Phthalein derivatives as a new tool for selectivity in thymidylate synthase inhibition

Article abstract of DOI:10.1021/jm9900016

A new set of phthalein derivatives stemming from the lead compound, phenolphthalein, were designed to specifically complement structural features of a bacterial form of thymidylate synthase (Lactobacillus casei, LcTS) versus the human TS (hTS) enzyme. The new compounds were screened for their activity and their specificity against TS enzymes from different species, namely, L. casei (LcTS), Pneumocystis carinii (PcTS), Cryptococcus neoformans (CnTS), and human thymidylate synthase (hTS). Apparent inhibition constants (K(i)) for all the compounds against LcTS were determined, and inhibition factors (IF, ratio between the initial rates of the enzymatic reaction in the presence and absence of each inhibitor) against each of the four TS species were measured. A strong correlation was found between the two activity parameters, IF and K(i), and therefore the simpler IF was used as a screening factor in order to accelerate biological evaluation. Compounds 5b, 5c, 5ba, and 6bc showed substantial inhibition of LcTS while remaining largely inactive against hTS, illustrating for the first time remarkable species specificity among TSs. Due to sequence homology between the enzymes, several compounds also showed high activity and specificity for CnTS. In particular, 3-hydroxy-3-(3-chloro-4-hydroxyphenyl)-6-nitro-1H,3H-naphtho[1,8-c,d]pyran- 1-one (6bc) showed an IF < 0.04 for CnTS (K(i) = 0.45 μM) while remaining inactive in the hTS assay at the maximum solubility concentration of the compound (200 μM). In cell culture assays most of the compounds were found to be noncytotoxic to human cell lines but were cytotoxic against several species of Grampositive bacteria. These results are consistent with the enzymatic assays. Intriguingly, several compounds also had selective activity against Cr. neoformans in cell culture assay. In general, the most active and selective compounds against the Gram-positive bacteria were those designed and found in the enzyme assay to be specific for LcTS versus hTS. The original lead compound was least selective against most of the cell lines tested. To our knowledge these compounds are the first TS inhibitors selective for bacterial TS with respect to hTS.

Full text of DOI:10.1021/jm9900016

2112
J . Med. Chem. 1999, 42, 2112-2124
P h th a lein Der iva tives a s a New Tool for Selectivity in Th ym id yla te Syn th a se
In h ibition
Paola M. Costi,*,† Marcella Rinaldi,† Donatella Tondi,† Piergiorgio Pecorari,† Daniela Barlocco,*,‡ Stefano Ghelli,§
Robert M. Stroud, Daniel V. Santi, Thomas J . Stout, Chiara Musiu,| Elena M. Marangiu,| Alessandra Pani,|
Donatella Congiu,| Giulia A. Loi,| and Paolo La Colla*,|
Dipartimento Scienze Farmaceutiche and Dipartimento Scienze Chimiche, Universita` di Modena e Reggio Emilia,
Via Campi 183, 41100 Modena, Italy, Istituto Chimica Farmacologia e Tossacologia, Universita` Milano, Viale Abruzzi 42,
20131 Milano, Italy, Departments of Biochemistry and Biophysics and of Pharmaceutical Chemistry, University of California,
San Francisco, California 94143, and Dipartimento Biologia Sperimentale, Universita` di Cagliari, v.le Regina Margherita 45,
09214 Cagliari, Italy
Received J anuary 4, 1999
A new set of phthalein derivatives stemming from the lead compound, phenolphthalein, were
designed to specifically complement structural features of a bacterial form of thymidylate
synthase (Lactobacillus casei, LcTS) versus the human TS (hTS) enzyme. The new compounds
were screened for their activity and their specificity against TS enzymes from different species,
namely, L. casei (LcTS), Pneumocystis carinii (PcTS), Cryptococcus neoformans (CnTS), and
human thymidylate synthase (hTS). Apparent inhibition constants (Ki) for all the compounds
against LcTS were determined, and inhibition factors (IF, ratio between the initial rates of
the enzymatic reaction in the presence and absence of each inhibitor) against each of the four
TS species were measured. A strong correlation was found between the two activity parameters,
IF and Ki, and therefore the simpler IF was used as a screening factor in order to accelerate
biological evaluation. Compounds 5b, 5c, 5ba , and 6bc showed substantial inhibition of LcTS
while remaining largely inactive against hTS, illustrating for the first time remarkable species
specificity among TSs. Due to sequence homology between the enzymes, several compounds
also showed high activity and specificity for CnTS. In particular, 3-hydroxy-3-(3-chloro-4-
hydroxyphenyl)-6-nitro-1H,3H-naphtho[1,8-c,d]pyran-1-one (6bc) showed an IF < 0.04 for CnTS
(Ki ) 0.45 µM) while remaining inactive in the hTS assay at the maximum solubility
concentration of the compound (200 µM). In cell culture assays most of the compounds were
found to be noncytotoxic to human cell lines but were cytotoxic against several species of Gram-
positive bacteria. These results are consistent with the enzymatic assays. Intriguingly, several
compounds also had selective activity against Cr. neoformans in cell culture assay. In general,
the most active and selective compounds against the Gram-positive bacteria were those designed
and found in the enzyme assay to be specific for LcTS versus hTS. The original lead compound
was least selective against most of the cell lines tested. To our knowledge these compounds
are the first TS inhibitors selective for bacterial TS with respect to hTS.
In tr od u ction
TS would prevent cell multiplication. The primary
structure of TS from various sources has been assessed.5
In some cases, crystal structures of apoenzymes and/or
of binary and ternary TS complexes with substrates and/
or ligands have also been solved.6-12 In particular, the
structures of the enzymes from Lactobacillus casei,
Escherichia coli, Pneumocystis carinii,6,9,10 and, more
recently, human cells have been reported.12 These
studies give information on the enzyme’s structure and
function, on the binding sites, and on the modes of
binding of different TS inhibitors, thus providing the
setting for structure-based drug design.13,14 Several TS
inhibitors able to bind either at the pyrimidine substrate
or at the folate cofactor sites, most of which have been
developed as anticancer agents, are known.1 The clas-
sical folate analogues, such as CB3717, and especially
the more recent ones, such as BW1843U89 (Figure 1),
are very potent inhibitors of human TS.15-23 They are
powerful antitumor agents with acknowledged adverse
side effects. Some problems are related to their meta-
bolic pathway:24,25 they require a carrier (reduced folate
Thymidylate synthase (TS), an enzyme crucial to
DNA synthesis in both prokaryotic and eukaryotic cells,
is a target for the development of antibacterial, anti-
mycotic, anticancer, and recently, antiviral agents.1-3
TS catalyzes the conversion of deoxyuridine monophos-
phate (dUMP) to deoxythymidine monophosphate (dTMP)
by a reductive methylation involving N5,N10-methyl-
enetetrahydrofolate (mTHF) as a cofactor.4 In the
absence of the additional formation of dTMP via thy-
midine kinase (salvage pathway), the reaction catalyzed
by TS is the rate-limiting step in DNA synthesis since
it is the sole de novo pathway for the synthesis of dTTP
(deoxythymidine triphosphate). Therefore, inhibition of
* To whom correspondence should be addressed.
† Dipartimento Scienze Chimiche, Universita` di Modena e Reggio
Emilia.
‡ Universita` Milano.
§ Dipartimento Scienze Farmaceutiche, Universita` di Modena e
Reggio Emilia.
University of California.
| Universita di Cagliari.
10.1021/jm9900016 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/29/1999

