Ligand-based discovery of N-(1,3-dioxo-1H,3H-benzo[de]isochromen-5-yl)- carboxamide and sulfonamide derivatives as thymidylate synthase A inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.11.117

Phenolnaphthalein derivatives show potential for pharmacological activity as inhibitors of thymidylate synthase (TS) but difficulties in their synthesis and derivatization hinder their development. A deconstruction approach aimed at identifying a suitable new scaffold was proposed. A new scaffold was identified and two compound libraries based on this scaffold were designed. The carboxamide library (Library B) showed specific inhibition activity against Escherichia coli TS, whereas the sulfonamide library (Library C) showed a non-specific inhibition profile against hTS. N-(1,3-Dioxo-1H,3H-benzo[de]isochromen-5-yl)- sulfonamide derivatives, 1C and 9C, showed one order of magnitude improvement in inhibition constant against hTS with respect to the starting lead and represent potential compounds for further lead development.

Full text of DOI:10.1016/j.bmcl.2012.11.117

Bioorganic & Medicinal Chemistry Letters 23 (2013) 663–668
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Ligand-based discovery of N-(1,3-dioxo-1H,3H-benzo[de]isochromen-5-yl)-car-
boxamide and sulfonamide derivatives as thymidylate synthase A inhibitors
Stefania Ferrari a, , Marco Ingrami a, Fabrizia Soragni a, Rebecca C. Wade b,c, M. Paola Costi a
⇑
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Modena e Reggio Emilia, Via Campi 183, 41126 Modena, Italy
b Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS), Schloss-Wolfsbrunnenweg 35, 69118 Heidelberg, Germany
c Zentrum für Molekulare Biologie (ZMBH), Heidelberg University, 69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Phenolnaphthalein derivatives show potential for pharmacological activity as inhibitors of thymidylate
synthase (TS) but difficulties in their synthesis and derivatization hinder their development. A decon-
struction approach aimed at identifying a suitable new scaffold was proposed. A new scaffold was iden-
tified and two compound libraries based on this scaffold were designed. The carboxamide library (Library
B) showed specific inhibition activity against Escherichia coli TS, whereas the sulfonamide library (Library
C) showed a non-specific inhibition profile against hTS. N-(1,3-Dioxo-1H,3H-benzo[de]isochromen-5-yl)-
sulfonamide derivatives, 1C and 9C, showed one order of magnitude improvement in inhibition constant
against hTS with respect to the starting lead and represent potential compounds for further lead
development.
Received 16 October 2012
Revised 27 November 2012
Accepted 29 November 2012
Available online 7 December 2012
Keywords:
Deconstruction approach
Enzyme inhibition
Thymidylate synthase
Antibacterial compound
Anticancer compound
Ó 2012 Elsevier Ltd. All rights reserved.
Thymidylate synthase (TS) (EC: 2.1.1.45) catalyzes the reductive
methylation of 20-deoxyuridine-50-monophosphate (dUMP) to 20-
deoxythymidine-50-monophosphate (dTMP) and is assisted by
the cofactor N5,N10-methylene tetrahydrofolate (MTHF).1 Because
TS is essential for the only synthetic source of dTMP in human cells,
it is a major target for the design of chemotherapeutic agents.2
Among the drugs known to target TS, 5-fluorouracil, raltitrexed
and pemetrexed are used therapeutically as anticancer agents.
Previous studies of phenolnaphthalein derivatives led to the dis-
covery of compounds with species-specific inhibition profiles,
to human cells. X-ray crystallographic studies of this class of com-
pounds [ 156, PDB ID: 1TSL; MR20 (3,3-bis(4-hydroxy-phenyl)-6-
a
nitro-3H-benzo[de]isochromen-1-one), PDB ID: 1TSM; GA9, PDB
ID: 2A9W] have shown that their interactions with TS are charac-
terized by multiple binding modes3,6 (Supplementary Fig. SI-1).
This observation has been corroborated by molecular docking
studies.7
In general, phenolnaphthalein derivatives show appreciable
pharmacological potential in terms of species specificity but exhi-
bit low chemical tractability and require improvement to be con-
sidered as drug candidates.4 To develop new inhibitors targeting
TS, we applied a ligand-based approach to phenolnaphthalein
derivatives, which were then deconstructed into fragments8
(Scheme 1). A novel scaffold was sought and identified (Library
A, Table 1), two libraries (Libraries B and C, Table 2) were synthe-
sized and screened against hTS and EcTS.
including
[de]isochromen-1-one) and GA9 (3,3-bis(3-bromo-4-hydroxy-phe-
nyl)-7-chloro-3H-benzo[de]isochromen-1-one).3–6
156 is fifty
times more active toward Escherichia coli TS (EcTS) than human
TS (hTS) (Ki = 0.6 and 30 M, respectively), GA9 shows a Ki versus
EcTS of 4.1 M with no inhibition of hTS at 50 M concentration.
