Supporting Information

Synthesis and evaluation of 2-(3-arylureido)pyridines and 2-(3-

arylureido)pyrazines as potential modulators of Aβ-induced mitochondrial

dysfunction in Alzheimer’s disease

Ahmed Elkamhawya,b,1, Jung-eun Parka,c,1, Ahmed H. E. Hassand,e, Ae Nim Paef,g, Jiyoun Leeh,

Beoung-Geon Parkf,i, Eun Joo Roha,g,*

a Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea

b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt

c Department of Chemistry, Sogang University, Seoul 04107, Republic of Korea

d Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt

e Medicinal Chemistry Laboratory, Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea

f Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology (KIST), Seoul

02792, Republic of Korea

g Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul, Republic of Korea

h Department of Global Medical Science, Sungshin Women's University, Seoul 142-732, Republic of Korea

i School of Life Sciences and Biotechnology, Korea University, Seoul 02792, Republic of Korea

Contents

1- Evaluation of drug likeness, pharmacokinetics, inhibition of/and metabolism by CYP

2- Details for LC and MS applied Conditions

3- High resolution Mass spectra and/or HPLC purity data of final compounds (7aa-7hc)

4- References

1- Evaluation of drug likeness, pharmacokinetics, inhibition of/and metabolism by CYP

Despite the in vitro effectiveness of CsA and non-immunosuppressant CsA analogs as mPTP

blockers, they are not suitable candidates for treatment of neurodegenerative diseases due to their

poor bioavailability to CNS. Their high molecular weight and polypeptidic nature hinder their

penetration of the blood brain barrier (BBB). Therefore, evaluation of drug likeness and

pharmacokinetic properties of promising candidate hits are important for successful development

of effective therapy of neurodegenerative diseases. Consequently, an in silico predictions for

compliance of the six hit compounds and CsA to drug likeness, oral bioavailability, penetration

of blood brain barrier, as well as, CYP inhibition and metabolism were performed. The web-

based application PreADMET was employed in these calculations and the results are presented

in Table S1 [1]. The calculations predicted all of the tested hits as suitable for Lipinski's rule of

five [2], as well as, qualified in likeness to the compounds of comprehensive medicinal

chemistry (CMC) database [3]. In addition, all of the tested hits were predicted to be mid-

structures in likeness to drugs in the MDL Drug Data Report (MDDR). Furthermore, out of 18

calculated properties in likeness to compounds of world drug index (WDI), the tested six

compounds violated only one property (Kier shape index order 3) which is a 2D topological

descriptors [4]. On the opposite side, CsA violated Lipinski's rule of five and did not qualify for

likeness to CMC. Despite predicted as mid-structure in MDDR likeness, it violated 16 properties

out of the 18 calculated properties in prediction of WDI likeness. In contrast to CsA, the

combined results of these calculations anticipated a high probability for hit compounds to act like

drugs.

Table S1. Results of in silico evaluation of drug-likeness and ADME properties

Property 7ec 7ed 7fa 7fb 7fc 7ff CSA

Lipinski's Rule Se S S S S S Ve

CMC likea Qe Q Q Q Q Q NQe

MDDR likeb MSe MS MS MS MS MS MS

No. of Violations of likeness to WDIc 1 1 1 1 1 1 16

% Human Intestinal Absorption 94.97 95.49 94.96 94.97 94.98 95.09 85.29

BBB penetrationd 2.18 3.27 1.78 2.15 2.18 2.85 0.03

CYP2D6 inhibition NIe NI NI NI NI NI NI

CYP3A4 inhibition NI NI NI NI NI NI Inhibitor

CYP2D6 substrate NSe NS NS NS NS NS NS

CYP3A4 substrate WSe WS WS WS WS WS WSa Likeness to compounds of Comprehensive Medicinal Chemistry (CMC) database.b Likeness to compounds in MDL Drug Data Report (MDDR)c World Drug Index (WDI)d Blood Brain Barrier penetration was calculated as predicted ratio between concentration of drug in the brain to concentration of drug in the blood.e Abbreviations in the table stands for: S = Suitable; V = Violated; Q = Qualified; NQ = Not Qualified; MS = Mid-structure; NI = No inhibition; NS = Non-metabolizable substrate; WS = A weakly metabolized substrate.

