Design, synthesis and biological evaluation of Helicobacter pylori inosine 5′-monophosphate dehydrogenase (HpIMPDH) inhibitors. Further optimization of selectivity towards HpIMPDH over human IMPDH2

Article abstract of DOI:10.1016/j.bioorg.2019.04.001

Inosine 5′-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes a crucial step in guanine nucleotide biosynthesis, thereby governing cell proliferation. In contrast to mammalian IMPDHs, microbial IMPDHs are relatively less explored as potential tar

Full text of DOI:10.1016/j.bioorg.2019.04.001

Accepted Manuscript
Design, synthesis and biological evaluation of Helicobacter pylori inosine 5′-
monophosphate dehydrogenase (HpIMPDH) inhibitors. Further optimization of
selectivity towards HpIMPDH over human IMPDH2
Chetan P. Shah, Gayathri Purushothaman, Vijay Thiruvenkatam, Sivapriya
Kirubakaran, Kapil Juvale, Prashant S. Kharkar
PII:
S0045-2068(18)31562-1
DOI:
https://doi.org/10.1016/j.bioorg.2019.04.001
YBIOO 2907
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
28 December 2018
20 March 2019
1 April 2019
Please cite this article as: C.P. Shah, G. Purushothaman, V. Thiruvenkatam, S. Kirubakaran, K. Juvale, P.S. Kharkar,
Design, synthesis and biological evaluation of Helicobacter pylori inosine 5′-monophosphate dehydrogenase
(
HpIMPDH) inhibitors. Further optimization of selectivity towards HpIMPDH over human IMPDH2, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.04.001
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Design, synthesis and biological evaluation of Helicobacter pylori inosine 5′-
monophosphate dehydrogenase (HpIMPDH) inhibitors. Further optimization
of selectivity towards HpIMPDH over human IMPDH2
a
b
b,c
b,d
,
Chetan P. Shah , Gayathri Purushothaman , Vijay Thiruvenkatam , Sivapriya Kirubakaran
a
a
Kapil Juvale , Prashant S. Kharkar *
a
Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’s
NMIMS (Deemed to be University), V. L. Mehta Road, Vile Parle (West), Mumbai 400 056,
India.
b
Biological Engineering, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar
3
82355, India.
c
Physics, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar 382355, India.
d
Chemistry, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar 382355, India.
*
Corresponding author at: Shobhaben Pratapbhai Patel School of Pharmacy and Technology
Management, SVKM’s NMIMS, V. L. Mehta Road, Vile Parle (West), Mumbai 400 056, India.
E-mail: Prashant.Kharkar@nmims.edu (P. S. Kharkar).
2

Abstract
Inosine 5′-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes a crucial step in
guanine nucleotide biosynthesis, thereby governing cell proliferation. In contrast to mammalian
IMPDHs, microbial IMPDHs are relatively less explored as potential targets for antimicrobial drug
discovery. In continuation with our previous work, here we report the discovery of moderately
potent and highly selective Helicobacter pylori IMPDH (HpIMPDH) inhibitors. The present study
is mainly focused around our previously identified, modestly potent and relatively nonselective (for
HpIMPDH over human IMPDH2) hit molecule IX (16i). In an attempt to optimize the selectivity
for the bacterial enzyme, we screened a set of 48 redesigned new chemical entities (NCEs)
belonging to 5-aminoisobenzofuran-1(3H)-one series for their in vitro HpIMPDH and human
IMPDH2 inhibition. A total of 12 compounds (hits) demonstrated ≥70% HpIMPDH inhibition at 10

