Synthesis of sulfonamide-containing N-hydroxyindole-2-carboxylates as inhibitors of human lactate dehydrogenase-isoform 5

Article abstract of DOI:10.1016/j.bmcl.2011.10.031

N-Hydroxyindole-2-carboxylates possessing sulfonamide-substituents at either position 5 or 6 were designed and synthesized. The inhibitory activities of these compounds against isoforms 1 and 5 of human lactate dehydrogenase were analysed, and K_i values of the most efficient inhibitors were determined by standard enzyme kinetic studies. Some of these compounds displayed state-of-the-art inhibitory potencies against isoform 5 (K_i values as low as 5.6 μM) and behaved as competitive inhibitors versus both the substrate and the cofactor.

Full text of DOI:10.1016/j.bmcl.2011.10.031

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7331–7336
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of sulfonamide-containing N-hydroxyindole-2-carboxylates
as inhibitors of human lactate dehydrogenase-isoform 5
Carlotta Granchi a, Sarabindu Roy a, Marco Mottinelli a, Elisa Nardini a, Fabio Campinoti a,
Tiziano Tuccinardi a, Mario Lanza b, Laura Betti b, Gino Giannaccini b, Antonio Lucacchini b,
Adriano Martinelli a, Marco Macchia a, Filippo Minutolo a,
⇑
a Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Hydroxyindole-2-carboxylates possessing sulfonamide-substituents at either position 5 or 6 were
designed and synthesized. The inhibitory activities of these compounds against isoforms 1 and 5 of
human lactate dehydrogenase were analysed, and Ki values of the most efficient inhibitors were
determined by standard enzyme kinetic studies. Some of these compounds displayed state-of-the-art
Received 1 September 2011
Revised 6 October 2011
Accepted 7 October 2011
Available online 17 October 2011
inhibitory potencies against isoform 5 (Ki values as low as 5.6
l
M) and behaved as competitive inhibitors
versus both the substrate and the cofactor.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Lactate dehydrogenase
Glycolysis
N-Hydroxyindoles
Enzyme inhibitors
Docking
Tumor cells require a high production of energy (ATP) and anab-
olites, even under hypoxic conditions, to ensure their proliferation
and survival. For this purpose, glucose metabolism in cancer cells is
shifted from normal oxidative phosphorylation (OXPHOS) to gly-
colysis, leading to the final production of lactate. This metabolic
switch, also known as the Warburg effect,1 is very often associated
with invasive tumor phenotypes,2 as well as with stem cells.3,4 The
peculiar metabolism of carbohydrates occurring in tumors is cur-
rently being considered as a novel target for non-toxic and selec-
tive therapeutic interventions against cancer.5,6 In fact, therapies
resulting from this approach should be directed specifically against
cancer cells and, therefore, devoid of important undesirable side
effects.7
Some of the enzymes involved in the glycolytic process are cur-
rently being considered as anti-cancer targets, and there are sev-
eral small molecules at the preclinical stage which are reported
to act as metabolic modulators in cancer cells,8 although there
are only a few examples in clinical trials. Lonidamine, an inhibitor
of hexokinase (HK), the enzyme that catalyzes the phosphorylation
of the 6-position of glucose, thus starting the glycolytic process,
has completed a phase 3 trial, but its efficacy was mined by pan-
creatic and hepatic toxicity.9 Among HK inhibitors, it is worthwhile
to mention 2-deoxyglucose (2-DG),10 and 3-bromopyruvate,11
although their clinical results still need to be definitely assessed.
