A combine approach of chemical synthesis, biological evaluation and structural dynamics studies revealed thiazole substituted arylamine derivatives as potent FabH enzyme inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104426

Bacterial FabH enzyme is a broad-spectrum antimicrobial target and can be used in the design of novel antibiotics. This study reports chemical synthesis of thiazole based amine compounds as FabH inhibitors, followed by biological evaluation, and computati

Full text of DOI:10.1016/j.bioorg.2020.104426

Journal Pre-proofs
A Combine Approach of Chemical Synthesis, Biological Evaluation and
Structural Dynamics Studies Revealed Thiazole Substituted Arylamine Deriv-
atives as Potent FabH Enzyme Inhibitors
Haseen Ahmad, Faisal Ahmad, Shaista Parveen, Sajjad Ahmad, Syed
Sikander Azam, Abbas Hassan
PII:
S0045-2068(20)31724-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.104426
YBIOO 104426
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
20 December 2019
8 June 2020
21 October 2020
Please cite this article as: H. Ahmad, F. Ahmad, S. Parveen, S. Ahmad, S. Sikander Azam, A. Hassan, A
Combine Approach of Chemical Synthesis, Biological Evaluation and Structural Dynamics Studies Revealed
Thiazole Substituted Arylamine Derivatives as Potent FabH Enzyme Inhibitors, Bioorganic Chemistry (2020),
doi: https://doi.org/10.1016/j.bioorg.2020.104426
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A Combine Approach of Chemical Synthesis, Biological Evaluation and
Structural Dynamics Studies Revealed Thiazole Substituted Arylamine
Derivatives as Potent FabH Enzyme Inhibitors
Authors: Haseen Ahmad,a, 1 Faisal Ahmad,b, 1 Shaista Parveen,a Sajjad Ahmad,b Syed Sikander
Azam,*b Abbas Hassan*a
1 Both authors contributed equally to this work.
Address: aDepartment of Chemistry, Quaid-i-Azam University Islamabad 45320 Pakistan
bComputational biology lab, National Center for Bioinformatics, Quaid-i-Azam University,
Islamabad, Pakistan
*Corresponding Author: Dr. Syed Sikander Azam, ssazam@qau.edu.pk; Dr. Abbas Hassan: ahassan@qau.edu.pk;
1

Abstract
Bacterial FabH enzyme is a broad-spectrum antimicrobial target and can be used in the
design of novel antibiotics. This study reports chemical synthesis of thiazole based amine
compounds as FabH inhibitors, followed by biological evaluation, and computational drug
designing analysis with ultimate objective to guide further biological optimization of the identified
hits. The compounds were synthesized through Pd-PEPPSI catalyzed cross coupling strategy for
the Buchwald-Hartwig amination of thiazole-substituted aryl bromide. Pd-PEPPSI pre-catalysts
were utilized for the cross couple with the diverse range of functionalized electron-deficient and
electron-rich anilines and aliphatic amines. The thiazole based heteroaryl bromide coupling was
found to be challenging and only specialized Pd-PEPPSI-IPr and Pd-PEPPSI-IPent catalysts were
found to be effective providing the coupling product yield in the range of 78% to 99%. Biological
investigation depicted compound 3f to be effective against Bacillus subtilis, Staphylococcus
aureus, Staphylococcus epidermis, and Escherichia coli with mean + standard deviation value of
9.6 ± 0.4, 11.6 ±0.4, 15.6± 0.4, and 11.6 ±0.4, respectively. This compound is also active against
free radicals with EC90 value of 39.45 µg/ml. Comparative docking predictions unravel the 3f
binding mode at FabH active tunnel as such to block complete access for the natural substrate and
involved balanced hydrogen and hydrophobic interactions. FabH-3f complex dynamics in solution
found the docked conformation between the protein and compound of higher stability with mean
carbon alpha deviation of 1.87 Å and mean residual deviation of 0.88 Å. Intermolecular
interactions analysis depicted Asn274 from FabH active pocket to be significant in compound
holding and strengthening of interaction as the simulation progresses. This was supported further
by radial distribution function (RDF) and axial frequency distribution (AFD) that demonstrated
the high distribution of compound atoms in close proximity of Asn274 residue and decrease in
interaction distance. Further, the docking and simulation findings were validated through
MMPB/GBSA methods that complements the compound affinity for the said target. In a nutshell,
the identified hit could be subjected to structure, biological and pharmacokinetic optimization for
development of effective FabH inhibitors.
Keywords: FabH Enzyme; Thiazole Substituted Arylamine Derivatives; Molecular
Dynamics Simulation; Antibacterial Activity; Binding free energy calculation.
2

1. Introduction
Though numerous antibiotics are currently available and in routine use for treating bacterial
infections, the fight between humans and bacterial pathogens is ongoing and will be as such for
the foreseeable future [1]. Adding to this, since the first use of antibiotics, the steady rise of
bacterial resistance witnessed and is accelerated to an alarming pace in the recent past leading to
the urgent need of developing newer antibiotics [2]. Towards this, computer aided drug design
(CADD) can be used in combination with experimental techniques to elucidate drug resistant
mechanisms, identification of novel drug targets and the design of novel antibiotics for both new
and known targets [3].
The fatty acid synthesis in bacteria has been studied extensively as an attractive target for
the design of new antibiotics [4, 5]. The 3-oxoacyl-[acyl-carrier-protein] synthase 3 (FabH) in this
pathway catalyzes the first step of condensation reaction by adding two carbon acyl acceptors from
malonyl-ACP and therefore vital in regulating the total rate of fatty acid production [6]. Given to
the fact that FabH activity is essential for cell viability and because of the detail structural
information availability, make it an excellent platform to facilitate novel antimicrobial discovery
hence minimizing cost and time [4, 5, 6]. Thiazole is one of the top five aromatic heterocycles
which are basic core of 9% of the total number of U.S. FDA approved small drug molecules [7].
Thiazole or 1,3-thiazole is an important heterocycle and among the most intensively investigated
class of five-membered aromatic heterocycles [8,9]. It is present in thiamine (vitamin B1) [10], in
various derivatives of penicillins [11] as well as many other natural products [12-15]. About 67%
of all thiazole containing pharmaceuticals belong to this important class. Some examples of
marketed drug with thiazole core are depicted in Figure 1.
3

