Discovery and Modification of in Vivo Active Nrf2 Activators with 1,2,4-Oxadiazole Core: Hits Identification and Structure-Activity Relationship Study

Article abstract of DOI:10.1021/acs.jmedchem.5b00170

Induction of phase II antioxidant enzymes by activation of Nrf2/ARE pathway has been recognized as a promising strategy for the regulation of oxidative stress-related diseases. Herein we report our effort on the discovery and optimization of Nrf2 activators with 1,2,4-oxadiazole core. Screening of an in-house collection containing 7500 compounds by ARE-luciferase reporter assay revealed a moderate Nrf2 activator, 1. Aimed at obtaining more derivatives efficiently, molecular similarity search by the combination of 2D fingerprint-based and 3D shape-based search was applied to virtually screening the Chemdiv collection. Three derivatives with the same core were identified to have better inductivity of Nrf2 than 1. The best hit 4 was selected as starting point for structurally optimization, leading to a much more potent derivative 32. It in vitro upregulated gene and protein level of Nrf2 as well as its downstream markers such as NQO1, GCLM, and HO-1. It remarkably suppressed inflammation in the in vivo LPS-challenged mouse model. Our results provide a new chemotype as Nrf2-ARE activators which deserve further optimization with the aim to obtain active anti-inflammatory agents through Nrf2-ARE pathway.

Full text of DOI:10.1021/acs.jmedchem.5b00170

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Article
Discovery and Modification of in vivo active Nrf2 Activators with 1, 2, 4-
oxadiazole Core: Hits Identification and Structure-Activity Relationship Study
Lili Xu, Junfeng Zhu, Xiaoli Xu, Jie Zhu, Li Li, Meiyang Xi, Zhengyu Jiang, Mingye Zhang, Fang Liu,
Mengchen Lu, Qichao Bao, Chao Zhang, Jinlian Wei, Xiaojin Zhang, Qi-Dong You, and Haopeng Sun
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 25 Jun 2015
Downloaded from http://pubs.acs.org on June 25, 2015
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Discovery and Modification of in vivo active Nrf2 Activators with 1, 2,
4
ꢀoxadiazole Core: Hits Identification and StructureꢀActivity Relationship Study
a,b
a,b
a,b
a,b
a,b
a,b
LiꢀLi Xu, JunꢀFeng Zhu, XiaoꢀLi Xu, Jie Zhu, Li Li, MeiꢀYang Xi,
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a,b
a,b
a,b
a,b
ZhengꢀYu Jiang, MingꢀYe Zhang, Fang Liu, Mengꢀchen Lu, QiꢀChao Bao,
a,b
a,b
a,b
a,b,d
a,b,e,*
Chao Zhang, JinꢀLian Wei, XiaoꢀJin Zhang,
QiꢀDong You,
and
a,b,c,*
HaoꢀPeng Sun,
a
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical
University, Nanjing, 210009, China
b
State Key Laboratory of Natural Medicines, China Pharmaceutical University,
Nanjing 210009, China
c
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical
University, Nanjing, 210009, China
d
Department of Organic Chemistry, School of Science, China Pharmaceutical
University, Nanjing, 210009, China
e
National Engineering and Research Center for Target Drugs, Jiangsu Hengrui
Medicine Co. Ltd., Lianyungang, 222000, China
Corresponding author:
Haoꢀpeng Sun: Fax & Tel: +86ꢀ25ꢀ83271216, Eꢀmail: sunhaopeng@163.com;
Qiꢀdong You: Fax & Tel: +86ꢀ25ꢀ83271351, Eꢀmail: youqidong@gmail.com.
