107 (7), 78 (5). HR-EI-MS: 411.0731 (Mþ, C16H17N3O8Sþ ; calc. 411.0736).
4-[(3-Methoxybenzyl)amino]-3-nitro-5-sulfamoylbenzoic Acid (4k). A mixture of 9 (500 mg,
1.78 mmol) and 3-methoxybenzylamine (0.93 ml, 7.1 mmol, 4.0 equiv.) in H2O (2.5 ml) was stirred
overnight (ca. 15 h) at r.t. The formed solid product was dissolved in hot H2O and treated with 4m HCl,
resp. After cooling the soln. to 08, the precipitated crude product was collected and purified through
recrystallization from MeOH. Yield: 433 mg (1.14ꢂ10ꢁ3 mol, 64%). Yellow fibroid solid. M.p. 200.9–
201.78. 1H-NMR (300 MHz, (D6)DMSO): 3.68 (s, MeO); 4.27 (d, J¼5.5, CH2(7)); 6.81–6.84 (m,
HꢁC(4’), HꢁC(6’)); 6.84 (s, HꢁC(2’)); 7.20 (t, J¼7.9, HꢁC(5’)); 7.40 (t, J¼5.1, NH); 8.04 (s, H2NSO2);
8.27 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.48 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 13.25 (br. s, COOH). 13C-NMR
(75 MHz, (D6)DMSO): 164.96 (COOH); 159.35 (C(3’)); 142.04 (C(4)); 138.33 (C(1’)); 136.95 (C(5)/
C(3)); 133.10 (C(6)/C(2)); 131.34 (C(6)/C(2)); 130.99 (C(3)/C(5)); 129.66 (C(5’)); 119.96 (C(6’)); 117.21
(C(1)); 113.52 (C(4’)/C(2’)); 113.18 (C(4’)/C(2’)); 54.98 (MeO); 49.55 (C(7)). ESI-MS: 380.1 (100, [Mꢁ
H]þ ). HR-EI-MS: 381.0619 (Mþ, C15H15N3O7Sþ ; calc. 381.0631). Anal. calc. for C15H15N3O7S: C 47.24, H
3.96, N 11.02; found: C 47.19, H 3.89, N 10.87.
4-[(4-Methoxybenzyl)amino]-3-nitro-5-sulfamoylbenzoic Acid (4l). Compound
9 (500 mg
1.78 mmol, 1.00 equiv.) and 4-methoxybenzylamine (0.90 ml 7.1 mmol, 4.0 equiv.) were combined in
H2O (2.5 ml) and stirred for 3 h at r.t. After diluting the resulting soln. with H2O (30 ml), 1m NaOH was
added (pH ca. 9), and the aq. phase was extracted with Et2O (3ꢂ10 ml). After acidification with 4m HCl,
the product was extracted with AcOEt (3ꢂ20 ml) and crystallized. Yield: 411 mg (1.08ꢂ10ꢁ3 mol,
61%). Yellow solid. M.p. 178.1–179.58. 1H-NMR (300 MHz, (D6)DMSO): 3.71 (s, MeO); 4.21 (d, J¼5.1,
CH2(7)); 6.83–6.86 (m, HꢁC(3’), HꢁC(5’)); 7.14–7.18 (m, HꢁC(2’), HꢁC(6’)); 7.36 (t, J¼5.5, NH); 8.01
(s, H2NSO2); 8.27 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 8.47 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 13.25 (br. s, COOH).
13C-NMR (75 MHz, (D6)DMSO): 164.96 (COOH); 158.83 (C(4’)); 141.92 (C(4)); 136.82 (C(5)/C(3));
133.11 (C(6)/C(2)); 131.33 (C(2)/C(6)); 130.85 (C(3)/C(5)); 129.37 (C(2’), C(5’)); 128.31 (C(1’)); 117.05
(C(1)); 113.96 (C(3’), C(5’)); 55.03 (MeO); 49.09 (C(7)). EI-MS: 381 (13, Mþ ), 261 (13), 136 (15), 135
(6), 122 (9), 121 (100), 78 (6), 77 (5). HR-EI-MS: 381.0634 (Mþ, C15H15N3O7Sþ ; calc. 381.0631).