N
S
CH3
O
H
N
H
N
H
NH
H2N
S
S
N
N
H
H
OH
N
O
N
N
N
N
N
Cl
N
S
NO2
HO2C
O
O
N
NH
CO2H
H3C
S
Cefixime
Dasatinib
Nizatidine
anti-biotic
anti-cancer
histamine H2 receptor antagonist
H2N
S
Ph
N
OH
O
NH2
N
S
S
O
CH3
N
HO
CH3
H
H
N
S
O
N
N
NH2
N
O
S
N
N
H
N
S
CH3
O
i-Pr O
S
NH2
O
Ph
O
O
Ritonavir
anti-retroviral
Famotidine
histamine H2 receptor antagonist
Meloxicam
anti-inflammatory
Figure 1. Commercially available Thiazole based drugs.
Several different groups of FabH inhibitors and their derivatives have been described in the recent
past including alkyl-CoA disulfides, thiolactomycin, 1,2-Dithiole-3-(thi)ones, indole,
alkylsulfonyl, cerulenin, benzoylaminobenzoic acid, and thiazolidine-2-one 1,1-dioxide [16].
Methyl-CoA, butyl-CoA, sec-butyl-CoA, octyl-CoA, and decyl-CoA disulfides of the alkyl-CoA
disulfides have been shown to inhibit Escherichia coli FabH up to maximum IC50 value of
57.1±4.5 mM [17]. Compound 4,5-dichloro-1,2-dithiole3-one from 1,2-Dithiole-3-(thi)ones is
considered a potent Staphylococcus aureus FabH inhibitor with IC50 of 0.16 μM [18]. Series of
acylhydrazone derivatives with deoxygenated rings revealed to exhibit anti-FabH activity of 1.56–
3.13 μg mL−1 [19]. Previously, it has been reported that thiazole and their derivatives are potent
inhibitors of FabH [20, 21]. In a study, Lv et al., 2009 described a series Schiff bases containing
thiazole template [20]. In the synthesized series, they found two potent inhibitors with MIC of 0.78
μg/mL and 1.56 μg/mL against E. coli and five other bacterial pathogens showing their broad-
spectrum potency. Molecular docking illustrated the compounds engagement in strong hydrogen
bonding with Asn247 of FabH substrate binding pocket. In another study, series of compounds are
reported with amine bond between aromatic acid and 4-(4-bromophenyl)thiazol-2-amine or 4-
phenylthiazol-2-amine and have anti-FabH activity (IC50, 5.8 μM - 48.1 μM )[21]. Thiazole
amines are also reported to shown inhibitory potential of FabH with MIC value of 5.33 μM [22].
Designing and synthesis of heterocycles is an important research areas in pharmaceuticals,
agriculture sciences and drug discovery. A wide-ranging array of these parent heterocyclic
4

compounds can be easily prepared by conventional synthetic routes[23] together with thiazole
derivatives [14,24]. A valuable alternative approach includes transition metal catalyzed synthesis
of heterocyclic synthesis [25-28]. However, the transition metal catalyzed synthesis and
derivatization of these heterocycles present a real challenge to the researchers due to competing
side reaction that may inhibit the catalytic activity of the catalyst system [29-31]. One of the most
important inhibitory phenomena caused by nitrogen and sulfur containing heterocycles is the
catalyst poisoning [32-35]. A similar challenge can be envisioned in the transition metal based
derivatization of thiazole based nucleus. Therefore, the current investigation is important as it deals
with cross coupling of aryl derivatives of thiazoles with amine, as these compounds can inhibit the
catalytic behavior of the metal involved.
The Pd-PEPPSI family of pre-catalysts system has been developed for highly challenging
cross coupling reactions and was found to be highly superior for the Buchwald-Hartwig amination
reactions. The systematic investigated into the catalyst lead to the discovery of highly effective
Pd-PEPPSI-IPent and IPentCl catalysts (Figure 2) [36-39]. The Pd-PEPPSI catalysis provides a
quantifying protocol for the cross couplings of most challenging substrates like electron rich alkyl-
aryl chlorides and electron deficient amine counterparts. Recently, the modification of the ligand
in Pd-PEPPSI-IPentCl found to be the most efficient catalyst leading to the amination of deactivated
coupling partners using carbonate base at room temperature. Due to immense importance of aryl
amination, we investigated the synthesis of thiazole substituted biarylamines. The investigation
involves the C-N cross coupling with variety of alkyl and aryl amine.
5

PCy2
iPr
iPr
PPh2
PPh2
N
N
O
PPh2
PPh2
iPr
Cl
rac-BINAP
Xanthphos
XPhos
IMesCl
Cl
Cl
N
N
N
N
N
N
Pd
N
Pd
Pd
N
Cl
Cl
Cl
Cl
Cl
Cl
N
Cl
Pd-PEPPSI-IPr
Cl
Pd-PEPPSI-IPent
Cl
Pd-PEPPSI-IPentCl
Figure 2. Commonly employed ligands and Pd-PEPPSI pre-catalysts for C-N coupling reactions.
2. Results and discussion
2.1. Chemistry
Initial reaction involving coupling of 4-(4-bromophenyl)-2-methylthiazole and N-
methylbenzylamine using Pd2(dba)3/BINAP system gave only 21% yield of the desired coupling
product. We optimize the catalyst system by screening various combinations of Pd-precursors with
mono and bidentate ligands. Using DPPF and Xanthphos, the yield did not improve (Table 1,
entries 2 and 3). Screening of monodentate ligands such as P(o-Tol)3 or P(Cy)3, the isolated yields
were 21% and 15%, respectively (entries 4 and 5). Sterically hindered biarylphosphine ligands
such as, JohnPhos and XPhos were also found to inferior under these conditions (entries 6 and 7).
We also investigated N-heterocyclic carbene based ligand i.e IMes, providing a strong δ-donor
carbene as ligand to the in situ generated Pd-NHC catalyst, resulted in the desired product in only
16% yield.
6

