Symmetric molecules with 1,4-triazole moieties as potent inhibitors of tumour-associated lactate dehydrogenase-A

Article abstract of DOI:10.1080/14756366.2017.1404593

A series of symmetric molecules incorporating aryl or pyridyl moieties as central core and 1,4-substituted triazoles as a side bridge was synthesised. The new compounds were investigated as lactate dehydro-genase (LDH, EC 1.1.1.27) inhibitors. The cancer associated LDHA isoform was inhibited with IC₅₀ = 117–174?μM. Seven compounds exhibited better LDHA inhibition (IC₅₀ 117–136?μM) compared to known LDH inhibitor–galloflavin (IC₅₀ 157?μM).

Full text of DOI:10.1080/14756366.2017.1404593

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Symmetric molecules with 1,4-triazole moieties
as potent inhibitors of tumour-associated lactate
dehydrogenase-A
Abdul-Malek S. Altamimi, Ahmed M. Alafeefy, Agnese Balode, Igor Vozny,
Aleksandrs Pustenko, Mohey Eldin El Shikh, Fatmah A. S. Alasmary, Sherif A.
Abdel-Gawad & Raivis Žalubovskis
To cite this article: Abdul-Malek S. Altamimi, Ahmed M. Alafeefy, Agnese Balode, Igor Vozny,
Aleksandrs Pustenko, Mohey Eldin El Shikh, Fatmah A. S. Alasmary, Sherif A. Abdel-Gawad &
Raivis Žalubovskis (2018) Symmetric molecules with 1,4-triazole moieties as potent inhibitors
of tumour-associated lactate dehydrogenase-A, Journal of Enzyme Inhibition and Medicinal
Chemistry, 33:1, 147-150, DOI: 10.1080/14756366.2017.1404593
To link to this article: https://doi.org/10.1080/14756366.2017.1404593
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Group.
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Date: 07 December 2017, At: 04:22

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 33, NO. 1, 147–150
https://doi.org/10.1080/14756366.2017.1404593
SHORT COMMUNICATION
Symmetric molecules with 1,4-triazole moieties as potent inhibitors of
tumour-associated lactate dehydrogenase-A
Abdul-Malek S. Altamimia, Ahmed M. Alafeefyb, Agnese Balodec,d, Igor Voznyc, Aleksandrs Pustenkoc,d
,
c
Mohey Eldin El Shikhe, Fatmah A. S. Alasmaryf, Sherif A. Abdel-Gawada,g and Raivis Zalubovskis
ꢀ
b
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Alkharj, Saudi Arabia; Department of
c
d
Chemistry, Kulliyyah of Science, International Islamic University Malaysia; Latvian Institute of Organic Synthesis, Riga, Latvia; Institute of
e
Technology of Organic Chemistry, Faculty of Materials Science and Applied Chemistry, Riga Technical University, Riga, Latvia; Experimental
Medicine and Rheumatology, William Harvey Research Institute, Queen Mary University of London, London, UK; Chemistry Department, College
of Science, King Saud University, Saudi Arabia, Riyadh; Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
f
g
ABSTRACT
ARTICLE HISTORY
Received 2 September 2017
Revised 7 November 2017
Accepted 7 November 2017
A series of symmetric molecules incorporating aryl or pyridyl moieties as central core and 1,4-substituted
triazoles as a side bridge was synthesised. The new compounds were investigated as lactate dehydro-
genase (LDH, EC 1.1.1.27) inhibitors. The cancer associated LDHA isoform was inhibited with
IC50 ¼ 117–174 mM. Seven compounds exhibited better LDHA inhibition (IC50 117–136 mM) compared to
known LDH inhibitor – galloflavin (IC50 157 mM).