4-[(2-Methoxybenzyl)amino]-3-nitro-5-sulfamoylbenzoic Acid (4m). A mixture of 9 (300 mg,
1.07 mmol) and 2-methoxybenzylamine (0.56 ml, 4.3 mmol, 4.0 equiv.) in H2O (2.5 ml) was stirred
overnight (ca. 15 h) at r.t. The formed solids were dissolved in large amounts of hot H2O and treated with

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2m NaOH after cooling to r.t. The soln. was extracted with Et2O (3ꢂ10 ml). The aq. layer was strongly
acidified with 4m HCl, and the crude product was obtained through extraction with AcOEt (3ꢂ20 ml)
and subsequently purified by flash CC to yield 4m (208 mg, 5.45ꢂ10ꢁ3 mol, 51%). Yellow solid. Rf
(AcOEt/MeOH 9 :1) 0.30. M.p. 237.3–239.88 (dec.). 1H-NMR (300 MHz, (D6)DMSO): 3.72 (s, MeO);
4.16 (d, J¼5.5, CH2(7)); 6.88 (dt, J¼7.5, 0.9, HꢁC(5’)); 6.99 (dd, J¼8.1, 0.9, HꢁC(3’)); 7.19 (dd, J¼7.5,
1.5, HꢁC(6’)); 7.28 (dt, J¼8.1, 1.5, HꢁC(4’)) 7.37 (t, J¼5.5, NH) 7.93 (s, H2NSO2); 8.31 (d, J¼2.1, HꢁC(2)/
HꢁC(6)); 8.47 (d, J¼2.1, HꢁC(2)/HꢁC(6)); 13.26 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO):
165.74 (COOH); 157.20 (C(2’)); 142.26 (C(4)); 136.72 (C(5)/C(3)); 133.36 (C(6)/C(2)); 131.11 (C(6)/
C(2)); 130.52 (C(3)/C(5)); 129.08 (C(4’)); 124.42 (C(1’)); 119.37 (C(1)); 110.98 (C(3’)); 55.34 (MeO);
45.77 (C(7)). ESI-MS: 380 (100, [MꢁH]þ ). HR-EI-MS: 381.0641 (Mþ, C15H15N3O7Sþ ; calc. 381.0631).
General Procedure for the Synthesis of 2,3-Diphenylacrylacetic Acids 5a–5d and 5i–5k. A mixture of
aldehyde 10 (200 mg) and of the appropriate 2-phenylacetic acid 11 (1.1 equiv.) in Ac2O (2 ml) and Et3N
(1 ml) was refluxed for 24 h. H2O was added, and the soln. was cooled to r.t. and extracted with CH2Cl2
(3ꢂ10 ml). The combined org. layers were washed with 0.1m NaOH (10 ml) and brine (10 ml), resp.
After drying (MgSO4), the crude products were concentrated in vacuo and purified by flash CC.
(2Z)-2-(4-Chlorophenyl)-3-(4,5-dimethoxy-2-nitrophenyl)prop-2-enoic Acid ((Z)-5a). Yield:
132 mg (3.63ꢂ10ꢁ4 mol, 38%). Yellow solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2% AcOH) 0.21. M.p. 193.9–
1
195.08. H-NMR (300 MHz, (D6)DMSO): 3.86 (s, Me(8’’)); 3.88 (s, Me(7’’)); 7.07 (s, HꢁC(6’’)); 7.46 (s,
HꢁC(3)); 7.48–7.56 (m, HꢁC(2’), HꢁC(3’), HꢁC(5’), HꢁC(6’)); 7.72 (s, HꢁC(3’’)); 13.29 (br. s, COOH).
13C-NMR (75 MHz, (D6)DMSO): 169.16 (COOH); 152.76 (C(5’’)); 148.20 (C(4’’)); 139.62 (C(2’’));
135.22, 135.08, 133.02, 129.02 (C(3)); 128.72 (C of C6H4Cl); 128.04 (C of C6H4Cl); 126.57 (C(1’)); 111.92
(C(6’’)); 107.76 (C(3’’)); 56.15 (MeO); 56.10 (MeO). EI-MS: 365/363 (3/8, Mþ ), 319 (37), 318 (28), 317
(100), 302 (13), 287 (10), 164 (33), 151 (10), 141 (24), 139 (76), 137 (5), 111 (11). HR-EI-MS: 363.0502
(Mþ, C17H14ClNOþ6 ; calc. 363.0510).
Isomer (E)-5a. Yield: 177 mg (4.87ꢂ10ꢁ4 mol, 51%). Yellow solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2%
AcOH) 0.44. M.p. 204.1–206.78. 1H-NMR (300 MHz, (D6)DMSO): 3.41 (s, Me(8’’)); 3.83 (s, MeC(7’’));
6.40 (s, HꢁC(6’’)); 7.10–7.15 (m, HꢁC(2’), HꢁC(6’)); 7.30–7.34 (m, HꢁC(5’), HꢁC(3’)); 7.63 (s,
HꢁC(3’’)); 8.06 (s, HꢁC(3)); 12.95 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 167.43 (COOH);
152.18 (C(5’’)); 148.25 (C(4’’)); 140.52 (C(2’’)); 138.05 (C(3)); 134.20 (C(1’)); 133.32 (C(4’)/C(2)); 132.29
(C(4’)/C(2)); 132.05 (C(2’), C(6’)); 127.95 (C(3’), C(5’)); 125.30 (C(1’’)); 113.48 (C(6’’)); 107.41 (C(3’’));
56.03 (C(7’’)); 55.71 (C(8’’)). EI-MS: 365/363 (5/13, Mþ ), 319 (36), 318 (27), 317 (100), 302 (12), 164
(31), 151 (10), 141 (21), 139 (68), 136 (25), 111 (10). HR-EI-MS: 363.0506, (Mþ, C17H14ClNO6þ ; calc.
363.0510).