Table 1.
Ligands and catalyst screening for amination of 4-(4-bromophenyl)-2-
methylthiazole.
Pd2(dba)3 (2.5 mol%)
CH3
S
H3C NH
Ligand
(10 mol%)
S
N
Br
N
H3C
N
H3C
K2CO3 (2 equiv.)
Toluene, 100 °C, 24 h
1a
(1 equiv.)
2m
(2 equiv.)
3m
S. No
1.
Ligand and Catalystb
BINAP
Yield (%)a
21
21
22
21
15
19
21
16
20
85
95
99
2.
DPPF
3.
Xanthphos
4.
P(o-Tol)3
5.
P(Cy)3
6.
JohnPhos
7.
XPhos
8.
IMesCl
9.
Pd-PEPPSI-IMes
Pd-PEPPSI-IPr
Pd-PEPPSI-IPent
Pd-PEPPSI-IPentCl
10.
11.
12.
Reaction conditions: 4-(4-Boromophenyl)-2-methylthiazole 622 (0.31 mmol, 1
equiv.), N-methylbenzylamine 623 (0.62 mmol, 2 equiv.), K2CO3 (0.62 mmol, 2
equiv.), toluene (0.3 M) at 100 °C for 24h; [a] isolated yields. [b] Entries 9-12 are
performed with pre-catalyst instead of Pd2(dba)3.
The efficiency of in situ active catalyst formation is, however difficult to control under the
usual reaction conditions may lead to potential waste of Pd-metal as well as ligand precursor but
7

essentially results in poor reproducibility [35-39]. Using Pd-PEPPSI-IMes catalyst we observed
slight increase in the yield in comparison to in situ. However, when we utilized the well-defined,
air and moisture-stable state of the art bulkier Pd-PEPPSI pre-catalyst (Table 1, entries 10-12) we
observed a dramatic increase in the efficiency of the catalyst system. The Pd-PEPPSI-IPr catalyst
resulted in 85% isolated yield (Table 1 entry 10). This dramatic increase in the yield may be
attributed to the steric bulk of IPr-NHC ligand assistances in reductive elimination. Increasing
trend in the reactivity was found in going to more sterically demanding NHC-backbone such as
Pd-PEPPSI-IPent and Pd-PEPPSI-IPentCl, leading to final coupled product in nearly quantitative
yields (Table 1, entries 11 and 12).
A similar trend was observed for the coupling of anilines. When we coupled the 4-(4-
bromophenyl)-2-methylthiazole 1a with p-toluidine in the presence of Pd-PEPPSI-IMes catalyst
and sodium tert-butoxide as base we observed only 10% of the diarylamine product 3c. Using Pd-
PEPPSI-IPr, the product was formed in 54% yield. Further increase in yield was possible with the
more efficient catalysts, the Pd-PEPPSI-IPent resulted in 68% while Pd-PEPPSI-IPentCl resulted
in 88% (Table 2).
Table 2.Pd-PEPPSI Catalysts screening for arylation of anilines using 4-(4-bromophenyl)-2-
methylthiazole.
Pd-PEPPSI Precatalyst
(2 mol%)
S
H
S
Br
H2N
CH3
N
CH3
N
NaOtBu (2 equiv)
toluene (0.3 M)
100 °C, 24h
H3C
N
H3C
1a
(1 equiv.)
2c
3c
(2 equiv.)
S. No.
Pd-Precatalyst
Yield (%)a
1.
2.
3.
Pd-PEPPSI-IMes
Pd-PEPPSI-IPr
Pd-PEPPSI-IPent
10
54
68
8

4.
5.
Pd-PEPPSI-IPentCl
88
99
Pd-PEPPSI-IPentCl (4 mol%)
Reaction conditions: 4-(4-Boromophenyl)-2-methylthiazole (0.31 mmol, 1
equiv.), 4-methylaniline (0.62 mmol, 2 equiv.), NaOtBu (0.628 mmol, 2 equiv.),
toluene (0.3 M) at 100 °C for 24h; [a] Isolated yield.
Initially cross coupling of 4-methylaniline with model substrate 4-(4-bromophenyl)-2-
methylthiazole using Pd-PEPPSI-IPr catalyst led to the formation of final amination product in
54% yield. Anilines are less basic than aliphatic amine, exhibiting anomalous behavior during
addition/coordination process with Pd due to its electron poor nature relative to alkyl amines.
Various substituted thiazole substrates were coupled with diverse range of amine counterpart in
order to synthesize heterocyclic amine moieties which could be lead precursors in the synthesis of
biologically active entities.
Table 3. Reaction scope for Pd-PEPPSI catalyzed amination using 1a and 1b.
9

Pd-PEPPSI-IPentCl (4 mol%)
S
H
S
H2N R2
Br
R2
N
N
NaOtBu (300 mol%)
toluene (0.3 M)
100 °C, 24h
R1
N
R1
1a,1b
(100 mol%)
R1 = Methyl, Phenyl
2a-o
3a-u
(200 mol%)
H3C
H
N
S
S
S
H
N
H
N
CH3
H3C
H3C
N
H3C
N
N
a
a
3c
, 96%
3a
3d
3b
3e
, 89%
, 98%
H3CO
H
N
OCH3
S
S
S
H
N
H
N
OCH3
H3C
H3C
N
H3C
N
N
N
b
b
a,c
, 96%
, 96%
3f
, 99%
Cl
Cl
S
S
S
H
N
H
N
H
N
Cl
Cl
H3C
H3C
N
H3C
N
N
3g
3h
3k
3n
3i
, 90%
, 99%
, 99%
, 98%
, 99%
CF3
S
CH3
N
S
S
H
N
H
N
H3C
F
H3C
H3C
N
N
a
3j
, 97%
3l
, 78%
CH3
S
CH3
N
S
S
S
S
H
N
N
O
H3C
N
N
N
H3C
H3C
a
3m
, 99%
3o
, 79%
CF3
S
S
S
H
N
H
N
H
N
CH3
F
Ph
N
Ph
Ph
Ph
N
N
N
3p
3q
3r
, 89%
, 92%
, 82%
CH3
N
CH3
N
S
N
O
Ph
N
N
Ph
3s
3t
3u,
80%
, 84%
, 90%
Reaction conditions: 4-(4-Boromophenyl)-2-methylthiazole (0.31 mmol, 100 mol%), aryl/alkyl-
amine (0.62 mmol, 200 mol%), NaOtBu (0.93 mmol, 300 mol%), toluene (0.3 M) at 100 °C for
10