Selective Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2113
in the human enzyme. Several of these derivatives had
considerable specificity for LcTS versus hTS and also
showed selective toxicity in bacterial versus human cell
line culture assays.
The present paper deals with the design, the synthe-
sis, the characterization, and the biological evaluation
of compounds designed to be selective against bacterial
TS (obtained from the Gram-positive organism L. casei)
and not against hTS. To our knowledge these are the
first inhibitors of TS to show such species selectivity.
The bases for structural recognition of the phthalein
analogues are discussed; detailed analysis of the mode
of binding through computational chemistry and mu-
tagenesis will be described elsewhere.27
Design
The crystal structure of the phenolphthalein-LcTS
complex has been solved at 2.3 Å resolution.26 Phenol-
phthalein is found to occupy the top of the folate binding
site (Figure 2). The ligand makes hydrogen bonds with
Asp221, Glu60, and Arg23. In particular, one phenol
hydroxyl group makes a hydrogen bond with Glu60
through a conserved water molecule (Wat404), while the
other hydroxyl group makes a hydrogen bond with
Asp221 (L. casei numbering) (Oδ1 2.95 Å). The carbonyl
of phenolphthalein is directed toward the -NH of Arg23
(at a distance of 4.02 Å). The phthalidic ring points
toward the large empty space present in the folate
pocket and does not interact directly with any other
residue. Specifically, position 4 of the phthalidic benzene
ring is 3.54 Å from Trp85. Positions 5 and 6 are 4.21
and 4.25 Å from Glu84, respectively, while position 7
points completely out of the folate site. The orientation
of the phthalidic ring points toward a previously unex-
plored region of the active site. Specifically, the benzene
ring approaches residues ordinarily uninvolved in ligand
binding: Val314 (4.58 Å), Ile81 (5.80 Å), and Ala194 (7.7
Å).
On the basis of this novel ligand-binding mode, we
designed a new series of phthalein analogues with an
expanded phthalidic ring. This ring expansion was
intended to bridge the empty space found in the PTH
complex and make novel interactions with distal resi-
dues which are specific to bacterial forms of TS. On the
basis of graphical modeling, the new 1,8-naphthalidic
derivative appeared to fit well into the binding pocket,
substantially maintaining its binding mode with respect
to phenolphthalein. The phenolic rings of the 1,8-
naphthalidic derivative superimposed well with those
of phenolphthalein. Modeling also indicated that intro-
duction of halogen substituents at the ortho-position on
the phenol ring would not cause large steric clashes,
would improve the binding energy with additional
interactions, and would influence the hydrogen bond
strength of the phenolic function.
F igu r e 1. Folate cofactor and TS antifolates.
carrier, RFC) to carry a prodrug thorough the cell
membrane and enter the cells, where they subsequently
need to be activated to produce a more effective inhibitor
of the enzyme. These prodrugs are substrates of folylpoly-
glutamyl synthetase (FPGS), which attaches additional
glutamic acid residues to the one already present.24,25
The resulting polyglutamylated form often binds more
tightly to TS than the monoglutamylated form. More-
over, these compounds bind in nearly the same mode
as the natural folate cofactor.19 This site is well-
conserved in both the primary and the tertiary struc-
tures among TS enzymes from different species. This
could explain why no selectivity has been obtained to
date: compounds that inhibit eukaryotic TS usually also
inhibit the prokaryotic enzyme.
To our knowledge, the potential capability of a
compound to bind bacterial TS with higher affinity than
the human enzyme has not been deeply investi-
gated.19,21,26 In most cases, compounds designed to be
selective have failed.19,20 In principle, the knowledge of
structural details of enzymes from different organisms
presents the opportunity to rationally design new
compounds exploiting the structural differences existing
in the active sites of the different species.
Ch em istr y
The syntheses of compounds 1 and 2 from phthalic
or 2,3-naphthalic anhydride, respectively, are depicted
in Scheme 1. Accordingly, heating the required anhy-
dride at 180 °C for 5 h with the appropriate (substituted)
phenol in the presence of a few drops of sulfuric acid
gave the desired phthaleins, which were subsequently
purified by silica gel chromatography (Table 1). The
Starting from the X-ray crystal structure of the
complex of the novel inhibitor phenolphthalein (PTH,
1a ) (Figure 2), bound to LcTS,26 new compounds were
designed and synthesized. These phthalein analogues
were designed to take advantage of differences in
structural aspects of the bacterial active site not found

2114 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Costi et al.
F igu r e 2. Detail of the X-ray crystal structure of the binary complex between LcTS and phenolphthalein.
Sch em e 1a
a
A ) H, phenyl. (a) H2SO4, 180 °C, 5 h.
unsymmetrically substituted 1c was obtained from the
commercially available 2-(4′hydroxybenzoyl)benzoic acid
by treatment with thionyl chloride and, in succession,
R-naphthol (Scheme 2). Attempts to apply the method
described above to 1,8-naphthalic anhydride for the 2,3-
isomer failed, resulting in starting material being
recovered in high amounts in addition to traces of
compound 3 or 4, depending on the starting phenol
(Scheme 3).
Sch em e 2a
An alternative pathway was therefore devised by
adapting the procedure of Hubacher.28 As shown in
Scheme 4, the 1,8-naphthalic anhydride was heated at
130-140 °C with the appropriate phenol and aluminum
a
(a) SO2Cl, 80 °C, 8 h; (b) R-naphthol.
chloride for 3 days. The still hot mixture was then
poured onto ice and extracted with dichloromethane.