M concentration; none of the hits were active against hIMPDH2. Compound 24 was found to be
the most potent and selective molecule (HpIMPDH IC50 = 2.21 µM) in the series. The study
reaffirmed the utility of 5-aminoisobenzofuran-1(3H)-one as a promising scaffold with great
potential for further development of potent and selective HpIMPDH inhibitors.
Keywords: Helicobacter pylori, IMPDH, HpIMPDH, hIMPDH2, 5-aminoisobenzofuran-1(3H)-one
Abbreviations: PUD: Peptic ulcer disease; MALT: gastric mucosa-associated lymphoid tissue;
XMP: xanthosine 5′-monophosphate; IMP: inosine 5′-monophosphate; DPPA: diphenylphosphoryl
azide; TEA: triethyl amine; EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; HBTU:
hexafluorophosphate benzotriazole tetramethyl uranium; NMM: N-methylmorpholine; EDCI: 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide; DMAP: 4-(N,N-dimethylamino)pyridine; DIPEA:
Di-isopropylethylamine
3

1
. Introduction
Approximately 50% of the world’s population is infected with Helicobacter pylori (H.
pylori), one of the most prevalent gram-negative bacterial pathogens, mostly residing in
human gastric mucosa [1]. The bacillus finds a home in the highly acidic environment of the
stomach, where very few other microorganisms can survive. The deadly pathogen is
associated with various gastric complications such as chronic gastritis, peptic ulcer disease
(
[
(
PUD), gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric carcinoma
1-3]. H. pylori is classified as a class I gastric carcinogen by the World Health Organization
WHO). Around 2.9% gastric cancers occur in H. pylori-infected individuals [4]. Treatment
of the infection is extremely challenging due to its survival in a hostile acidic environment.
Various methodo-logies investigated in the recent past such as drug repurposing campaigns
of approved drugs [4,5], randomized controlled trials [6], natural products therapy [7,8], and
many others are likely to offer potential alternatives for the treatment of H. pylori. Newer
molecules with novel mechanism(s) of action are needed for contributing significantly in, not
only treating but also eliminating, H. pylori from the body.
Inosine 5′-monophosphate dehydrogenase (IMPDH) presents an interesting, yet largely
unexploited target for antimicrobial drug development. Rapid proliferation is an important
characteristic of many microbial infections, which is supported by the expansion of guanine
nucleotide pool by the rapidly-dividing cells. The enzyme IMPDH (EC 1.1.1.205) catalyzes
the oxidation of inosine 5′-monophosphate (IMP) to xanthosine 5′-monophosphate (XMP)
+
with concomitant reduction of nicotinamide adenine dinucleotide (NAD ), which is a crucial
step in the de novo biosynthesis of guanine nucleotides. Inhibition of IMPDH certainly leads
to a fall in the supply of XMP and reduces the formation of GMP and supplementary guanine
derivatives (guanine nucleotide pool). The major consequence of IMPDH inhibition is an
overall reduction in intracellular GTP and the ‘guanylate pool’ (GMP, GDP, GTP and dGTP)
4

which blocks proliferation. Also, IMPDH has historically been a fascinating target for
immunosuppressive, antiviral and anticancer drug development. The differences in the
affinity for the substrates, structural features and kinetic properties dictate the design of
selective and potent inhibitors for prokaryotic and eukaryotic IMPDHs [9-11].
Just like other microbes, Cryptosporidium parvum (C. parvum), a protozoan parasite
causing diarrhoea and malnutrition especially in children, is dependent on IMPDH for its
supply of guanine nucleotides to spread and sustain the infection. In fact, C. parvum IMPDH
(
CpIMPDH) inhibitor development is an active area of research. Several selective and potent
CpIMPDH inhibitors are reported in the literature [12,13]. Also, it is well documented that
CpIMPDH, being structurally similar to many bacterial IMPDHs, is likely to be inhibited by
the similar, if not same, set of compounds inhibiting other bacterial IMPDHs [9,12]. Working
on the similar lines in reverse direction, five series of CpIMPDH-selective inhibitors (I-V,
Fig. 1; linker with 3-4 atom spacer containing amide, urea, etc., between the two aryl groups)
were screened for in vitro enzyme inhibitory activity against IMPDHs from Bacillus
anthraces [13], Mycobacterium tuberculosis (Mtb) [14] and Francisella tularensis [15]. In
addition, few CpIMPDH inhibitors were found to be potent H. pylori IMPDH (HpIMPDH)
inhibitors; C91 (VI, Fig. 1) was the most potent HpIMPDH inhibitor reported so far (IC50 = 8
and 1.1 nM for CpIMPDH and HpIMPDH, respectively) [12,16]. Very recently, our group
has reported 3-aryldiazenyl indoles (VII, Fig. 1) [17] and 3-carboxamido indoles (VIII, Fig.
1
) as new series of HpIMPDH inhibitors [18].
Known Hp- and CpIMPDH inhibitors possess common structural features, namely, two
1
2
aromatic rings (Ar and Ar ) connected with a suitable linker. All the reported HpIMPDH
inhibitors have a three-to-four C spacer (mostly acetamide). This information is crucial for
the
5