24 h. [a] Pd-PEPPSI-IPentCl (2 mol%); [b] Pd-PEPPSI-IPent (2 mol%) and NaOtBu (200 mol%)
[c] NaOtBu (200 mol%)
The optimized reaction condition was found to be general for a variety of anilines and
aliphatic amines. Initially we performed the amination of 4-(4-bromophenyl)thiazole 1a. Simple
aniline was found to work better with only 2 mol% of Pd-PEPPSI-IPentCl resulting in the desire
product 3a in 89% isolated yield. A similar high reactivity was found with o-toluidine resulting in
the product 3b in 98% yield. Electron rich anilines, o-anisidine and m-anisidine were highly
reactive and worked well with only 2 mol% of Pd-PEPPSI-IPent catalyst, resulting in products 3d
and 3e each in 96% yields. p-Anisidine in the standard condition gave product 3f in quantitative
yield. Subsequently, we look in to electron poor aniline in anticipation that they will show slightly
lower conversion. On the contrary, chloro and fluoro substituted anilines works well and resulted
in excellent yields. o-Chloroaniline, p-chloroaniline and 2,4-dichloroaniline resulted in product
3g, 3h and 3i in 99%, 99% and 90% yields respectively. p-Fluoroaniline gave the product 3j in
97% isolated yield. In addition, 3-(trifluoromethyl)aniline resulted in products 3k in 98% yield.
When a N-substituted aniline, N-Methylaniline was treated in similar conditions, the tertiary
aniline 3l was formed in 78% yield. Similarly, aliphatic amines were found to be good coupling
partners, even with Pd-PEPPSI-IPr as a catalyst. N-Methylbenzylamine and morpholine were
proved to be nice coupling counterparts in this system, yielding the desired coupled products 3m
and 3n in range of 85-88%, while with Pd-PEPPSI-IPent as a catalyst, the reaction gave the cross
coupled products in nearly quantitative yields. n-Butylamine behaved different in comparison to
the other aliphatic amine as utilizing PEPPSI-IPr catalyst it led to desired coupled product in just
15% yield but changing the catalyst system to Pd-PEPPSI-IPentCl, it furnished the cross coupled
product 3o in 79% yield (Table 3).
The scope of the reaction was further explored using phenyl substituted thiazole substrate 1b,
which was used to cross coupled with electron rich and electron deficient anilines as well as
aliphatic amine counterparts, demonstrating the generality and efficiency Pd-PEPPSI catalysis for
C-N bond forming reaction. Therefore, when 1b was cross coupled with p-toluidine, the product
3p was isolated in 92% yield. Furthermore, 4-flouroaniline and 3-triflouromethylaniline led to the
desired products 3q and 3s in 82% and 89% yield respectively. Other amines such as N-
methylaniline, N-methylbenzylamine and morpholine were also successfully coupled furnishing
11

the desired cross couple products in good to excellent yields (Table 3). The reaction scope
exploration for amination of thiazole derivatives with variety of aromatic and aliphatic amine,
utilizing Pd-PEPPSI catalyzed amination protocol proved the practicality of the procedure and
enabled us to synthesized diverse thiazole substituted amine derivatives.
2.2. Biological Evaluation of the Compounds
Compounds 3f, 3k, 3s and 3c were the most active both against gram-positive and gram-negative
producing inhibitory zone of 16.6 mm against S. aureus, 22 mm against Staphylococcus epidermis,
14 mm against Bacillus subtilis and 15 mm against E. coli.
Figure 3. Antibacterial activity of synthesized compounds.
The percent free radical scavenging activity (% FRSA) was measured based on discoloration of
2,2-diphenyl-1-picrylhydrazyl (DPPH). Compounds with EC90 value < 90 µg/ml were considered
as best antioxidants whereas those with EC90 value > 90 were categorized as less active in term of
antioxidant activity. Considering this, 3a, 3d, 3f, 3g, 3h, 3k, 3l, 3m, 3n, 3o, 3p, 3q, 3s, 3t, 3u were
found as good antioxidants. Our best active antibacterial compound 3f has EC90 value of 39.45
µg/ml. Compound 3h has the lowest EC90 value of 13.73 µg/ml while compound 3c has the highest
EC90 value 1083 µg/ml showing no response against free radicals. Compounds antioxidant activity
can be found in Table 4.
12

Table 4. Optical density of compounds at different dosage concentration.
20%
15%
10%
5%
Compound
% scavenging activity
EC90 (µg/ml)
(µg/ml) (µg/ml) (µg/ml) (µg/ml)
3a
0.27
0.29
0.93
0.21
0.46
0.15
0.21
0.17
0.28
0.24
0.27
0.13
0.42
0.25
0.5
0.38
0.29
0.93
0.27
0.46
0.23
0.18
0.25
0.34
0.27
0.26
0.14
0.45
0.29
0.54
0.32
0.19
0.28
0.21
0.1
0.32
0.32
0.94
0.3
0.32
0.35
0.94
0.4
70.44 65.34 65.34 59.22
49.5
69.64
1083
30.56
188
3b
68.4
3.1
68.4 65.34 62.28
3.1 2.08 2.08
3c
3d
76.57 70.44 67.38 57.18
51.06 51.06 52.08 46.97
82.69 74.53 71.46 64.32
81.67 80.65 79.63 76.57
3e
0.45
0.26
0.17
0.29
0.37
0.27
0.39
0.17
0.5
0.5
3f
0.33
0.16
0.29
0.5
39.54
44.27
13.73
59.9
3g
3h
80.65 72.48 68.4
68.4
3i
69.42 63.3 60.24 46.97
73.51 70.44 70.44 67.38
71.46 70.44 58.2 56.16
84.73 83.71 80.65 77.59
55.14 52.08 46.97 44.93
72.48 68.4 61.26 58.2
46.97 42.89 38.81 37.79
73.51 65.34 62.28 56.16
79.63 78.61 75.55 71.46
76.57 69.42 64.32 59.22
79.63 76.57 72.48 60.24
87.79 87.79 86.77 78.61
92.38 91.57 91.36 90.95
3j
0.3
65.6
3k
0.41
0.2
47.4
3l
29.49
32.85
37.38
36.69
44.14
37.32
43.54
44.49
20.8
3m
0.52
0.39
0.59
0.41
0.26
0.33
0.37
0.19
0.069
0.98
3n
0.36
0.58
0.35
0.22
0.38
0.25
0.11
0.065
0.98
3o
3p
0.24
0.18
0.21
0.18
0.1
3q
3s
3t
3u
Positive control
Negative control
0.055
0.98
0.063
0.98
0
In general, increasing concentration of the compounds is directly related to high mortality of the
nauplii as shown in Table 5. All the compounds were noticed toxic; 3c and 3o being the most toxic
among all having LD50 value of 145 µg/ml followed by compounds 3i, 3k and 3n that have LC50
value of 156 µg/ml each. Less cytotoxic activity was measured for compounds 3f, 3h, 3j, 3l, 3p,
3q and 3s having LD50 value of 291 µg/ml each.
Table 5. Brine shrimp assay at different dosage concentration and LC50.
Compoun Number
Number of live nauplii per concentration
13