Selective Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2115
Sch em e 3
The insoluble residue was filtered off, the filtrate
concentrated under vacuum, and the residue purified
by silica gel chromatography to give, in elution order,
the unreacted anhydride, compounds 5, and the mono-
substituted compounds 6, whose presence was strongly
dependent on the starting material. When phenol was
used, together with the corresponding para-substituted
5a , the o-phenol derivative 6a was obtained. It should
be noted that when using thymol the para-substituted
compound 5 was not formed and only the ortho-mono-
the mode of inhibition. Most of the compounds were
shown to interfere with the folate cofactor activity and
therefore behave as antifolates, as reported in Table 3.
However, while most showed competitive inhibition with
respect to the folate cofactor, two compounds (6c, 5ba )
displayed mixed-type inhibition. Compound 6bc dis-
played noncompetitive inhibition. Analysis of the Ki’s
of these compounds (Table 3) suggests that the semi-
naphthalenic compound 6bc is the best inhibitor of
LcTS (Ki of 0.25 µM), while compounds 5c, 5a a , and 5ba
show Ki’s of 0.7 µM. Many other compounds (1b, 2b,
2c, 5a , 5b, 5a b, 5a c, 7) show Ki’s in the range of 1-2.8
µM. The least potent inhibitors are compounds 1c, 5d ,
and 6a -6d (Ki values ranging from 20.8 to higher than
290 µM). All of the compounds can be grouped into
phthaleinic (1a -1c), 2,3-naphthalenic (2a -2c), 1,8-
naphthalenic (5a -5d , 5a a -5bb, 7), and semi-naphtha-
lenic structures (6a -6d , 6bc). Among the phthaleinic
compounds, the o-chloro substituent (compound 1b)
enhances the inhibitory activity of phenolphthalein (1a )
2.5-fold against LcTS, while compound 1c, bearing an
R-naphthol group, is about 4 times less active than PTH.
This is likely due to the steric hindrance of the naphthol
ring. In the 2,3-naphthalenic series, the o-halogen on
the phenolic ring (compounds 2b, 2c) enhances the
inhibitory potencies with respect to the unsubstituted
compound (2a ). As a group, the 1,8-naphthalenic com-
pounds form the most interesting series: the semi-
naphthalenic derivatives unsubstituted on the naph-
thalenic ring (6a -6d ) showed low or very low inhibitory
potencies (6d , Ki ) 26 µM; 6a , Ki ) 104.7 µM; 6c, Ki )
295.6 µM), while the compound substituted at the
naphthalenic ring is the most active compound among
all those studied (6bc, Ki ) 0.25 µM). It should be noted
that while compounds 6a -6c have the phenolic hy-
droxyl in position 2, compound 6bc has the phenolic
hydroxyl in the 4-position, an o-chlorine group in the
3-position (ortho with respect to the phenolic hydroxyl),
and a nitro group on the naphthalenic ring. The 1,8-
naphthalenic derivatives (5a -5d ), unsubstituted on the
naphthalenic ring, show Ki values ranging between 0.7
µM and 47% inhibition at 27 µM. The chlorine atom on
the phenolic ring enhances the inhibitory activity: 5c
shows the lowest Ki value (0.7 µM) in this series of
1
substituted 6d was seen through H NMR (Scheme 4).
The alkoxy derivatives 5d and 6c were synthesized by
reacting 5a and 6a , respectively, with the appropriate
alkyl halide in refluxing acetone and in the presence of
potassium carbonate (Scheme 4, Table 1). Attempts to
prepare 5d starting from 1,8-naphthoic anhydride and
anisole failed, since a concomitant demethylation took
place, leading to 5a as the sole reaction product.
Following the same procedure described for compounds
5, compound 7 was prepared starting from acenaph-
thene-5,6-dicarboxylic anhydride (Table 2 and Experi-
mental Section).
Finally, it should also be noted that when starting
from the 4-chloro- or 4-bromo-substituted 1,8-naphthalic
anhydride, an approximately 60:40 mixture of the two
possible isomers, 5a a and 5a b, 5ba and 5bb (Table 2),
1
was obtained, as clearly indicated by H NMR spectra,
while no monosubstituted productssas compounds 6s
were detected. Tables 3-5 report the biological activity
of the above-mentioned compounds measured as iso-
meric mixtures. Future work involves the resolution of
these isomers and testing their TS inhibition properties.
In contrast, the presence of a 4-nitro group on the 1,8-
naphthalic anhydride led to the formation of a single
isomer for both 5 (5a c) and 6 (6bc) (Scheme 5 and Table
2).
Resu lts a n d Discu ssion
En zym e In h ibition . All of the compounds were
tested for their inhibitory properties against TS en-
zymes from different species: LcTS, PcTS, CnTS, and
hTS. First, Ki’s were determined for each of the com-
pounds against LcTS. Lineweaver-Burk (LB) analyses
were performed for many of them in order to identify

2116 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Costi et al.
Sch em e 4a
Sch em e 5a
a
(a) AlCl3, ∆ (130/140 °C), 72 h.
6/7-substituted 3,3-bis(4-hydroxyphenyl)-1H,3H-naph-
tho[1,8-cd]pyran-1-one derivatives implying that the
substituents do not give specific interactions with any
active site residue. It should be noted that the isomeric
mixtures of 6/7-substituted compounds (5a a and 5a b,
5ba and 5bb) were tested without separating the two
isomers. Work in progress involves the separation of the
isomeric mixtures and the testing of the two isomers.
Finally, compound 7 shows a Ki value of 1.5 µM. This
is in the same range as compounds 5a a , 5a b, 5a c, and
5ba , which is in keeping with the above-described lack
of specific interactions for this region of the compounds
with active site residues.
Before conducting cell culture assays, inhibition fac-
tors (IF) were determined at a single inhibitor concen-
tration (27 µM) against each of the four enzymes (LcTS,
PcTS, CnTS, and hTS)29,30 (Table 4). This approach
consisted of measuring the single concentration inhib-
ited and uninhibited initial rates of the enzyme reaction
to give the IF value (see Experimental Section). This
rapid method is utilized for efficient compound screen-
ing/profiling and to direct more detailed kinetic and in
vitro experiments.
a
(a) AlCl3, 140 °C, 72 h; (b1) CH3I, K2CO3, acetone; (b2)
(CH3)NCH2CH2I, K2CO3, acetone.
compounds. That compound 5d is much less active than
5a (47% inhibition at 27 µM for 5d , 2.8 µM for 5a )
implies that the substitution of the phenolic hydroxyl
with a methoxy group blocks a key hydrogen bond
interaction within the active site. 1,8-Naphthalenic
derivatives such as chloro (5a a , 5ba ), bromo (5a b, 5bb),
or nitro (5a c) show promising inhibition, with Ki values
ranging between 0.7 and 1.7 µM. The exception to this
trend is compound 5bb, which has a slightly higher Ki
of 6.3 µM.
Since many of these compounds have one or two
electron-withdrawing groups (halogen or nitro group)
on the phenolic and/or naphthalenic rings but no large
difference in inhibitory potency, it would seem that
these functional modifications alone are not critical for
the activity toward the LcTS enzyme. In particular, no
improvement in the binding energy was observed in the
To evaluate the reliability of IF as an inhibitor of the
biological activity of the compounds during the screen-
ing phase and to gain a significant biological profile, we
studied the relationships for each compound between
the Ki measured against LcTS (Table 3) and the IF