(2Z)-3-(4,5-Dimethoxy-2-nitrophenyl)-2-phenylprop-2-enoic Acid ((Z)-5b). Yield: 85 mg (2.58ꢂ
10ꢁ4 mol, 27%). Yellow solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2% AcOH) 0.17. M.p 189.3–190.58 (M.p. lit.
1958 [69]). 1H-NMR (300 MHz, (D6)DMSO): 3.86 (s, MeC(8’’)); 3.89 (s, Me(7’’)); 7.11 (s, HꢁC(6’’)); 7.39
(s, HꢁC(3)); 7.35–7.47 (m, HꢁC(3’’), HꢁC(3’), HꢁC(5’)); 7.51–7.53 (m, HꢁC(2’), HꢁC(6’)); 7.72 (s,
HꢁC(3’’)); 13.29 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 169.16 (COOH); 152.73 (C(5’’));
148.10 (C(4’’)); 139.68 (C(2’’)); 136.96 (C(2)); 136.17 (C(1’)); 128.71 (C(3’), C(5’)); 128.39 (C(4’)); 127.23
(C(3)); 126.49 (C(2’), C(6’)); 111.95 (C(6’’)); 107.79 (C(3’’)); 56.11 (MeO); 56.09 (MeO). EI-MS: 329 (5,
Mþ ), 284 (26), 283 (100), 268 (9), 256 (6), 253 (8), 240 (7), 239 (8), 164 (19), 153 (7), 152 (9), 136 (17),
77 (17). HR-EI-MS: 329.0899 (Mþ, C17H15NO6þ ; calc. 329.0899).
Isomer (E)-5b. Yield: 215 mg (6.53ꢂ10ꢁ4 mol, 69%). Yellow solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2%
AcOH) 0.33. M.p. 214.1–215.88 ([70]: 217–2208). 1H-NMR (300 MHz, (D6)DMSO): 3.32 (s, HꢁC(8’’));
3.82 (s, HꢁC(7’’)); 6.35 (s, HꢁC(6’’)); 7.10–7.12 (m, HꢁC(2’), HꢁC(6’)); 7.25–7.27 (m, HꢁC(3’), HꢁC(4’),
HꢁC(5’)); 7.62 (s, HꢁC(3’’)); 8.02 (s, HꢁC(3)); 12.88 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO):
167.79 (COOH); 152.00 (C(5’’)); 148.13 (C(4’’)); 140.61 (C(2’’)); 137.04 (C(3)); 135.38 (C(1’)); 134.63
(C(2)); 132.10 (C(2’), C(6’)); 127.97 (C(3’), C(5’)); 127.46 (C(4’)); 125.44 (C(1’’)); 113.53 (C(6’’)); 107.34
(C(3’’)); 55.70 (C(7’’)); 55.57 (C(8’’)). EI-MS: 329 (5, Mþ ), 284 (26), 283 (100), 268 (10), 253 (8), 239
(9), 238 (6), 224 (6), 165 (7), 164 (19), 153 (8), 152 (10), 136 (18), 105 (69), 77 (18). HR-EI-MS:
329.0897 (Mþ, C17H15NOþ6 ; calc. 329.0899).
(2Z)-3-(4,5-Dimethoxy-2-nitrophenyl)-2-(4-methylphenyl)prop-2-enoic Acid ((Z)-5c). Yield:
102 mg (2.97ꢂ10ꢁ4 mol, 31%). Slightly yellow solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2% AcOH) 0.17. M.p.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2523
209.7–211.88. 1H-NMR (300 MHz, (D6)DMSO): 2.33 (s, MeꢁC(4’)); 3.86 (s, Me(8’’)); 3.89 (s, MeC(7’’));
7.08 (s, HꢁC(6’’)); 7.36 (s, HꢁC(3)); 7.23–7.26 (m, HꢁC(3’), HꢁC(5’)); 7.39–7.42 (m, HꢁC(2’), HꢁC(6’));
7.71 (s, HꢁC(3’’)); 13.18 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 169.80 (COOH); 152.72
(C(5’’)); 148.07 (C(4’’)); 139.67 (C(2’’)); 137.99 (C(4’)/C(2)); 136.72 (C(4’)/C(2)); 133.22 (C(1’)); 129.31
(C(3’), C(5’)); 126.76 (C(1’’)); 126.35 (C(3), C(2’), C(6’), 3C), 111.92 (C(6’’)); 107.82 (C(3’’)); 56.1
(MeO); 20,75 (Me)). EI-MS: 343 (14, Mþ ), 298 (30), 297 (100), 282 (18), 270 (14), 267 (9254 (10), 253
(13), 179 (18), 165 (14), 152 (8), 136 (31), 120 (9), 119 (93), 91 (24). HR-EI-MS: 343.1056 (Mþ,
C18H17NOþ6 ; calc. 343.1056).
