Discovery of novel human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2) inhibitors as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2018.09.016

The enzyme inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the de novo biosynthesis of guanine nucleotides, and thus regulates the guanine nucleotide pool required for cell proliferation. Of the two isoforms, human IMPDH type 2 (hIMPDH2) is a validated molecular target for potential immunosuppressive, antiviral and anticancer chemotherapy. In search of newer hIMPDH2 inhibitors as potential anticancer agents, three novel series (A: 5-aminoisobenzofuran-1(3H)-one, B: 3,4-dimethoxyaniline and C: benzo[d]-[1,3]dioxol-5-ylmethanamine) were synthesized and evaluated for in vitro and cell-based activities. A total of 37 molecules (29–65) were screened for their in vitro hIMPDH2 inhibition, with particular emphasis on establishing their structure–activity relationship (SAR) trends. Eight compounds (hits, 30, 31, 33–35, 37, 41 and 43) demonstrated significant enzyme inhibition (>70% @ 10 μM); especially the A series molecules were more potent than B series (<70% inhibition @ 10 μM), while C series members were found to be inactive. The hIMPDH2 IC₅₀ values for the hits ranged from 0.36 to 7.38 μM. The hits displaying >80% hIMPDH2 inhibition (30, 33, 35, 41 and 43) were further assessed for their cytotoxic activity against cancer cell lines such as MDA-MB-231 (breast adenocarcinoma), DU145 (prostate carcinoma), U87 MG (glioblastoma astrocytoma) and a normal cell line, NIH-3T3 (mouse embryonic fibroblast) using MTT assay. Most of the compounds exhibited higher cellular potency against cancer cell lines and notably lower toxicity towards NIH-3T3 cells compared to mycophenolic acid (MPA), a prototypical hIMPDH2 inhibitor. Two of the series A hits (30 and 35) were evaluated in human peripheral blood mononuclear cells (hPBMC) assay and found to be better tolerated than MPA. The calculated/predicted molecular and physicochemical properties were satisfactory with reference to drug-likeness. The molecular docking studies clearly demonstrated crucial interactions of the hits with the cofactor-binding site of hIMPDH2, further providing critical information for refining the design strategy. The present study reports the design and discovery of structurally novel hIMPDH2 inhibitors as potential anticancer agents and provides a guide for further research on the development of safe and effective anticancer agents, especially against glioblastoma.

Full text of DOI:10.1016/j.ejmech.2018.09.016

Accepted Manuscript
Discovery of novel human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2)
inhibitors as potential anticancer agents
Chetan P. Shah, Prashant S. Kharkar
PII:
S0223-5234(18)30788-8
10.1016/j.ejmech.2018.09.016
EJMECH 10727
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 May 2018
Revised Date: 13 August 2018
Accepted Date: 5 September 2018
Please cite this article as: C.P. Shah, P.S. Kharkar, Discovery of novel human inosine 5′-
monophosphate dehydrogenase 2 (hIMPDH2) inhibitors as potential anticancer agents, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.09.016.
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Graphical abstract

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Discovery of novel human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2)
inhibitors as potential anticancer agents
Chetan P. Shah and Prashant S. Kharkar*
Department of Pharmaceutical Chemistry, Shobhaben Pratapbhai Patel School of Pharmacy
and Technology Management, SVKM’s NMIMS, V. L. Mehta Road, Vile Parle (West),
Mumbai 400 056. India.
* Corresponding Author:
Prashant S. Kharkar, PhD
E-mail: Prashant.kharkar@nmims.edu

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Abstract
The enzyme inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes an
essential step in the de novo biosynthesis of guanine nucleotides, and thus regulates the
guanine nucleotide pool required for cell proliferation. Of the two isoforms, human IMPDH
type 2 (hIMPDH2) is a validated molecular target for potential immunosuppressive, antiviral
and anticancer chemotherapy. In search of newer hIMPDH2 inhibitors as potential anticancer
agents, three novel series (A: 5-aminoisobenzofuran-1(3H)-one, B: 3,4-dimethoxyaniline and
C: benzo[d]-[1,3]dioxol-5-ylmethanamine) were synthesized and evaluated for in vitro and
cell-based activities. A total of 37 molecules (29-65) were screened for their in vitro
hIMPDH2 inhibition, with particular emphasis on establishing their structure–activity
relationship (SAR) trends. Eight compounds (hits, 30, 31, 33-35, 37, 41 and 43) demonstrated
significant enzyme inhibition (>70% @ 10 µM); especially the A series molecules were more
potent than B series (<70% inhibition @ 10 µM), while C series members were found to be
inactive. The hIMPDH2 IC50 values for the hits ranged from 0.36 to 7.38 µM. The hits
displaying >80% hIMPDH2 inhibition (30, 33, 35, 41 and 43) were further assessed for their
cytotoxic activity against cancer cell lines such as MDA-MB-231 (breast adenocarcinoma),
DU145 (prostate carcinoma), U87 MG (glioblastoma astrocytoma) and a normal cell line,
NIH-3T3 (mouse embryonic fibroblast) cell lines using MTT assay. Most of the compounds
exhibited higher cellular potency against cancer cell lines and notably lower toxicity towards
NIH-3T3 cells compared to mycophenolic acid (MPA), a prototypical hIMPDH2 inhibitor.
Two of the series A hits (30 and 35) were evaluated in human peripheral blood mononuclear
cells (hPBMC) assay and found to be better tolerated than MPA. The calculated/predicted
molecular and physicochemical properties were satisfactory with reference to drug-likeness.
The molecular docking studies clearly demonstrated crucial interactions of the hits with the
cofactor-binding site of hIMPDH2, further providing critical information for refining the
design strategy. The present study reports the design and discovery of structurally novel
hIMPDH2 inhibitors as potential anticancer agents and provides a guide for further research
on the development of safe and effective anticancer agents, especially against glioblastoma.
Keywords: IMPDH; hIMPDH2 inhibitors; 5-aminoisobenzofuran-1(3H)-one; MTT assay;
anticancer agents

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1. Introduction
Cancer is a swiftly growing disease of the current era and represents one of the
biggest healthcare issues for the human race. It is the leading cause of death irrespective of
the developments in the disease diagnosis, treatment and prevention methods [1–3]. Cancer is
characterized by uncontrolled growth and spread of abnormal cells due to their high
metabolic state, i.e., increased cellular metabolism. Enzymes that are critical for cell viability
and that exhibit markedly different activities or expression levels in the disease state tissues
are especially promising molecular targets for cancer therapeutics [4]. It is a genetic disease
(genetic instabilities) that progresses via development of multistep carcinogenesis with
participation from various cell signalling pathways and apoptosis, and as a result, cancer is
extremely intricate to fight [5]. Mostly, cancer begins as a local disease, but it metastasizes
over time in most cases, making it difficult to cure. These cells construct hostile
microenvironment and are not only adapted to this reserve environment but also, grow and
migrate to distant tissues [6]. Mutations in DNA repair genes (p21, p22, p27, p51 and tool
box for DNA), tumor suppressor genes (p53, NF1, NF2, RB and biological breaks),
oncogenes [myc, raf, bcl-2, ras (biological accelerators)] and genes involved in cell growth
metabolism are thought to be responsible for the uncontrolled proliferation of normal cells
which are transformed into malignant cells [7].
Despite the availability of several anticancer drugs, it is a challenging job for a
medicinal chemist to design newer agents that target tumor-specific pathways due to
complicated problems like tumor heterogeneity, frequent oncogenic mutations, multidrug
resistance (MDR), less therapeutic efficacy, debilitating adverse effects and poor
bioavailability issues. This necessitates the development of safer and targeted anticancer
agents [8,9]. Rapid proliferation is an important characteristic of cancer cells. This ultimately
requires, in addition to other factors, an expansion of the guanine nucleotide pool that
generally cannot be sustained by salvage pathways. This is where the importance of inosine
5'-monophosphate dehydrogenase (IMPDH, an enzyme linked with proliferation and
malignancy) comes in picture. The enzyme IMPDH (EC 1.1.1.205) catalyzes a rate-limiting
step in the de novo synthesis of guanine nucleotides which are essential for cell proliferation,
cell signalling, and as an energy source [10–12].
The enzyme IMPDH catalyzes the oxidation of inosine 5'-monophosphate (IMP) to
xanthosine 5'-monophosphate (XMP), which is then converted into guanosine 5'-
monophosphate (GMP) by GMP synthase. IMP also serves as a substrate for the biosynthesis