Finally, dichloroacetate (DCA) is a promising pyruvate dehydroge-
nase kinase (PDK) inhibitor under phase 2 clinical study, whose ac-
tion restores the normal oxidative demolition of pyruvate by
reactivating PDH and, as a consequence, indirectly diverting anaer-
obic glycolysis.12 As of yet, there are no clinical trials involving
anti-cancer agents that inhibit lactate dehydrogenase (LDH). LDH
catalyzes the reduction of pyruvate into lactate and constitutes a
major checkpoint in the glycolytic pathway for the switch from
OXPHOS to anaerobic glycolysis. Human isoforms of LDH are tetra-
meric, and may be composed of three types of subunits: LDH-A (or
LDH-M, muscle), LDH-B (or LDH-H, heart) and LDH-C.13 Subunit C
has been found only in a homotetrameric isoform LDH-C4 (or
hLDHX), which is involved in male fertility.14 On the other hand,
subunits A and B form five isoforms deriving from their various
possible combinations in the following way: hLDH1 = LDH-B4,
hLDH2 = LDH-AB3, hLDH3 = LDH-A2B2, hLDH4 = LDH-A3B, hLDH5 =
LDH-A4. Whereas hLDH1 is mostly represented in the heart, hLDH5
is prevalent in liver and skeletal muscle. Isoform 5, containing only
the A-subunit, was found to be overexpressed in highly invasive
and hypoxic carcinomas,15,16 and it was clearly associated with hy-
poxia inducible factor 1
a (HIF-1a
).17 Moreover, an augmented
expression of the LDH-A subunit was found in several tumor lines,
together with the overproduction of glucose transporter GLUT1,
after exposure to oxygen deprivation,18 due to the increased glu-
cose consumption required by anaerobic glycolysis.
⇑
Corresponding author. Tel.: +39 050 2219 557; fax: +39 050 2219 605.
E-mail address: filippo.minutolo@farm.unipi.it (F. Minutolo).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.031

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C. Granchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7331–7336
The reduced oxygen supply often found in invasive hypoxic tu-
R1
N
COOH
mors increases the vulnerability of their cells to hLDH5-inhibition
because of the glycolysis upregulation and the inability to switch
back to oxidative phosphorylation in oxygen-deprived conditions.
In fact, hLDH5 has been acknowledged as one of the most promis-
ing targets for anti-cancer therapy, because repression of its
expression cuts the main energy production process in hypoxic tu-
mors, as shown by a reduced invasiveness in metastatic cell
lines.19,20 Most importantly, hLDH5-inhibition should not give rise
to important side-effects in humans, since hereditary LDH-A defi-
ciency causes myopathy only after intense anaerobic exercise,
but does not provoke any symptoms under ordinary circum-
stances.21 Unfortunately, only a few hLDH5-inhibitors are now dis-
cussed in the scientific literature, mainly because there had been
no evidence for health benefits deriving from this action until re-
cent times.22 About a year ago, compound FX-11, an efficient inhib-
R2
S
N
OH
O O
3a-q
O O
S
Ph
COOH
N
R1
N
N
OH
COOH
Me
N
R2
S
O O
OH
4
5a-d
R1
R2
CH3
Ph
R1
R2
a
CH3
CH3
n-Bu
i
j
CH3
CH3
CH3
CH3
CH3
3-Cl-Ph
2-Cl-Ph
itor of hLDH5 (Ki = 8 lM), was reported to block tumor progression
both in vitro and in vivo.23 Furthermore, a phenylbutyric acid
derivative linked to an adenosine-like fragment was shown to inhi-
b
c
bit hLDH5 with an IC50 value of 14.8 l
M,24 although no data rela-
tive to cancer cell proliferation assays were reported.