Isomer (E)-5c. Yield: 138 mg (4.02ꢂ10ꢁ4 mol, 42%). Yellow solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2%
AcOH) 0.33. M.p. 203.3–204.98. 1H-NMR (300 MHz, (D6)DMSO): 2.24 (s, MeꢁC(4’)); 3.34 (s, Me(8’’));
3.82 (s, Me(7’’)); 6.36 (s, HꢁC(6’’)); 6.96–6.99 (m, HꢁC(2’), HꢁC(6’)); 7.05–7.08 (m, HꢁC(5’), HꢁC(3’));
7.61 (s, HꢁC(3’’)); 7.98 (s, HꢁC(3)); 12.82 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 167.90
(COOH); 151.99 (C(5’’)); 148.09 (C(4’’)); 140.58 (C(2’’)); 136.83 (C(4’)); 136.73 (C(3)); 134.51 (C(2));
132.32 (C(1’)); 129.98 (C(2’), C(6’)); 128.50 (C(3’), C(5’)); 125.60 (C(1’’)); 113.53 (C(6’’)); 107.28 (C(3’’));
55.98 (C(7’’)); 55.53 (C(8’’)); 20.67 (Me(4’)). EI-MS: 343 (14, Mþ ), 298 (30), 297 (100), 282 (18), 270
(14), 267 (11), 254 (10), 253 (13), 179 (19), 165 (15), 164 (44), 136 (32), 119 (91), 91 (23). HR-EI-MS:
343.1057 (Mþ, C18H17NOþ6 ; calc. 343.1056).
(2Z)-3-(4,5-Dimethoxy-2-nitrophenyl)-2-(4-methoxyphenyl)prop-2-enoic Acid ((Z)-5d). Yield:
73 mg (1.85ꢂ10ꢁ4 mol, 20%). Yellow solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2% AcOH) 0.15. M.p. 192.7–
194.28 ([38]: 178–1808). 1H-NMR (300 MHz, (D6)DMSO): 3.79 (s, MeOꢁC(4’)); 3.86 (s, Me(8’’)); 3.89
(s, Me(7’’)); 6.98–7.01 (m, HꢁC(3’), HꢁC(5’)); 7.07 (s, HꢁC(6’’)); 7.30 (s, HꢁC(3)); 7.44–7.46 (m,
HꢁC(2’), HꢁC(6’)); 7.71 (s, HꢁC(3’’)); 13.19 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 169.93
(COOH); 159.48 (C(4’)); 152.70 (C(5’’)); 147.99 (C(4’’)); 139.65 (C(2’’)); 136.46 (C(2)); 128.38 (C(1’));
127.80 (C(3’), C(5’)); 126.89 (C(1’’)); 125.15 (C(3)); 114.18 (C(3’), C(5’)); 111.92 (C(6’’)); 107.83 (C(3’’));
56.08 (MeO); 55.23 (MeOꢁC(4’)). EI-MS: 359 (31, Mþ ), 314 (18), 313 (36), 299 (13), 298 (39), 286
(30), 283 (17), 282 (12), 270 (14), 269 (17), 268 (12), 254 (13), 179 (43), 165 (15), 164 (84), 151 (17), 136
(64), 135 (100), 108 (11), 77 (12). HR-EI-MS: 359.1003 (Mþ, C18H17NO7þ ; calc. 359.1005).
Isomer (E)-5d. Yield: 184 mg (5.12ꢂ10ꢁ4 mol, 54%). Yellow solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2%
1
AcOH) 0.30. M.p. 169.8–172.38 ([38]: 170–1728). H-NMR (300 MHz, (D6)DMSO): 3.37 (s, Me(8’’));
3.70 (MeOꢁC(4’)); 3.82 (s, Me(7’’)); 6.39 (s, HꢁC(6’’)); 6.81–6.84 (m, HꢁC(2’), HꢁC(6’)); 7.00–7.03 (m,
HꢁC(5’), HꢁC(3’)); 7.61 (s, HꢁC(3’’)); 7.95 (s, HꢁC(3)); 12.81 (br. s, COOH). 13C-NMR (75 MHz,
(D6)DMSO): 168.07 (COOH); 152.08 (C(5’’)); 158.71 (C(4’)); 148.08 (C(4’’)); 140.61 (C(2’’)); 136.44
(C(3)); 134.16 (C(2)); 131.40 (C(2’), C(6’)); 127.29 (C(1’)); 125.79 (C(1’’)); 113.51 (C(6’’)); 113.45 (C(3’),
C(5’)); 107.29 (C(3’’)); 55.98 (Me(7’’)); 55.61 (Me(8’’)); 55.08 (MeOꢁC(4’)). EI-MS: 359 (31, Mþ ), 314
(18), 313 (35), 299 (12), 298 (38), 286 (30), 283 (13), 270 (13), 269 (16), 254 (12), 179 (43), 165 (17), 164
(81), 151 (16), 137 (10), 136 (62), 135 (100), 108 (10), 77 (11). HR-EI-MS: 359.0998 (Mþ, C18H17NO7þ ;
calc. 359.1005).