Selective Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2117
Ta ble 1. Physicochemical Data of Compounds 1, 2, 5, and 6
compd
R
R1
% yield
mp (°C)
formulaa
1b
2a
2b
2c
5a
5b
5c
Cl
H
F
Cl
H
F
Cl
H
H
H
50
29
30
35
18
25
9
88-90
C20H12Cl2O4
C24H16O4
C24H14F2O4
C24H14Cl2O4
C24H16O4
C24H14F2O4
C24H14Cl2O4
C26H20O4
298-300
135-140
200-205
265-267
250-253
162-165
167-168
175-178
160-163
192-193
H
H
H
CH3
H
5d
6a c
6c
83b
15
40
12
C18H12O4
C23H23NO4
C22H20O4
(CH2)3
H
6d c
3-CH(CH3)2 and 6-CH3
a
b
Elemental analyses for the compounds were within (0.4% of the theoretical values. From 5a . c 1H NMR spectra (DMSO-d6) indicate
the existence of a corresponding open form (see Supporting Information.
Ta ble 2. Physicochemical Data of Compounds 5a a -bb, 6bc,
and 7
Ta ble 3. Inhibition Constants (µM) against L. casei
Thymidylate Synthase for Compounds 1, 2, and 5-7
compda
Kiapp
type of inhibitionb
1a c (1)
1b (6)
1c
4.7 (33.1)
1.9
20.8
M/C
C
C
2a (2)
2b
7.0
1.5
C
C
2c
1.5
C
5a (3)
5b
2.8
1.6
C
C
5c (4)
5d
6a
6c
6d
0.7
C
47% at 27 µMd
104.7
295.6 (764.0)
26
C
M
C
compd
R
R1
% yield
formulaa
5a a e
5a be
5a c (5)
5ba e (7)
5bbe
6bc (8)
7
0.7
1.7
1.0
0.73 (1.0)
6.3
C
C
C
M
C
NC
C
C
5a a b
5a bb
5a c
5ba b
5bbb
6bc
7
H
H
H
Cl
Cl
6(7)-Cl
6(7)-Br
6-NO2
6(7)-Cl
6(7)-Br
50
12
10
50
14
10
10
C24H15ClO4
C24H15BrO4
C24H15NO6
C24H13Cl3O4
C24H13BrCl2O4
C18H10ClNO6
C26H18O4
0.25
1.5
CB3717
0.095f
a
Elemental analyses for the test compounds were within (0.4%
a
b
Number in parentheses refers to the corresponding compound
of the theoretical values. As a 60/40 mixture of two isomers. See
b
Supporting Information for 1H NMR data.
in ref 27 for the convenience of the reader. C, competitive
inhibition; M, nonlinear mixed-type inhibition; NC, noncompeti-
tive. Numbers in parentheses indicate RKi for mixed-type inhibitor.
c 1a is phenolphthalein. Percentage of inhibition at the indicated
d
against LcTS. The plot of log(Ki) against log(IF) gives a
linear relationship with a strong correlation factor (R
) 0.8122) between the two parameters (data not shown).
This strong correlation between the two parameters
prompted us to screen all the compounds using simple
IF data as inidicators of the inhibitory properties.
Inhibition factors for all the compounds against LcTS,
PcTS, CnTS, and hTS are shown in Figure 3 where they
are grouped by selectivity with respect to hTS. The
selectivity is given by the ratio IFhTS/IFother•species•TS
(data not shown). In the top panel of Figure 3, the IF
values against LcTS (blue) with respect to hTS (red) are
reported and the compounds are ranked by increasing
selectivity.
concentration. e 60/40 mixture of two isomers (see Table 2). f Ref-
erence 22.
2c showed appreciable inhibitory activity with IF values
between 0.103 and 0.182. The other derivatives were
weakly active (1b, 2a , 2b, 5a , 5a a , 5a b, 5a c, 7) with
IF values between 0.424 and 0.670 or inactive (5b, 5c,
5d , 6a , 6c, 6d , 5ba , 5bb, 6bc) ranging between 0.814
and 1. On the contrary, very interesting results were
observed in the inhibition of LcTS. In particular,
compounds 2c, 5b, 5c, 5a a , 5a b, 5ba , and 6bc displayed
IFs ranging from 0.070 to 0.129, while others (1a , 1b,
2a , 2b, 5a , 5a c, 5bb, 7) had inhibition factors < 0.5.
Structure-selectivity analysis suggests that the 2,3-
naphthalic anhydride derivative 2a has the same in-
The majority of the compounds were ineffective
toward hTS; only 1a , 1c, the phthalein derivatives, and

2118 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Costi et al.
Ta ble 4. Inhibition Factors (IF)a for Compounds 1, 2, and 5-7
the naphthalenic ring. However, 5ba and 5bb are
equipotent against LcTS; this suggests that these effects
are neither additive nor synergistic.
against Thymidylate Synthease from Different Species
compd
LcTS
PcTS
CnTS
hTS
1a b
1b
1c
2a
2b
2c
5a
5b
5c
5d
6a
6c
6d
0.444
0.470
0.529
0.247
0.298
0.093
0.309
0.096
0.070
0.624
0.781
0.768
0.966
0.092
0.112
0.250
0.129
0.360
0.086
0.409
<0.04
0.608
0.725
0.498
0.459
0.582
0.252
0.704
0.389
0.215
0.507
0.182
0.670
0.172
0.594
0.521
0.103
0.272
1
0.814
1
0.957
1
Compounds 6bc, 5c, and 5ba , characterized by the
presence of an o-chlorine on the phenolic rings, are the
most selective against LcTS versus hTS (Figure 3). It
seems that the o-halogen substitution with chlorine is
an important prerequisite for bacterial selectivity; the
o-fluoro derivative of 5a showed similar selectivity in
the only molecule synthesized (5b).
0.179
0.283
0.240
0.749
0.151
0.928
0.788
0.632
0.536
0.645
0.488
0.788
0.836
0.576
0.666
0.868
0.256
0.243
0.365
0.120
The selective activities of these compounds against
the TS from P. carinii are reported in the middle panel
of Figure 3 with respect to hTS and are ranked by
increasing selectivity. Only 5a , 5c, and 6a showed
interesting properties toward PcTS with IFs of 0.179,
0.240, and 0.151, respectively, while the others were
devoid of significant activity. Compounds 1c, 2a , 5ba ,
6bc, and 7 show IF values ranging between 0.344 and
0.498; compounds 1a , 1b, 2b, 5d , 6d , 5a a , 5a b, and 5a c
show low inhibitory activity with IF values ranging
between 0.536 and 0.788, while 6c is almost inactive
(Table 4 and Figure 3). The majority of the compounds
(1a , 1c, 5a c, 5a a , 5a b, 2b, 1b) are less active against
PcTS with respect to hTS. However, compounds 6c, 6d ,
2a , 5d , 7, and 5a are slightly better inhibitors of PcTS
than hTS, while compounds 5ba , 6bc, 5c, and 6a show
some selectivity and significantly inhibit PcTS while
remaining almost inactive against hTS (Figure 3). Also,
in this regard, compounds 5c, 5ba , and 6bc are among
the most selective compounds: the ortho-position chlo-
rine on the phenolic ring and the nitro group on the
naphthalenic ring in both the 1,8-naphthalenic and 1,8-
semi-naphthalenic derivatives confer selectivity to the
compounds toward bacterial/pathogenic TSs with re-
spect to the human enzyme.
1
5a a c
5a bc
5a c
5ba c
5bbc
6bc
7
0.438
0.453
0.424
1
1
1
0.418
0.344
<0.04
<0.04
0.344
<0.04
0.475
<0.04
CB3717
a
IF ) Vii/Vi, ratio between the initial rate of the enzymatic
reaction in the presence (Vii) and absence (Vi) of the inhibitor at
27 µM. LcTS, L. casei TS; PcTS, P. carinii TS; CnTS, Cr.
neoformans TS; hTS, human TS. 1a is phenolphthalein. c 60/40
b
mixture of two isomers (see Table 2).
hibitory activity against hTS (IF ) 0.59) and microbial
TS (IF ) 0.21-0.45). Similar behavior is observed with
the addition of a fluorine atom to the 3-position on
phenolic rings (2b), while a chlorine atom at the
3-position (2c) gave increased activity against hTS while
leaving the bacterial TS inhibition almost unchanged.
Among the 1,8-naphthalic anhydride derivatives, the
methylation of the phenolic hydroxyl gave an inactive
compound (5d ) against both LcTS and hTS, while the
addition of an o-chlorine atom on the phenolic rings led
to the derivative 5c, which is 4-fold more potent and
more selective against LcTS than the corresponding
unsubstituted 5a . Similar results were obtained when
a chlorine (5a a ) or bromine (5a b) atom was added to
In the bottom panel of Figure 3, IF values for CnTS
versus hTS are reported and the compounds are ranked
by increasing selectivity. Compounds 6bc and 5a b show
IF values < 0.04 and 0.120, respectively. Compounds
1a , 1c, 2a , 5a , 5a a , 5a b, 5a c, and 7 range from 0.215
Ta ble 5. In Vitro Activity (µM) of Compounds 1, 2, and 5-7
MIC/MGCb
a
compd
CC50 MT-4
S. aureus
Streptococcus
Cr. neoformans
SIc CC50/MICStr.
1a d
1b
1c
2a
2b
2c
5a
5b
5c
5d
6a
6c
6d
58
77
54
79
94
120
41
117
46
21
263
70
89
69
76
41
63
154
66
>58
32/64
17/54
17/68
15/30
3.1/6.2
34/41
25/50
7.0/14
>21
38/58
32/32
8/34
34/34
15/15
3.1/6.2
17/34
25/25
1.8/3.5
>21
171/263
>70
72/>89
8/8
7.0/28
>41
>58
>77
>54
>79
>94
>200
>41
>200
9.37
>21
18.7
>70
>89
>69
37.5
>41
9.37
>200
>66
>53
1.5
2.4
6.7
2.3
6.2
39.6
2.4
4.6
25.5
>263
>70
1.5
>89
1.2
8.6
10.8
5a a e
5a be
5a c
5ba e
5bbe
6bc
7
8/16
7.0/28
15/30
3/3
3.1/12.5
4/17
1.6/1.6
3.1/3.1
8/8
39.4
49.6
8.25
3.3
53
16/32
16/32
a
b
CC50, compound dose necessary to reduce the viability of mock-infected cells at 50%. MIC/MGC, minimum compound dose able to
inhibit bacterial growth/lowest compound dose with germicidal effect. c SI, selectivity index given by CC50/MICStreptococcus (CC50/MICStr.).
1a is phenolphthalein. e 60/40 mixture of two isomers (see Table 2).
d