Abstract
Induction of phase II antioxidant enzymes by activation of Nrf2/ARE pathway has
been recognized as a promising strategy for the regulation of oxidative stressꢀrelated
diseases. Herein we report our effort on the discovery and optimization of Nrf2
activators with 1,2,4ꢀoxadiazole core. Screening of inꢀhouse collection containing
7
500 compounds by AREꢀluciferase reporter assay revealed a moderate Nrf2 activator,
1. Aimed at obtaining more derivatives efficiently, molecular similarity search by the
1
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combination of 2D fingerprintꢀbased and 3D shapeꢀbased search was applied to
virtually screening the Chemdiv collection. Three derivatives with the same core were
identified to have better inductivity of Nrf2 than 1. The best hit 4 was selected as
starting point for structurally optimization, leading to a much more potent derivative
32. It in vitro upꢀregulated gene and protein level of Nrf2 as well as its downstream
markers such as NQO1, GCLM and HOꢀ1. It remarkably suppressed inflammation in
the in vivo LPSꢀchallenged mouse model. Our results provide a new chemotype as
Nrf2ꢀARE activators, which deserve further optimization with the aim to obtain active
antiꢀinflammatory agents through Nrf2ꢀARE pathway.
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Introduction
Nuclear factor erythroid 2 p45ꢀrelated factor 2 (Nrf2), a basicꢀleucine zipper
(bꢀZIP) transcription factor normally located in the cytoplasm of cells, regulates the
transcription of protective genes by interacting with a cisꢀacting element (antioxidant
response element, 5’ꢀGTGACnnnGCꢀ3’) / (Electrophile Response Element)
1
(ARE/EpRE) in their promoters to ameliorate oxidative and environmental stresses.
When cells are exposed to inflammatory stimulus or environmental stress, such as
toxicant, oxidation and electrophilic attack, Nrf2 detaches from its endogenous
inhibitory partner Kelchꢀlike ECHꢀassociated protein 1 (Keap1), subsequently
translocates to the nucleus and binds to the antioxidant response element (ARE) of
2
target genes, results in transcriptional induction and chemoprotection effects. As a
result, The Keap1ꢀNrf2 proteinꢀprotein interaction (PPI) is recognized as the major
regulatory pattern of cytoprotective gene expression against oxidative and/or
3
electrophilic stresses. Keap1 shepherds Nrf2 toward the polyubiquitination and
degradation machinery of the cell before it translocates into the nucleus, leading to
4
negatively regulation of Nrf2 transcriptional function. In addition to Keap1, several
protein kinases have been confirmed to activate Nrf2, thus are considered to be the
5
regulatory nodes in the Nrf2 related pathways.
Nrf2 can regulate almost all of the relevant antioxidants and cytoprotective genes
such as NAD(P)H/quinone oxidoreductase 1(NQO1), heme oxygenaseꢀ1 (HOꢀ1),
2
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glutamateꢀcysteine ligase modifier subunit (GCLM), γꢀglutamyl cysteine synthase,
glutathione peroxidase (GPx), glutathione Sꢀtransferases (GSTs), and several
members of the glutathione Sꢀtransferase family which express the ARE in their
6
ꢀ8
promoter. As a result, activation of Nrf2 is considered to be an efficient strategy to
protect the cells from abnormal stimulus or chemical attacks, which are usually
existed in the chronic and degenerative syndromes, such as uncontrolled inflammation,
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carcinogenesis, and Alzheimer’s disease.
So far, many compounds have shown Nrf2 inductive activities. Dimethyl fumarate
(DMF), a very simple compound, can covalently bind to Cys151 of Keap1 and
upregulate the activity of Nrf2. It has been approved for the treatment of Multiple
1
0
Sclerosis by FDA in 2013. Bardoxolone methyl (also named as CDDOꢀMe), is
11
considered to act with the similar mechanism to dimethyl fumarate, though that
1
2
13
notion has recently been challenged. Other natural products including curcumin,
14
15
16
sulforaphane (SFN), chalcone, resveratrol and caffeic acid phenethyl ester
1
7
(
CAPE) also exhibit chemopreventive effects through activating Nrf2 related
pathway. Additionally, small molecules directly targeting Keap1ꢀNrf2 PPI have been
18
disclosed by many groups. These compounds, either peptides or synthetic
19
20
compounds such as cpd16, (SRS)ꢀ5, are proposed to block the binding of Keap1 to
the ETGE or DLG motifs of Nrf2. They provide a novel strategy for the development
of highly efficient Nrf2 activators as chemopreventive agents (Figure 1).
Our laboratory has previously reported several small compounds as Nrf2
21, 22
activators,
which provide promising chemotypes for the development of active
chemopreventive agents (Figure 1).