KEYWORDS
Lactate dehydrogenase;
triazole; inhibitors
Introduction
number of LDHA inhibitors is reported in the literature so far8. In
several papers, very promising LDHA inhibition results have been
reported. As a one of the first inhibitors with low-micromolar
inhibition activity (Ki ¼ 13.5 lM) compound FX11 (Figure 1) along
with two similar compounds was reported in 20109. Utilising NMR
and SPR (surface plasmon resonance) fragment based hit identifi-
cation technique and further optimisation of structure a series
with low- and submicromolar LDHA inhibition was obtained with
best lead A (IC50 ¼ 0.27 lM)10. In the other study, also using frag-
ment-based hit identification, series of new LDHA inhibitors was
obtained. In this series, IC50 values ranged from 59 to 0.12 lM,
where the best inhibition was observed for compound B.11 It was
noted that in this series carbohydrate moiety in the middle of the
molecule and its stereochemistry had significant influence on the
inhibition activity. In very recent work through docking-based vir-
tual screening, several potential LDHA inhibitors were identified.
One of the compounds identified (C) showed very good LDHA
inhibition potency in vitro with an IC50 value of 0.33 lM.12
The lactate dehydrogenase (LDH, EC 1.1.1.27) is one of the most
abundant proteins and it is expressed in all tissues1. The main
function of LDH is interconversion of lactate and pyruvate with
accompanying interconversion of NADþ and NADH.
Three LDH isoforms are present in humans – LDHA, LDHB and
LDHC. LDHA and LDHB are expressed in all cells, whereas LDHC is
produced only in testis1. In the active form, LDH is a tetramer
formed of LDHA and LDHB in various ratios making five tetramers:
LDH1 (4 ꢀ LDHB), LDH2 (1 ꢀ LDHA/3 ꢀ LDHB), LDH3 (2 ꢀ LDHA/
2 ꢀ LDHB), LDH4 (1 ꢀ LDHA/3 ꢀ LDHB) and LDH5 (4 ꢀ LDHA)2.
In normal cells, predominant is LDHB where it converts lactate
to pyruvate with interconversion of NADþ into NADH, which
allows cells to use lactate as a nutrient source for oxidative metab-
olism, and/or for gluconeogenesis2. LDHA is the predominant iso-
form found in skeletal muscle and other highly glycolytic tissues.
In contrast to LDHB, LDHA has a higher affinity for pyruvate, that
is, LDHA and LDH5 tetramer in particular predominantly converts
pyruvate to lactate with consumption of one NADH molecule to
produce NADþ which in turn is essential in glycolysis3. Cancer cells
mainly generate energy through glycolysis even in the presence of
normal oxygen pressure4. Since the LDHA is the final enzyme in
glycolysis pathway where generated NADþ is necessary for contin-
ued high glycolysis rate in cancer cells (Warburg effect)4, LDHA is
an important supporter of glucose metabolism in cancer cells and
Results and discussions
Here, we report the synthesis of symmetric molecules incorporat-
ing aryl or pyridyl moieties as central core and 1,4-substituted tria-
zoles as a side bridge and they evaluation as LDHA inhibitors.
At the beginning of our study based on literature data11, we
can affect tumourigenesis and metastasis5. Additionally, elevated assumed that V-shape structures are beneficiary for good LDHA
levels of LDHA are markers of many tumours, the majority of them inhibition. Our assumption was based on published X-ray struc-
are highly glycolytic, and high LDHA levels are related with poor tures, for instance for B–LDHA complex (PDB code 4I9H). It was
prognosis, for instance in several human malignancies6. Therefore, also obvious that besides V-shape of the molecule and appropriate
LDHA is defined as anticancer drug target6,7. Notably, a limited length of V-“arms” the terminal groups have to be able to make
ꢀ
CONTACT Raivis Zalubovskis
raivis@osi.lv
Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006, Riga, Latvia; Ahmed M. Alafeefy
alafeefy@hot-
mail.com
Department of Chemistry, Kulliyyah of Science, International Islamic University, P.O. Box 141, 25710 Kuantan, Malyasia
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

148
A. S. ALTAMIMI ET AL.
HO
HO
H
N
H
HO
N
O
S
S
O
O
O
N
HO
O
HO
A
FX11
F
OH OH
O
O
N
H
OH OH
N
OH
N
O
HN
Cl
O
O
O
O
N
S
O
OH
HO
NH2
O
B
C
Figure 1. Examples of chemical structures of known LDHA inhibitors.