(2E)-2-(4-Chlorophenyl)-3-phenylprop-2-enoic Acid ((E)-5e). The pure (E)-isomer was obtained
by refluxing the mixture of 2-(4-chlorophenyl)acetic acid and PhCHO, as described in the General
Procedure for synthesis of the 2-phenylacetic acids, for 30 min. The product was recrystallized from
AcOEt, and the mother liquor was further purified by flash CC (hexane/AcOEt 2 :1, 0.1% AcOH to
hexane/AcOEt 1:1, 0.1% AcOH). Yield: 218 mg (8.45ꢂ10ꢁ4 mol, 45%). Colorless needles. Rf (hexane/
AcOEt 2 :1) 0.31. M.p. 174.6–175.78 ([71]: 148–1538). 1H-NMR (300 MHz, (D6)DMSO): 7.05–7.08 (m,
HꢁC(2’’), HꢁC(6’’)); 7.17–7.20 (m, HꢁC(2’), HꢁC(6’)); 7.22–7.24 (m, HꢁC(3’’), HꢁC(4’’), HꢁC(5’’));
7.41–7.44 (m, HꢁC(3’), HꢁC(5’)); 7.79 (s, HꢁC(3)); 12.79 (br. s, COOH). 13C-NMR (75 MHz,
(D6)DMSO): 167.96 (COOH); 139.54 (C(3)); 135.15 (C(1’)); 134.17 (C(4’)/C(2)); 132.30 (C(4’)/C(2));
132.11 (C(1’’)); 131.53 (C(2’), C(6’)); 130.11 (C(2’’), C(6’’)); 129.14 (C(4’’)); 128.52 (C(3’), C(5’)); 128.38
(C(3’’), C(5’’)). EI-MS: 260 (34), 259 (14), 258 (100, Mþ ), 179 (23), 178 (52), 177 (16), 176 (22), 152
(43), 107 (17). HR-EI-MS: 258.0449 (Mþ, C15H11ClO2þ ; calc. 258.0448).
(2E)-2-(4-Chlorophenyl)-3-(4-methoxyphenyl)prop-2-enoic Acid ((E)-5f). A mixture of 4-methoxy-
benzaldehyde (200 mg, 1.47 mmol) and 2-(4-chlorophenyl)acetic acid (250 mg, 1.47 mmol) was heated to
reflux in Ac2O/Et3N 2 :1 (1.5 ml) for 1 h. H2O was added, and the aq. soln. was extracted with CH2Cl2
(3ꢂ10 ml). The org. layer was first washed with 0.1m NaOH (10 ml) and afterwards with brine (10 ml),

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
and recrystallization from CH2Cl2 gave (E)-5f (2.89ꢂ10ꢁ4 mol, 86 mg, 20%). White needles. M.p. 223.8–
224.98 ([71]: 225–2288). 1H-NMR (400 MHz, (D6)DMSO): 3.71 (s, MeO); 6.79–6.82 (m, HꢁC(3’’),
MeOꢁC(5’’)); 7.01–7.03 (m, HꢁC(2’’), HꢁC(6’’)); 7.18–7.21 ((m, HꢁC(2’), HꢁC(6’)); 7.44–7.47 (m,
HꢁC(3’), HꢁC(5’)); 7.74 (s, HꢁC(3)); 12.64 (br. s, COOH). 13C-NMR (101 MHz, (D6)DMSO): 168.19
(COOH); 160.05 (C(4’’)); 139.39 (C(3)); 135.65 (C(1’)); 132.20, 132.01 (C(3’’), C(5’’)); 131.58 (C(2’),
C(6’)); 129.44, 128.67 (C(3’), C(5’)); 126.51 (C(1’’)); 113.99 (C(3’’), C(5’’)); 55.22 (MeO). EI-MS: 290/
288 (32/100, Mþ ), 243 (6), 208 (9), 199 (6), 165 (20), 152 (14), 137 (41). HR-EI-MS: 288.0547 (Mþ,
C16H13ClOþ3 ; calc. 288.0553).
(2E)-2-(4-Chlorophenyl)-3-(3-methoxyphenyl)prop-2-enoic Acid ((E)-5g). 3-Methoxybenzalde-
hyde (0.74 mmol, 90 ml) and 2-(4-chlorophenyl)acetic acid (182 mg, 1.07 mmol, 1.50 equiv.) were heated
to reflux for 20 min in Ac2O/Et3N 2 :1 (3 ml). Recrystallization of the product from AcOEt gave (E)-5g
(92 mg, 3.19ꢂ10ꢁ4 mol, 43%). Off-white needles. M.p. 202.1–203.58. 1H-NMR (300 MHz, (D6)DMSO):
3.54 (s, MeOꢁC(5’’)); 6.54–6.62 (m, HꢁC(6’’)); 6.69–6.72 (m, HꢁC(2’’)); 6.83 (ddd, J¼8.3, 2.7, 1.0,
MeOꢁC(4’’)); 7.16 (t, J¼8.0, HꢁC(3’’)); 7.19–7.23 (m, HꢁC(2’), HꢁC(6’)); 7.43–7.48 (m, HꢁC(3’),
HꢁC(5’)); 7.77 (s, HꢁC(3)); 12.78 (COOH). 13C-NMR (75 MHz, (D6)DMSO): 168.02 (COOH); 158.81
(C(5’’)); 139.35 (C(3)); 135.47 (C(1’’)); 135.33 (C(1’)); 132.47 (C(2)/C(4’)); 132.31 (C(2)/C(4’)); 131.53
(C(2’), C(6’)); 129.45 (C(3’’)); 128.52 (C(3’’), C(5’’)); 122.91 (C(2’’)); 115.20 (C(4’’)); 114.90 (C(6’’));
54.66 (C(7’’). EI-MS: 291/289 (6/19, Mþ ), 288 (100), 253 (16), 245 (7), 244 (5), 243 (18), 235 (6), 209
(10), 208 (21), 199 (7), 194 (7), 178 (8), 177 (5), 176 (6), 166 (7), 165 (28), 164 (6), 163 (7), 154 (7), 152
(21), 137 (9), 124 (5), 109 (14). HR-EI-MS: 288.0552 (Mþ, C16H13ClO3þ ; calc. 288.0553).