d
of nauplii
used
75
(µg/ml)
100
(µg/ml)
LD50
25 (µg/ml)
50 (µg/ml)
(µg/ml)
LogC
(1.87)
LogC
(2)
LogC (1.39)
LogC (1.69)
3a
3b
3c
3d
3e
3f
3g
3h
3i
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
8
8
8
8
8
8
8
8
7
8
7
8
8
7
8
8
8
8
8
7
8
8
8
7
8
8
8
8
8
7
8
7
8
8
7
8
7
8
8
8
7
8
7
8
7
8
7
8
8
8
7
8
7
8
7
7
7
7
8
8
8
7
8
7
6
6
6
7
7
6
7
6
7
6
7
7
6
7
6
7
7
7
6
6
218
166
145
166
218
291
166
291
156
291
156
291
218
156
218
145
291
291
291
156
166
3j
3k
3l
3m
3n
3n
3o
3p
3q
3s
3t
3u
Negative
control A
10
10
9
9
9
9
8
9
9
8
1000
375
Control B
2.3. Computational Studies of the compounds
The computational drug likeness and pharmacokinetics of the compounds are presented in
supplementary information.
2.3.1. Molecular Docking
14

Modification of thaizoles ring are reported to be a promising approach for enhancing antibacterial
activity of thaizole containing compounds. In previous reported studies, thaizoles derivatives were
found potent inhibitor of E. coli FabH enzyme, therefore, considered as a receptor in docking
evaluation [20-22]. The substrate binding site information was derived from the published E. coli
FabH co-crystalized ligand structure (PDB id: IHNJ) [40]. Only one monomer of the enzyme was
used in docking as both shared the same identical active pocket. The docking scores of the
compounds can be found in Figure 4. Comparative evaluation of the docking results and anti-
bacterial activity of the compounds revealed compound 3f as the most active derivative against the
enzyme. The compound has Genetic Optimization for Ligand Docking (GOLD) fitness score of
54.62 and AutoDock binding energy of -6.5 kcal.mol-1. Although the binding site is same in both
GOLD and Autodock Vina, yet the bounded orientation and interactions of the compound was
found different (Figure 5). In both GOLD and AutoDock Vina, only one hydrogen bond was
observed between oxygen atom of the anisole ring and Arg36 residue in GOLD and Phe304 in
AutoDock Vina. In addition to hydrogen bonding, several residues of protein active site are
involved in hydrophobic interactions. These includes: Asn247, Asn210, Gly209, and Thr37. The
binding interactions are different in both GOLD and AutoDock Vina and this could be assumed
due to different binding mode and deep binding of the compound in AutoDock Vina compared to
GOLD. In GOLD, the compound interacts with major area of the substrate binding site and
orientated along the channel of the active pocket. The anisole ring is positioned at active pocket
interface to ease formation of hydrogen bond with Arg36 of the enzyme at the surface. Arg36 is
one of the key residue of the enzyme and play major role towards substrate binding and catalysis.
The 2-methylthaizole allows the compound to interact with bottom floor of the active cavity. In
AutoDock Vina, the anisole ring is placed deep into the cavity, where oxygen of the anisole ring
is attached hydrophilically to the Phe304 residue of the enzyme. The 2-methylthaizole is placed at
the center of the pocket. Like in GOLD, the compound in Autodock Vina binds along the channel
of the enzyme active pocket. Unlike AutoDock Vina, in GOLD the compound is attached along
the wall of the pocket rather than in the center. Concluding, based on docking studies the
compounds can be theoretically regarded as potent inhibitors of FabH enzyme.
15

80
70
60
50
40
30
20
10
0
0
-1
-2
-3
-4
-5
-6
-7
-8
GOLD fitness score
Autodock Vina binding energy (Kcal.mol-1)
Figure 4. GOLD score and binding energy of the compounds.
Figure 5. Docked compound 3f at FabH enzyme active pocket.
16

2.2. Molecular Dynamics (MD) Simulation
Fluctuations of the enzyme-inhibitor complex and the ligand associated movements over the
course of simulation were investigated to infer complex stability and provide illustrations about
the affinity of the inhibitor for the enzyme active pocket. Deciphering the inhibitor movements
and inhibitor induced structural movements in the enzyme structure could provide myriad of future
directions for lead optimization; yielding novel inhibitors with unique mechanism of action [41].
Different simulation analysis of the complex is provided in Figure 6.The average root mean square
deviation (RMSD) deduced for the system is 1.87 Å with maximum RMSD of 2.70 Å observed at
80th ns, indicating high system stability. The system was observed with high stability for the first
25th ns where the average RMSD value of 1.29 Å was reported. Moving afterward, a slight upward
shift of RMSD graph was observed that continued with the same pattern towards the end of
simulation period. Superimposing the structure obtained at the 25th ns over that of 1st ns revealed
no major structural variations (Figure 7). The higher RMSD correlating to the displacement of the
loop regions after 25th ns is due to ligand induced movement within the active pocket of the
enzyme. These movements may be an attempt to achieve the stable pose in the pocket. This rotating
movement along the length of the inhibitor for achieving deep cavity binding was observed till the
end of simulation and could possibly explain the increased affinity of the inhibitor as the time
progress. Information about structural flexibility of each residue Cα atoms in the system was
investigated using root mean square fluctuation (RMSF) [41]. The average RMSF of the system is
0.88 Å, the highest RMSF value above than 3 Å is observed for residues Asn193-Glu200. These
residues lie in the loop regions of the enzyme and such loops are flexible to accommodate the
inhibitor within the pocket and allow strong interactions with the active residues. As discussed
earlier, at the 25th ns, the change in RMSD was noticed due to fluctuations in the loop region
involving residues5tf Asn193-Glu200. The equilibrium and structural compactness of the system
was unveiled by calculating the values of radius of gyration (Rg) over the simulation time [42].
The mean Rg value for the complex is 20.55 Å. The Rg value support the RMSD value of the
system and is the depiction of high system stability. Beta factor (β-factor) of the system represent
a collective term for thermal disorderness and each residue atoms structure stability [42]. The
finding of the β-factor is consistent with that of RMSF of the system. The mean β-factor found for
the system is 32.12 Ų, with the highest detected for the loop region involving the residues Asn193-
17