design and development of species-selective and potent microbial IMPDH inhibitors. Several
investigators are working on the IMPDH inhibitor development programmes [12-24]. Our
research group has contributed recently on the design and development of potent and
selective prokaryotic (including H. pylori) and eukaryotic IMPDH inhibitors [10,17-24].
Fig. 1. Reported C. parvum and H. pylori IMPDH inhibitors
Our previous report on HpIMPDH inhibitors led to the identification of moderately
potent hit 16i (IX, Fig. 1) displaying higher HpIMPDH (99.89% inhibition at 10 M) and
moderate human IMPDH2 (hIMPDH2) inhibition (56.81% at 10 M). The hit 16i was
relatively nonselective for HpIMPDH [22]. We started off with the sole objective of
modulating the selectivity of 16i for the bacterial enzyme over the human counterpart by
systematic structure-activity and selectivity exploration. In the present study, we report the
design and synthesis of 5-aminoisobenzofuran-1(3H)-one derivatives possessing similar
6

pharmacophoric features as the reported HpIMPDH inhibitors (Fig. 1). The synthesized
compounds were further screened in in vitro biochemical assays (HpIMPDH and hIMPDH2
enzyme inhibition). In addition, we believe that the availability of structurally diverse, potent
and selective HpIMPDH inhibitors would stimulate the scientific community’s interest in this
field, attempting to eliminate deadly H. pylori from the masses. Here, we report our initial
stint with the design and development of structurally novel, moderately potent and highly
selective HpIMPDH inhibitors. The hits are likely to be explored further for improving
potency, molecular, physicochemical, pharmacokinetic and toxicity profiles.
2
. Results and Discussion
2
.1. Chemistry
In the present study, two series of phthalide derivatives – series I and II – were
designed and synthesized as outlined in Schemes 1 and 2, respectively. The emphasis was
placed on the pharmacophoric requirements for IMPDHs in general, and microbial IMPDHs
1
2
in particular. The two aryl groups (Ar and Ar ) with varying linkers, conformationally
flexible versus rigid, short (2-3 atoms) versus long (4-5 atoms), etc., were tried. One of the
1
aryl groups (Ar ) was fixed as a 5-substitued phthalide substructure in all the designed
molecules, based on our previous experience with HpIMPDH inhibitors design strategy [22].
Several derivatives of 5-aminoisobenzofuran-1(3H)-one (1) were synthesized using
conventional amide bond formation reaction [25]. Numerous amide-coupling reagents were
employed, namely, diphenylphosphoryl azide (DPPA)/triethylamine (TEA), N-
ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline
benzotriazole tetramethyl uronium (HBTU)/N-methylmorpholine (NMM), TBTU/HOBt,
HATU/DIPEA, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)/4-(N,N-
(EEDQ)/pyridine,
hexafluorophosphate
dimethylamino)pyridine (DMAP)/HOBt, etc. None of these reagents worked. Hence, we
7