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Figure 1. Active Nrf2 activators from different publications.
Herein we report our effort on the discovery of new Nrf2 activators with
1,2,4ꢀoxadiazole core. Compound 1 was firstly identified from screening of a
collection of 7500 compounds by AREꢀluciferase reporter assay. To obtain more
derivatives efficiently, molecular similarity search by the combination of 2D
fingerprintꢀbased and 3D shapeꢀbased search was applied to virtually screening the
Chemdiv collection. The resulted compound 4, which was more potent than 1, was
selected as starting point for optimization. Compound 32, which was much more
potent than 4, was revealed from the structureꢀactivity relationship (SAR) study.
Pharmacological study showed the compound in vitro upꢀregulated gene and protein
level of Nrf2 as well as its downstream markers such as NQO1, GCLM and HOꢀ1. It
also remarkably suppressed inflammation in the in vivo LPSꢀchallenged mouse model.
Therefore, our results provide a new chemotype as Nrf2ꢀARE activators, which
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deserve further optimization with the aim to obtain potent antiꢀinflammatory agents
through Nrf2ꢀARE pathway.
Results
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1. Hits identification from virtual screening (VS) using shapeꢀbased similarity
search.
Virtual screening. The initial compound 1 was screened out from an inꢀhouse
collection containing 7500 compounds by using AREꢀluciferase reporter assay in
HepG2ꢀAREꢀC8 cells. It exhibited a mild inductivity, with 4.10 ± 0.35 fold and 5.47
±
0.14 fold upregulation of the ARE level at 20 ꢁM and 50 ꢁM, respectively. To
obtain more activators efficiently, compound 1 was used as the template for the
subsequent molecular similarity search to identify potential hits from Chemdiv
collection. 2D molecular fingerprint FCFC_6 method (‘Find Similar Molecules by
Fingerprints’ module in Discovery Studio 3.0) was first used for its high efficiency in
the screening of large compound database. The tanimoto coefficient was set as 0.7.
3
7104 hits were screened out to have the similarity to compound 1, however, the data
only suggested a similarity of chemical structure and atom connectivity in the 2D
level between the reference molecule and the hits, while the 3D conformational
similarity can not be evaluated by this method. Therefore, we subsequently performed
a shapeꢀbased 3D similarity search (‘Search 3D database’ using molecular shape in
Discovery Studio 3.0). The best 50 hits were output for the manual selection. 12 hits
were retained and purchased from Topscience (http://www.tsbiochem.com/). Their
Nrf2 inductivity was then evaluated by the AREꢀluciferase reporter assay. Finally, 3
hits with 1, 2, 4ꢀoxadiazole core (2~4, Figure 2B) were confirmed to have improved
Nrf2 inductivity compared to the template molecule 1. The screening strategy was
summarized in Figure 2A.
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Figure 2. (A) The screening strategy combining 2D and 3D molecular similarity search. (B) The
structure of the four Nrf2 activators with 1,2,4ꢀoxadiazole core (Compound 1~4).
Hit compounds exhibited ARE inductivity in luciferase reporter assay. The
ARE inductivity from luciferase reporter assay shows the compound 2~4 can
doseꢀdependently induce the ARE response, with much higher potency than the
positive compound tBHQ. Compared to tBHQ which showed 5.27ꢀfold induction at
8
0 ꢁM, the most potent compound 4 significantly increased the level of ARE to
1
2.41ꢀfold at 40 ꢁM (Figure 4A). To avoid false positive data, we also determined the
luminescent signal of the hits themselves using the same experimental condition to
luciferase reporter assay. No luminescent signals of the compounds were determined,
indicating that the inductivity of the Nrf2ꢀARE pathway was due to the real activities
of the hits. Next we observed the cell toxicity of 2~4 using MTT assay. The three
compounds had no obvious antiꢀproliferative effects on HCT116 cells (Figure S1).
Interestingly, compound 4 exhibited a doseꢀdependently proliferative inductivity,
remarkably at a high concentration of 40 ꢁM. This may be attributed to its cell
protective effect through the activation of Nrf2 signaling. These observations
indicated a potential safety tolerance in the usage of the compounds.