In particular, 156 has been proposed to be an antibacterial agent
a156 (3,3-bis(3-chloro-4-hydroxy-phenyl)-3H-benzo-
a
l
l
l
a
against vancomycin-resistant infections caused by Gram-positive
bacteria, such as Staphylococcus aureus, because of its low toxicity
Abbreviations: DHFR, dihydrofolate reductase; DMSO, dimethylsulfoxide; dTMP,
20-deoxythymidine-50-monophosphate; dUMP, 20-deoxyuridine-50-monophos-
phate; EcTS, Escherichia coli TS; hTS, human TS; IC50, concentration of inhibitor
required for inhibition of 50% of target enzyme activity; Ki, inhibition constant;
LcTS, Lactobacillus casei TS; MTHF, N5,N10-methylene tetrahydrofolate; Py, pyridine;
TS, thymidylate synthase.
⇑
Corresponding author. Tel.: +39 059 205 5125; fax: +39 059 205 5131.
E-mail address: stefania.ferrari@unimore.it (S. Ferrari).
Scheme 1. Deconstruction approach.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.117

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S. Ferrari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 663–668
Table 1
Inhibition activity profiles of compounds from Library A
Code
Structure
Ki EcTS (
l
M)a
Ki hTS (
l
M)a
Code
Structure
Ki EcTS (
l
M)a
Ki hTS (l
M)a
O
O
O
O
O
O
>>265
NI (54)
>>294
NI (60)
1A
4.2
10A
2.0
O
O
O
S
>>196
NI (40)
2A
3.1
11A
105
7.3
Cl
O
O
O
O
O
>>103
NI (20)
>>98
NI (20)
3A
12A
26.8
2.0
O
O
O
OH
O
NO2
O
O
O
N
>>294
NI (60)
>>309
NI (60)
>>784
NI (160)
>>824
NI (160)
4A
13A
O
S
OH
O
N
O
O
O
O
O
N
>>294
NI (60)
>>309
NI (60)
>>451
NI (92)
>>474
NI (92)
5A
14A
S
HO
O
O
O
>>294
NI (60)
>>588
NI (120)
6A
3.1
15A
12
CH3
S
O
O
O
O
O
O
O
O
>>304
NI (62)
>>152
NI (31)
>>160
NI (31)
7A
8A
47
33
16A
17A
NH2
O
O
O
O
O
O
>>588
NI (120)
>>196
NI (40)
>>206
NI (40)
NO2
NH2
O
O
>>294
NI (60)
>>309
NI (60)
>>412
NI (80)
9A
18A
6.8
a
NI indicates no detectable inhibition at the concentration reported in brackets ([I]). In these cases Ki was estimated considering an inhibition percentage (I%) of 2% at this
21
concentration and applying the formulas IC50 = (100 – I%) ꢀ [I]/I% and Ki = IC50/(1 + [S]/Km
)
where [S] is the concentration of folate used in the assay and Km is the affinity
constant of the enzyme for that substrate.5

S. Ferrari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 663–668
665
Table 2
Inhibition activity profiles of compounds from Libraries B and C
O
O
O
O
O
O
Code
Ki EcTS (
l
M)a
Ki hTS (
l
M)a
Code
Ki EcTS (
l
M)a
Ki hTS (l
M)a
R
R
N
H
N
H
R chain
R chain
O
S
O
NO2
1B
2B
7.3
2.3
1C
2C
4.2
2.1
0.3
1.1
O
NO2
NO2
O
S
O
17
17.3
O
O
NH2
CF3
O
S
O
NO2
>>823
NI (160)
3B
21.6
3C
1.2
1.3
O
S
S
>>206
NI (40)
O
4B
5B
6B
6.4
4.4
4.0
4C
5C
6C
2.2
1.3
1.2
0.5
0.9
1.3
O
O
O2N
O
S
CF3
44.6
25.4
CF3
O
O
Cl
O
S
NO2
NO2
S
O
S
O
O
Cl
O
S
O
O
S
7B
8B
6.7
5.3
12.3
13.8
7C
8C
4.6
1.1
1.2
0.8
Cl
N
O
O
O
S
O
S
O
S
O
Cl
>>411
NI (80)
9B
6.5
7.1
9C
2.9
0.9
0.3
5.0
S
O
O
S
N
N
O
O
S
10B
16.2
10C
O
O
(continued on next page)

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S. Ferrari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 663–668
Table 2 (continued)
O
O
O
O
O
O
Code
Ki EcTS (
l
M)a
Ki hTS (
l
M)a
Code
Ki EcTS (
l
M)a
Ki hTS (l
M)a
R
R
N
H
N
H
R chain
R chain
O
O
S
O
O
11B
7.4
57.2
11C
12C
0.3
1.9
0.6
0.8
O
O
S
O
S
>>616
NI (120)
Cl
12B
13B
32.3
10.6
CF3
N
CH3
O
O
CF3
>>206
NI (40)
O
Cl
Cl
N
N
>>826
NI (160)
14B
15B
44.7
6.1
O
O
25.9
O
F
a
NI indicates no detectable inhibition at the concentration reported in brackets ([I]). In these cases Ki was estimated considering an inhibition percentage (I%) of 2% at this
21