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of adenosine 5'-monophosphate (AMP). Consequently, inhibition of IMPDH causes a variety
of biological responses, such as reduction in the guanine nucleotide pools resulting in cell
proliferation arrest (interruption of DNA and RNA syntheses) [13], a decline in intracellular
signalling (G-protein-mediated signal transduction) [14–16], down-regulation of c-myc and
K-ras oncogenes in vitro [17]. Also, IMPDH inhibition is associated with an up-regulation of
p53 (commonly mutated protein in human cancers) [18], p21, bax and a down-regulation of
bcl-2, survivin and p27 protein [19]. These observations pose IMPDH as an attractive target
to suppress tumor cell growth.
Human IMPDH enzyme exists in two isoforms - hIMPDH1 and hIMPDH2 with high
sequence similarity (84%) and similar kinetic properties. The hIMPDH1, expressed at
relatively constant levels in both normal and neoplastic cells, seems to play a general
housekeeping role while the expression of hIMPDH2 is up-regulated in human neoplastic
cells [20]. The disproportionate increase in hIMPDH2 activity in neoplastic cells has made it
a significant target for the development of anticancer drug discovery [4,21]. Also, hIMPDH2
has historically been a major drug target for immunosuppressive [22,23], antiviral [24] and
antimicrobial chemotherapy [10,25]. Mycophenolic acid (MPA) (A, Figure 1), a natural
product, is a reversible, potent and uncompetitive inhibitor of IMPDH and is a known
anticancer and immunosuppressive agent. Mycophenolate mofetil, a prodrug of MPA (MMF,
B, Figure 1), has been approved for the treatment of acute allograft rejection following
kidney transplant. The MPA and its related forms MMF or MPA sodium (C, Figure 1) (MPS)
cause dose-limiting gastrointestinal (GI) toxicities. However, adverse effects related to the
treatment with MPA-based drugs, such as diarrhea, leukopenia, sepsis and vomiting, are the
barriers to the administration of higher doses and thereby more effective treatment [26].
Similarly, the nucleoside analogs which are competitive IMPDH inhibitors, e.g., tiazofurin
(D), ribavirin (E) and mizoribine (F) (Figure 1) (bioprecursors which need intracellular
activation by phosphorylation) have unfavourable tolerability profiles too. Thus, there is an
urgent need for newer, safer, potent and orally bioavailable hIMPDH2 inhibitors with a broad
spectrum of anticancer activity [27,28].

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Figure 1. Clinically used IMPDH inhibitor drugs
In order to develop potent IMPDH inhibitors, significant efforts were focused on
various structural modifications of MPA using bio-isosteric replacements and innumerable
structural derivatives with different scaffolds but with limited success [4,29]. MPA has been
demonstrated to be an effective inducer of differentiation in a number of cancer cell lines and
displayed synergism with imatinib in the treatment of chronic myologenous leukemia (CML)
[30]. Furthermore, antitumor activities of several MPA derivatives are reported [31–35].
These outcomes strongly support the role of MPA as a potential anticancer drug. Moreover,
MPA, being an acid, is likely to be prevented from entering in the CNS. There are no reports
in the literature citing this fact that MPA could be useful for treating gliomas, dreadful
cancers of the CNS [36]. Earlier reports proposed that diminutive structural alteration of
MPA was damaging to its IMPDH inhibitory activity. Therefore, it was crucial to select the
design strategy leading to retention of the inhibitory potential against hIMPDH2. Also, it was
reported that the isobenzofuran-1(3H)-one part and the trans configuration of MPA were
important to retain the inhibitory potential against hIMPDH2 [12,37–44]. Working on similar
thought process, in this report, we have outlined the design, synthesis and biological
evaluation of three novel series of potential hIMPDH2 inhibitors (A, B and C, Figure 2). The
title compounds were evaluated for in vitro hIMPDH2 inhibition and further assessed for
their cellular potency against MDA-MB-231 (breast adenocarcinoma), DU145 (prostate
carcinoma), U87 MG (glioblastoma astrocytoma) and NIH-3T3 (mouse embryonic fibroblast)
cell lines. In addition, their structure–activity relationships (SAR) trends were studied
carefully throughout in order to guide further improvements in potency and other

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physicochemical properties. The results of the present study provide insights into the design
and development of novel hIMPDH2 inhibitors as potential anticancer agents.
Figure 2. Representative chemotypes (A-G) of novel hIMPDH2 inhibitors reported in this
study
2. Results and discussion
Mycophenolic acid is a well-known hIMPDH2 inhibitor with several pharmacological
activities [12]. Its antitumor activity decreased or completely lost when any structural
modification, not matter how subtle, was done [31,32]. Numerous classes of compounds such
as amides, oxazolyl indoles, phenylamino oxazoles, amino-oxazoles, guanidines, triazines,
isoquinolines, quinolones, quinazolinones, quinazolinethiones and quinazolinediones, indoles
and acridones have been synthesized using molecular modification approach [45].
Unfortunately, none of these molecules occurred to be advantageous as anticancer
compounds.

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Mycophenolic acid exhibits unfavourable metabolism (7-O-glucuronide, major
metabolite; acyl glucuronide, minor metabolite) which is responsible for its higher dose,
dosing frequency, GI side effects and variable exposure. The phenolic –OH on the iso-
benzofuranone substructure is the main culprit. It would be worthwhile to come up with
molecules with retained or improved potency but devoid of the issues with MPA. With this
thing in mind, in the present study, we attempted to discover potent and novel hIMPDH2
inhibitors. The increasing knowledgebase of the IMPDH structure laid foundation for
summarizing essential chemical features, which may inspire the development of novel
IMPDH inhibitors and their spatial alignment. Three substructures namely, 5-
aminoisobenzofuran-1(3H)-one (23), 3,4-dimethoxyaniline (24), benzo[d][1,3]dioxol-5-
ylmetha- namine (25) linked with substituted chalcones, were used as cores. These scaffolds
were selected to mimic the isobenzofuran-1(3H)-one part and the trans configuration of the
MPA.
2.1. Chemistry
The derivatives 29-65 (Series A-C) were synthesized as outlined (Schemes 1-3). The
general synthetic route to target chalcones (General procedure A, 1-22) is depicted in Scheme
1. All the chalcones were synthesized by Claisen-Schmidt condensation using substituted
acetophenones and (hetero)aryl aldehydes in presence of NaOH as base in EtOH as reported
earlier [46]. The chloroacetamides scaffolds (General procedure B, 26-28) were synthesized
using the starting amines (23-25) and chloroacetyl chloride in dry THF in the presence of
TEA at 0 °C (Scheme 2). This was followed by O-arylation reaction using K2CO3/DMF to
yield the title compounds (General procedure C) 29-65 (Scheme 3, Table 1) [47].
Scheme 1a. General scheme for synthesis of substituted chalcones (1-22)
aReagents and conditions: a. 20% NaOH, EtOH, 5 °C to RT, 24-72 hr

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2.2. Biological activity
2.2.1. In vitro hIMPDH2 inhibition assay
All 37 molecules were screened in-house for in vitro hIMPDH2 inhibition at 10 µM
concentration. The results are shown in Table 1 and Figure 1S (see Supplementary
Information section). Compounds 30, 31, 33-35, 37, 41 and 43 (hits) exhibited >70%
inhibition. The hits were further taken up for IC50 determination. Overall, series A molecules
(29-50) were more active than their series B counterparts (51-60), while molecules from C
series (61-65) were inactive at the concentration tested (Table 1). These observations
unequivocally highlighted the significance of isobenzofuran-1(3H)-one substructure from
MPA for mediating crucial interactions with enzyme residues for potent inhibition of
hIMPDH2.
Series A: The molecules were further divided in four chemotypes, A to D (Figure 2) for easy
interpretation of the SAR trends. There was gradual increase in % inhibition in case of
chemotype A when the size of R1 increased from phenyl (37, 79%, Table 1) to 3′-OMePh
(33, 91%). Shifting the 3′–OMe substituent to 4′-position increased the activity slightly (30,
96%), demonstrating the critical role of the H-bond acceptor functionality at the distal end of
the molecule. Increasing the size of the 4′-substituent from –OMe to –OEt (48, 65%), O-Prn
(49, 60%), –O-Bun (50, 58%) resulted in systematic decrease in % inhibition. Additional
increase in bulk led to substantial reduction in the inhibitory activity featuring limited space
available in the binding site of the enzyme (45-47, 37-52%). Further increasing the size of R1
by placing one or more additional substituent(s) on the Ph ring decreased the activity (29:
65%; 34:71%). Placing the electron-withdrawing substituents on the Ph ring such as 2′-Cl
(32, 60%), 4′-Cl (44, 62%), 4′-NO2 (36, 63%) led to reduction in activity over their
unsubstituted counterpart. Compound 35 with R1 = 2′,6′-diFPh substituent exhibited
increased % inhibition (94%). This may be due to smaller size of F and its H-bonding
potential leading to significant increase in binding energy with increase in % inhibition.