Glu200. This mean that the RMSF and β-factor values of the system complement each other in
depicting overall structural stability.
Figure 6. MD simulation trajectories analysis: A. RMSD, B. RMSF, C. Rg, and D. β-factor.
Figure 7. A. Superimposition of enzyme obtained at 25 ns (goldenrod) over that obtained after
1-ns (chartreuse). The RMSD obtained after superimposing was 1.06 Å. B. Close view of the
inhibitor movement. Inhibitor at 0-ns (gold) while at 25-ns (firebrick).
18

2.3. Radial Distribution Function (RDF) Analysis
RDF yields information that describes the variation of atom(s) density as a function of distance
from a reference particle [43]. An in-house visual molecular dynamics (VMD) script was first run
to find those residues of the enzyme involved in strong hydrogen bonds with the inhibitor. This
was vital in highlighting the role of residues from the active site that play significant role in
inhibitor binding and anchoring during simulation. This further signifies in keeping these
interactions for the synthesis of novel inhibitor derivatives with improved interaction and
mechanism of action. Analysis of the hydrogen bonds revealed Asn274 from the enzyme active
site as the vital residue. Asn274 is a critical residue of the FabH enzyme and it inactivation can
lead to enzyme function inactivation. Two atoms: ND2 and HD21 of this residue were unveiled
critical for inhibitor 4-methoxyaniline nitrogen recognition through the course of simulation
Figure 8. RDF plots were generated for both these atoms and are illustrated in Figure 9. RDF for
Asn274:HD21 and inhibitor N1, the highest density distribution of the interaction was found at
distance of 2.27 Å with g(r) value of 0.48 at the first 10-ns of simulation, while at the end 10-ns of
simulation, the maximum density distribution of these atoms interaction was observed at 2.32 Å
with g(r) value of 0.53. This plot further depicts that towards the simulation time, that the
interaction between these atoms are more stronger than that of beginning simulation and hence
suggest that as the time progress the inhibitor affinity for this active residue increases. In case of
Asn274:ND2 and inhibitor N1 atom, at start of simulation maximum atoms interaction was
reported at 3.07 Å with g(r) value of 0.24, while at last 10-ns of simulation, the distribution density
of the interaction was revealed at 3.28 Å with g(r) value of 0.27. This RDF plot is also the depiction
of close contact of the said interaction as simulation approaching the end. Overall, both the RDF
plots present strong agreements on the increasing affinity of the inhibitor for the enzyme Asn274
residue.
19

Figure 8. Atomic distance between Asn274 and inhibitor.
Figure 9. RDF plot for Asn274:HD21 and inhibitor N1 (A) and RDF plot for Asn274:ND2 and
inhibitor N1 (B)
20

2.4. Axial Frequency Distribution (AFD) Analysis
To understand further the pattern of hydrogen bonding between Asn274 residue of the enzyme and
the inhibitor, an analytical tool of AFD was employed [44]. In AFD, protein atom is used as a
reference, while the coordinates of inhibitor atom are mapped on the XY plane with respect to the
reference protein atom. As can be seen in Figure 10, AFD for Asn274: HD21 and inhibitor N1
atom, distribution of the inhibitor atom with respect to the protein reference atom is more dispersed
at the end of simulation compared to the simulation start. This distribution can be explained by the
inhibitor slow movement to achieve the more stable conformation and in close contact with the
Asn274: HD21 atom. In case of Asn274:ND2 and inhibitor N1 atom (Figure 11), the atoms were
seen more in close proximity at the end of simulation compared to the start of simulation. The
distribution area of the graph is reduced that depicts that in most of the frames towards the
simulation end, the protein and inhibitor atom spend time in close contacts and have increased
affinity for one another.
Figure 10. AFD plot for Asn274:HD21 and inhibitor N1 at start of simulation (A) and AFD plot
for Asn274:HD21 and inhibitor N1 towards end of simulation (B).
21

Figure 11. AFD plot for Asn274:ND2 and inhibitor N1 at start of simulation (A) and AFD plot
for Asn274:ND2 and inhibitor N1 towards end of simulation (B).
2.5. MMGB-PBSA Based Binding Free Energies Calculations
The MMGB-PBSA methods for estimating absolute free energy of the inhibitor to intended
biological macromolecule are popular approaches in computational aided drug designing (CAAD)
and applied to various system with vary degree of success [45]. Herein, both these methods were
utilized to calculate the inhibitor affinity for the enzyme and present critical evaluations that could
provide significant understanding for future potent derivative synthesis. The total binding free
energy in both approaches was found highly significant and favorable (Table 6). In case of
MMGBSA, the net binding energy is -39.94 kcal/mol, while for MMPBSA the total binding energy
is -4.85 kcal/mol. The van der Waals energy was found highly favorable and dominant the system
overall binding energy. The van der Waals energy found for the system is -41.95 kcal/mol. The
Columbus energy of the complex was also high and favorable i.e. -11.04 kcal/mol. Non-favorable
contributions from polar part of solvation free energy was witnessed for the enzyme-inhibitor
complex that in case of GB was 18.67 kcal/mol, while for PB it was 26.65 kcal/mol. The total
electrostatic energy in both the methods for the system was found non-favorable i.e. 7.62 kcal/mol
in case of GB and 15.61 kcal/mol in case of PB. The polar solvation energy inferred for the system
22

is 13.05 kcal/mol and 48.14 kcal/mol, respectively. The total binding energy of the complex was
further decomposed into enzyme residues. The main purpose of this exercise was to identify those
residues of the active pocket that contribute majorly in complex stabilization [46]. Additionally,
the knowledge of these residues could aid in identifying the chemical moiety of the inhibitor that
could be critical in inhibitor binding by the key residues of the enzyme active pocket and biological
activity. Moreover, the presence of such chemical moiety could be ensured in future synthesis of
derivative to achieve more potent leads. Enzyme active residues that contribution highly can be
referred as hot spot and are important contributor in ligand recognition and binding. It can be seen
in Figure 12 that the following residues: Met207, Val212, Hie244, Ala246, Asn247, Asn274 and
Phe304 are hot spot amino acids. All these residues are part of enzyme active site and are critical
from inhibitor catalysis point of view. The Met207 and Val212 interact hydrophilically with the
nitrogen of the dimethylamine, Hie244 and Ala246 through hydrogen bonding interacts with
sulphur and nitrogen of the 2-methythiazole ring of the inhibitor, Asn247 through its hydrogen and
nitrogen atoms interact with the oxygen of the anisole ring from the inhibitor and atoms of Asn274
and Phe304 interact with the 2-methythaizole of the inhibitor.
Table 6. Energies contribution of the complex.
Contribution Energy values (kcal·mol−1)
ΔEvdw
−41.95
−11.04
−52.99
−5.62
26.65
48.14
15.61
−4.85
18.67
ΔEele
ΔEgas
ΔGsol − np
ΔGPB
ΔGsolv,PB
ΔGele,PB
Htot,PB
ΔGGB
23

