Background: Leflunomide (LFM) and its active metabolite, teriflunomide (TFM), have
drawn a lot of attention for their anticancer activities, treatment of rheumatoid arthritis and malaria due
to their capability to inhibit dihydroorotate dehydrogenase (DHODH) and Plasmodium falciparum dihydroorotate
dehydrogenase (PfDHODH) enzyme. In this investigation, the strength of intramolecular
hydrogen bond (IHB) in five analogs of TFM (ATFM) was analyzed employing density functional theory
(DFT) using B3LYP/6-311++G (d, p) level and molecular orbital analysis in the gas phase and water
solution. A detailed electronic structure study was performed using the quantum theory of atoms in
molecules (QTAIM) and the hydrogen bond energies (EHB) of stable conformer obtained in the range of
76-97 kJ/mol, as a medium hydrogen bond. The effect of substitution on the IHB nature was studied by
natural bond orbital analysis (NBO). 1H NMR calculations showed an upward trend in the proton
chemical shift of the enolic proton in the chelated ring (14.5 to 15.7ppm) by increasing the IHB
strength. All the calculations confirmed the strongest IHB in 5-F-ATFM and the weakest IHB in 2-FATFM.
Molecular orbital analysis, including the HOMO-LUMO gap and chemical hardness, was performed
to compare the reactivity of inhibitors. Finally, molecular docking analysis was carried out to
identify the potency of inhibition of these compounds against PfDHODH enzyme.
TFM acts as an inhibitor of dihydroorotate dehydrogenase (DHODH) and Plasmodium falciparum dihydroorotate
dehydrogenase (PfDHODH) enzyme. Leflunomide and its active metabolite teriflunomide
have been identified as drugs for treatment of some diseases, such as multiple sclerosis (MS), rheumatoid
arthritis (RA), malaria, and cancer. Hydrogen bonds play a key role in the interaction between
drugs and enzymes.
Objectives: The aim of the present work is to investigate the effect of the strength of intramolecular hydrogen
bonds (IHBs) in the active metabolite analogs of leflunomide or analogs of teriflunomide (ATFMs)
and study the interaction of these inhibitors against the PfDHODH enzyme using quantum mechanical
Methods: At first, intramolecular hydrogen bonds in five ATFMs were evaluated by the DFT method,
quantum theory of atoms in molecules (QTAIM), nuclear magnetic resonance (NMR), natural bond orbital
(NBO), and molecular orbital (MO) analyses. Then, the interaction of these inhibitors against the
PfDHODH enzyme were compared using molecular docking study.
Results: All the computed results confirm the following trend in the intramolecular hydrogen bond
strength in five mono-halo-substituted 2-cyano-3-hydroxy-N-phenylbut-2-enamide (ATFM): 5-FATFM>
4-Br-ATFM ≈ 3-Br-ATFM>3-Cl-ATFM>TFM-Z>2-F-ATFM which is in agreement with
QTAIM, NMR, and NBO results. Docking results show that 5-F-ATFM (EHB=97kJ/mol) has the minimum
MolDock score due to its considerable IHB strength.
Conclusion: For strong IHBs (EHB>100kJ/mol), C=O and O–H group are involved in the intramolecular
interactions and do not contribute to the external interactions. Also, the docking study revealed maximum
binding energy between TFM-Z and PfDHODH enzyme.