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Scheme 2a. General scheme for synthesis of substituted chloroacetamides (26-28)
aReagents and conditions: a. Chloroacetyl chloride, TEA, THF, 0 °C to RT, overnight
Scheme 3a. General scheme for synthesis of final compounds (29-65)
aReagents and conditions: a. 1-22, DMF, K2CO3, RT, 16 hr
Substitution by the heteroaryl rings – 2′-thienyl (31, 76%) and 2′-furyl (38, 57%) –
clearly resulted in reduced activity. It is well-known that thiophene is very similar to benzene

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with respect to its aromatic character, while furan is the least aromatic and way different from
benzene. This was worth noting that sufficient aromatic π cloud was required for higher
activity. There may be underlying π-π stacking interactions with the enzyme residues.
For chemotypes B and C, similar SAR trends as above were seen. Compound 41
(94%, Table 1) was more active than its chemotype A counterpart (37), further hinting us
about the narrow cavity at the distal end of the molecule. Increased steric bulk in the form of
additional substituents on the aromatic ring led to more or less reduced % inhibition (39, 40
and 42). Compound 43 (chemotype D, Figure 2, Table 1) with reversed ‘enone’ functionality
exhibited slight reduction in potency (88%). Overall, the series A molecules exhibited
prominent SAR trends which would be helpful in future design strategies.
Series B: The proximal part of the molecule contained a 3,4-dimethoxyphenyl substructure in
this set of molecules. This single change led to drastic reduction in the % inhibition over their
series A counterparts (51 versus 30 – three-fold reduction in activity, Table 1), (52 versus 33
– three-fold reduction in activity) irrespective of the Chemotypes - E and F, except compound
55 which retained the activity (55 versus 48). This reiterated the crucial role played by the
nature of the proximal part, which is likely to interfere with IMP stacking during the binding
process.
Series C: All the molecules from this series with 3,4-methylenedioxy substructure as the
proximal part, were inactive. They completely failed to demonstrate any inhibition at the
concentrations tested. In summary, we have successfully identified a novel series of
molecules with appreciable hIMPDH2 inhibition. The insights gained from the initial data
could potentially drive the design and development of potent inhibitors.
Overall, among three series (A-C), all the identified promising hits were from series A
indicating the importance of 5-aminoisobenzofuran-1(3H)-one substructure for stacking with
IMP at the proximal end of the molecule and might be interacting with the active site residues
(Gly 326 and Thr 333) similar to MPA. While the other two series B (3,4-dimethoxyaniline)
and series C (benzo[d]-[1,3]dioxol-5-ylmethanamine) possibly lacked these interactions and
hence were less active or inactive.
The hit molecules - 30, 31, 33-35, 37, 41 and 43 - all from series A, with >70%
inhibition at 10 µM were used for IC50 determination, along with MPA, the positive control.
The IC50 values of the hits (0.36 to 7.38 µM) were comparable to MPA (0.25 µM).
Compounds 31, 34 and 37 with <80% enzyme inhibition were not persuaded further for cell-
based assays; rest of the hits were.

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Table 1. Structural features and hIMPDH2 % inhibition of derivatives 29-65 at 10 µM
hIMPDH2
Compound
Chemotype
R1
% Inhibitiona
99.90 ± 1.20
65.45 ± 3.56
95.63 ± 4.36
76.30 ± 2.12
59.52 ± 2.11
91.45 ± 1.67
71.35 ± 1.75
94.15 ± 3.22
63.18 ± 1.49
79.08 ± 4.83
57.31 ± 3.92
56.97 ± 2.33
63.66 ± 1.39
93.68 ± 1.67
19.88 ± 3.07
87.86 ± 1.22
62.15 ± 2.56
52.23 ± 1.85
36.67 ± 2.42
39.12 ± 0.93
65.45 ± 1.45
60.14 ± 1.74
57.68 ± 3.27
33.56 ± 2.85
28.84 ± 1.31
23.29 ± 1.48
20.61 ± 3.16
63.50 ± 1.79
25.67 ± 2.53
30.12 ± 1.28
IC50 (µM)b
0.25 ± 0.03
n.d.c
-
-
3′,4′-dimethoxyphenyl
4′-methoxyphenyl
2′-thienyl
MPA
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
A
A
A
A
A
A
A
A
A
A
B
B
B
C
D
A
A
A
A
A
A
A
E
0.66 ± 0.38
4.54 ± 1.70
n.d.c
2′-chlorophenyl
3′-methoxyphenyl
3′,4′,5′-trimethoxyphenyl
2′,6′-difluorophenyl
4′-nitrophenyl
0.36 ± 0.04
7.38 ± 0.74
0.36 ± 0.12
n.d.c
Phenyl
0.79 ± 0.25
n.d.c
n.d.c
n.d.c
2′-furyl
3′,4′-dimethoxyphenyl
3′,4′,5′-trimethoxyphenyl
Phenyl
0.43 ± 0.04
3′,4′,5′-trimethoxyphenyl
4-fluorophenyl
n.d.c
0.57 ± 0.03
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
4′-chlorophenyl
4′-benzyloxyphenyl
4′-(2,4-dichlorobenzyloxy)phenyl
4′-(4-chlorobenzyloxy)phenyl
4′-ethoxyphenyl
4′-propoxyphenyl
4′-butoxyphenyl
4′-methoxyphenyl
3′-methoxyphenyl
3′,4′-dimethoxyphenyl
3′,4′,5′-trimethoxyphenyl
4′-ethoxyphenyl
E
F
n.d.c
n.d.c
E
E
n.d.c
n.d.c
E
2′-chlorophenyl
E
4′-chlorophenyl

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n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
n.d.c
E
E
2′,6′-difluorophenyl
2′-furyl
35.93 ± 2.16
21.34 ± 1.61
24.52 ± 1.99
n.a.d
58
59
60
61
62
63
64
65
E
2′-thienyl
G
G
G
G
G
4′-methoxyphenyl
3′,4′-dimethoxyphenyl
3′,4′,5′-trimethoxyphenyl
4′-ethoxyphenyl
2′,6′-fluorophenyl
n.a.d
n.a.d
n.a.d
n.a.d
a All the data are expressed as ± SD (results are average of duplicate analysis)
b All the data are expressed as ± SD (results are average of triplicate analysis). IC50 value was determined when
the inhibitory rate of compound is higher than 70% at the concentration of 10 µM.
c n.d. = not determined; d n.a. = not active
2.2.2. Cell viability assay
Cytotoxic activity of hits 30, 33, 35, 41 and 43 (>80% hIMPDH2 inhibition at 10 µM)
and MPA was evaluated against cell lines: MDA-MB-231 (breast adenocarcinoma), DU145
(prostate carcinoma), U87 MG (glioblastoma astrocytoma) and NIH-3T3 (mouse embryonic
fibroblast cells) using colorimetric MTT assay. The results are reported in Table 2 and Figure
2S (see Supplementary Information section). The IC50 values were expressed in µM.
Cisplatin and doxorubicin HCl were used as positive controls in addition to MPA. Most of
the hits exhibited better activity than MPA in this assay. It might be difficult for MPA, an
acid, to cross the cell membrane and exhibit activity. In fact, the lowest potency for MPA was
found against U87 MG, a glioblastoma cell line. In contrast, the hits were more lipophilic
(average AlogP 3.98). Out of the five hits tested against different cell lines, 41 exhibited
greater potency compared to others against MDA-MB-231 and DU145 cell lines. Compound
41 was ~4-fold (MDA-MB-231) and 1.7-fold (DU145) more potent than MPA, respectively.
Compound 35 showed promising activity against U87 MG (~3-fold more potent than MPA)
(Table 2).
For any new chemical entity (NCE) to become a successful drug, it is not only
effective against the diseased cells but also does minimum harm to the normal cells. The hits
(30, 33, 35, 41 and 43) were evaluated against NIH-3T3 cell line (normal cell line) to
determine their selectivity. The hits were found to be relatively less toxic compared to MPA
and cisplatin and doxorubicin (standard chemotherapeutic drugs) and no significant
cytotoxicity was observed even at higher concentration (100 µM) (Figure 3 and Table 1S,
Supplementary Information section). Also, hits (30, 35) and MPA were tested in hPBMC