resorted to chloroacetyl chloride (series I, Scheme 1) and substituted acid chloride (series II,
Scheme 2) as the activated acid. The choice of chloroacetyl chloride as acyl halide was based
on the linker present in the known HpIMPDH inhibitor, C91 (VII, Figure 1).
In case of series I – ethers (3-26, Table 1) and amines (27-31, Table 1), the
chloroacetamide intermediate 2 was synthesized using 1 and chloroacetyl chloride in dry
THF in presence of TEA at 0 °C, followed by overnight alkylation using anhydrous
K
2
CO /DMF (General procedure A) as a solvent to yield the title compounds (3-31). The
3
series II amides (32-50, Table 2) were prepared using 1 and various substituted acid chlorides
in presence of pyridine and THF (General procedure B) as a solvent to yield the title
compounds (32-50). The reactions were quite clean and posed little or no problem for
purification by selective precipitation and/or column chromatography.
ο
Scheme 1. Reagents and conditions: (a) chloroacetyl chloride, TEA, THF, 0 C to RT, over-
night; (b) substituted phenol or substituted amine, K
2
CO , DMF, RT, 16 hrs.
3
8

Scheme 2. Reagents and conditions: (a) substituted acid chlorides, pyridine, THF, 0 ºC to
RT, overnight.
2
.2. Biological evaluation
All the synthesized compounds (3-50, Tables 1 and 2) were screened for in vitro
HpIMPDH and hIMPDH2 inhibition at 10 μM concentration. The % inhibition data are given
in Tables 1 and 2. Followed by the initial screening, hits with ~70% or higher inhibition
(
Figures 2 and 3) were further taken up for IC50 determination (3, 9, 10, 15, 16, 23, 24, 30,
7-50). To generate the concentration-response curves, initial rates of reaction were plotted
4
against their corresponding logarithmic concentrations in case of HpIMPDH. The IC50 data
for HpIMPDH are given in Tables 1 and 2. Concentration-response curve for the most potent
compound 24 is given in the Fig. 2.
From series I (ether derivatives, 3-26, Table 1), a total of eight compounds (3, 9, 10, 15,
1
6, 23, 24 and 30, Fig. S112, Supplementary Data section) exhibited ~70% HpIMPDH
inhibition. Compound 24 showed highest % inhibition. The IC50s of the hits ranged from 2.21
20.9 M. The substituents on the aryl ring (o-, m-, p-) ranged from very strongly
deactivating (-NO ), strongly deactivating (Ac, formyl, etc.) to activating (-CH ). The -
–
2
3
COOMe substituent at the p-position yielded slightly more potent compound (10) compared
to the one at the o-position (3). The corresponding n-propyl (11) and n-butyl (12) esters were
9

considerably less active, mostly emphasizing the limited size in the binding pocket of the
enzyme. Shifting m-CH substituent (6) to p-position (9) increased enzyme inhibitory
activity. Similarly, replacing p-CH (9) with p-Cl (15) retained the activity. Any further
3
3
combination of the two substituents o,m-, o,p- m,p- or o,m,p- led to either retention (16),
slight decrease (17-19, 21-22) or substantial decrease (20) in % HpIMPDH inhibition.
Replacing the substituted Ph ring on the ether O with naphthyl ring led to substantial increase
in enzyme inhibitory activity. Of the two such compounds, one with 2-naphthyl ether (24)
was slightly more potent than its 1-naphthyl counterpart (23). Further substituents such as
quinolin-8-yl (25) led to slight decrease in activity while additional substitution on the
quinoline ring (26) abolished the activity, further demonstrating limited tolerance to bulk at
this end of the molecule.
Interestingly, all the secondary (27-29), aromatic (30) and tertiary amines (31)
exhibited >50% HpIMPDH inhibition, with compound 30 consisting of 3-formyl-1H-indol-1-
yl as the second aryl feature being better than others in the series (% inhibition ~70% at 10