Ph
k
l
2,4-Cl2-Ph
3,4-Cl2-Ph
We have recently developed a new class of N-hydroxyindole-2-
carboxylates (NHIs), and found that those containing a phenyl ring
at either position 5 or 6 (1, Fig. 1) possess efficient inhibitory prop-
erties of hLDH5, with Ki values reaching the low micromolar range
d
(as low as 4.7 l
M).25 These compounds displayed a marked selec-
N
m
n
o
p
q
4-COOH-Ph
tivity for isoform hLDH5, since their inhibition of the other homo-
tetrameric isoform, hLDH1, was either undetectable or very low
(611% at 125 lM). The introduction of triazole linkers between
e
f
CH3
CH3
CH3
CH3
4-CH3-Ph
4-CF3-Ph
4-F-Ph
N
the aryl substituent and the NHI scaffold did not produce signifi-
cant improvements of the inhibition potency.26 The generally low-
er activity found with these triazole-substituted NHIs (2, Fig. 1)
was presumed to be due to the desolvation energy associated with
the presence of these additional polar heterocycles,27 which is not
sufficiently counterbalanced by the nice fit in the enzyme active
site that these compounds show by computational methods.26
For the purpose of expanding the chemical classes of hLDH5-
inhibitors, and in an attempt to find new potent and isoform
-selective derivatives, which are still scarcely represented in the
literature in spite of the growing interest in this emerging target,
we decided to extend this NHI series by introducing sulfonamide-
based substituents (3–5, Fig. 2). Specifically, we have designed
and synthesized a series of 22 sulfonamide-bearing NHIs in which
the aminosulfonyl group is either directly bound to the indole scaf-
fold in position 6 or 5 (compounds 3a–q or 4, respectively) or linked
to the central scaffold through a phenyl spacer (compounds 5a–d).
The substituents present on the sulfonamide nitrogen atom (R1 and
R2) are either independent groups (3a–c,e–m, 4, 5a–c) or part of the
same cycle (3d,n–q, 5d).
N
g
h
N
O
4-Cl-Ph
N
Figure 2. Structures of newly developed sulfonamide-containing NHIs 3–5.
This last step required a fine optimization of the reaction condi-
tions, since various amounts of over-reduced indole side-products
were also obtained. The simplest member of this class (8a) was
prepared by reaction with SnCl2 in DME.28,29 In the synthesis of
8b, the same conditions caused the formation of considerable
amounts of indole side-product; therefore, this compound was in-
stead obtained by using milder reducing conditions (sodium hypo-
phosphite in the presence of palladium over charcoal).30
A
The synthesis of 5-sulfonamido-substituted NHIs 3a–q is dis-
played in Scheme 1. Condensation of 4-methyl-3-nitrobenzene-1-
sulfonyl chloride (6) with the appropriate amine was carried out
under various reaction conditions in the presence of different bases
(see Supplementary data for details). The resulting sulfonamides
(7a–q) were treated with sodium hydride/dimethyl oxalate to
combination of SnCl2 and PhSH/Et3N31,32 proved to be the optimal
reagent for the preparation of compounds 8c,d,i–q. The remaining
NHI-esters (8e–h) were instead obtained by a reduction of the
appropriate precursors in the presence of lead and triethylformate
in hot methanol.33 Final hydrolysis of the COOMe group was ob-
tained upon treatment with an aqueous 2 N solution of LiOH in a
1:1 mixture of THF and methanol.
generate
a-ketoester intermediates, which were then directly sub-
mitted to a reductive cyclization step, leading to NHI-esters 8a–q.
An analogous synthetic pathway was followed for the prepara-
tion of compound 4, bearing the N-methyl-N-phenylsulfonamide
group in position 5 (Scheme 1), starting from 3-methyl-4-nitroben-
zene-1-sulfonyl chloride (9).34 It is worth mentioning that, in this
case, the most efficient reductive cyclization step was promoted
by the SnCl2/PhSH/Et3N system.31,32
A slightly modified reaction sequence was applied to the prep-
aration of the ‘phenyl-spaced’ series of sulphonamido-NHIs (5a–d,
Scheme 2). Sulfonamide 13a was commercially available, whereas
13b,c were prepared by direct condensation of an excess of the
4
N
N
N
5
6
5
6
COOH
COOH
Ar/Alk
N
N
OH
OH
1
2
Figure 1. Structures of reference phenyl- (1) and triazole-substituted (2) NHIs.

C. Granchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7331–7336
7333
cyclization operated by the SnCl2/PhSH/Et3N system,31,32 and final
ester hydrolysis, to produce NHIs 5a–d.