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proliferation assay (see Supplementary Information section for protocol). The % cell viability
was found to be 63% for MPA, 86% for 30 and 77% for 35 which clearly demonstrated that
30 and 35 were less toxic to hPBMCs in comparison to MPA. The possibility of the title
compounds exhibiting higher activity than MPA could be due to their ability to penetrate the
cells owing to higher AlogP values. The higher cellular potency of these derivatives over
MPA was definitely a headway in the direction of the goal, i.e., to discover a novel
hIMPDH2 inhibitor as potential anticancer agent.
Table 2. Cytotoxicity data of the hits in MTT assay
IC50 (µM) ± SDa
Sr. No.
Compound
MDA-MB-231b
50.40 ± 1.10
0.54 ± 0.08
4.38 ± 0.06
3.03 ± 0.27
3.42 ± 0.19
2.41 ± 0.10
1.10 ± 0.09
5.42 ± 0.14
DU145c
34.47 ± 0.78
0.21 ± 0.06
2.94 ± 0.09
2.13 ± 0.18
2.55 ± 0.28
1.90 ± 0.15
1.75 ± 0.08
6.88 ± 0.13
U87 MGd
21.32 ± 0.39
0.11 ± 0.02
10.69 ± 0.21
4.90 ± 0.18
5.75 ± 0.24
3.67 ± 0.14
3.97 ± 0.12
6.05 ± 0.24
1
2
3
4
5
6
7
8
Cisplatin
Doxorubicin HCl
MPA
30
33
35
41
43
aAll the data are expressed as ± SD (results are average of triplicate analysis). bMDA-MB-231 (breast
adenocarcinoma); cDU145 (prostate carcinoma); dU87 MG (glioblastoma astrocytoma).
NIH-3T3
**
**
*
*
100
50
0
*
MPA
30
33
35
41
43
Compound
Figure 3. % Cell viability of the compounds against NIH-3T3 cell line (data are mean ± SD,

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n=3) (Asterisk above columns indicate statistically significant difference
compared to MPA. * = p< 0.05, ** = p< 0.01, *** = p< 0.001)
2.3 Computational studies
2.3.1. In silico physicochemical property prediction
The molecular properties play crucial role in the random-walk and the drug-receptor
interactions. At the design stage, we ensured that the compounds passed all the drug-like
filters (data not shown). Although the presence of aromatic –NO2, -Cl, as well as heterocycles
like thiophene and furan are a strict no-no in molecule structures, we still synthesized these
molecules as a part of SAR investigations. The interesting results from the in vitro enzyme
inhibition and cell-based assays compelled us to investigate the physicochemical property
profiles of the synthesized compounds in detail. The initial part of the study dealt with the
molecular properties calculation/prediction using Canvas module of Schrödinger Small-
Molecule Drug Discovery Suite [49]. The detailed analysis of the property profiles of all the
compounds (data not shown) led to interesting outcome. It was observed that the limited size
at the distal end of the molecule supposedly contributed to increased hIMPDH2 activity. A
bubble plot of these properties is shown for clear understanding (Figure 4).
Figure 4. Bubble plot of volume by % inhibition sized by RB shaded by SASA
We further calculated/predicted molecular and physicochemical parameters of the hits
(30, 33, 35, 41 and 43) and MPA using QikProp module of Schrödinger Small-Molecule
Drug Discovery Suite [49]. The results are shown in Table 3. The AlogP values of the hits
were in the range 3.81 to 4.24 (MPA AlogP: 3.11). All the hits passed Lipinski's rule of five
filter. The properties related to membrane permeability such as QPPCaco and QPPMDCK as
well as % human oral absorption were better than MPA and found satisfactory. In summary,

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the molecular/ physicochemical properties calculation/prediction confirmed the drug-likeness
and acceptable pharmacokinetic parameters of the hits.
The present study finally culminated in a set of newer, relatively potent hIMPDH2
inhibitors which have shown higher anticancer activity in vitro. The hits are likely to exhibit
acceptable pharmacokinetic profiles and in vivo activity, as predicted from the in silico
studies. Further work in this direction will yield novel and potent hIMPDH2 inhibitors ready
to enter further development stages.
Table 3. Predicted physicochemical properties
Compound
Sr. No.
Property
MPA
320.34 443.45 443.45
3.11 3.81 3.81
30
33
35
41
413.42 431.41
3.82 4.03
43
449.4
1
2
3
4
5
6
7
8
MW
AlogP
4.24
117.29 122.79 122.80 114.66 114.63 114.60
PSA
-3.41
2.5
-5.87
3.50
-5.77
3.49
-6.27
3.83
-5.54
3.40
-5.99
3.65
QPlogS
QPlogPo/w
QPPCaco
QPlogBB
QPPMDCK
36.63
-1.59
17.64
238.26 245.62 246.69
-1.97 -1.93 -1.68
245.5
-1.82
245.77
-1.74
195.9
104.96 108.47 284.63 108.41
% Human
Oral
69.57
89.98 90.16 92.19 89.64
91.09
9
Absorption
2.3.2. Molecular docking
To identify the potential structural features of the ligand and its interaction with the
cofactor-binding site of hIMPDH2, molecular docking studies were performed. The results of
these studies i.e., docking scores and interaction energy of the ligands with the hIMPDH2
residues are listed in Table 4. Further, the docked poses of MPA and two most potent hits (35
and 41) are shown in Figure 5. It is clear observed that MPA, 35 and 41 nicely occupied the
hIMPDH2 cofactor-binding site lined by Asp274, Ser276, Gly326, Thr333 and Gln441. The
2-oxo group of the isobenzofuran-1-(3H)-one of MPA, 35 and 41 formed strong H-bond with
side-chain -OH of Thr333; 35 exhibited additional H-bond with backbone -NH of Gly326.
MPA -COOH group formed a strong H-bond with side-chain -OH of Ser276. The hit 41

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demonstrated similar interaction involving distal chalcone -C=O group. The aromatic -NH of
35 formed a H-bond with -COO- of Asp274. The chalcone portion of the hits was found in a
region surrounded by several hydrophobic residues. Overall, the binding modes of 41 and 35
clearly revealed additional interactions of the hits with the residues lining the cofactor-
binding pocket of hIMPDH2. These studies clearly demonstrated that isobenzofuran-1-(3H)-
one substructure was responsible for efficient binding (i.e., anchoring) of the hits in the
cofactor-binding site of hIMPDH2.
a
b
d
c
Figure 5. Binding mode of MPA and the hits into the cofactor-binding site of hIMPDH2
(PDB ID: 1JR1). The ligand is shown as ball-and-stick model while the hIMPDH2 residues
are shown as line models coloured by the element. Yellow dotted-lines indicate H-bonding
interactions. a) MPA formed H-bond with Ser276, Gly326, Thr333 and Gln441; b) 35
showed H-bonding interactions with Asp274 and Gly326; c) 41 was involved in H-bonding
interactions with Ser276, Gly326 and Thr333; d) overlay of MPA (orange), 35 (purple) and
41 (green).

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Table 4a. Results of the docking studies of the hits
Glide Emodel
(kcal/mol)
Compound
XP GScore
-8.144
-6.713
-5.325
-5.307
-4.991
-4.914
-75.421
MPA
41
-77.132
-76.915
-72.782
-72.020
-76.495
35
30
33
43
a MPA docking results are shown for comparison
2.4. Solubility and Log D7.4
Molecules with sufficient solubility (logS) and lipophilicity (e.g., logD7.4) are obliging
and seem to have a greater impact on ADMET properties during the drug discovery process.
Solubility is an important parameter for the human intestinal absorption and subsequently to
oral bioavailability and mostly command the route of drug administration. It is often
expressed as log units of molar solubility (mol/L) or logS. The commended range for a
molecule to be good oral bioavailable is -6 to 0.5 [50]. The most promising hits – 35 and 41,
exhibited experimental logS of -5.81. The distribution coefficient, on the other hand, has a
crucial role to play in drug overall drug disposition. Hydrophilic molecules have higher
solubility, but are less equipped to cross the cell membrane readily. A compound is
hydrophilic, if logD < 0 and lipophilic, if logD > 0. Nearly ~85 % of the anticancer drugs
have logD value ranging from -0.86 to 4.88 with a median of 2.57, while maximum lead
candidates are hydrophobic in nature (logD = 3.15, median value) [50]. Given the absence of
ionizable functional groups in the most promising hits, logD at physiological pH, i.e., pH 7.4
(logD7.4) was determined to represent lipophilicity. The logD7.4 values for compounds 35 and
41 were found 2.99 and 2.79, respectively.
3. Conclusions
The design strategy used in the present study, i.e., mimicking isobenzofuranone
substructure and the trans configuration of MPA side chain, crucial for retaining the chemical
features required for the hIMPDH2 inhibitory potential, yielded fruitful outcome. The
synthesized compounds exhibited systematic SAR trends with respect to in vitro %