(2E)-2-(4-Chlorophenyl)-3-(3,4-dimethoxyphenyl)prop-2-enoic Acid ((E)-5h). The pure (E)-iso-
mer was obtained by refluxing the starting materials for 3 h and purifying the crude product by flash CC
(hexane/AcOEt 2 :1, 1% AcOH). Fractions containing the product were freed from trace amounts of the
(Z)-isomer by extracting the org. layers with 2m NaOH (3ꢂ10 ml), acidifying the aq. layer with 1m HCl
(pH ca. 5), and extraction with CH2Cl2 (3ꢂ10 ml). Yield: 274 mg (8.60ꢂ10ꢁ4 mol, 47%). Colorless
needles. Rf (Hexane/AcOEt 2 :1, 1% AcOH) 0.35. M.p. 210.7–211.68. 1H-NMR (300 MHz,
(D6)DMSO): 3.38 (s, Me(8’’)); 3.68 (s, Me(7’’)); 6.50 (d, J¼1.9, HꢁC(6’’)); 6.79 (dd, J¼8.5, 1.9,
HꢁC(2’’)); 6.85 (d, J¼8.5, HꢁC(3’’)); 7.20–7.23 (m, HꢁC(6’), HꢁC(2’)); 7.45–7.48 (m, HꢁC(3’),
HꢁC(5’)); 7.72 (s, HꢁC(3)); 12.59 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 168.10 (COOH);
149.83 (Me(7’’)); 147.93 (Me(8’’)); 139.70 (C(3)); 135.79 (C(1’)); 132.19 (C(4’)); 131.67 (C(2’), C(6’));
129.46 (C(2)); 128.63 (C(3’), C(5’)); 126.55 (C(1’’)); 124.58 (C(2’’)); 112.86 (C(6’’)); 111.35 (C(3’’)); 55.43
(C(7’’)); 54.58 (C(8’’)). EI-MS: 320/318 (35/100, Mþ ), 167 (8), 152 (8), 139 (5). HR-EI-MS: 318.0656
(Mþ, C17H15ClOþ4 ; calc. 318.0659).
(2Z)-2-(4-Chlorophenyl)-3-(2-nitrophenyl)prop-2-enoic Acid ((Z)-5i). The reaction mixture was
heated to reflux for 15 min. Yield: 140 mg (4.61ꢂ10ꢁ4 mol, 35%). Yellow crystals. Rf (CH2Cl2/AcOEt
1
2 :1, 0.2% AcOH) 0.22. M.p. 159.8–161.38. H-NMR (75 MHz, (D6)DMSO): 7.49–7.57 (m, HꢁC(3),
HꢁC(6’’), C6H4Cl); 7.62 (td, J¼7.6, 1.4, HꢁC(4’’)); 7.78 (td, J¼7.9, 1.3, C(5’’)); 8.16 (dd, J¼7.6, 1.3); 13.26
(s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 168.63 (COOH); 147.27 (C(2’’)); 135.68, 135.11, 133.90,
133.16 (C(5’’)); 132.09 (C(1’’)); 130.32, 129.34, 129.26, 128.71 (C6H4Cl); 128.66 (C6H4Cl); 124.60 (C(3’’)).
EI-MS: 305/303 (3/9, Mþ ), 258 (22), 257 (13), 256 (11), 255 (14), 243 (17), 242 (14), 230 (35), 229 (32),
228 (25), 227 (91), 214 (12), 213 (25), 199 (15), 191 (11), 190 (10), 178 (16), 177 (11), 176 (24), 166 (14),
165 (54), 164 (14), 163 (13), 151 (11), 141 (33), 140 (10), 139 (100), 119 (92), 113 (17), 111 (28), 104 (10),
96 (11), 92 (74), 91 (23), 89 (16), 83 (12), 82 (10), 76 (14), 75 (16). HR-EI-MS: 303.0306 (Mþ,
C15H10ClNOþ4 ; calc. 303.0298).