The inhibitory activities of compounds 3a–q, 4 and 5a–d on the
two purified human enzyme isoforms hLDH5 and hLDH1 were
measured by standard enzyme kinetics experiments. Initially, we
evaluated the percent inhibition of hLDH5 relative to control at a
compound concentration of 125 lM in the presence of 25 lM
NADH. Enzyme activity was determined by measuring the absor-
bance decrease at 340 nm, due to the consumption of NADH. Com-
pounds displaying an inhibition level higher than 50% at 125 lM
were then submitted to a full enzyme kinetics characterization,
to establish their type of inhibition versus both NADH and pyru-
vate, as well as their isoform selectivity (hLDH5 versus hLDH1).
Therefore, we initially measured the Michaelis–Menten constants
(Km) relative to the cofactor (NADH) and the substrate (pyruvate)
for hLDH5, derived from Lineweaver–Burk plots. Next, we evalu-
ated the K0m values in the presence of the selected compounds
CH3
NO2
Cl
S
a
a
O
O
6
CH3
NO2
CH3
NO2
R1
N
CH3
N
R2
S
S
O
O
O
O
MeOOC
7a-l,n-q
7m
b,c
b,c
R1
N
CH3
N
COOMe
COOMe
N
OH
R2
N
S
S
O
O
O
O
OH
MeOOC
8a-l,n-q
8m
d
d
(concentration range = 25 ꢀ 100
lM). Final Ki values (Table 1) were
then determined by double-reciprocal Lineweaver–Burk plots ob-
tained from the values of K0m, obtained as described above for each
compound.
3a-q
O
O
S
O
O
The smallest member of the 6-sulfonamido-substituted series,
bearing only two methyl groups (3a), did not show any appreciable
activity, whereas the introduction of one phenyl ring in the sulfon-
amide nitrogen atom produced a potent inhibitor (3b) displaying Ki
CH3
NO2
CH3
NO2
S
Ph
Cl
a
N
b,c
Me
9
10
values of 17 lM versus NADH and 9.2 lM versus pyruvate. When
O
the same N-methyl-N-phenylsulfamoyl group was moved from po-
sition 6 of 3b to position 5, the resulting compound (4) completely
lost the hLDH5-inhibitory activity. Other inactive compounds were
produced when the sulfonamide nitrogen atom was part of cyclic
systems, such as indole (3d), piperidine (3n), pyrrolidine (3o), mor-
pholine (3p), or isoindoline (3q). Replacement of the N-methyl
group with larger alkyl substituents, such as a n-butyl (3c), did
not modify the competitive inhibition potencies versus the cofactor
O
Ph
S
N
Me
d
COOMe
4
N
OH
11
Scheme 1. Reagents and conditions: (a) R1R2NH (see Supplementary data); (b)
(COOMe)2, NaH 60%, dry DMF, ꢁ15 °C to rt; (c) 8a: SnCl2ꢂ2H2O, dry DME, 4 Å MS,
0 °C to rt; 8b: H2PO2NaꢂH2O, Pd-C 10%, H2O/THF (1:1), rt; 8c,d,i–q, 11: SnCl2ꢂ2H2O,
C6H5SH, Et3N, CH3CN, rt; 8e–h: Pb, TEAF, MeOH, 55 °C; (d) aqueous 2 N LiOH, THF/
MeOH (1:1), rt.
(Ki = 18
lM), whereas it substantially reduced the efficiency of its
competition with the substrate (Ki = 90
l
M). An explorative series
of N-aryl-substituted analogs of 3b were then analyzed (3e–l).
Among these compounds, 4-trifluoromethylphenyl-substituted
derivative 3f showed a certain improvement of the pyruvate-com-
petitive hLDH5-inhibition, whereas its competition versus NADH
was less efficient than that of its non-substituted counterpart 3b.