Table 1. The Nrf2 inductivity of the hit compounds in luciferase reporter assay
a
Nrf2 inductivity (ꢁM)
Cpd.
0.02
0.2
2
20
40
80
160
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
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5
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6
1
2
3
4
1.18±0.12 1.22±0.10 1.47±0.14 3.31±0.49
4.05±0.49
5.96±0.31
7.34±0.11
5.88±0.61
5.48±0.65
6.00±0.81
8.36±0.99
1.46±0.13 1.83±0.11 3.17±0.55 3.88±0.76
1.18±0.10 1.12±0.08 4.48±1.42 5.65±1.29
7.67±1.34 11.70±2.75
1.09±0.06 1.50±0.01 3.35±0.28 7.61±0.24 12.41±0.54 12.13±0.20 8.73±0.57
2.14±0.10 5.27±0.41 8.46±1.37
tBHQ 1.18±0.08 1.25±0.01 1.24±0.04 1.41±0.07
a
The inductivity of the compound is calculated compared to the blank control, and data are presented as
mean ± SEM of three separate experiments;
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Compound 4 upregulates Nrf2ꢀARE pathway in both gene and protein level.
According to above results, the expression of Nrf2 and several phase II enzymes
including HOꢀ1 and NQO1 were further determined in the compound 4 treated
HCT116 cells. The compound maximized the expression of HOꢀ1 and NQO1 at 4 h
and 6 h, respectively, while induction of Nrf2 reached a stable state after 2 h (Figure
3
A). Additionally, 4 doseꢀdependently enhanced the expression of Nrf2 and NQO1
(
Figure 3B) after 6 h treating with 4 in the HCT116 cells, these data indicated that
compound 4 can induce the expression of Nrf2 and its downstream proteins in the
cellꢀbased level. The semiꢀquantification of the western blot of compound 4 was
provided in Figure S2.
Figure 3. (A) Timeꢀdependent expression of HOꢀ1, NQOꢀ1 and Nrf2 affected by the compound 4.
After treatment with 4 (10 ꢁM), cell lysates were prepared from HCT116 cells and subjected to
Western blot analysis. (B) Doseꢀdependent expression of NQOꢀ1 and Nrf2 affected by the compound 4.
After treatment with 4 for 6 hours, cell lysates were prepared from HCT116 cells and subjected to
western blot analysis.
To confirm the above results, we further measured the transcription of Nrf2 and
antioxidant genes GCLM, HOꢀ1 and NQO1 using quantitative RTꢀPCR (qRTꢀPCR).
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For the timeꢀcourse studies, we determined the expression of these genes at 0, 1, 2, 4,
6
, 16, 24 h after treated with 10 ꢁM of 4 in HCT116 cells. The compound
significantly increased the Nrf2 expression in a timeꢀdependent manner (Figure 4A),
with about 5ꢀfold increase at 4 h. It also remarkably induced the expression of GCLM,
HOꢀ1 with the maximum capacity at 6 h (about 2.5ꢀfold and 2.4ꢀfold respectively).
But in the case of NQO1, 4 did not show obvious inductivity, with only about 1.5ꢀfold
at all the timeꢀpoints. It was partly because that the constitutive expression of NQO1
in HCT116 cells was at a high level.
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The gene expression of Nrf2, GCLM and NQO1 were measured at 4 h after
treatment with various concentrations (0, 1, 5, 10, 20, 40 ꢁM) of compound 4 in
HCT116 cells (Figure 4B). The compound significantly increased the Nrf2, GCLM,
HOꢀ1 gene expression in a doseꢀdependent manner, with about 3.4ꢀfold, 3.8ꢀfold and
7.0ꢀfold increase in the expression at the highest concentration, respectively. While
for NQO1, the induction fold was not so remarkable, with only about 2.0ꢀfold at the
concentration of 40 ꢁM. These data were in accordance with the protein expression
level of NQO1.