(i)
(ii)
Br
Br
TMS
TMS
1
2
3
N3
Cl
O
NH2
HN
O
HN
(iv)
(iii)
(v)
R
R
R
5a-c
6a-c
4a-c
N
N
N
N
HN
N
NH
N
O
O
R = a SO2NH2; b CO2H; c NO2.
R
R
7a-c
Scheme 1. Reagents and conditions: (i) trimethylsilylacetylene, Pd2(PPh3)2, CuI, i-Pr2NH, THF, 70 ꢁC; (ii) KF, MeOH/THF, rt, 69% for two steps; (iii) chloroacetyl chloride,
.
K2CO3, THF, 0 ꢁC, 5a (98%), 5b (84%), 5c (89%); (iv) NaN3, DMF, rt, 6a (97%), 6b (68%), 6c (95%); (v) CuSO4 5H2O, sodium ascorbate, AcOH, DMF/H2O, rt, 7a (32%), 7b
(71%), 7c (41%). All detailed experimental procedures are provided in the Supplemental data.
hydrogen bonding, therefore we chose carboxylic, sulphonamide intermediates 9a and 9b, which were converted into correspond-
ing azides 10a,b by treatment with NaN3 (Scheme 2). Following
reaction of azides 10a,b with building block 3 under acidic click
reaction conditions provided desired inhibitors 11a and 11b.
Bis-acetylenylpyridine building block 14 was prepared in similar
way to compound 3, where 3,5-dibromopyridine 12 was first
reacted with TMS-acetylene under Sonogashira reaction conditions
and then TMS were eliminated in obtained intermediate 13 by
treatment with K2CO3 proving building block 14 in 67% yield over
two steps (Scheme 3)18.
The reaction between building blocks 13 and 6a-c under acidic
click reaction conditions provided inhibitors 15a-c (Scheme 3). In
turn, treatment of 13 by azides 10a,b under the same condition
provided inhibitors 16a,b with extended bridge.
and nitro groups as terminal ones for this study.
Chemistry
The synthesis of desired inhibitors 7a-c was started from commer-
cially available 1,3-dibromobenzene (1) which was reacted with tri-
methylsilylacetylene in Sonogashira reaction to provide bis-TMS
protected derivative 2 (Scheme 1). Following deprotection with KF
afforded building block 3 in good yield over two steps13. Azides
6a-c necessary for Cu-mediated click reaction were prepared in
two steps from commercially available anilines 4a-c. Acylation of
anilines 4a-c with chloroacetyl chloride afforded chlorides
5a-c14–16 in good yields and following treatment with NaN3 pro-
vided azide building blocks 6a-c also in good yields. Reaction of
building block 3 with 6a-c under acidic click reaction condition17
provided inhibitors 7a-c.
And finally, for the synthesis of inhibitors 20a-c and 21a,b, ani-
sole building block 19 was synthesised in analogy to 3 and 14 as
above, starting synthesis from dibromoanisole 17. Bis-TMS pro-
tected intermediate 18 was obtained under Sonogashira reaction
The same strategy as described earlier was utilised for the syn- conditions and following deprotection by potassium hydroxide
provided building block 19 in 61% yield over two steps
thesis of inhibitors 11a and 11b. First, aminomethylphenyl deriva-
tives 8a,b were acylated with chloroacelyl chloride to obtain
(Scheme 4)19.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
149
O
O
Cl
N3
NH2
N
N
NH
NH
N
N
HN
N
NH
N
(i)
(ii)
(iii)
O
O
R
R
R
R
R
8a,b
11a,b
9a,b
10a,b
a
b
R = SO2NH2; CO2H.
.
Scheme 2. Reagents and conditions: (i) chloroacetyl chloride, K2CO3, THF, 0 ꢁC to rt, 9a (33%), 9b (69%); (ii) NaN3, DMF, rt, 10a (78%), 10b (77); (iii) 3, CuSO4 5H2O,
sodium ascorbate, AcOH, DMF/H2O, rt, 11a (17%), 11b (59%).
N
Table 1. Inhibition data of human LDHA.