The introduction of a para-chloro atom generated the most potent
compound of this series (3h), which displayed very efficient inhibi-
appropriate aniline with 4-bromobenzene-1-sulfonyl chloride (12).
Indole-containing sulfonamide 13d required different conditions
for preparation, such as the use of finely powdered NaOH and tet-
rabutylammonium hydrogensulfate in CH2Cl2. These aryl bromides
were then subjected to Pd-catalyzed cross-coupling reactions to
obtain nitro-toluene intermediates 14a–d. As seen in the previous
scheme, the last steps of this synthesis also involved sodium hy-
dride-promoted condensation with dimethyl oxalate, reductive
tion levels versus both the cofactor (Ki = 6.6
lM) and the substrate
(Ki = 5.6
lM). The shift of the chlorine atom from the para-position
Table 1
Results of enzyme (hLDH5) inhibition assay for selected compounds
Entry
Compound
Kia
(
l
M) (versus NADH)b
Kia
9.2
90
20
6.0
28
5.6
43
25
40
36
22
(l
M) (versus Pyr)c
CH3
Br
a
Br
1
2
3
4
5
6
7
8
9
3b
3c
3e
3f
3g
3h
3i
3j
3k
3l
17
18
20
26
125
6.6
48
14
30
18
16
R1
N
R1
N
b
NO2
14a-d
Cl
R2
S
S
R2
O
O
O
O
S
12
13a-d
O
O
COOMe
c,d
R1
N
e
N
OH
10
11
12
5a-d
5b
5d
R2
S
7.7
8.8
15a-d
O
O
a
Values are reported as the average of three or more measurements; the error in
Scheme 2. Reagents and conditions: (a) R1R2NH, DCM, rt (13b,c) or NaOH, TBAHS,
DCM, 0 °C to rt (13d); (b) 4-methyl-3-nitrobenezeneboronic acid, Pd(OAc)2, TBAB,
these values is within 30% of the average.
b
Saturating concentration (2.0 mM) of sodium pyruvate and competitive
increasing concentrations (12.5 ꢀ 150
l
M) of NADH.
M) of NADH and competitive increasing con-
M ꢀ 1.0 mM) of sodium pyruvate.
Na2CO3, H2O,
lW, 5 min (14a) or Pd(OAc)2, PPh3, aqueous 2 M Na2CO3, toluene,
c
Saturating concentration (200
centrations (25
l
EtOH, 100 °C, 24 h (14b–d); (c) (COOMe)2, NaH 60%, dry DMF, ꢁ15 °C to rt; (d)
l
SnCl2ꢂ2H2O, C6H5SH, Et3N, CH3CN, rt; (e) aqueous 2 N LiOH, THF/MeOH (1:1), rt.

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C. Granchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7331–7336
of 3h to the meta- (3i) and ortho- (3j) positions generally produced
reductions of the inhibition potencies. The same negative trend was
observed when an additional chloro-group was inserted either in
the ortho- (3k) or meta- (3l) positions of compound 3h. The inser-
tion of other types of para-substituents caused either a reduction
(3e: 4-CH3; 3g: 4-F) or a complete loss (3m: 4-COOH) of the inhib-
itory activity. The restricted series of ‘phenyl-spaced’ derivatives
5a–d generally afforded weaker inhibitors. In fact, compounds 5a
and 5c were completely inactive. Compound N-methyl-N-phenyl-
substituted 5b proved to be a slightly poorer inhibitor than its
non-spaced counterpart 3b. However, the most potent inhibitor of
this ‘phenyl-spaced’ subclass was the indole-sulfonamide 5d, in
sharp contrast to its inactive close analog 3d belonging to the
‘non-phenyl-spaced’ family. In fact, 5d displayed Ki values (7.7
above) with the loss of the H-bonds involving the N-hydroxy
group, and a consequent distortion of the interactions between
the carboxylic substituent and R169, as shown by representative
example 3n in Figure 4A. In order to further clarify why these com-
pounds behaved so differently from their ‘non-cyclic’ counterparts,
a manual docking of representative ‘cyclic’ sulfonamide 3n was
carried out, in which this derivative was forced to assume the bind-
ing pose previously obtained with active inhibitors, such as 3h (cf.