M concentration). The modest activity (~50% inhibition) may arise from the protonated
form (wherever possible) of the amine compensating the binding energy via electrostatic
interaction. This is particularly important in view of the fact that the H. pylori resides in a
highly acidic environment where most of these amines will be highly protonated. This
subseries containing
the amino functional group needs further exploration in terms of potency and selectivity for
HpIMPDH over hIMPDH2.
In series II of phthalide derivatives (32-50, Table 2, Fig. S113, Supplementary Data
section), the spacer between 5-substituted phthalide substructure and other aryl
(
pharmacophoric) feature was reduced to 2-3 atoms (Scheme 2) which helped in
understanding the importance of the spacer length. In the first subgroup (32-43, Table 2), a
10

conformationally rigid, two-atom spacer was tried. Compound 32 displayed moderated
inhibition (61.34%). Addition of substituents, irrespective of electron-donating (e.g., 36) or
withdrawing (e.g., 33), smaller (e.g., 35) or bulky (e.g., 34) nature of the substituent,
maintained similar, albeit less, potency compared to 32 (50-57%). Compared to 3- or 4-
methoxy substituents (36 and 37), the 2,5-(OCH
enzyme inhibitory activity (40).
3
) substituents resulted in complete loss of
2
Table 1. Biological activities of compound 3-31 against HpIMPDH
HpIMPDH
hIMPDH2
Code
C91
-R
a
a
a
%
Inhibition
IC50 (µM)
% Inhibition
b
c
-
-
-
0.197 ± 0.08
1.88 ± 0.07
11.9 ± 0.05
n.d.
Inactive
d
IX (16i)
99.89 ± 2.12
69.7 ± 2.65
1.2 ± 1.03
4.4 ± 1.06
50.6 ± 1.85
52.8 ± 2.35
6.2 ± 1.13
70.4 ± 1.74
78.8 ± 3.82
14.1 ± 2.69
12.6 ± 2.12
3.3 ± 1.06
22.6 ± 1.36
71.6 ± 2.65
56.81 ± 0.95
< 0.1
3
4
5
6
7
8
9
2-methylcarboxylate
2-nitro
< 0.1
2-acetyl
n.d.
< 0.1
3-methyl
n.d.
< 0.1
4-nitro
n.d.
< 0.1
4-acetyl
n.d.
< 0.1
4-methyl
12.9 ± 0.21
8.9 ± 0.03
n.d.
< 0.1
1
1
1
1
1
1
0
1
2
3
4
5
4-methylcarboxylate
4-n-propylcarboxylate
4-n-butylcarboxylate
4-formyl
< 0.1
< 0.1
n.d.
< 0.1
n.d.
< 0.1
4-acetamide
4-chloro
n.d.
< 0.1
11.5 ± 0.07
< 0.1
11

1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4-chloro-3-methyl
4-chloro-2-nitro
69.6 ± 1.78
61.6 ± 2.96
54.1 ± 3.32
60 ± 4.12
15.7 ± 0.06
n.d.
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
4-chloro-2,5-dimethyl
3-methyl-4-nitro
n.d.
n.d.
4-acetyl-2-methoxy
4-formyl-2-methoxy
2-isopropyl-5-methyl
naphthalen-1-yl
16.1 ± 2.09
55.1 ± 3.78
64.6 ± 2.31
76.9 ± 4.8
87.2 ± 3.96
62.3 ± 2.6
19.1 ± 1.02
51.6 ± 2.89
56.3 ± 1.96
54.2 ± 4.89
71.2 ± 2.95
58.4 ± 2.59
n.d.
n.d.
n.d.
12.7 ± 0.07
2.21 ± 0.02*
n.d.
naphthalen-2-yl
quinolin-8-yl
5-chloro-7-iodoquinolin-8-yl
N-methyl-N-phenylamino
N-ethyl-N-phenylamino
N-(4-fluorophenyl)amino
3-formyl-1H-indol-1-yl
Morpholino
n.d.
n.d.
n.d.
n.d.
20.9 ± 0.11*
n.d.
a
All the data are expressed as ± SD (average of n=3). IC50 values were determined for compounds
b
with % inhibition ~70% or more at 10 µM concentration; n.d. = not determined; Ref. 17,18. The
c
d
literature IC50 as reported in Ref. 12 was 1.1 nM; Ref. 12; Ref. 22; * Inhibition data obtained by
c
monitoring absorbance of NADH; Ref. 22
Table 2. Biological activity of compound 32-50 against HpIMPDH
HpIMPDH
hIMPDH2
Code
-R
-R′
a
a
a
%
Inhibition
IC50 (µM)
% Inhibition
12