Figure 4. (A) Timeꢀdependent increase in Nrf2 and Nrf2ꢀregulated genes after treatment with
compound 4. HCT116 cells were treated with compound 4 (10 ꢁM) at various time points. Nrf2,HOꢀ1，
GCLM and NQO1 were determined using βꢀactin as loading control. Values shown are mean ± SD (n =
3
). (B) Expression of Nrf2ꢀregulated genes after treatment with compound 4. HCT116 cells were
treated with 4 under the indicated concentrations at 4 h. Nrf2, HOꢀ1，GCLM and NQO1 were
determined using βꢀactin as loading control. Values shown are mean ± SD (n = 3).
2. Preliminary SAR of derivatives containing 1, 2, 4ꢀoxadiazole core.
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Design and synthesis of the derivatives. Considering that compound 4 exhibited
the most potent inductivity in the initial hits from VS, we next selected this compound
as the starting point for SAR study. Firstly, the indole ring A was firstly converted into
other aromatic rings such as naphthalene (5) and benzene (6) to evaluate whether this
group was important for the ARE inductivity. Secondly, the function of methyl groups
on benzo[d]imidazole ring C of 4 was analyzed by removing it, resulting in compound
0
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9
0
7
and 8. Finally, ring A of compound 4 was replaced by various substituted phenyl
groups and other aromatic rings, leading to target compounds 9~41.
Derivatives were synthesized using the route summarized in scheme 1. Briefly,
1
Hꢀbenzo[d]imidazoleꢀ5ꢀcarbonitrile (43) was cyclized from 3,4ꢀdiaminobenzonitrile
(42) refluxed in formic acid with a high yield (94%). The nitrile group of 43 was
reacted with H NOH·HCl and converted into (Z)ꢀhydroxylcarboximidamide (44) in
2
ethanol with a yield of 50%. Target compounds 7ꢀ37 were obtained through the
cyclization reaction of 44 with varied substituted benzoic acid in DMF under the
condition of 110 °C. Compound 38~41 were synthesized from 44 cyclized with
2
ꢀnaphthoic acid, picolinic acid or quinolineꢀ2ꢀcarboxylic acid, respectively.
3
Methylation of 1Hꢀbenzo[d]imidazole using CH I in DMF resulted in the target
compound 5 and 6.
Scheme 1. Synthetic route of the 1, 2, 4ꢀoxadiazole derivatives.
Reagents and conditions: (a) 5N HCl, HCOOH, reflux, 4h, 94%; (b) K
2
CO
3
, EtOH, H
2
NOH·HCl,
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reflux, 12h, 50%; (c) CDI, DMF, 110 °C, 12~18 h; (d): DMF, NaOMe, CH I, rt, 4h.
3
SAR study. ARE inductivities of all the synthesized derivatives were evaluated by
luciferase reporter assay in HepG2ꢀAREꢀC8 cells. When the indole (ring A) of
compound 4 was replaced by naphthalenꢀ2ꢀyl (5), the ARE inductivity was reduced,
especially at a high concentration. But when the indole was substituted by phenyl ring
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18
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21
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23
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(6), a more potent inductivity was observed, indicating that mono rings with smaller
steric effect was preferable at this position. However, the activity of 6 remarkably
reduced at high concentrations, which may be attributed to its poor solubility. As a
result, we tried to improve the solubility by removing the methyl group at
benzo[d]imidazole ring C of 4. The resulted compound 7 did not show obvious
activity improvement compared to 4, but compound 8, whose methyl group was
removed from 6, exhibited remarkably enhanced inductivity. The result suggested that
this methyl group was not the determinant for the activity of this series of compounds.
Based on the results, we started further optimization of 8 by introducing various
substituted phenyl groups to replace ring A. Compounds with electronꢀdonating group
at different positions of the phenyl ring, such as ꢀOCH
3
(9~14), ꢀOH (15~17) and
ꢀ
CH (27~29), show reduced Nrf2ꢀARE inductivity more or less. Additionally, the
3
stronger electronꢀdonating ability of the group was, the more poor activity of the
compound was, for example, we can see the Nrf2ꢀARE inductivity of compound
ꢀ
OCH
groups were not preferable.