ꢂ
Compound
Inhibition, IC50 (lM)
N
N
N
HN
NH
N
N
N
7a
7b
7c
128
120
172
158
156
136
125
156
174
117
165
127
163
173
128
157
0
O
O
R
15a-c
R
11a
11b
15a
15b
15c
16a
16b
20a
20b
20c
21a
21b
Galloflavin
Blank
ꢂ
(iii)
N
N
N
(i)
(ii)
Br
Br
TMS
TMS
14
13
12
(iv)
R = a SO2NH2; b CO2H; c NO2.
N
N
N
N
N
HN
N
NH
N
O
O
R
Mean from three different assays, by colorimetric assay
measuring the absorbance at 450 nm (errors were in the
range of 5–10% of the reported values).
16a,b
R
Scheme 3. Reagents and conditions: (i) trimethylsilylacetylene, Pd2(PPh3)2, CuI,
.
Et3N, 75 ꢁC; (ii) K2CO3, MeOH/THF, rt, 67% for two steps; (iii) 6a-c, CuSO4 5H2O,
Treatment of 19 with azides 6a-c under the same condition as
described above provided inhibitors 20a-c (Scheme 4). In turn,
treatment of 19 with azides 10a,b afforded desired inhibitors
21a,b.
sodium ascorbate, AcOH, DMF/H2O, rt, 15a (85%), 15b (39%), 15c (73%); (iv)
.
10a,b, CuSO4 5H2O, sodium ascorbate, AcOH, DMF/H2O, rt, 16a (63%), 16b (46%).
The structures of all new compounds synthesised were fully
approved by 1H and 13C NMR, IR and HRMS data (see
Supplemental data).
OCH3
N
N
N
N
HN
NH
N
N
O
O
Inhibition studies
R
R
All 15 symmetrical compounds were evaluated for their ability to
inhibit LDHA, for the comparison galloflavin as a known isoform
nonselective LDHA inhibitor20 was used. Our choice of galloflavin
was based on the fact that it is extensively studied as potent anti-
cancer agents in recent years21–25; additionally, galloflavin is
already commercially available.
Even though all compounds exhibited similar LDHA inhibition
and it is difficult to perform structure-activity-relationship (SAR)
analysis, we can divide this compound into two groups. First one,
the most active compounds (7a, 7b, 15a, 15b, 16b, 20b and 21b)
showed better LDHA inhibition compared to galloflalvin, ranging
IC50 values from 117 to 136 mM for compounds in first group,
whereas galloflavin has IC50 ¼ 157 mM (Table 1). The most active
compound in this group was 16b (IC50 ¼ 117 mM), which incorpo-
rated pyridyl moiety as a central core and carboxylic groups as a
terminal ones. Second group of compounds (7c, 11a, 11b, 15c,
16a, 20a, 20c and 21a) exhibited equal or weaker inhibition com-
pared to galloflavin, ranging IC50 values from 156 to 174 mM. The
most active compounds in this group were 11a, 11b and 15c
(IC50 158, 156 and 156 mM, respectively). Similar activity of this
20a-c
(iii)
OCH3
OCH3
OCH3
(ii)
(i)
Br
Br
TMS
TMS
17
18
19
(iv)
OCH3
R = a SO2NH2; b CO2H; c NO2.
N
N
N
HN
N
N
NH
N
O
O
21a,b
R
R
Scheme 4. Reagents and conditions: (i) trimethylsilylacetylene, Pd2(PPh3)2, CuI,
.
Et3N, THF 65 ꢁC; (ii) KOH, MeOH/THF, rt, 61% for two steps; (iii) 6a-c, CuSO4 5H2O,
sodium ascorbate, AcOH, DMF/H2O, rt, 20a (64%), 20b (56%), 20c (47%); (iv)
10a,b, CuSO4 5H2O, sodium ascorbate, AcOH, DMF/H2O, rt, 21a (31%), 21b (85%).
.

150
A. S. ALTAMIMI ET AL.
three compounds is hard to explain, where compounds 11a and
11b have phenyl ring as a central core and sulphonamide and
carboxyl groups as terminal ones on aminomethylphenyl moieties,
but compounds 15c has pyridyl central moiety and nitro groups
as terminal one on shorter, aniline containing, bridge.