3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
H
-
61.3 ± 1.50
57.1 ± 2.53
50.1 ± 2.95
51.3 ± 3.56
56.3 ± 3.62
57.1 ± 1.68
57.5 ± 1.35
55 ± 1.65
n.d.
n.d.
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
4-nitro
-
4-phenyl
-
n.d.
4-methyl
-
n.d.
3-methoxy
-
n.d.
4- methoxy
-
n.d.
3-benzyloxy
3-trifluoromethyl
2,5-dimethoxy
2-chloro-5-fluoro
2,4-dichloro
3-bromo-4-methoxy
Phenyl
-
n.d.
-
n.d.
-
-
Inactive*
n.d.
-
51.9 ± 3.12
54.5 ± 2.65
50.9 ± 1.23
54.9 ± 3.96
56 ± 3.25
-
n.d.
-
H
n.d.
n.d.
Phenyl
cyclopentyl
n.d.
Phenyl
-CH
2
-CH
2
-
52.2 ± 2.24
76.6 ± 4.60
86.2 ± 1.99
78.5 ± 2.10
73.2 ± 2.78
n.d.
(
(
(
±)-47
±)-48
±)-49
4-isobutylphenyl
3-benzoylphenyl
2-fluorobiphenyl-4-yl
4-fluoronaphth-1-yl
CH
3
10.7 ± 0.12
7.2 ± 0.07
8.6 ± 0.03
11.4 ± 0.09
CH
3
CH
3
5
0
H
a
All the data are expressed as ± SD (average of n=3). IC50 values were determined for compounds
with % inhibition ~70% or more at 10 µM concentration; n.d. = not determined. * Inhibition data
obtained by monitoring absorbance of NADH.
13

In the second subgroup of series II (44-50), the spacer length was maintained at three
atoms with addition of one more (cyclo)alkyl group, especially -CH (in order to search for
3
potential ‘magic’ methyl effect), was attempted. The base compound (44) exhibited moderate
inhibition of HpIMPDH. The placement of a cycloalkyl group on the -carbon next to amide
(
45-46) did not have any impact on the enzyme inhibition. Placing bulky substituents on the
aryl ring along with the -methyl group (racemic mixture) increased the activity notably (47
versus 44). Placing 3-benzoyl group on the second aryl ring led to further increase in %
HpIMPDH inhibition yielding relatively potent compound in this subgroup of series II (48,
8
6.21%, IC50 = 7.20 ± 0.07 M). Other two compounds 49 and 50, in line with series I
results, maintained higher HpIMPDH potency.
Fig. 2. Concentration response curve of compound 24. The data was fitted using four-
parameter Hill equation and each data point represents mean of triplicate
On the similar lines, all the synthesized molecules were evaluated for hIMPDH2
inhibition at 10 M concentration, to address the issue of selectivity for the microbial
14