Fig. 3). The results obtained by this procedure with 3n are shown in
Figure 4B: the resulting complex shows that the piperidine cyclic
substituent of 3n is oriented towards the alkyl chain of V31 and
displays an intense steric clash (red arrow, Fig. 4B) with this resi-
due that prevents this type of compound from assuming an
efficient interaction with the enzyme active site.
and 8.8
so far, compound 3h.
As for isoform selectivity, it is important to notice that none of
these active hLDH5-inhibitors displayed any detectable inhibition
of hLDH1 (<3%) at 125 lM, thus confirming the generally high
l
M) approaching those of the best sulfonamide inhibitor
The addition of a phenyl spacer between the indole central scaf-
fold and the sulfonamide portion of the ligands is tolerated in two
examples (5b and 5d). Our docking results confirm the good puta-
tive binding of indole-substituted ‘spaced’ derivative 5d (Fig. 5). In
fact, 5d participates in an additional H-bond with the backbone of
R99 by means of one of its sulfonamide oxygen atoms.
Furthermore, the indole portion connected to the sulfonamide
group shows a strong aromatic interaction with the aryl side-chain
of Y247.
The loss of the inhibitory activity found with compound 4,
which is caused by the shift of the sulfonamide group from posi-
tion 6 to 5, can be explained only partially by a simple docking
analysis of its putative binding pose. In fact, as shown in Figure
6, the indole central scaffold of 4 is sensibly shifted from the posi-
specificity of NHI-based inhibitors for hLDH5.
All the reported compounds were analyzed by means of docking
studies. Active inhibitors 3b,c,e–l are characterized by a common
interaction scheme. As shown in Figure 3, and in agreement with
our previously published results,25,26 the carboxylic group of the
most potent inhibitor of this series, compound 3h, shows strong
polar interactions with R169 and T248; moreover, its N-hydroxy
group displays an H-bond with the backbone nitrogen atom of
T248, as well as with a water molecule that mediates the interac-
tion with H193. The indole central scaffold of 3h is placed in a cleft
mainly delimited by N138, A238, V241, I242, T248, and I252. The
sulfonamide group does not seem to participate in specific polar
interactions; rather, it places the aromatic substituent of 3h in a
lipophilic cleft mainly constituted by V31, A98, and Y247. Similar
binding poses were found for all the other active inhibitors. On
the other hand, the low activity of carboxy-substituted NHI 3m
could be explained by the fact that the additional COOH group of
this compound does not participate in any significant interaction
with the enzyme (Fig. S1, Supplementary data).
The insertion of the sulfonamide nitrogen atom into a cycle
such as indole (3d), piperdine (3n), pyrrolidine (3o), morpholine
(3p), or isoindoline (3q) causes a marked decrease of activity.
The docking of these derivatives highlights the overturning of the
indole central scaffold, when compared to active inhibitors (see
Figure 4. Docking analysis of 3n into the LDH-A subunit (A) and manual docking of
3n using the results obtained for compound 3h as a reference disposition (B),
displaying the steric clash occurring in this case.
Figure 3. Docking analysis of 3h into the LDH-A subunit.

C. Granchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7331–7336
7335
interactions with R169, T248, and the structural water molecule.
The strength of the interaction occurring between residue
p–p
Y247 and either the phenyl group of 3h or the indole substituent
of 5d is further confirmed by this MD analysis. Furthermore, the
H-bond between the sulfonamide oxygen atom of 5d and the nitro-
gen backbone of R99 is also maintained (Fig. S3, Supplementary
data).