Oppositely, the electronꢀwithdrawing group, such as ꢀNO
3 3
(11) ≈ ꢀOH (17) < ꢀCH (29). The results indicated that electron donating
2
3
(18~20), ꢀCF (24~26),
can improve the activity. It was observed that the Nrf2ꢀARE inductivity was increased
accompanied by the electronꢀwithdrawing ability of the substituents. For example, the
2 3
activity of 20 (ꢀNO ) was better than that of 26 (ꢀCF ). Besides, the substituted
position also impacted on the Nrf2ꢀARE inductivity. Compound with oꢀsubstitution
was slightly better than pꢀsubstitution (e.g. 18 vs 20, 24 vs 26), while mꢀsubstitution
caused obviously decreased activity (e.g. 19, 25). However, ꢀCN was an exception
(21~23), only pꢀsubstitution maintained moderate activity, oꢀsubstitution and
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5
6
mꢀsubstitution completely led to the loss of the inductivity. We infer that this group
may bring some critical penalty when the compound binds to its target, or lead to the
mechanical change. Similar manner was also observed in the halogen substituted
compounds (30~32). The stronger electronꢀwithdrawing ability of the halogen atom
was, the better Nrf2ꢀARE inductivity of the compound was, for example, the
Nrf2ꢀARE inductivity of compound ꢀF (32) > ꢀCl (31) > ꢀBr (30). Among these
compounds, 32 showed a stable and doseꢀdependent inductivity of Nrf2ꢀARE.
We propose a hypothesis to explain this phenomenon: the microenvironment of
inflammatory condition is prone to contain negative charged contents or materials
such as free radical and unsaturated chemical groups. If the electron density of a
compound is high, then it may be repulsive by the environment, leading to reduced
binding affinity to the target or issue. The scaffold of our compounds is consisted of
phenyl and heterocycles, which are electron enriched structure, therefore,
electronꢀdonating groups undoubtedly will further enhance the electron density of the
compounds, so they are not preferred. Oppositely, the electronꢀwithdrawing groups
can help disperse the electron density to the whole skeleton, making the
physicochemical property more proper for the inflammatory microenvironment, thus
improve the Nrf2ꢀARE inductivity.
0
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8
9
0
To examine the influence of the size of the groups on the activity, ꢀEt (33),
ꢀnꢀpropyl (34), ꢀnꢀbutyl (35), ꢀnꢀpentyl (36), phenyl (37), naphthalenꢀ2ꢀyl (38),
pyridine (39, 40), quinoline (41) derivatives were designed and synthesized. The
activity of ꢀnꢀbutyl > ꢀnꢀpropyl > ꢀEt indicated that group with large size was
preferred, and the best size was fourꢀcarbon group, larger size caused decreased
3
activity (e.g. 35 vs 36). However, ꢀCH compounds (27~29) showed the different
manner, and that could be explained by that inductive effect of methyl played a more
important role than size effect in affecting the Nrf2ꢀARE inductivity. When the
substituent was replaced by aromatic rings, the activity decreased more or less. We
inferred that the aromatic rings increased the electron density, leading to the repulsion
of the compound by inflammatory microenvironment, and that was similar to the
conclusion that electronꢀwithdrawing groups enhanced the activity.
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In summary, the SAR can be described as: 1) For ring C, the ꢀNHꢀ of indole ring is
not necessary to be methylated; 2) For ring A, phenyl exhibits more potent Nrf2ꢀARE
inductivity than naphthyl; 3) For the substituents at phenyl ring, electronꢀdonating
groups are not preferable to the ARE inductivity, while electronꢀwithdrawing groups
can enhance the activity; 4) Halogens with strong electronꢀwithdrawing ability can
increase the activity; 5) Flexible alkyl chains are more preferable than rigid aromatic
rings, while heterocycles have no more advantages than phenyl ring.
10
11
12
13
14
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16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
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33
34
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36
37
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39
40
41
42
43
44
45
46
47
48
49
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52
53
54
55
56
57
58
59
60
Table 2. The Nrf2 inductivity of all the derivatives in luciferase reporter assay
a,b
ARE inductivity in luciferase reporter assay (ꢁM)
Cpd.