In addition to drug likeness calculations, predictions of pharmacokinetics are vital for

anticipation of oral bioavailability, and more importantly, penetration of blood brain barrier

which is a perquisite for central nervous system active drugs. The calculated results retrieved for

these predictions anticipated excellent oral bioavailability for all of the six hits with estimated

human intestinal absorption around 95% which is significantly higher than that estimated for

CsA (nearly 85%). The calculations predicted excellent ability of the six compounds to cross

BBB with a predicted ratio of compound’s concentration in the brain tissue to that in the blood

stream within a range of 1.78~3.27. These predicted high values are incomparable to the

calculated extremely low ratio of concentration of CsA in brain tissue to that in the blood stream

which predicted to be 0.03 (Table S1).

Cytochrome P450 (CYP) is the major enzyme responsible for metabolism of xenobiotics within

the body. CYP family includes several isoforms with different localization tissues and diverse

substrates. In drug discovery, the metabolism of compounds by CYP and the inhibitory effect of

compounds on CYP are two important considerations. Metabolism of candidate molecule is one

of the determining factors of its in vivo effective dose and duration of action. In addition,

inhibition of a certain CYP member by a candidate molecule would affect rate of metabolism of

other substrates, as well as, other biological functions exerted by this CYP isoform. CYP2D6 and

CYP3A4 are two CYP isoforms known to be localized in brain tissues and are responsible for

metabolizing central nervous system active drugs in addition to other biological functions in the

central nervous system. It is known that CYP isoform 2D6 metabolizes endogenous

neurochemicals and hence, reduction of activity level of CYP2D6 might present some risk in

neurological diseases. Also, CYP isoform 3A4 has been reported to participate in cell protection

in diseased brain, in addition to its role in drug metabolism [5-9]. Therefore, the six hit

compounds and CsA were submitted for an in silico prediction of their impact on/and

metabolism by these two CYP isoforms. As Table S1 indicates, all of the six hits were predicted

to be devoid from inhibitory effect on both CYP isoforms. In addition, they were predicted as

non-substrates for CYP2D6 and as weakly metabolized substrates of CYP3A4. In these

calculations, CsA was predicted as a weakly metabolized substrate and an inhibitor of CYP3A4.

These results suggest that normal CYP functions and metabolism within brain tissues will be

maintained in presence of these hit compounds, in addition, they would be slowly metabolized

by CYP3A4.

2- Details for LC and MS applied Conditions

3- High resolution Mass spectra and/or HPLC purity data of final compounds (7aa-7hc)

References:

[1] O. Trott, A.J. Olson, AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, Journal of Computational Chemistry, 31 (2010) 455-461.[2] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Advanced Drug Delivery Reviews, 23 (1997) 3-25.[3] A.K. Ghose, V.N. Viswanadhan, J.J. Wendoloski, A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases, Journal of combinatorial chemistry, 1 (1999) 55-68.[4] T.I. Oprea, Property distribution of drug-related chemical databases, Journal of computer-aided molecular design, 14 (2000) 251-264.[5] J. Cheng, Y. Zhen, S. Miksys, D. Beyoglu, K.W. Krausz, R.F. Tyndale, A. Yu, J.R. Idle, F.J. Gonzalez, Potential role of CYP2D6 in the central nervous system, Xenobiotica; the fate of foreign compounds in biological systems, 43 (2013) 973-984.[6] C. Ghosh, N. Marchi, N.K. Desai, V. Puvenna, M. Hossain, J. Gonzalez-Martinez, A.V. Alexopoulos, D. Janigro, Cellular localization and functional significance of CYP3A4 in the human epileptic brain, Epilepsia, 52 (2011) 562-571.[7] C.S. Ferguson, R.F. Tyndale, Cytochrome P450 enzymes in the brain: emerging evidence of biological significance, Trends in pharmacological sciences, 32 (2011) 708-714.[8] V. Agarwal, R.P. Kommaddi, K. Valli, D. Ryder, T.M. Hyde, J.E. Kleinman, H.W. Strobel, V. Ravindranath, Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite, PloS one, 3 (2008) e2337.[9] I. Siegle, P. Fritz, K. Eckhardt, U.M. Zanger, M. Eichelbaum, Cellular localization and regional distribution of CYP2D6 mRNA and protein expression in human brain, Pharmacogenetics, 11 (2001) 237-245.