ΔGsolv,GB
ΔGele,GB
Htot,GB
13.05
7.62
−39.94
Internal
Polar Solvation
van der Waals
Electrostatic
Total
Non-Polar Solvation
MET 207
1.5
1
0.5
0
PHE 304
VAL 212
-0.5
-1
-1.5
-2
ASN 274
HIE 244
ASN 247
ALA 246
Figure 12. Binding free energies of the most stable residues involved in interactions with the
compound.
24

3. Methodology Flow
3.1. Chemical Synthesis of the Compounds
The detailed experimental procedure for the synthesis of all compounds and analytical data can be
found in the associated supporting information.
3.2. Biological Activities of the compounds
3.2.1. Antibacterial Assay
The antibacterial potency of compounds was characterized using agar disc diffusion method [47]
on Mueller Hinton Agar (MHA, Oxoid, England) media. Stock solutions were made by dissolving
1mg of compounds in 1ml of dimethyl sulfoxide (DMSO) (Duksan pure chemicals, Korea).
Positive control as Irgasan (triclosan) was prepared by same procedure described for the
compounds. Four test bacteria strains were used: S. aureus (gram-positive), S. epidermis (gram-
positive) and B. subtilis (gram-positive) and E. coli (gram-negative). Bacterial inoculum was
prepared by inoculating overnight cultured test strains inoculum in 1 ml sterilized normal saline
followed by turbidity adjustment to 0.5 McFarland (1% BaCl2 and 1% H2SO4). A bacterial lawn
was prepared and sterilized filter paper discs (6 mm in diameter, Whatman International Ltd,
England) were placed at an angle of 90º on the plates. Finally, 5 µl compound, positive control
(final concentration 5µg/5 µl), and negative control (DMSO impregnated disc) were applied on
the discs and incubated for overnight at 37 ºC. Antibacterial activity was measured by measuring
zone of inhibition in millimeter (mm). The experiment was run three time and readings were
statistically analyzed.
3.2.2. Antioxidant Assay
Free radical scavenging activities of the compounds were assessed using DPPH (Sigma-Aldrich,
United States) method [48]. DPPH stock solution of 0.06 mM was prepared by dissolving 0.0024g
DPPH in 100 ml methanol (Merck Chemicals, Germany). MULTISKAN GO spectrophotometer
(Thermo Fisher Scientific, United States) was used to adjust the optical density of DPPH to ~ 0.98
at 517 nm. Ascorbic acid (Sigma-Aldrich, United States) 1 mg per ml DMSO was used and its
calibrated standard curve was set as a reference. Compounds at different volumes: i.e. 5, 10, 15
and 20 μl (5 µl, final concentration 5µg/5 µl) were added to the 96 well plates. The wells were
25

filled with DPPH to achieve volume of 200 μl in each well. The plate was incubated at room
temperature in dark for 5-15 min. Absorbance of the reaction was analyzed at 517 nm. For a
negative control, DPPH was used, while ascorbic acid mixed with DPPH was used as a positive
control. The percent radical scavenging activity was determined by,
=
%푭푹푺푨 (푨풃풔 풄풐풏풕풓풐풍 ― 푨풃풔 풔풂풎풑풍풆 /푨풃풔 풄풐풏풕풓풐풍) ∗ ퟏퟎퟎ]………………………....(풊)
IC50 values were calculated and compared to the positive control.
3.2. 3. Brine Shrimp Lethality Assay
Toxicity of the compounds was investigated against brine shrimps following the protocol proposed
by Bibi et al. (2011) [49,50]. A bi-compartment perforated tank was filled with seawater containing
Artemia salina eggs (1.5 gm/L) at one end. The tank compartment with Artemia salina eggs
covered with aluminum foil and placed under light source and allowed to incubate at 30 ºC for 48
hours for hatching. After incubation, shrimps were collected and transferred to a sterilized test
tubes filled with seawater. The compounds were analyzed at different dilutions: 100 ug/ml, 75
ug/ml, 50 ug/ml, 25 ug/ml. The tubes were then incubated at 37°C for 24 hours. Doxorubicin
(positive control) was also used in different concentrations described for compounds. Dead nauplii
was counted in each tube. Compounds showing ≤ 50% of mortality was analyzed for LC50 (lethal
dose 50) in Table Curve 2D v4 software. The percent mortality was estimated using formula,
% 풎풐풓풕풂풍풊풕풚 = 푫풆풂풅 풔풉풓풊풎풑풔 / 푻풐풕풂풍 풔풉풓풊풎풑풔 × ퟏퟎퟎ…………….…(풊풊)
3.3. Computational Studies
3.3.1. Computational Calculation of Drugllikenss and Pharmacokinetics Properties
Computational drugllikenss of the compounds was predicted using SwissADME [51], while
pharmacokinetics
properties
of
the
compounds
were
predicted
using
http://biosig.unimelb.edu.au/pkcsm/prediction.
3.3.2. Molecular Docking Studies
Compounds 2D structure were drawn and minimized in ChemOffice 2012 package [52]. A single
unit of the FabH enzyme was selected, assigned with Gasteiger charges and minimized for 1500
26