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inhibition. The efforts successfully mapped the binding site of the enzyme in terms of the
electronic nature, size and shape. The hits exhibited potent cytotoxicity against MDA-MB-
231, DU145 and U87 MG and were relatively less toxic towards normal cells (NIH-3T3).
Also, the most potent hits were significantly less toxic than MPA against hPBMC. The
binding modes of the hits with hIMPDH2 cofactor-binding site clearly demonstrated
conservation of the essential interactions of the core isobenzofuranone moiety while
additional interactions by the distal end of the hits, i.e., chalcone portion. The identified hits
illustrated improved cellular potency over MPA against the tested cancer cell lines. The series
A hits were better than other two series as hIMPDH2 inhibitors. The interesting outcome of
the present study is likely to guide further design and development of potential hIMPDH2
inhibitors as newer anticancer agents. Our concerted medicinal chemistry efforts thus, yielded
potent lead molecules.
4. Experimental
4.1. General
All the chemicals utilized for the present study were purchased from the producers
such as Acros Organics (Geel, Belgium), Alfa Aesar (Karlsruhe, Germany), Spectrochem
(Mumbai, India), Sigma Aldrich (Steinheim, Germany) or Merck (Darmstadt, Germany) and
used without further purification unless otherwise indicated. Solvents were used directly
unless specified. All the reactions were carried out under dry N2 atmosphere and progress of
the reaction was monitored by thin layer chromatography (TLC) using an aluminium plate
coated with silica gel 60 F254 (Merck Millipore, Billerica, MA, USA). All the title compounds
were purified by column chromatography packed with silica gel of 230-400 mesh, 60 Å of
Merck using a mixture of DCM/MeOH as eluent and the TLCs analyzed in a UV cabinet with
an excitation wavelength of 254 nm. Melting points of the compounds was determined on
Veego VMP DS (General Trading Co., India) and are uncorrected. FT-IR spectra were
recorded on a Perkin Elmer RX1 instrument (Waltham, MA). The purity of all biologically
evaluated compounds was determined using HPLC and found to be >95%. HPLC analysis
was performed on Agilent 1220 Infinity system (Santa Clara, CA, USA). An isocratic mobile
phase consisting of (A) Acetonitrile and (B) Water (80:20, v/v) was used with a C18
Kromasil® column (15 cm × 4.6 mm, 5 µ particle size, 100 Å pore size) (Bohus, Sweden),
flow rate of 1 mL/min. The NMR spectra were recorded in DMSO-d6 and tetramethylsilane
1
(TMS) as internal standard. H-NMR spectra were obtained on Bruker Advance 400 (400

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MHz); chemical shifts are expressed in δ (ppm) Mass spectra (MS) were recorded on a
Shimadzu 8040 LC-MS/MS system (Japan), using electrospray ionization (ESI) mode.
4.2. Synthesis
4.2.1. General procedure A: Synthesis of substituted chalcones (1-22) [46]. An aqueous
solution of NaOH (20%, 5 mL) was added dropwise to a previously cooled mixture of
selected acetophenone (5 mmol) and selected (hetero)aryl aldehydes (5 mmol) in EtOH (25
mL) under vigorous stirring. The mixture was stirred at RT for 24–72 hr. After completion of
the reaction (as indicated by TLC), the mixture was poured onto crushed ice and acidified
with dilute HCl. The precipitated product was filtered at suction and washed to neutral
filtrate. The solid was recrystallized from EtOH to get crystalline product.
(E)-3-(3,4-dimethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (1): The title compo-
und was synthesized from 4-hydroxyacetophenone and 3,4-dimethoxybenzaldehyde as
described in the general procedure A. Yield: 68%; mp: 204-206 °C.
(E)-1-(4-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (2): The title compound
was synthesized from 4-hydroxyacetophenone and 4-methoxybenzaldehyde as described in
the general procedure A. Yield: 72%; mp: 188-190 °C.
(E)-1-(4-hydroxyphenyl)-3-(thiophen-2-yl)prop-2-en-1-one (3): The title compound was
synthesized from 4-hydroxyacetophenone and thiophene-2-carbaldehyde as described in the
general procedure A. Yield: 69%; mp: 172-174 °C.
(E)-3-(2-chlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (4): The title compound was
synthesized from 4-hydroxyacetophenone and 2-chlorobenzaldehyde as described in the
general procedure A. Yield: 78%; mp: 172-174 °C.
(E)-1-(4-hydroxyphenyl)-3-(3-methoxyphenyl)prop-2-en-1-one (5): The title compound
was synthesized from 4-hydroxyacetophenone and 3-methoxybenzaldehyde as described in
the general procedure A. Yield: 65%; mp: 162-164 °C.
(E)-1-(4-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (6): The title comp-
ound was synthesized from 4-hydroxyacetophenone and 3,4,5-trimethoxybenzaldehyde
as3described in the general procedure A. Yield: 80%; mp: 238-240 °C.

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(E)-3-(2,6-difluorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (7): The title compound
was synthesized from 4-hydroxyacetophenone and 2,6-difluorobenzaldehyde as described in
the general procedure A. Yield: 77%; mp: 198-200 °C.
(E)-1-(4-hydroxyphenyl)-3-(4-nitrophenyl)prop-2-en-1-one (8): The title compound was
synthesized from 4-hydroxyacetophenone and 4-nitrobenzaldehyde as described in the
general procedure A. Yield: 85%; mp: 250-252 °C.
(E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one (9): The title compound was
synthesized from 4-hydroxyacetophenone and benzaldehyde as described in the general
procedure A. Yield: 70%; mp: 174-176 °C.
(E)-3-(furan-2-yl)-1-(4-hydroxyphenyl)prop-2-en-1-one (10): The title compound was
synthesized from 4-hydroxyacetophenone and furan-2-carbaldehyde as described in the
general procedure A. Yield: 68%; mp: 162-164 °C.
(E)-3-(3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (11): The title compo-
und was synthesized from 3-hydroxyacetophenone and 3,4-dimethoxybenzaldehyde as
described in the general procedure A. Yield: 70%; mp: 86-88 °C.
(E)-1-(3-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (12): The title com-
pound was synthesized from 3-hydroxyacetophenone and 3,4,5-trimethoxybenzaldehyde as
described in the general procedure A. Yield: 80%; mp: 174-176 °C.
(E)-1-(3-hydroxyphenyl)-3-phenylprop-2-en-1-one (13): The title compound was synthe-
sized from 3-hydroxyacetophenone and benzaldehyde as described in the general procedure
A. Yield: 67%; mp: 130-132 °C.
(E)-1-(4-hydroxy-3-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one(14):
The title compound was synthesized from 4-hydroxy-3-methoxyacetophenone and 3,4,5-
trimethoxybenzaldehyde as described in the general procedure A. Yield: 64%; mp: 188-190
°C.
(E)-1-(4-fluorophenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (15): The title compound
was synthesized from 4-fluoroacetophenone and 4-hydroxybenzaldehyde as described in the
general procedure A. Yield: 73%; mp: 188-190 °C.

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(E)-3-(4-chlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (16): The title compound
was synthesized from 4-hydroxyacetophenone and 4-chlorobenzaldehyde as described in the
general procedure A. Yield: 63%; mp: 186-188 °C.
(E)-3-(4-(benzyloxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (17): The title com-
pound was synthesized from 4-hydroxyacetophenone and 4-(benzyloxy)benzaldehyde as
described in the general procedure A. Yield: 68%; mp: 180-182 °C.
(E)-3-(4-(2,4-dichlorobenzyloxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (18): The
title compound was synthesized from 4-hydroxyacetophenone and 4-(2,4-dichloro-
benzyloxy)benzaldehyde as described in the general procedure A. Yield: 70%; mp: 202-204
°C.
(E)-3-(4-(4-chlorobenzyloxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (19): The title
compound was synthesized from 4-hydroxyacetophenone and 4-(4-chlorobenzyloxy)-
benzaldehyde as described in the general procedure A. Yield: 67%; mp: 200-202 °C.
(E)-3-(4-ethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (20): The title compound
was synthesized from 4-hydroxyacetophenone and 4-ethoxybenzaldehyde as described in the
general procedure A. Yield: 62%; mp: 144-146 °C.
(E)-1-(4-hydroxyphenyl)-3-(4-propoxyphenyl)prop-2-en-1-one (21): The title compound
was synthesized from 4-hydroxyacetophenone and 4-propoxybenzaldehyde as described in
the general procedure A. Yield: 65%; mp: 162-164 °C.
(E)-3-(4-butoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (22): The title compound
was synthesized from 4-hydroxyacetophenone and 4-butoxybenzaldehyde as described in the
general procedure A. Yield: 64%; mp: 148-150 °C.
4.2.2. General procedure B: Synthesis of substituted chloroacetamides (26-28) [47]. To a
mixture of amine (1 eq.) and TEA (1.2 eq.) in dry THF was added chloroacetyl chloride (1.1
eq.) dropwise at 0 °C. After completion of reaction, triethylammonium chloride was filtered
off and the filtrate was concentrated under vacuum and quenched with water. The solid
obtained was filtered at suction and washed to neutral filtrate, air dried and used without any
further purification.