Selective Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2119
F igu r e 3. Plot of the inhibition factors (IF) at 27 µM of compounds 1, 2, and 5-7 toward TS from L. casei. P. carinii, Cr. neoformans,
and human. IFs are grouped by selectivity with respect to human TS, and the compounds are ranked by increasing selectivity.
to 0.389, while 1b, 2b, 6a , and 6c show IFs ranging
between 0.507 and 0.704. Finally, 5c, 5d , and 6d display
IF values higher than 0.789. In general, the compounds
are more active against CnTS than PcTS (Table 4). With
regard to selectivity, compounds 1c, 1a , 1b, 5a , 2b, and
5c display essentially the same inhibition potency
toward both PcTS and hTS (Figure 3). Compounds 6d ,
5a c, 5d , and 7 are slightly less potent against hTS with
respect to CnTS, while 6c, 6a , 5a a , 5a b, and 2a show
higher selectivity. Finally, compound 6bc showed an IF

2120 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Costi et al.
derivatives were evaluated in vitro in two CD4+ lym-
phocyte cell lines: MT-4 and C8166. Compounds were
also evaluated for antiviral activity against two different
RNA viruses, human immunodeficiency virus (HIV) and
human rhinovirus (HRV), as well as against two DNA
viruses, such as vaccinia (VV) and herpes simplex
(HSV). The antibacterial activity was tested against
Gram-positive (Staphylococcus aureus, group D Strep-
tococcus), Gram-negative (Pseudomonas aeruginosa,
Salmonella sp.), and acid-fast (Mycobacterium avium,
M. tuberculosis), “microorganisms”, whereas the anti-
mycotic activity was evaluated against Candida albi-
cans, C. parapsilosis, C. paratropicalis, and Cryptococ-
cus neoformans. All of the studied compounds, with the
exception of the more active 5d and of the less active
2c, 5b, 5bb, and 6a , showed a similar low degree of
cytotoxicity for MT-4 cells (Table 5), with CC50 values
ranging from 41 to 94 µM. Compound 5d showed a CC50
value of 21 µM, and compounds 2c, 5b, 5bb, and 6a
were only slightly cytotoxic or not cytotoxic at all.
Similar results were obtained with C8166 cells (data not
shown). When evaluated for their ability to prevent
virus-induced cytopathogenicity in cell cultures infected
at low multiplicities of infection, none of the compounds
were found to inhibit the multiplication of HIV-1 (data
not shown). On the other hand, 1a was active against
two HRV strains, HRV-2 and HRV-14, at concentrations
(45 µM) that, although close to the CC50 value for MT-
4, were not cytotoxic for HeLa cells, suggesting a
selective inhibition of rhinoviruses (data not shown).
Plaque reduction assays revealed that the compounds
were inactive against both vaccinia and HSV (data not
shown). With respect to antibacterial activity, all of the
compounds were inactive against P. aeruginosa and
Salmonella sp., whereas some of them inhibited the
growth of both Staphylococcus and Streptococcus (Table
5). Compounds 5ba , 5c, 5bb, 2c > 5a a , 6bc, 5a b were,
in the order of decreasing potency, the most active
compounds. These compounds showed minimum inhibi-
tory concentrations (MIC) ranging between 1.6 and 8
µM and minimum germicidal concentrations (MGC)
ranging between 1.6 and 28 µM. These concentrations
were well below those found to be cytotoxic for T-cell
lines. This suggested a selective mode of inhibition.
When evaluated for antimycotic activity, none of the
compounds inhibited the multiplication of the various
Candida strains. However, several phenolphthalein
derivatives were selective inhibitors of Cr. neoformans,
the most potent compounds being 5c, 5ba , 5a b, and 6a
active at doses ranging between 9.4 and 37.5 µM.
F igu r e 4. Superimposition of the X-ray crystal structures of
the binary complexes of LcTS-PTH (in white, yellow residues)
and LcTS-5c (in gray, red residues).
< 0.04 against CnTS, while being inactive in the hTS
assay, thus displaying a potentially high selectivity with
respect to these two enzymes. To a lesser extent,
compound 5ba also showed some selectivity with respect
to CnTS.
The most potent and selective compound against
CnTS is the nitro derivative 6bc whose structure is
“semi-naphthalenic” with the substitution of a hydroxyl
group for one of the phenolic rings. Since compounds
6a , 6c, 6d , and 7 showed no particular selectivity or
potency, it is apparent that both the hydroxyl substitu-
tion for the phenol ring and the nitro group are crucial
to the activity and selectivity of this molecule. Finally,
compounds 3 and 4, whose structures are symmetric,
were devoid of activity.
The biological activity profile of the classic antifolate
inhibitor CB3717 was compared to that of the present
compounds: Ki values against LcTS, PcTS, CnTS, and
hTS were all very similar (0.095,22 0.09,22 0.075, 0.18
µM22), indicating that no selectivity is observed for this
classical TS inhibitor. As a very potent inhibitor of TS,
the IF values for CB3717 are lower than 0.04.
The molecular recognition of these unusual anti-
folates, examined through the analyses of the X-ray
crystal structures of the binary complexes of compound
5c with LcTS and of compound 5a c with LcTS,27 showed
that these naphthalene derivatives occupy different
binding sites than has been observed for compounds
1a 26 (PTH) and CB3717.9 Figure 4 shows details of the
crystal structure of the LcTS-5c binary complex (5c in
gray, residues in red) superimposed on the LcTS:PTH
binary complex (PTH in white, residues in yellow). It
can be noted that a different conformation must be
adopted by the enzyme to accommodate compound 5c
in this binding site, where it forms close interactions
with Arg23, His106, and Val316. These interactions are
discussed in detail elsewhere.27
The qualitative structure-activity relationship in
Staphylococcus and Streptococcus cell culture shows
that the introduction of a chlorine atom on the phenolic
ring of 1,8-naphthalene derivatives (cf. 5a with 5c, 5a a
with 5ba , 5a b with 5bb) increases either the activity
(i.e., 17/34 µM (5a ) 1.8/3.5 µM (5c)) or the selectivity
(i.e., 2.4 µM (5a ) 25.5 µM (5c)). The same behavior is
found with 2,3-naphthalene derivatives. Compound 2c
is more active and selective than 2a . The substitution
of the phenolic OH with a -OCH3 group (cf. 5a , 5d , 6a ,
6d ) and of the phenol ring with an OH group (cf. 5a ,
6a ) reduces the activity.
In Vitr o Assa ys. Following the initial enzymatic
screen, the cytotoxicity of phenolphthalein (1a ) and its