steps (750 of conjugate and 750 of steepest descent) in UCSF Chimera [53]. Molecular docking
of the compounds with the FabH was performed with GOLD 5.2 software [54] and Autodock Vina
[55]. During docking, coordinates of Asn74: ND2 atom were set as a center. For each compound,
10 interactions were generated around 10 Å radius around Asn74: ND2. Docking affinity of
compound for protein was measured by GOLD fitness score. For comparative analysis, in addition,
the compounds were docked with protein through Autodock Vina using the same coordinates and
parameters used in GOLD. In Autodock Vina, the compounds affinity for protein was measured
in term of binding energy. Pencillin G was used as a positive control. The binding conformation
and interactions of compounds within protein active cavity were visualized using UCSF Chimera
and Discovery Studio [56].
3.3.3. Molecular Dynamics Simulation Protocol
The docked complex of the best characterized compound unveiled by both antibacterial assay and
molecular docking studies was then analyzed in 100-ns of molecular dynamics simulations. This
analysis could not only provide molecular insights of inhibition mechanism but in addition could
sum up behavioral significance of the compound for further potential lead optimization and
discovery [57]. The initial parameter files for compound were produced through Antechamber
program of AMBER14 simulation package [58]. The leap module aided in integrating the complex
into a TIP3p water box with padding distance of 12 Å between the protein and water box
boundaries. Force field, ff14SB, was used to describe system interactions. The system was
neutralized by adding 12 Na+ ions to it. The system was then processed through a series of phases.
The first phase of minimization split into hydrogen atoms minimization for 500 cycles, water box
minimization for 1000 cycles, carbon alpha atoms minimization for 1000 cycles and all non-heavy
atoms minimization for 300 cycles. In heating phase, NVT ensemble was used to heat the system
and langevin dynamics was employed for maintaining system temperature at 300 K for 20
picosecond (ps). The time step was set to 2 femtoseconds (fs), while restraint of 5 kcal/mol-A2 on
carbon alpha atoms were employed. In the equilibration step, the system was equilibrated for 100
ps with langevin dynamics for temperature scaling and SHAKE algorithm for constraint on
hydrogen bonds. In the pressure phase, pressure on the system was maintained for 50 ps. The
system was further equilibrated for 1 ns. Lastly, Berendsen temperature coupling algorithm
coupled with NVT ensemble for production run of 100 ns was accomplished at a time step of 2 fs.
27

Trajectories of simulation were analyzed for structural parameters using CCPTRAJ program of
the AMBER. For visualization, VMD software [59] was used.
3.3.4. RDF and AFD Analysis
A Perl written script was employed in VMD to extract enzyme residues involved in close distance
hydrogen bonding with the inhibitor. This is vital to highlight enzyme residues that are involved
in inhibitor binding during simulation. Once the interactions were identified, RDF [43] and a novel
analytical tool, AFD [44], which is indigenously developed to extract the role of inhibitor and
enzyme hot spot residues vital for inhibitor catalytic mechanism.
3.3.5. MMGB-PBSA Based Binding Free Energies Calculations
In total of 500 frames were picked from simulation trajectories and analyzed for binding free
energies estimation using AMBER MMPBSA module [60]. The net binding energy was calculated
through the following equation,
∆푮풃풊풏풅풊풏품 = ∆푮풆풏풛풚풎풆 ― 풊풏풉풊풃풊풕풐풓 풄풐풎풑풍풆풙 – [∆푮풆풏풛풚풎풆 + ∆푮풊풏풉풊풃풊풕풐풓].(풊풊풊)
In the above equation, ∆G is the Gibbs free energy and can be split into the following terms:
∆푮 = 푬품풂풔 + ∆푮풔풐풍풗 ― 푻푺풔풐풍풖풕풆………………………………………………………..(풊풗)
In the equation, Egas stands for gas phase energy and is the collective term for van der Waals,
electrostatic and internal energies:
푬품풂풔 = 푬풗풅풘 + 푬풆풍풆 + 푬풊풏풕…………………………………………………………………(풗)
The term, ∆Gsolv, is calculated implicitly and includes non-polar and electrostatic contributions:
∆푮풔풐풍풗 = ∆푮풏풑 + ∆푮풆풍풆………………………………………………………………………(풗풊)
∆푮풏풑 =
휸풔풐풍풗풆풏풕 풂풄풄풆풔풔풊풃풍풆 풔풖풓풇풂풄풆 풂풓풆풂 + 휷……………………………………(풗풊풊)
Estimation of ∆Gnp in MMPBSA is done using Linear Combinations of Pairwise Overlaps (LCPO)
with water probe radius set to 1.4 Å. In MMGBSA, the standard value of γ and β are 0.0072
kcal/mol-Å2 and 0 kcal/mol, respectively. For MMPBSA, the value of γ is 0.0052 kcal/mol-Å2
while that for β the value is set to 0.92 kcal/mol.
28

∆Gnp is non-electrostatic energy and is directly proportional to molecule solvent accessible area
while Gele involves the addition of polar solvation component and electrostatic energy:
∆푮풆풍풆 = ∆푮푮푩/푷푩 + 푬풆풍풆…………………………………………………………………(풗풊풊풊)
4. Conclusions
This study details synthesis of a novel series of thiazole based amine compounds as promising
bacterial FabH enzyme inhibitors. Biological investigation revealed compound 3f as promising
active molecule against both tested gram-positive and gram-positive bacterial pathogens. Docking
studies indicated deep binding of the compound in the substrate binding pocket of the enzyme
where anisole ring possess unique positioning at the active pocket interface interacting with
Arg36 and 2-methylthaizole with bottom floor. Molecular dynamics simulation further affirms
the docking studies in deciphering stable binding of the compound at the docked site. RMSD and
RMSF fluctuation analysis depicted FabH enzyme local conformational movements with no
impact on compound binding. RDF and AFD shown the stability of complex mainly due to
Asn274 active residues in holding the compound at the docked site. The binding free energies
predicted for the system demonstrated dominant role of van der Waals interaction towards
intermolecular stability as dynamics of the complex progresses. Systemic structure modification
of this lead molecule may possible lead novel antibacterial discovery.
Conflict of Interest
The authors declare that they have no conflict of interest.
Acknowledgments
Dr. Syed Sikander Azam is highly grateful to the Pakistan-United States Science and Technology
Cooperation Program (Grant No. Pak-US/2017/360) and the Higher Education Commission
(HEC) for granting the financial assistance. Dr. Abbas Hassan is grateful to HEC for research grant
number 20-3885/R&D/HEC/14. We are thankful to Prof. Michael G. Organ, University of Ottawa
for kind donation of Pd-PEPPSI-IPr and Pd-PEPPSI-IPent.
Authors Contribution
29