concentration and applying the formulas IC50 = (100 ꢁ I%) ꢀ [I]/I% and Ki = IC50/(1 + [S]/Km
)
where [S] is the concentration of folate used in the assay and Km is the affinity
constant of the enzyme for that substrate.5
A deconstruction approach was applied to three known phenol-
(1,3-dioxo-1H,3H-benzo[de]isochromen-5-yl)-carboxamide deriv-
atives (Library B, 1B–15B) (Table 2) and N-(1,3-dioxo-1H,3H-
benzo[de]isochromen-5-yl)-sulfonamide derivatives (Library C,
1C–12C) (Table 2), were designed, synthesized and screened. Frag-
ments considered for addition at the amino group at position 5
were selected for their drug-like properties and their adherence
to Lipinski’s rules,13 with the aim of exploring molecular diversity
while focusing on aromatic and hetero-aromatic compounds. Sub-
stituents included small aliphatic chains (10B); aromatic systems
with zero, one, or two substituents (1B–7B, 9B, 11B, 1C–5C and
7C–10C); longer chains with a variable-size aliphatic bridge (8B,
12B, 13B and 11C); two aromatic systems with variable substitu-
ents (14B, 15B, 6C and 12C). MW values ranged from 327 to
506 Da; LogD values at pH 7.4 ranged from 0.37 to 4.76. Except
for compound 3B, which had three H-bond donor atoms, all of
the other derivatives had only one H-bond donor center. The num-
ber of H-bond acceptor atoms varied from 4 to 7.
naphthalein derivatives:
a156, MR20 and GA9. Two simple frag-
ments were identified: phenol (1) and benzo[de]isochromene-
1,3-dione (known also as 1,8-naphthalic anhydride) (2) (Scheme 1).
We focused on fragment 2 and an initial library of commercially
available compounds (Library A, 1A–18A) (Table 1) that repre-
sented potentially new scaffolds was compiled and screened
against hTS and EcTS. The library was designed using ChemFinder
from the ChemOffice program suite9; the ACXProd database and
ChemFinder were searched using the substructure search (see Sup-
plementary Fig. SI-2 for queries used). The 306 retrieved results
were filtered based on each candidate’s suitability for further
derivatization, commercial availability and cost. All of the com-
pounds were commercially available, with the exception of com-
pound 18A, which was obtained by the reduction of compound
17A.10,11
With the exception of three compounds (11A, 12A and 18A;
Ki = 105, 26.8 and 6.8
l
M, respectively) (Table 1), none of the com-
Compounds 1B–15B were obtained by reaction of 18A with the
corresponding acyl chloride as depicted in Scheme 2.10 After reac-
tion termination, ice was added to the solution and a precipitate
was formed. In the case of 1B, 2B, 4B–7B, 9B and 12B–14B, the
reaction mixture was first filtered to separate a precipitate consist-
ing of the starting material and then treated with ice. The filtered
precipitate was suspended in water and treated with sodium bicar-
bonate (pH to 8 and 9); the reaction product was then separated
out by filtration (Scheme 2).
pounds inhibited EcTS.12 Almost half of the compounds in Library A
(3A–5A, 9A, 13A–14A and 16A–18A) did not show inhibition
against hTS. The most active compounds against hTS were 10A
and 12A, with Ki values of 2.0 lM (Table 1, Fig. 1).
From Library A, compound 18A was selected because it has a
chemical handle which allows easy structural variation. This com-
pound was active at low micromolar levels against EcTS and
showed no inhibition of the human enzyme. Two libraries, N-

S. Ferrari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 663–668
667
Figure 1. Inhibition activity profile of Libraries A, B and C.