enzyme. None of the tested molecules exhibited significant hIMPDH2 inhibition. The results
of % hIMPDH2 inhibition are listed in Tables 1 and 2. This undoubtedly confirmed the gain
in selectivity of the hits for HpIMPDH over hIMPDH2 due to extensive SAR investigations.
Compound 24 turned out to be the most, yet moderately potent and selective, hit of the series.
C91, the most potent HpIMPDH known so far, was also selective over the human enzyme.
Compound 24 shared 2-naphthyl substructure with C91, along with similar four-atom linker.
The flexible alignment of 24 onto C91 is shown in Fig. 3. As seen from the alignment, the 5-
aminoisobenzofuran-1(3H)-one substructure was aligned onto the 2-naphthyl substituent in
C91 and vice versa for the 2-naphthyl group of 24, which was found aligned onto the
benzimidazole substructure of C91 in an attempt to align the –NHCO- functionality of the
linkers. The ether O in 24 linker was nicely aligned onto the pyridine N from C91 (H-bond
acceptor feature). Additionally, the pyridine ring was protruding from the superimposed bulk
from both the molecules. Nonetheless, the alignment gave us the insight for future design
strategies based on 5-aminoisobenzofuran-1(3H)-one core structure. In addition, the docked
pose of 24 into the cofactor-binding site of HpIMPDH homology model revealed crucial
drug-receptor interactions (Figure 4). While the proximal end of 24, i.e., the
aminoisobenzofuranone ring O, formed one H-bond with Val224 (aminoisobenzofuranone
core) and a cation- interaction (Lys71), the rest of 24 interacted with the cofactor-binding
site residues via mostly hydrophobic interactions, particularly the distal 2-naphthyl group.
The - stacking interactions with IMP underlined the importance of 2-napthyl group in
potency and possibly, selectivity. The lower activity of 23 (Table 1), in view of the proposed
binding mode, could be attributed to the disturbed - stacking interaction with IMP. The
methodical details of the docking analyses along with the relevant references in the
Supplementary Data section. The clues obtained from the flexible alignment and the docking
studies are crucial in driving further design of potent and selective IMPDH inhibitors.
15

Overall, our systematic and concerted efforts in developing potent and selective
HpIMPDH inhibitors yielded fruitful outcomes in the form of moderately potent and selective
hits. These hits can certainly act as structurally novel starting points for the design of newer
and selective HpIMPDH inhibitors.
Fig. 3. Flexible alignment of 24 onto C91. The capped-stick structures are shown in element
color.
Fig 4.
Binding mode of 24 into the cofactor-binding pocket of HpIMPDH homology
model. The ligand is shown as ball-and-stick model, substrate (IMP) as stick
model while the enzyme residues are shown as line models colored by the
element. Yellow dotted lines indicate H-bond interaction with Val224, dark-
16

green dotted lines indicate cation- interaction of Lys71 and the amino-
isobenzofuranone core while the light-green dotted lines indicate the -
stacking interaction between the naphthyl ring of 24 and the purine ring of
IMP.
2
.3. Computational studies
.3.1. Calculation/prediction of molecular and physicochemical property profiles of the title
2
compounds
All the designed compounds (series I and II) were evaluated for their
calculated/predicted molecular and physicochemical property profiles to check their drug
likeliness and identify potential issues, if any, related to potentially poor pharmacokinetics
well before the synthesis. Table 1S (Supplementary Data) lists all the abovementioned
property categories for all the molecules. As given in Table S1, none of the designed
molecules exhibited abnormal values and were within the commonly accepted ranges for
drugs and drug-like molecules. These molecules were then taken up for syntheses and
biological evaluation.
2
.3.2. In silico predicted toxicity profiles of the hits
Given the importance of toxicity issues in drug discovery and development, it is
imperative that one should avoid problematic substructures contributing to the toxicity issues
such as carcinogenicity, mutagenicity, genotoxicity, reproductive toxicity, etc. The field of
predictive toxicity has come a long way. Keeping this in mind, we initiated the predicted
toxicity profiling of the hits to eliminate problematic structures right before they are
evaluated further down the drug discovery funnel. The details of the predictive toxicity
models, as implemented in Case Ultra ICH M7 bundle are provided in Table 2S
17