Isomer (E)-5i. Yield: 222 mg (7.31ꢂ10ꢁ4 mol, 55%). Pale yellow crystals. Rf (CH2Cl2/AcOEt 2 :1,
1
0.2% AcOH) 0.36. M.p. 192.8–194.68 ([72]: 186–1878). H-NMR (300 MHz, (D6)DMSO): 6.92–7.04
(m, 1 arom. H); 7.04–7.15 (m, HꢁC(2’), HꢁC(6’)); 7.23–7.34 (m, HꢁC(3’), HꢁC(5’)); 7.43–7.58 (m, 2
arom. H); 8.06 (s, HꢁC(3)); 8.07–8.16 (m, 1 arom. H); 13.12 (COOH). 13C-NMR (75 MHz,
(D6)DMSO): 167.36 (COOH); 147.79 (C(2’’), 137.72 (C(3)); 133.89, 133.68, 133.63, 132.39, 132.01
(C(2’), C(6’)); 131.65, 131.31, 129.51, 127.96 (C(3’), C(5’)); 124.57. EI-MS: 305/303 (8/3, Mþ ), 258 (22),
243 (11), 242 (14), 232 (10), 230 (33), 227 (20), 214 (9), 199 (10), 178 (12), 177 (13), 176 (36), 166 (10),
165 (34), 163 (10), 151 (12), 141 (14), 139 (100), 120 (10), 119 (94), 113 (10), 111 (25), 104 (11), 92 (69),
91 (23), 88 (11), 77 (11), 76 (11), 75 (14). HR-EI-MS: 303.0293 (Mþ, C15H10ClNO4þ ; calc. 303.0298).

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2525
(2Z)-2-(4-Chlorophenyl)-3-(4-methoxy-2-nitrophenyl)prop-2-enoic Acid ((Z)-5j). The reaction was
heated to reflux for 45 min. Yield: 33 mg (9.89ꢂ10ꢁ5 mol, 9%). Yellow crystals. Rf (CH2Cl2/AcOEt 2 :1,
0.2% AcOH) 0.15. M.p. 179.5–180.88. 1H-NMR (300 MHz, (D6)DMSO): 3.89 (s, MeO); 7.36 (s,
HꢁC(3)); 7.38 (dd, J¼2.7, 8.7, HꢁC(5’’)); 7.48 (d, J¼8.7, HꢁC(6’’), 7.50–7.54 (m, Ph-Cl); 7.66 (d, J¼2.6,
HꢁC(2’’)); 13.22 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 168.94 (COOH); 159.31 (C(4’’));
148.16 (C(2’’)); 135.31, 135.27, 133.02, 131.34 (C(6’’)); 128.71 (Ph-Cl); 128.51 (Ph-Cl); 128.32 (C(3));
123.92 (C(1’’)); 120.02 (C(5’’)); 109.37 (C(3’’)); 56.11 (MeO). EI-MS: 335/333 (6/21, Mþ ), 289 (10), 288
(23), 287 (32), 286 (24), 285 (10), 273 (20), 272 (14), 260 (34), 259 (16), 258 (26), 257 (46), 256 (29), 245
(15), 244 (26), 243 (45), 242 (45), 163 (13), 153 (7), 152 (24), 151 (10), 149 (28), 141 (30), 139 (100), 122
(18), 111 (14), 106 (13), 63 (10). HR-EI-MS: 333.0399 (Mþ, C16H12ClNO5þ ; calc. 333.0404).
Isomer (E)-5j. Yield: 112 mg (3.36ꢂ10ꢁ4 mol, 31%). Yellow crystals. Rf (CH2Cl2/AcOEt 2 :1, 0.2%
AcOH) 0.38. M.p. 183.4–185.78. 1H-NMR (300 MHz, (D6)DMSO): 3.87 (s, MeO); 6.99 (dd, J¼8.6, 2.5,
HꢁC(6’’), 7.03 (d, J¼2.5, HꢁC(3’’), 7.50–7.53 (m, HꢁC(6’), HꢁC(2’)); 7.73–7.77 (m, HꢁC(3’), HꢁC(5’));
8.25 (s, HꢁC(3)); 13.09 (COOH). 13C-NMR (75 MHz, (D6)DMSO): 163.42 (COOH); 160.64 (C(4’’));
155.76 (C(2’’)); 141.99 (C(3)); 134.60, 133.69 (C(6’’)); 130.90 (C(2’), C(6’)); 130.67, 129.06 (C(5’), C(3’));
122.71 (C(5’’)); 113.79, 113.61, 101.11, 56.86 (MeO). EI-MS: 335/333 (2/7, Mþ ), 288 (39), 287 (26), 286
(100), 258 (20), 257 (14), 245 (24), 244 (19), 243 (69), 242 (16), 178 (10), 152 (27), 141 (11), 139 (35).
HR-EI-MS: 333.0402 (Mþ, C16H12ClNO5þ ; calc. 333.0404).
(2Z)-2-(4-Chlorophenyl)-3-(5-methoxy-2-nitrophenyl)prop-2-enoic Acid ((Z)-5k). Yield: 114 mg
(3.42ꢂ10ꢁ4 mol, 31%). White solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2% AcOH) 0.29. M.p. 167.8–168.58.