9. Le A, Cooper CR, Gouw AM, et al. Inhibition of lactate
dehydrogenase A induces oxidative stress and inhibits tumor
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10. Ward RA, Brassington C, Breeze AL, et al. Design and
synthesis of novel lactate dehydrogenase A inhibitors by
fragment-based lead generation.
55:3285–306.
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We hypothesise that the putative interaction of the inhibitors
described in this work with LDHA is similar to the published one,
that is, inhibitors bound in the active centre close to conserved
11. Kohlmann A, Zech SG, Li F, et al. Fragment growing and
linking lead to novel nanomolar lactate dehydrogenase
inhibitors. J Med Chem 2013;56:1023–40.
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dehydrogenase A (LDHA) inhibitors as anticancer agents to
inhibit the proliferation of MG-63 osteosarcoma cells. Med
Chem Commun 2017;8:1720–6.
13. Neenan TX, Whitesides GM. Synthesis of high carbon materi-
als from acetylenic precursors. Preparation of aromatic
monomers bearing multiple ethynyl groups. J Org Chem
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residues involved in the catalytic processing of LDHA substrates26
.
We also assume that at the same time the NADH cofactor is
bound in the active centre near the inhibitor molecule. This bind-
ing region of the protein is known to undergo considerable con-
formational changes during the catalytic cycle, that is, it has rather
high flexibility. Therefore, inhibitor binding might be unstable and
this reflects in IC50 values obtained.
Conclusions
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zenes as fluorescent photoaffinity probes for human car-
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water. Tetrahedron Lett 2006;47:6321–4.
16. Deepkumar J, Kalpesh P. Synthesis and evaluation of novel
benzimidazole derivatives as antimicrobial agents. Med
Chem Res 2014;23:1290–9.
17. Shao C, Wang X, Xu J, et al. Carboxylic acid-promoted
copper(I)-catalyzed azide-alkyne cycloaddition. J Org Chem.
2010;75:7002–5.
18. Goto H, Heemstra JM, Hill DJ, et al. Single-site modifcation
and their effect on the folding stability of m-phenylene ethy-
nylene oligomers. Org Lett 2004;6:889–92.
In conclusion, a series of new symmetric molecules have been
designed and synthesises as potent LDHA inhibitors. The com-
pounds synthesised exhibited promising in vitro LDHA inhibition
activity, where seven compounds were better inhibitors (IC50
117–136 mM) as known LDH inhibitor – galloflavin (IC50 157 mM),
and other eight showed equal or slightly lower inhibitory activity
(IC50 156–174 mM) as galloflavin. The results obtained are promis-
ing base for further development of novel LDH inhibitors.
Disclosure statement
The authors declare no conflict of interest. The authors alone are
responsible for the content and writing of this article.
19. Beves JE, Blanco V, Blight BA, et al. Towards metal com-
plexes that can directionally walk along tracks: controlled
stepping of a molecular biped with a palladium(II) foot.
J Am Chem Soc 2014;136:2094–100.
Funding
This project was supported by the National Plan of Science,
Technology and Innovation [Grant No. 12-MED2980–54], Prince
Sattam bin Abdulaziz University, Alkharj, PO Box 173, 11942.
20. Manerba M, Vettraino M, Fiume L, et al. Galloflavin (CAS
568-80-9):
a novel inhibitor of lactate dehydrogenase.
ChemMedChem 2012;7:311–17.
21. Farabegoli F, Vettraino M, Manerba M, et al. Galloflavin, a
new lactate dehydrogenase inhibitor, induces the death of
human breast cancer cells with different glycolytic attitude
by affecting distinct signaling pathways. Eur J Pharm Sci
2012;47:729–38.
22. Vettraino M, Manerba M, Govoni M, et al. Galloflavin sup-
presses lactate dehydrogenase activity and causes MYC
downregulation in Burkitt lymphoma cells through NAD/
NADH-dependent inhibition of sirtuin-1. Anticancer Drugs
2013;24:862–70.
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