Selective Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2121
F igu r e 5. Superimposition of the X-ray complexes of PTH (black), CB3717 (yellow), 5a c (white), and 5c (gray).
Con clu sion s
validated the specificity effects shown by modifications
of the phenol moieties and explained the promiscuity
of derivitizations on the naphthalenic core.27 These
structures showed that the C6-C7 edge of the naph-
thalenic ring system is directed toward a large cavity
within the active site where there are no close contacts
made with the protein, while the phenol rings make
distinct, tight interactions with residues from an amino
acid insertion unique to bacterial and mycotic forms of
the enzyme.
With the LcTS:PTH crystal structure as a starting
point, we have been able to design a new class of
naphthalenic TS inhibitors which specifically target
interactions within the enzyme’s active site which are
unique to bacterial and mycotic species. As a result,
several rounds of structure-guided inhibitor design have
led to compounds with both enhanced potency of a high
degree of species specificity. The best compounds in this
series show nanomolar inhibition of bacterial TSs, while
leaving hTS unimpeded even at the solubility limits of
the compounds (200 µM). Structure-activity analysis
of these compounds against LcTS showed that many
functional variations can be made on the naphthalenic
core without significantly altering the compounds’ bind-
ing affinities, from which we conclude that this portion
of the chemical scaffold is not crucial to inhibitory
activity. In contrast, small modifications on the phenol
ring moieties, such as the introduction of chlorine at the
ortho-position, significantly alter the selectivity alter the
selectivity alter the selectivity properties of the mol-
ecules. Crystal structures of two binary complexes of
LcTS with compounds in this series (5c, 5a c) both
Since this new class of TS inhibitors was designed to
be selective against human TS, these compounds were
evaluated for their antibacterial activity as well as for
efficacy against opportunistic pathogens whose infec-
tions are often lethal in disorders arising from HIV and
cancer therapies. All of the compounds were initially
screened for biological activity using the relatively
simple inhibition factor (IF) assay. The results of this
first-pass analysis were used to direct more detailed
kinetic experiments, greatly accelerating the design,
synthesis, and evaluation stages while also minimizing
the expenditure of material and resources. Enzymatic
assays show that this class of compounds is unusually