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2-chloro-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (26): The title compound
was synthesized from 23 (3 g, 20.1 mmol), TEA (3.36 mL, 24.12 mmol) and chloroacetyl
chloride (1.76 mL, 22.11 mmol) as described in the general procedure B to yield 26 as a light
brown solid. Yield: 90%; mp: 218-220 °C.
2-chloro-N-(3,4-dimethoxyphenyl)acetamide (27): The title compound was synthesized
from 24 (3 g, 19.6 mmol), TEA (3.28 mL, 23.5 mmol) and chloroacetyl chloride (1.71 mL,
21.54 mmol) as described in the general procedure B to yield 27 as a dark grey solid. Yield:
88%; mp: 182-184 °C.
N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-chloroacetamide (28): The title compound was
synthesized from 25 (3 g, 19.85 mmol), TEA (3.32 mL, 23.82 mmol) and chloroacetyl
chloride (1.74 mL, 21.83 mmol) as described in the general procedure B to yield 28 as a
brown solid. Yield: 85%; mp: 106-108 °C.
4.2.3. General procedure C: Synthesis of final compounds (29-65) [47].
A solution
chloroacetamides (26 or 27 or 28, 1 eq.), anhydrous K2CO3 (3 eq.), and appropriate chalcone
(1-22, 1 eq.) in dry DMF (4 mL) was stirred for 16 hr at 25 °C. After completion of the
reaction, the reaction mixture was quenched with brine (50 mL). The precipitated product
was filtered off, washed with water and dried under vacuum. The crude product was further
purified by column chromatography using DCM:MeOH (95:5) as mobile phase.
(E)-2-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzo-
furan-5-yl)acetamide (29): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33
mmol), and 1 (0.12 g, 0.44 mmol) as described in the general procedure C to yield 29 as a white
solid. Yield: 68%; TLC Rf = 0.46 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp: 202-204
1
°C; H-NMR (DMSO-d6, 400 MHz) δ 10.7 (brs, 1H), 8.17 (d, J = 8.4 Hz, 2H), 8.05 (s, 1H),
7.87-7.76 (m, 2H), 7.73-7.62 (m, 2H), 7.52 (s, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 8.5
Hz, 2H), 7.01 (d, J = 8.3 Hz, 1H), 5.36 (s, 2H), 4.91 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H); MS
(ESI) m/z: 474 [M+H]+.
(E)-2-(4-(3-(4-methoxyphenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-
yl)acetamide (30): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol),
and 2 (0.11 g, 0.44 mmol) as described in the general procedure C to yield 30 as a white solid.
Yield: 65%; TLC Rf = 0.48 (DCM:MeOH, 95:5); purity (HPLC): 98.03%; mp: 218-220 °C;
1H-NMR (DMSO-d6, 400 MHz) δ 10.68 (s, 1H), 8.16 (d, J = 8.6 Hz, 2H), 8.06 (s, 1H), 7.87-

ACCEPTED MANUSCRIPT
7.76 (m, 4H), 7.75-7.63 (m, 2H), 7.14 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 5.37 (s,
13
2H), 4.92 (s, 2H), 3.81 (s, 3H); C-NMR (DMSO-d6, 100 MHz) δ 187.25, 170.14, 166.89,
161.46, 161.21, 148.91, 143.63, 143.23, 131.27, 130.69, 130.64, 127.39, 125.79, 120.16,
119.69, 119.43, 114.61, 114.35, 112.34, 69.56, 67.05, 55.33; MS (ESI) m/z: 444 [M+H]+.
(E)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-(4-(3-(thiophen-2-yl)acryloyl)phenoxy)-
acetamide (31): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol), and
3 (0.1 g, 0.44 mmol) as described in the general procedure C to yield 31 as a white solid. Yield:
62%; TLC Rf = 0.51 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp: 248-250 °C; 1H-NMR
(DMSO-d6, 400 MHz) δ 10.7 (brs, 1H), 8.11 (d, J = 8.8 Hz, 2H), 8.05 (s, 1H), 7.87 (d, J =
15.2 Hz, 1H), 7.81-7.74 (m, 2H), 7.72-7.62 (m, 2H), 7.57 (d, J = 15.3 Hz, 1H), 7.22-7.09 (m,
3H), 5.36 (s, 2H), 4.91 (s, 2H); MS (ESI) m/z: 420 [M+H]+.
(E)-2-(4-(3-(2-chlorophenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-
yl)acetamide (32): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol),
and 4 (0.11 g, 0.44 mmol) as described in the general procedure C to yield 32 as a white solid.
Yield: 65%; TLC Rf = 0.52 (DCM:MeOH, 95:5); purity (HPLC): 98.97%; mp: >300 °C; 1H-
NMR (DMSO-d6, 400 MHz) δ 10.82 (brs, 1H), 8.22-8.18 (m, 3H), 8.07 (s, 1H), 8.00 (s, 2H),
7.81 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 6.7 Hz, 1H), 7.60-7.51 (m, 1H), 7.57-7.44 (m, 2H),
7.17-7.15 (m, 2H), 5.37 (s, 2H), 4.95 (s, 2H); MS (ESI) m/z: 448 [M+H]+.
(E)-2-(4-(3-(3-methoxyphenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-
yl)acetamide (33): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol),
and 5 (0.11 g, 0.44 mmol) as described in the general procedure C to yield 33 as a white solid.
Yield: 67%; TLC Rf = 0.46 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp: 196-198 °C;
1H-NMR (DMSO-d6, 400 MHz) δ 10.74 (brs, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.05 (s, 1H),
7.94 (d, J = 15.0 Hz, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 10.8 Hz, 2H), 7.55-7.27 (m,
3H), 7.14 (d, J = 8.6 Hz, 2H), 7.04-6.97 (m, 1H), 5.36 (s, 2H), 4.92 (s, 2H), 3.82 (s, 3H); 13C-
NMR (DMSO-d6, 100 MHz) δ 187.38, 170.16, 166.91, 161.69, 159.61, 148.90, 143.87,
143.24, 136.16, 130.98, 130.89, 129.86, 125.77, 122.23, 121.56, 120.17, 119.59, 116.48,
114.66, 113.29, 112.37, 69.54, 67.10, 55.26; MS (ESI) m/z: 444 [M+H]+.
(E)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-(4-(3-(3,4,5-trimethoxyphenyl)acryloyl)
phenoxy)acetamide (34): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33
mmol), and 6 (0.14 g, 0.44 mmol) as described in the general procedure C to yield 34 as a white