1H-NMR (300 MHz, (D6)DMSO): 3.89 (s, Me(8’’)); 7.03 (d, J¼2.7, HꢁC(6’’)); 7.14 (dd, J¼9.2, 2.8,
HꢁC(4’’)); 7.50 (s, HꢁC(3)); 7.52–7.57 (m, HꢁC(2’’), HꢁC(3’’), HꢁC(4’’), HꢁC(5’’)); 8.21 (d, J¼9.2,
HꢁC(3’’)); 13.29 (s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 168.77 (COOH); 162.99 (C(5’’)); 140.19
(C(2’’)); 135.45, 134.97, 134.95, 133.16, 129.30 (C(3)); 128.74 (C(2’), C(6’)/C(3’), C(5’)); 128.55 (C(2’),
C(6’)/C(3’), C(5’)); 127.49 (C(3’’)); 115.25 (C(6’’)); 113.94 (C(4’’)); 56.19 (Me(8’’)). EI-MS: 335/333 (4/13,
Mþ ), 290 (11), 289 (26), 288 (32), 287 (61), 273 (10), 272 (23), 257 (12), 243 (11), 164 (11), 163 (15), 152
(20), 149 (56), 141 (31), 140 (10), 139 (100), 134 (17), 121 (47), 113 (12), 111 (24), 107 (10), 106 (95), 76
(10), 63 (11). HR-EI-MS: 333.0396 (Mþ, C16H12ClNO5þ ; calc. 333.0404). Anal calc. for C16H12ClNO5
(333.72): C 57.58, H 3.62, N 4.20; found: C 57.87, H 3.26, N 4.44.
Isomer (E)-5k. Yield: 160 mg (4.75ꢂ10ꢁ4 mol, 50%). White solid. Rf (CH2Cl2/AcOEt 2 :1, 0.2%
AcOH) 0.47. M.p. 168.5–169.78. 1H-NMR (300 MHz, (D6)DMSO): 3.61 (s); 6.46 (d, J¼2.9, HꢁC(6’’));
7.00 (dd, J¼9.2, 2.8 HꢁC(4’’)); 7.06–7.17 (m, HꢁC(2’), HꢁC(6’)); 7.25–7.36 (m, HꢁC(3’), HꢁC(5’)); 8.08
(s, HꢁC(3)); 8.13 (d, J¼9.2, HꢁC(3’’)); 13.07 (br. s, COOH). 13C-NMR (75 MHz, (D6)DMSO): 167.32
(COOH); 162.61 (C(5’’)); 140.69 (C(2’’)); 138.38 (C(3)); 134.13, 133.80, 133.60, 132.31, 131.92 (C(2’),
C(6’)); 127.91 (C(3’), C(5’)); 127.27 (C(3’’)); 116.44 (C(6’’)); 114.48 (C(4’’)); 55.95 (MeO). EI-MS: 335/
333 (6/17, Mþ ), 290 (10), 289 (24), 288 (30), 287 (54), 272 (22), 257 (10), 243 (11), 208 (6), 165 (10), 164
(12), 163 (18), 152 (20), 150 (11), 149 (64), 141 (33), 139 (99), 134 (18), 121 (56), 113 (10), 111 (25), 107
(10), 106 (100), 63 (10). HR-EI-MS: 333.0405 (Mþ, C16H12ClNO5þ ; calc. 333.0404).
Methyl 4-(Methylamino)-3-nitrobenzoate (7). MeNH2 (40% soln. in H2O, 1.27 ml, 16.38 mmol,
13.00 equiv.) was added dropwise to a suspension of 13 (250 mg, 1.26 mmol) and K2CO3 (347 mg,
2.52 mmol, 2.00 equiv.) in CH2Cl2 (1.50 ml), and the resulting mixture was stirred 15 h at r.t. The reaction
was quenched with H2O (10 ml), and the aq. layer was extracted with CH2Cl2 (3ꢂ10 ml). The combined
org. layers were washed with brine (10 ml) and dried (MgSO4). The crude product was concentrated and
filtered over a plug of SiO2 using CH2Cl2 as eluent. Evaporation of the solvent afforded 7 (252 mg, 1.20ꢂ
1
10ꢁ3 mol, 95%). Bright yellow solid. Rf (CH2Cl2) 0.35. M.p. 145.2–146.78 ([73]: 142–1438). H-NMR
(300 MHz, CDCl3): 3.06 (d, J¼5.1, MeN); 3.89 (s, MeO); 6.84 (d, J¼9.0, HꢁC(5)); 8.05 (dd, J¼9.0, 1.9
HꢁC(6)); 8.32 (br. s, NH); 8.85 (d, J¼1.9, HꢁC(2)). 13C-NMR (75 MHz, CDCl3): 165.63 (COOMe);
148.49 (C(4)); 136.40 (C(6)); 131.35 (C(3)); 129.42 (C(2)); 117.17 (C(1)); 113.11 (C(5)); 52.07 (C(8));
29.90 (C(7)). EI-MS: 210 (100, Mþ ), 193 (7), 180 (7), 179 (47), 177 (6), 164 (8), 163 (8), 162 (9), 149
(18), 133 (16), 132 (19), 131 (12), 121 (9), 120 (7), 118 (8), 106 (12), 105 (81), 104 (26), 103 (11), 92 (5),
91 (5), 85 (5), 78 (11), 77 (14), 71 (5), 66 (6), HR-EI-MS: 210.0633 (Mþ, C9H10N2O4þ ; calc. 210.0641).

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Received September 14, 2012