2122 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Costi et al.
followed by the desired 1 or 2 (analytical data, Table 1; 1H
NMR data, Supporting Information).
selective for bacterial and mycotic TS versus human TS,
being able to inhibit LcTS and CnTS at much lower
concentrations than required for hTS. To our knowledge,
no selective TS inhibitors have been reported to date
with the exception of some patented compounds.21 The
in vitro assays are, in general, in good agreement with
the enzymatic results. The naphthalene derivatives are
only slightly or not cytotoxic and show antibacterial
activity against S. aureus and Streptococcus. Unexpect-
edly, compounds 5c and 5ba were also active against
Cr. neoformans. The most divergent result involves
compound 6bc, which, though very active against CnTS
in the enzyme assay, as reported above, did not retain
this activity in the in vitro assay. This inconsistency
may be due to poor membrane penetration or, possibly,
metabolic transformations.
Unlike the classic antifolates, all of these compounds
lack the pteridine-like moiety and the glutamate “tail”
present in the common antifolate-based TS inhibitors.
Since these compounds do not resemble folates chemi-
cally, in the biological setting they are also not con-
strained to interactions with the RFC-FPGS system
thereby reducing the potential for the development of
resistance mechanisms. Given the dramatic rise in
antibiotic resistance and the problems of opportunistic
infections among the immunocompromised, new chemi-
cal classes of antimicrobials, such as these compounds,
are much needed.
Compounds 3 and 4, respectively, were obtained using the
same method, by condensing 1,8-naphthoic anhydride with
phenol or o-chlorophenol (Scheme 3). For 3: yield 10%; 1H
NMR (DMSO-d6) δ 6.95 (d, 4H), 7.80 (d, 4H), 8.00 (t, 2H), 8.60
1
(d, 4H). For 4: yield 8%; H NMR (DMSO-d6) δ 7.01 (t, 2H),
7.11 (d, 2H), 7.28 (dd, 2H), 7.37 (dd, 2H), 7.42 (dd, 2H), 7.57
(d, 2H), 9.29 (s, 2H, exch with D2O), 10.37 (s, 2H, exch with
D2O).
3,3-Bis(4-h ydr oxyph en yl)-1H,3H-n aph th o[1,8-cd]pyr an -
1-on e Der iva tives (5a , 5b, 5c), 3-Hyd r oxy-3-(h yd r oxy-
p h en yl)-1H,3H-n a p h th o[1,8-cd ]p yr a n -1-on e Der iva tives
(6a , 6c), a n d Th eir An a logu es Su bstitu ted a t th e Na p h -
th alen e Rin g [5(5*)aa,ab,ac,ba,bb; 6bc]: Gen er al Meth od.
To a mixture of 1,8-naphthalic anhydride (0.01 mol) and the
appropriately substituted phenol (0.02 mol) in 1,1,2,2-tetra-
chloroethane (100 mL) was added aluminum chloride (0.02
mol) portionwise at room temperature, and the suspension was
stirred for 3 days at 110-115 °C. The still-hot mixture was
then poured onto ice to which was added 200 mL of dichlo-
romethane. After stirring for 0.5 h, the precipitate was filtered
off and the organic layer separated and dried over sodium
sulfate. After evaporation of the solvent, the residue was puri-
fied by silica gel chromatography, eluting with dichloromethane/
methanol (95/5 v/v). The unreacted anhydride eluted first,
followed by the bis- (5) and mono- (6) substituted phthaleins.
Compounds substituted at the naphthalenic ring were ob-
tained from the required 1,8-naphthalic anhydride and the
appropriate phenol, following the same procedure. It should
be noted that in this case a mixture of 6- or 7-substituted
compounds (5, 5*) was obtained, as clearly evidenced by their
1H NMR spectra. Though attempts are in progress to separate
the isomers, in this study they were evaluated as such.
Finally, compound 6d was obtained according to the same
method, starting from 1,8-naphthalic anhydride and thymol
(analytical data, Tables 1 and 2; 1H NMR data, Supporting
Information).
Exp er im en ta l Section
Ch em istr y. Phenolphthalein (PTH), 1,8-naphthalic anhy-
dride, 2,3-naphthalic anhydride, and 4-hydroxybenzoylbenzoic
acid were purchased from Aldrich-Chemie. Melting points were
determined on a Bu¨chi 510 capillary melting points apparatus
and are uncorrected. Elemental analyses for the test com-
7,7-Bis(4-h yd r oxyp h en yl)-5H,7H-1,3-d ih yd r o-6-oxa cy-
clop en ta [cd ]p h en a len -5-on e (7). Following the method
described above, compound 7 was obtained starting from 1,2-
dihydro-6-oxacyclopenta[cd]phenalene-5,7-dione (acenaphthene-
5,6-didecarboxyanhydride): mp 232-235 °C (analytical data,
1
pounds were within (0.4% of the theoretical values. H NMR
spectra were recorded on a Bruker MX400 WB spectrometer.
DMSO-d6 was used as the solvent, unless otherwise noted. UV
spectra were determined using a Perkin Elmer UV spectro-
photometer model lambda 16, equipped with a multicell system
which was temperature regulated with a Haake F3C circulat-
ing bath. All of the compounds were characterized through
1
Table 2; H NMR data, Supporting Information).
3,3-Bis(4-m eth oxyph en yl)-1H,3H-n aph th o[1,8-cd]pyr an -
1-on e Der iva tives (5d ) a n d 3-Hyd r oxy-3-[(3-d im eth yla m i-
n opr op -2-yl)oxy]-1H,3H-n a ph th o[1,8-cd]pyr a n -1-on e (6c).
A mixture of the required 5a or 6a (1.35 mmol), the appropri-
ate alkyl halide (2.7 mmol), and potassium carbonate (2.7
mmol) in acetone (5 mL) was refluxed for 4 h. The solvent was
evaporated and the residue treated with water (3 mL) and
extracted with diethyl ether (3 × 5 mL). After evaporation of
the solvent, the residue was purified by silica gel chromatog-
raphy eluting with dichloromethane/methanol (98/2 v/v), fol-
lowed by crystallization from chloroform (Table 1 for data).
3-(4-Hyd r oxyp h en yl)-3-(4-h yd r oxyn a p h th yl)-1(3H)-iso-
ben zofu r a n on e (1c). A mixture of 2-(4-hydroxybenzoyl)-
benzoic acid (8.2 mmol) and thionyl chloride (5 mL) was
refluxed for 8 h. After cooling, the excess thionyl chloride was
evaporated under vacuum and the residue dissolved in toluene
(5 mL) and added dropwise to a solution of R-naphthol (8.2
mmol) in toluene (5 mL). After stirring for 2 h at 40 °C, the
mixture was cooled and the precipitate filtered under vacuum
and purified by silica gel chromatography, eluting with cyclo-
hexane/ethyl acetate (70/30 v/v): yield 70%; mp 255-258 °C;
1H NMR (DMSO-d6) δ 6.8 (dd, 3H), 7.0 (d, 2H), 7.2 (d, 1H),
7.3-7.4 (m, 2H), 7.5-7.6 (m, 2H), 7.8-8.0 (m, 3H), 8.2 (d, 1H),
9.6 (s, 1H, exch with D2O), 10.6 (s, 1H, exch with D2O).
Bioch em istr y a n d Biology. 1. En zym ology. P u r ifica -
tion of th e en zym e: Plasmids that express L. casei TS in the
Thy E. coli strain ø2913 have been previously described.31 The
enzyme was purified by column chromatography using phos-
phocellulose (P11, Biorad) and hydroxyapatite (HAP, Biorad)
resin, using phosphate buffer as the eluent.32 TS enzymes
1
mono- and bidimensional H NMR, and the data are enclosed
as Supporting Information. TLC on silica gel plates was used
to check product purity. Silica gel 60 (Merck; 70-230 mesh)
was used for column chromatography. Flash chromatography
was performed using silica gel 60, 200-400 mesh (Merck art.
9385). The structures of all compounds were consistent with
their analytical and spectroscopic data. The stock solutions of
the inhibitors were prepared in 100% DMSO and stored at
-20 °C until use. The stability of the stock solutions of the
compounds was monitored with time by spectrophotometric
UV analysis; the spectra all remained unchanged throughout
the course of these experiments. The solvent systems used in
the enzymatic assays include DMSO and TES buffer (N-tris-
(hydroxymethyl)methyl-2-aminoethanesulfonic acid) (50 mM)
at pH 7.4, MgCl2 (25 mM), formaldehyde (6.5 mM), EDTA
(ethylenediaminetetraacetic acid) (1 mM), and 2-mercapto-
ethanol (75 mM).
3,3-Bis(4-h yd r oxyp h en yl)-1(3H)-isoben zofu r a n on e De-
r iva tives (1), 3,3-Bis(4-h yd r oxyp h en yl)-3H-n a p h th o[2,3-
c]fu r a n -1-on e Der iva tives (2), 1,3-Bis(p-qu in on yl)-1H,3H-
ben zo[de]isoch r om on (3), an d 1,3-Bis(3-ch lor o-4-h ydr oxy-
p h en yl)-1,3-d ih yd r oxy-1H,3H-ben zo[d e]isoch r om on (4):
Gen er a l Meth od . A mixture formed by the required naph-
thalic or 2,3-naphthalic anhydride (0.01 mol), the appropriate
phenol (0.02 mol), and several drops of H2SO4 was heated while
stirring at 180 °C for 5 h. After cooling, the residue was
purified by silica gel chromatography eluting with dichloro-
methane/methanol (95/5 v/v) to give the unreacted anhydride