ACCEPTED MANUSCRIPT
solid. Yield: 68%; TLC Rf = 0.39 (DCM:MeOH, 95:5); purity (HPLC): 97.70%; mp: 238-240
1
°C; H-NMR (DMSO-d6, 400 MHz) δ 10.75 (brs, 1H), 8.22-8.15 (m, 2H), 8.04 (s, 1H), 7.89
(d, J = 15.5 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.72-7.61 (m, 2H), 7.21 (s, 2H), 7.14 (d, J =
8.8 Hz, 2H), 5.36 (s, 2H), 4.91 (s, 2H), 3.85 (s, 6H), 3.70 (s, 3H); MS (ESI) m/z: 504 [M+H]+.
(E)-2-(4-(3-(2,6-difluorophenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-
5-yl)acetamide (35): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol),
and 7 (0.11 g, 0.44 mmol) as described in the general procedure C to yield 35 as a white solid.
Yield: 65%; TLC Rf = 0.57 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp: 242-244 °C;
1H-NMR (DMSO-d6, 400 MHz) δ 10.06 (brs, 1H), 8.07-8.03 (m, 3H), 7.86 (d, J = 16.0 Hz,
1H), 7.77 (d, J = 8.4 Hz, 1H), 7.67-7.63 (m, 2H), 7.58-7.50 (m, 1H), 7.25 (t, J = 8.9 Hz, 2H),
7.15 (d, J = 8.4 Hz, 2H), 5.34 (s, 2H), 4.90 (s, 2H); 13C-NMR (DMSO-d6, 100 MHz) δ
187.24, 170.17, 166.86, 162.31, 161.97, 159.72, 148.90, 144.00, 132.28, 130.81, 130.45,
128.56, 127.37, 125.75, 120.20, 119.52, 114.90, 112.46, 112.37, 112.22, 112.07, 69.55,
67.08; MS (ESI) m/z: 450 [M+H]+.
(E)-2-(4-(3-(4-nitrophenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)
acetamide (36): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol), and
8 (0.11 g, 0.44 mmol) as described in the general procedure C to yield 36 as a white solid.
Yield: 70%; TLC Rf = 0.39 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp: 250-254 °C;
1H-NMR (DMSO-d6, 400 MHz) δ 10.71 (brs, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.21 (d, J = 8.5
Hz, 2H), 8.17-8.103 (m, 3H), 8.05 (s, 1H), 7.80-7.76 (m, 2H), 7.70 (d, J = 8.4 Hz, 1H), 7.16
(d, J = 8.5 Hz, 2H), 5.36 (s, 2H), 4.93 (s, 2H); MS (ESI) m/z: 459 [M+H]+.
(E)-2-(4-cinnamoylphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide
(37):
The title compound was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol), and
9 (0.09 g, 0.44 mmol) as described in the general procedure C to yield 37 as a white solid.
Yield: 66%; TLC Rf = 0.60 (DCM:MeOH, 95:5); purity (HPLC): 98.34%; mp: 228-230 °C;
1H-NMR (DMSO-d6, 400 MHz) δ 10.78 (brs, 1H), 8.18 (d, J = 8.8 Hz, 2H), 8.05 (s, 1H),
7.95 (d, J = 15.6 Hz, 1H), 7.87 (d, J = 6.6 Hz, 2H), 7.79 (d, J = 8.4 Hz, 1H), 7.75-7.66 (m,
2H), 7.50-7.40 (m, 3H), 7.14 (d, J = 8.8 Hz, 2H), 5.36 (s, 2H), 4.92 (s, 2H); MS (ESI) m/z:
414 [M+H]+.
(E)-2-(4-(3-(furan-2-yl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)
acetamide (38): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol), and

ACCEPTED MANUSCRIPT
10 (0.09 g, 0.44 mmol) as described in the general procedure C to yield 38 as a white solid.
1
Yield: 67%; TLC Rf = 0.49 (DCM;MeOH, 95:5); purity (HPLC): >99%; mp: >300 °C; H-
NMR (DMSO-d6, 400 MHz) δ 10.70 (s, 1H), 8.10-8.06 (m, 3H), 7.90 (s, 1H), 7.81 (d, J = 8.3
Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.54 (s, 2H), 7.13 (d, J = 8.9 Hz, 2H), 7.08 (d, J = 3.4 Hz,
1H), 6.68 (d, J = 3.5 Hz, 1H), 5.37 (s, 2H), 4.92 (s, 2H); MS (ESI) m/z: 404 [M+H]+.
(E)-2-(3-(3-(3,4-dimethoxyphenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzo-
furan-5-yl)acetamide (39): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33
mmol), and 11 (0.12 g, 0.44 mmol) as described in the general procedure C to yield 39 as a
white solid. Yield: 69%; TLC Rf = 0.47 (DCM:MeOH, 95:5); purity (HPLC): 99%; mp: 184-
1
186 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.71 (brs, 1H), 8.07 (s, 1H), 7.82-7.77 (m, 3H),
7.71-7.67 (m, 3H), 7.52 (d, J = 2.9 Hz, 2H), 7.38 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.4 Hz,
1H), 7.00 (d, J = 8.4 Hz, 1H), 5.36 (s, 2H), 4.89 (s, 2H), 3.84 (s, 3H), 3.81 (s, 3H); MS (ESI)
m/z: 474 [M+H]+.
(E)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-(3-(3-(3,4,5-trimethoxyphenyl)acryloyl)-
phenoxy)acetamide (40): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33
mmol), and 12 (0.14 g, 0.44 mmol) as described in the general procedure C to yield 40 as a
white solid. Yield: 70%; TLC Rf = 0.41 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp:
1
222-224 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.72 (brs, 1H), 8.06 (s, 1H), 7.88-7.78 (m,
3H), 7.70-7.68 (m, 3H), 7.53 (t, J = 7.9 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.23 (s, 2H), 5.36
(s, 2H), 4.89 (s, 2H), 3.85 (s, 6H), 3.70 (s, 3H); MS (ESI) m/z: 504 [M+H]+.
(E)-2-(3-cinnamoylphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (41): It
was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol), and 13 (0.09 g, 0.44
mmol) as described in the general procedure C to yield 41 as a white solid. Yield: 67%; TLC
Rf = 0.59 (DCM;MeOH, 95:5); purity (HPLC): >99%; mp: 160-162 °C; 1H-NMR (DMSO-d6,
400 MHz) δ 10.75 (brs, 1H), 8.07 (s, 1H), 7.93-7.86 (m, 3H), 7.82-7.78 (m, 2H), 7.75-7.69
(m, 3H), 7.52 (t, J = 7.9 Hz, 1H), 7.45-7.42 (m, 3H), 7.32 (d, J =8.2 Hz, 1H), 5.36 (s, 2H),
4.90 (s, 2H); 13C-NMR (DMSO-d6, 100 MHz) δ 188.80, 170.19, 167.25, 158.07, 148.92,
144.24, 143.87, 138.95, 134.58, 130.69, 130.04, 128.90, 125.78, 122.07, 121.76, 120.22,
119.84, 119.60, 113.92, 112.40, 69.57, 67.21; MS (ESI) m/z: 414 [M+H]+.
(E)-2-(2-methoxy-4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-
dihydroisobenzofuran-5-yl)acetamide (42): It was synthesized from 26 (0.1 g, 0.44 mmol),

ACCEPTED MANUSCRIPT
K2CO3 (0.18 g, 1.33 mmol), and 14 (0.15 g, 0.44 mmol) as described in the general procedure C to
yield 42 as a white solid. Yield: 68%; TLC Rf = 0.38 (DCM:MeOH, 95:5); purity (HPLC):
1
>99%; mp: >300 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.91 (brs, 1H), 8.04 (s, 1H), 7.92-
7.84 (m, 2H), 7.80 (d, J = 8.4 Hz, 1H), 7.69-7.62 (m, 3H), 7.21 (s, 2H), 7.06 (d, J = 8.5 Hz,
1H), 5.36 (s, 2H), 4.94 (s, 2H), 3.90 (s, 3H), 3.84 (s, 6H), 3.69 (s, 3H); MS (ESI) m/z: 534
[M+H]+.
(E)-2-(4-(3-(4-fluorophenyl)-3-oxoprop-1-enyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzo-
furan-5-yl)acetamide (43): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33
mmol), and 15 (0.11 g, 0.44 mmol) as described in the general procedure C to yield 43 as a
white solid. Yield: 63%; TLC Rf = 0.58 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp:
232-234 °C; 1H-NMR (DMSO-d6, 400 MHz) δ 10.78 (brs, 1H), 8.23 (d, J = 8.7 Hz, 2H), 8.07
(s, 1H), 7.89 (d, J = 2.0 Hz, 1H), 7.86 (d, J = 5.7 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.73 (d, J
= 3.4 Hz, 1H), 7.70 (d, J = 3.8 Hz, 1H), 7.38 (t, J = 8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H),
5.37 (s, 2H), 4.88 (s, 2H); 13C-NMR (DMSO-d6, 100 MHz) δ 187.53, 170.17, 167.09, 166.19,
159.83, 148.91, 143.94, 143.74, 134.43, 131.42, 131.33, 130.79, 127.93, 125.78, 120.14,
119.67, 115.83, 115.61, 115.08, 112.34, 69.57, 67.06; MS (ESI) m/z: 432 [M+H]+.
(E)-2-(4-(3-(4-chlorophenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-
yl)acetamide (44): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol),
and 16 (0.11 g, 0.44 mmol) as described in the general procedure C to yield 44 as a white solid.
Yield: 61%; TLC Rf = 0.49 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp: 288-290 °C;
1H-NMR (DMSO-d6, 400 MHz) δ 10.78 (s, 1H), 8.19 (d, J = 8.9 Hz, 2H), 8.07 (s, 1H), 7.99-
7.90 (m, 3H), 7.81 (d, J = 8.4 Hz, 1H), 7.73-7.67 (m, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.15 (d, J
= 8.9 Hz, 2H), 5.37 (s, 2H), 4.94 (s, 2H); MS (ESI) m/z: 448 [M+H]+.
(E)-2-(4-(3-(4-(benzyloxy)phenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzo-
furan-5-yl)acetamide (45): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33
mmol), and 17 (0.15 g, 0.44 mmol) as described in the general procedure C to yield 45 as a
white solid. Yield: 65%; TLC Rf = 0.52 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp:
1
242-244 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.68 (s, 1H), 8.17 (d, J = 8.5 Hz, 2H), 8.07
(s, 1H), 7.84-7.79 (m, 4H), 7.72-7.65 (m, 2H), 7.46 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.4 Hz,
2H), 7.33 (t, J = 7.2 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 8.3 Hz, 2H), 5.37 (s, 2H),
5.17 (s, 2H), 4.93 (s, 2H); MS (ESI) m/z: 520 [M+H]+.