On the other hand, the analysis of the MD trajectory for the
complex obtained by molecular docking of 4 into the LDH-A active
site clearly supports the low activity of this 5-substituted deriva-
tive. In fact, as shown in Figure 7, during MD simulation compound
4 completely loses its crucial interaction with R169 of the enzyme
and the structural water molecule, whereas it maintained the H-
bond interaction only between its carboxylic group and the OH
group of the side chain of T248. This behavior would indicate a
much weaker interaction of compound 4 and the LDH-A active site
and, therefore, would justify the experimentally-found poor
inhibitory activity of 4.
In conclusion, the results presented here give the following SAR
indications concerning the hLDH5-inhibitory efficiency of this class
of sulfonamide-containing NHIs: (1) the sulfonamide substituent
must be placed in position 6 of the NHI scaffold, rather than in po-
sition 5 (compare compounds 3b and 4); (2) the sulfonamide-
nitrogen atom should bear one aryl substituent (R2) in addition
to the methyl group (R1) (in fact compound 3a is inactive); (3) large
R1-alkyl groups, such as n-butyl, partially (3c) or completely (5c)
compromise the ability of the resulting compounds to compete
with the small natural substrate pyruvate in the deepest cavity
of the enzyme active site; (4) cyclic ‘non-spaced’ sulfonamides
are not suitable for this purpose (3d,n–q); (5) an extra COOH group
in the phenyl substituent causes a loss of activity (3m), whereas
the introduction of a para-chlorine atom in the same position af-
fords the most potent inhibitor of this series (3h); (6) sulfonamide
groups linked to position 6 of the NHI scaffold through a phenylene
spacer maintain a good inhibition level and, in this case, indole-
substituted derivative 5d proves to be the most potent inhibitor,
in contrast to its ‘non-spaced’ counterpart (3d). Molecular model-
ing studies nicely confirm the experimental inhibition results ob-
tained by enzyme kinetics assays. Overall, this new chemical
class produced some of the most potent and selective
hLDH5-inhibitors, displaying Ki values in the low micromolar
range. In the near future, a full pharmacological evaluation will
be carried out on selected compounds from this series, in order
Figure 5. Docking analysis of 5d into LDH-A subunit.
tion occupied by the same portion of its 6-substituted analogs of
type 3 (see Fig. 3). This shift causes a weakening of the interaction
between the carboxylic group of 4 and R169, and the concomitant
loss of its interaction with the nitrogen backbone of T248 by means
of the N–OH group, although the polar link between its COOH and
the hydroxy group of T248 is preserved. The phenyl ring bound to
the sulfonamide group of 4 still shows lipophilic interactions with
Y239. Overall, this binding pose would not be sufficient to justify
the dramatic loss of activity displayed by compound 4. Therefore,
in order to further understand the different biological outcomes
obtained within this class of compounds and the presumed modes
of interaction with the enzyme active site, representative deriva-
tives 3h, 4, and 5d were submitted to 10 ns of molecular dynamic
(MD) simulations. An analysis of the root-mean-square deviation
(rmsd) from the initial model of the
a carbons of the enzyme,
showed that after an initial increase, the rmsd remained approxi-
mately constant around the value of 1.0–1.6 Å during the last
7 ns for all three complexes (Fig. S2, Supplementary data).
In agreement with the docking analysis, MD simulations con-
firmed that compounds 3h and 5d maintain their strong H-bond
Figure 6. Docking analysis of 4 into LDH-A subunit.
Figure 7. MD simulation results for the complex of LDH-A with 4.

7336
C. Granchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7331–7336
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The research leading to these results has received funding (to
F.M. and S.R.) from the European Union’s Seventh Framework
Programme (FP7-PEOPLE-IIF-2008) under grant agreement no
235016 (‘NOXYCANCERSTARV’). Siena Biotech S. p. A. - Italy, is
gratefully acknowledged for a research contribution (F.M.). C.G.
thanks the Italian Ministry for University and Research (MIUR)
for a PhD fellowship. We thank Dr. Giorgio Placanica and Dr. Cate-
rina Orlando for technical assistance in the analysis of the chemical
products.
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