R
1
R
2
0
.02
0.2
2
20
40
80
160
4
5
6
7
8
9
ꢀ
ꢀ
1.09±0.06
1.44±0.26
2.03±0.51
1.22±0.06
1.49±0.20
1.24±0.08
1.79±0.24
1.43±0.03
1.25±0.12
1.70±0.38
1.31±0.10
1.72±0.01
1.68±0.17
1.56±0.18
1.73±0.13
1.43±0.01
1.64±0.06
1.36±0.05
1.26±0.13
1.22±0.03
1.23±0.22
2.56±0.01
1.35±0.29
1.50±0.01
1.40±0.20
3.41±0.42
1.50±0.24
4.72±0.26
1.39±0.08
1.95±0.10
2.72±0.27
1.59±0.21
2.96±0.11
2.24±0.31
1.54±0.17
1.71±0.10
1.65±0.23
3.76±0.44
1.56±0.16
2.02±0.14
1.48±0.21
1.36±0.04
1.25±0.08
2.28±0.20
8.04±1.61
2.85±0.73
3.35±0.28
1.43±0.25
7.61±0.24
1.39±0.13
12.41±0.54 12.13±0.20
8.73±0.57
2.44±0.43
1.75±0.14
8.88±1.14
4.46±0.54
5.85±0.15
8.38±1.18
5.52±0.51
1.46±0.59
5.20±1.05
naphthalenꢀ2ꢀyl
phenyl
CH
CH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3
1.45±0.06
8.41±0.11
1.37±0.18
3.33±0.32
3
18.20±1.46 17.38±1.39
1.77±0.29
14.17±2.24 21.42±1.48 23.73±1.80
1Hꢀindolꢀ6ꢀyl
10.24±1.73 10.96±0.62 11.59±0.36
phenyl
3.19±0.71
8.46±0.68
2ꢀmethoxyphenyl
3ꢀmethoxyphenyl
4ꢀmethoxyphenyl
2,4ꢀdimethoxyphenyl
2,5ꢀdimethoxyphenyl
3,4,5ꢀtrimethoxyphenyl
2ꢀhydroxyphenyl
3ꢀhydroxyphenyl
4ꢀhydroxyphenyl
2ꢀnitrophenyl
1.62±0.06
3.16±0.56
3.27±0.17
2.84±0.45
3.21±0.40
2.41±0.36
1.53±0.06
2.44±0.18
2.19±0.07
5.96±0.30
10.70±0.18
1
0
12.37±1.86 14.42±0.36 12.41±1.12
11
9.84±0.79
8.57±1.56
4.48±1.29
3.23±0.12
2.67±0.21
8.62±1.30
6.23±0.43
13.03±0.17
11.61±0.22
4.24±1.24
3.24±0.25
2.75±0.12
4.18±0.39
8.67±1.23
4.92±0.31
4.35±0.55
3.70±0.49
1
1
2
3
14
13.94±0.48 22.49±1.72
1
1
1
5
6
7
1.34±0.03
4.05±0.13
8.81±1.02
1.17±0.10
3.93±1.08
7.35±0.71
18
12.07±1.22 36.61±1.70 53.62±3.26 42.65±3.42 38.35±1.57
1
2
9
0
3ꢀnitrophenyl
4.74±0.27
6.59±0.29
2.46±1.44
1.42±0.11
1.35±0.14
9.01±0.61
11.60±0.98 21.83±2.58 16.50±1.33 11.54±1.29
15.63±0.28 36.73±0.44 30.62±0.12 38.26±2.25
4ꢀnitrophenyl
21
2ꢀcyanophenyl
1.33±0.17
0.87±0.13
8.12±0.88
1.32±0.05
0.91±0.01
1.39±0.05
3.16±1.40
2.09±0.16
8.05±2.27
2
2
2
2
2
2
3
4
5
6
3ꢀcyanophenyl
4ꢀcyanophenyl
11.94±1.36 10.04±1.59 12.71±0.82
2ꢀ(trifluoromethyl)phenyl
3ꢀ(trifluoromethyl)phenyl
4ꢀ(trifluoromethyl)phenyl
19.08±1.26 24.24±0.73 17.92±0.76
3.73±0.74
2.94±0.30
1.13±0.12
13.92±0.23 10.53±1.91
5.65±0.21
3.45±0.21
6.56±1.67
6.91±0.26 17.73±1.29 21.63±0.99
12
ACS Paragon Plus Environment