ACCEPTED MANUSCRIPT
(E)-2-(4-(3-(4-(2,4-dichlorobenzyloxy)phenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydro-
isobenzofuran-5-yl)acetamide (46): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3
(0.18 g, 1.33 mmol), and 18 (0.18 g, 0.44 mmol) as described in the general procedure C to yield
46 as a white solid. Yield: 66%; TLC Rf = 0.54 (DCM:MeOH, 95:5); purity (HPLC): >99%;
1
mp: 228-230 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.69 (s, 1H), 8.17 (d, J = 8.5 Hz, 2H),
8.07 (s, 1H), 7.86-7.80 (m, 4H), 7.72-7.62 (m, 4H), 7.49 (d, J = 2.2, 8.3 Hz, 1H), 7.15-7.09
(m, 4H), 5.37 (s, 2H), 5.21 (s, 2H), 4.93 (s, 2H); MS (ESI) m/z: 588 [M+H]+.
(E)-2-(4-(3-(4-(4-chlorobenzyloxy)phenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydro-
isobenzofuran-5-yl)acetamide (47): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3
(0.18 g, 1.33 mmol), and 19 (0.16 g, 0.44 mmol) as described in the general procedure C to yield
47 as a white solid. Yield: 62%; TLC Rf = 0.52 (DCM:MeOH, 95:5); purity (HPLC): >99%;
1
mp: >300 °C; H-NMR (DMSO-d6, 400 MHz) ) δ 10.71 (s, 1H), 8.16 (d, J = 8.8 Hz, 2H),
8.07 (s, 1H), 7.85-7.80 (m, 4H), 7.72-7.65 (m, 2H), 7.50-7.42 (m, 4H), 7.14 (d, J = 8.8 Hz,
2H), 7.08 (d, J = 8.5 Hz, 2H), 5.37 (s, 2H), 5.18 (s, 2H), 4.93 (s, 2H); MS (ESI) m/z: 554
[M+H]+.
(E)-2-(4-(3-(4-ethoxyphenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzo-furan-5-
yl)acetamide (48): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol),
and 20 (0.12 g, 0.44 mmol) as described in the general procedure C to yield 48 as a white solid.
1
Yield: 58%; TLC Rf = 0.55 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp: >300 °C; H-
NMR (DMSO-d6, 400 MHz) δ 11.05 (s, 1H), 8.16 (d, J = 8.9 Hz, 2H), 8.09 (s, 1H), 7.83-7.75
(m, 5H), 7.67 (d, J = 15.5 Hz, 1H), 7.13 (d, J = 8.9 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 5.37
(s, 2H), 4.97 (s, 2H), 4.08 (q, J = 6.9 Hz, 2H), 1.33 (t, J = 7.0 Hz, 3H); MS (ESI) m/z: 458
[M+H]+.
(E)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)-2-(4-(3-(4-propoxyphenyl)acryloyl)-
phenoxy)acetamide (49): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33
mmol), and 21 (0.12 g, 0.44 mmol) as described in the general procedure C to yield 49 as a
white solid. Yield: 57%; TLC Rf = 0.57 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp:
1
>300 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.68 (s, 1H), 8.16 (d, J = 8.4 Hz, 2H), 8.06 (s,
1H), 7.82-7.78 (m, 4H), 7.72-7.65 (m, 2H), 7.14 (d, J = 8.5 Hz, 2H), 6.99 (d, J = 8.3 Hz,
2H), 5.37 (s, 2H), 4.92 (s, 2H), 3.98 (t, J = 6.5 Hz, 2H), 3.16 (d, J = 5.1 Hz, 1H), 1.73 (q, J =
7.0 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H); MS (ESI) m/z: 472 [M+H]+.

ACCEPTED MANUSCRIPT
(E)-2-(4-(3-(4-butoxyphenyl)acryloyl)phenoxy)-N-(1-oxo-1,3-dihydroisobenzo-furan-5-
yl)acetamide (50): It was synthesized from 26 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol),
and 22 (0.13 g, 0.44 mmol) as described in the general procedure C to yield 50 as a white solid.
1
Yield: 55%; TLC Rf = 0.57 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp: >300 °C; H-
NMR (DMSO-d6, 400 MHz) δ 10.73 (s, 1H), 8.16 (d, J = 8.6 Hz, 2H), 8.06 (s, 1H), 7.82-7.78
(m, 4H), 7.70-7.63 (m, 2H), 7.13 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.4 Hz, 2H), 5.36 (s, 2H),
4.91 (s, 2H), 4.02 (t, J = 6.5 Hz, 2H), 1.70 (d, J = 8.5 Hz, 2H), 1.43 (q, J = 7.5 Hz, 2H), 0.93
(t, J = 7.4 Hz, 3H).; MS (ESI) m/z: 486 [M+H]+.
(E)-N-(3,4-dimethoxyphenyl)-2-(4-(3-(4-methoxyphenyl)acryloyl)phenoxy)acetamide
(51): It was synthesized from 27 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol), and 2 (0.11 g,
0.44 mmol) as described in the general procedure C to yield 51 as a grey solid. Yield: 54%;
1
TLC Rf = 0.54 (DCM:MeOH, 95:5); purity (HPLC): 98.10%; mp: 188-190 °C; H-NMR
(DMSO-d6, 400 MHz) δ 10.03 (s, 1H), 8.17 (d, J = 8.4 Hz, 2H), 7.84-7.80 (m, 3H), 7.68 (d, J
= 15.5 Hz, 1H), 7.33 (d, J = 2.4 Hz, 1H), 7.18-7.13 (m, 3H), 7.01 (d, J = 8.3 Hz, 2H), 6.90
(d, J = 8.7 Hz, 1H), 4.80 (s, 2H), 3.81 (s, 3H), 3.71 (d, J = 2.3 Hz, 6H); MS (ESI) m/z: 448
[M+H]+.
(E)-N-(3,4-dimethoxyphenyl)-2-(4-(3-(3-methoxyphenyl)acryloyl)phenoxy)acetamide
(52): It was synthesized from 27 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol), and 5 (0.11 g,
0.44 mmol) as described in the general procedure C to yield 52 as a grey solid. Yield: 57%;
1
TLC Rf = 0.55 (DCM:MeOH, 95:5); purity (HPLC): 98.39%; mp: 290-292 °C; H-NMR
(DMSO-d6, 400 MHz) ) δ 10.02 (s, 1H), 8.19 (d, J = 8.5 Hz, 2H), 7.95 (d, J = 15.7 Hz, 1H),
7.68 (d, J = 15.5 Hz, 1H), 7.47 (s, 1H), 7.42 (d, J = 7.4 Hz, 1H), 7.37-7.32 (m, 2H), 7.15-
7.13 (m, 3H), 7.01 (d, J = 7.9 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 4.81 (s, 2H), 3.82 (d, J = 1.4
Hz, 3H), 3.71 (d, J = 1.7 Hz, 6H); MS (ESI) m/z: 448 [M+H]+.
(E)-N-(3,4-dimethoxyphenyl)-2-(3-(3-(3,4-dimethoxyphenyl)acryloyl)phenoxy)acetamide
(53): It was synthesized from 27 (0.1 g, 0.44 mmol), K2CO3 (0.18 g, 1.33 mmol), and 11 (0.13 g,
0.44 mmol) as described in the general procedure C to yield 53 as a grey solid. Yield: 55%;
TLC Rf = 0.52 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp: >300 °C; 1H-NMR (DMSO-
d6, 400 MHz) δ 10.25 (s, 1H), 7.83-7.79 (m, 2H), 7.71-7.67 (m, 2H), 7.54-7.52 (m, 1H), 7.51-
7.49 (m, 1H), 7.40-7.36 (m, 2H), 7.30 (d, J = 8.2 Hz, 1H), 7.20 (d, J = 2.4, 8.7 Hz, 1H), 7.00
(d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 4.81 (s, 2H), 3.85 (s, 3H), 3.80 (s, 3H), 3.70 (s,
6H); MS (ESI) m/z: 478 [M+H]+.