12B–14B) are species-specific, that is, they showed inhibitory
activity toward the bacterial enzyme without inhibiting the human
enzyme. The other compounds were also active against hTS, with
Ki values in the range of 2.3–58
Compounds 1C–12C are better inhibitors of TS and exhibit Ki
values in the narrower range of 0.3–5.0 M, but they are not spe-
lM (Table 2, Fig. 1).
l
cific inhibitors of the bacterial TS, as they show the same level of
inhibition against both bacterial and human enzymes12 (Table 2,
Fig. 1).
It is worth considering the change in the inhibition activity pro-
file resulting from the derivatization of the scaffold at position 5
with a carboxamidic bridge or a sulfonamidic bridge. The carbox-
amidic bridge does not modify the activity level or the species
specificity profile of the scaffold compound. In contrast, the sulfo-
namidic bridge leads to increased activity with respect to the scaf-
fold compound (18A) over non-specific compounds and enhances
the activity against hTS. Docking studies have been performed to
explain the difference in activity profiles between Library B and Li-
brary C (see Supplementary data). The results obtained show that
these molecules (4B, 1C, 4C and 9C) are predicted to bind in the
same region, that is, in the folate binding site where other folate-
analog TS inhibitors bind. The sulfonamidic derivatives show a dif-
ferent overall shape compared to the carboxamide compounds and
bind deeper in the active site (see Supplementary Figs. SI-2 and
SI-3). The interaction pattern is different: the sulfonamidic
Scheme 2. Synthesis of Libraries B and C.
Compounds 1C–12C were synthesized by reacting 18A with the
corresponding sulfonyl chloride as depicted in Scheme 2.10 After
reaction termination, the reaction solution was diluted with water
and the resulting precipitate was separated by filtration, dissolved
in a mixture of acetone and ethanol (50/50 v/v) and subsequently
dried under vacuum. This last step was repeated two or three times
to eliminate the residual Py (Scheme 2).
Compounds 1B–15B are active as inhibitors of EcTS, with Ki val-
ues in the range of 4.0–44.7 l
M.12 Six compounds (3B–4B, 9B and

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S. Ferrari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 663–668
5. Costi, M. P.; Gelain, A.; Barlocco, D.; Ghelli, S.; Soragni, F.; Raniero, F.; Rossi, T.;
Ruberto, A.; Guillou, C.; Cavazzuti, A.; Casolari, C.; Ferrari, S. J. Med. Chem. 2006,
49, 5958.
6. Finer-Moore, J. S.; Anderson, A. C.; O’Neil, R. H.; Costi, M. P.; Ferrari, S.;
Krucinski, J.; Stroud, R. Acta Crystallogr., Sect. D 2005, D61, 1320.
7. Ferrari, S.; Costi, M. P.; Wade, R. Chem. Biol. 2003, 10, 1183.
8. Babaoglu, K.; Shoichet, B. K. Nat. Chem. Biol. 2006, 2, 720.
9. ChemOffice Ultra 7.0, Cambridge Soft Corporation, http://www.
cambridgesoft.com.
derivatives interact with the non conserved residue, Trp 83 in EcTS
that corresponds to Asn 112 in hTS; the carboxamidic compound
4B is predicted to interact only with the bulky Trp. These results
can partly explain the observed biological activity profiles.
Some of the compounds in Library B were also tested against
human dihydrofolate reductase (hDHFR), which is an enzyme of
the thymidylate cycle, to identify potential off-target effects. All
of the tested compounds were inactive toward hDHFR (see Supple-
mentary Table SI-4), which demonstrates that they are selective
inhibitors of the TS enzyme.
By applying a ligand-based approach to the phenolnaphthalein
class of TS inhibitors, we identified a new scaffold (18A) that could
be easily modified through synthetic chemistry, thus allowing the
development of two compound libraries stemming from an initial
scaffold. Screening against hTS and EcTS revealed differences in the
species-specific inhibition activity of the two libraries. Most of the
carboxamide derivatives showed specificity toward the bacterial
TS. In particular, compounds 4B and 9B will be further developed.