Selective Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2123
purified from Cr. neoformans, P. carinii, and human were
purified as reported.33-35 The enzyme preparations were >95%
homogeneous as visualized by SDS (sodium dodecyl sulfate)-
polyacrylamide gel electrophoresis. The purified enzymes were
stored at -80 °C in 10 mM phosphate buffer, pH 7.0, and 0.1
mM EDTA.
consists in the formation of syncitia; therefore it is not possible
to calculate the vial titer as plaque-forming units (PFU). For
this reasons the titer of HIV stock solutions is referred to as
cell culture infectious dose 50 (CCID50). The titer of the HIV-1
stock solution used throughout this study had titer of 6 × 106
CCID50/mL. Stock solutions of HRV types 2 and 14 had titers
of 4 and 8 × 106 PFU/mL, respectively.
Enzyme activity was determined spectrophotometrically by
steady-state kinetic analysis, following the increasing absor-
An tivir a l a ssa ys: Activity of compounds against the HIV-1
and HRV-2/HRV-14 multiplication in acutely infected cells was
based on inhibition of virus-induced cytopathogenicity in MT-4
and HeLa Ohio cells, respectively. Briefly, 50 µL of culture
medium (RPMI 10% FCS in the case of HIV-1; D-MEM 2%
FCS, 30 mM MgCl2, 15 µg/mL DEAE-dextran in the case of
HRVs) containing 1 × 104 cells was added to each well of flat-
bottomed microtiter trays containing 50 µL of medium with
or without various concentrations of test compounds; 20 µL of
HIV-1, HRV-2, or HRV-14 suspensions containing 100 CCID50
was then added. After a 4-day incubation at 37 °C (3 days at
33 °C for the HRV-infected cultures), the number of viable cells
was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) method.40 The cytotoxicity of
compounds was evaluated in parallel with their antiviral
activity. It was based on the viability of mock-infected cells,
as monitored by the MTT method. The activity of test
compounds against VV and HSV was tested in classical plaque
reduction assays.41
bance at 340 nm due to the oxidation reaction of N5,N10
-
methylenetetrahydrofolate to dihydrofolate.36 1 mL of reaction
solution was formed by standard assay buffer pH 7.4, dUMP
(120 µM), 6(R,S)-1-CH2CH4-folate (140 µM), enzyme (0.07 µM).
Assays were performed at 20 °C in the standard assay buffer
formed by TES (N-tris(hydroxymethyl)methyl-2-aminoethane-
sulfonic acid) (50 mM) at pH 7.4, MgCl2 (25 mM), formaldehyde
(6.5 mM), EDTA (1 mM), and 2-mercaptoethanol (75 mM).
Assa ys: Stock solutions of the inhibitors were prepared in
DMSO (dimethyl sulfoxide) and stored at -20 °C until use.
The inhibition pattern for all of the compounds has been
determined by steady-state kinetic analysis of the dependence
of folate concentration from TS enzyme activity at varying
inhibitor concentrations. For the most part, the compounds
showed competitive or nonlinear intersecting mixed-type
inhibition. Therefore, Ki values have been measured in the first
case, and Ki and RKi values have been determined in the
second case.37 Only a few compounds showed noncompetitive
inhibition. For those compounds that showed competitive
inhibition, Ki values were obtained from the linear least-
squares fit of the residual activity as a function of inhibitor
concentration to the suitable equations for competitive inhibi-
tion.38 Each of the experiments were repeated at least three
times, and no individual measurement differed more than 10%
from the mean.
An tiba cter ia l a ssa ys: S. aureus, group D Streptococcus,
P. aeruginosa, and Salmonella sp. were recent clinical isolates.
Tests were carried out in nutrient broth, pH 7.2, with an
inoculum of 103 cells/tube. MICs were determined after 18 h
of incubation at 37 °C in the presence of serial dilutions of the
test compounds.
An tim ycotic a ssa ys: Yeast blastospores were obtained
from a 30-h-old shaken culture incubated at 30 °C in Sab-
ouraud dextrose broth. The dermatophyte inoculum was
scraped aseptically with a spatula from a 7-day-old culture
on agar, and the macerate was finely suspended in Sabouraud
dextrose broth using a glass homogenizer. Glycerol, final
concentration 10%, was added as a cryoprotective agent to both
yeast and dermatophyte suspensions, aliquots of which were
then stored in liquid nitrogen. Test tubes were inoculated with
103 blastospores or colony-forming units (CFU)/tube. The
minimal inhibitory concentration (MIC) was determined by
serial dilutions using Sabouraud dextrose broth (pH 5.7) and
incubating at 37 °C. The growth control for yeasts was read
after 1 day and for dermatophytes after 3 days (5 days for Cr.
neoformans). The MIC was defined as the compound concen-
tration at which no macroscopic signs of fungal growth were
detected. The minimal germicidal concentration (MGC) was
evaluated by subcultivating negative test tubes in Sabouraud
dextrose agar, without the inhibitors, and represents the
lowest dose of compound with germicidal effect.
The inhibition factors were determined as follows: the
experiments were performed on each compound at a single
concentration of 27 µM. The screening assay was done on the
enzymes from L. casei, P. carinii, Cr. neoformans, and human
to evaluate the species specificity of the synthesized compound.
The assay conditions were those of the standard TS assay.
Assays were performed at 20 °C in the standard assay buffer
described above. A new reaction mixture (1 mL) was formed
using standard assay buffer at pH 7.4 and the substrate,
dUMP, at 120 µM; however, the folate concentration used was
60 µM, because the Km’s of folate for each of the enzymes are
similar (15.7 µM for PcTS, 10 µM for LcTS, 20 µM for CnTS,
8 µM for hTS). TS assays were performed in the absence and
presence of each inhibitor, the respective initial rates were
considered, and the ratio between the initial rate in the
presence (Vii) and in the absence (Vi) of each inhibitor gave
the respective inhibition factors (IF ) Vii/Vi). Each of the
inhibition factors was determined a minimum of three times,
and no individual measurement differed more than 10% from
the mean.
Ack n ow led gm en t. This work was supported by
Consiglio Nazionale delle Richerche (CNR) (P.P.) and
Ministero della Ricerca Scientifica e Tecnologica Italiano
(MURST) (M.P.C.) (60%). Thanks are due to Centro
Interdipartimentale Grandi Strumenti Universita` di
Modena (CIGS) and Centro Interdipartimentale di
Calcolo Elettronico (CICAIA) for computing facilities,
to Dr. Brian Shoichet and Prof. I. D. Kuntz for critical
reading of the manuscript, to Rossella Gallesi of the
Microanalyses Laboratory, and to Fabrizia Soragni of
Dipartimento di Scienze Farmaceutiche Universita` di
Modena for her excellent technical assistance.
The effect of increasing DMSO concentration in the TS assay
mixture was studied, and it was observed that no change in
TS activity is seen at concentrations up to 10% DMSO.39
2. In Vitr o Assa ys. Com p ou n d s: Since the test compounds
have poor aqueous solubility, they were dissolved in DMSO
at an initial concentration of 300 mM and then serially diluted
in culture medium.
Cells: Cell lines were from American Type Culture Collec-
tion (ATCC); bacterial and fungal strains were either clinical
isolates (obtained from Clinica Dermosifilopatica, University
of Cagliari) or collection strains from ATCC. H9/IIIB, MT-4,
and C8166 cells (grown in RPMI 1640 containing 10% fetal
calf serum (FCS), 100 U/mL penicillin G, and 100 µg/mL
streptomycin) were used for anti-HIV assays. HeLa Ohio cells
were used for anti-HRV assays, whereas Vero cells were used
for anti-VV and anti-HSV assays. Cell cultures were checked
periodically for the absence of mycoplasma contamination with
a MycoTect kit (Gibco).
Su p p or tin g In for m a tion Ava ila ble: NMR characteriza-
tion of the compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
Refer en ces
Vir u ses: Human immunodeficiency virus type-1 (HIV-1,
IIIB strain) was obtained from supernatants of persistently
infected H9/IIIB cells. The cytopatic effect (CPE) of HIV-1
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