Their levels of activity and specificity are similar to that of GA9 but
the chemical structure of this series of compounds allows further
exploration of the neighbouring chemical space to obtain promis-
ing antibacterial lead compounds. In contrast, the sulfonamide
derivatives are promising as anticancer compounds, as they inhibit
10. Chemistry: Reagents were purchased from Sigma–Aldrich. Reaction progress
was monitored by TLC on pre-coated silica gel 60 F254 plates (Merck) and
visualization was accomplished using UV light (254 nm). The purity of all
materials was determined to be at least 95% by TLC, 1H NMR and elemental
analyses. Compounds 1A–17A were purchased from different vendors and
their purity was evaluated to be higher than 95% by elemental analyses
(Supplementary Table SI-2). Reaction yields refer to purified products and are
not optimized (Supplementary Table SI-3). The purity of all synthesized
compounds was determined by elemental analyses performed on a Perkin–
Elmer 240C instrument and the results for C, H and N microanalysis were
within 0.4% of theoretical values (Supplementary Table SI-3). All synthesized
compounds were characterized by 1H NMR on a Bruker FT-NMR AVANCE 200.
Spectra were recorded in hexadeuterodimethylsulfoxide (DMSO-d6). Chemical
shifts are reported as d values (ppm) and referenced to tetramethylsilane as an
internal standard. When peak multiplicities are given, the following
abbreviations are used: s, singlet; d, doublet; t, triplet; dd, double doublet;
m, multiplet (see Supplementary data).
11. Synthesis of 5-amino-benzo[de]isochromene-1,3-dione (18A): The scaffold of
Libraries B and C was obtained through the catalytic reduction of 17A (5-nitro-
benzo[de]isochromene-1,3-dione). The nitro compound, 17A (5 g, 20 mmol),
was dissolved in dimethylformamide (40 mL) and 10% Pd/C (0.5 g) was added.
The mixture was stirred under H2 (P = 2 atm, T = 40 °C), for 18 h. The catalyzer
was then filtered off and the product 18A was precipitated by addition of
water.
12. Inhibition activity profile evaluation: Proteins were purified as described
previously.14–18 Folate cofactors and substrates were a gift from Merck & Co
(Switzerland); all other substrates, cofactors and reagents were purchased
from different companies at the highest purity grade possible. Kinetic
experiments with TS were conducted under standard conditions.19 Inhibition
experiments were conducted by measuring the effects of different inhibitor
concentrations on the initial rate of the enzymatic reaction, in the presence of a
limited concentration of the folate substrate. Reactions were initiated by
addition of the enzyme. IC50 (concentration of inhibitor required to inhibit 50%
of enzymatic activity) values were determined and Ki values were detected,
showing competitive inhibition with respect to MTHF for all of the
compounds.20,21 Kinetic measurements using human DHFR were performed
at 25 °C in standard enzyme buffer.22,23 For the determination of reductase
activity, NADPH oxidation was followed at 340 nm. The kinetic experiments
were performed in triplicate and no individual measurements differed by >20%
from the mean. Stock solutions of each inhibitor were freshly prepared in
dimethylsulfoxide (DMSO). The DMSO concentration was kept below the
concentration known to affect enzymatic activity (5% for TS and human DHFR).
13. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
2001, 46, 3.
hTS with Ki values in the range of 300 nM–5 lM. In particular,
three compounds (1C, 4C and 9C) with Ki values from 300 to
500 nM will be proposed for the development of drug leads that
are useful against human tumor cells.
Acknowledgments
This study was supported by MIUR-PRIN 2009 to Costi M.P.
200925BPZ5_004. We wish to thank the Cassa di Risparmio di
Modena (CDRM) Foundation. We thank CIGS (Centro Interdiparti-
mentale Grandi Strumenti) of the University of Modena and Reggio
Emilia for the use of their instrumentation facilities. R.C.W. grate-
fully acknowledges the support of the Klaus Tschira Foundation.
We also thank Professor M. Rinaldi for assistance with chemical
synthesis.
Supplementary data
14. Maley, G. F.; Maley, F. J. Biol. Chem. 1988, 263, 7620.
15. Davisson, V.; Sirawaraporn, W.; Santi, D. V. J. Biol. Chem. 1989, 264, 9145.
16. Davisson, V. J.; Sirawaraporn, W.; Santi, D. V. J. Biol. Chem. 1994, 269, 30740.
17. Pedersenlane, J.; Maley, G. F.; Chu, E.; Maley, F. Protein Expr. Purif. 1997, 10, 256.
18. Dann, J. G.; Ostler, G.; Bjur, R. A.; King, R. W.; Scudder, P.; Turner, P. C.; Roberts,
G. C.; Burgen, A. S. Biochem. J. 1976, 157, 559.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
11.117.
19. Pogolotti, A. L., Jr.; Danenberg, P. V.; Santi, D. V. J. Med. Chem. 1986, 29, 478.
20. Chou, T. Mol. Pharmacol. 1974, 10, 235.
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