(
Supplementary Data). The results of the in silico toxicity predictions for the mutagenicity,
genotoxicity and carcinogenicity end-points are given in Table 3.
The results compare the hits (3, 9, 10, 15, 16, 23, 24, 30, 47-50) from the two series
along with the most potent HpIMPDH inhibitor C91 in terms of mutagenicity, genotoxicity
and carcinogenicity, three major preclinical toxicity end-points. In case of bacterial
mutagenicity (ICH M7) models, most of the hits, except C91 and 23, were negative in at least
three of the five models, with the overall result negative. Further investigation revealed the
presence of benzimidazole (C91) and 1-naphthyl (23) substructures led to either positive of
inconclusive outcome. For all practical purposes, the inconclusive was considered positive. In
case of genotoxicity models, the results were mixed. Models such as GT3_A8J predicted all
hits as positive for potential genotoxicity. These results may be interpreted cautiously and can
be of great help in identifying potential trouble-makers early in the discovery phase.
Similarly, the carcinogenicity predictions were also mixed, e.g., for hit 40, of the six models,
three predicted it as positive and the remaining ones as negative. Compound C91 has been
predicted as positive by all the models. Nonetheless, the medicinal chemist’s experience and
intuition are likely to help in interpreting these predictions in combination with the structural
analysis (presence/absence of a potential alert, such as Michael acceptor).
3
.
Conclusions
In the present study, a novel series of HpIMPDH inhibitors was designed, synthesized
and evaluated for potency and selectivity over the human enzyme. Systematic structure-
activity trends in the series led to the discovery of moderately potent and selective HpIMPDH
inhibitors. The probing of the microbial IMPDH binding site and the results thereof, could go
a long way in HpIMPDH inhibitor discovery given the relative dearth of chemotypes in this
field. Overall, a total of 13 hits displayed ~70% inhibition or more. The top-ranked
18

compound of the series 24 exhibited moderate potency (IC50 = 2.21 µM) and high selectivity
over human IMPDH2. The outcome of the present investigation led to further refinement of
the microbial,
in particular, HpIMPDH inhibitor requirements. The results clearly disclosed the potential of
5
-aminoisobenzofuran-1(3H)-one as a lead scaffold which can be exploited for further design
and development of safer, selective and potent, not only H. pylori but also, other microbial
IMPDH inhibitors.
Table 3. In silico toxicity predictions for the hit molecules
Sr. No.
Model Name
C91 3 9 10 15 16 23 24 30 40 47 48 49 50
Bacterial Mutagenicity Models (ICH M7)
1
2
3
4
5
GT1_A7B
GT1_AT_ECOLI
GT_EXPERT
PHARM_ECOLI
PHARM_SALM
#
#
-
$
-
-
-
-
#
-
-
-
-
#
-
-
-
-
#
-
-
-
+
+
+
-
#
+
-
-
-
-
#
+
-
-
#
-
-
-
-
#
-
-
-
-
#
-
-
-
#
#
-
-
-
-
# #
#
+
$
+
-
-
-
-
-
-
-
-
Genotoxicity Models
6
7
8
9
GT2_A7U
GT2_A7V
GT2_A7W
GT2_A7X
GT2_A8H
GT3_A7S
GT3_A7T
GT3_A8J
GT4_A7N
+
$
+
#
$
$
#
+
+
+ #
# #
+ -
+ -
#
#
-
-
-
#
#
-
-
-
#
#
#
-
#
#
+
+
#
+
+
+
+
$
-
+
+
+
+
#
+
#
-
-
-
+
+
+
+
+
+
#
-
#
+
+
#
-
-
-
$
-
-
+
+
#
-
-
-
#
-
-
+
#
#
#
-
-
-
-
-
+
#
#
-
#
-
-
+
-
#
$
+
$
$
$
+
+
+
1
1
1
1
1
0
1
2
3
4
-
-
-
-
-
-
-
-
#
+
#
+
+
#
+ +
# #
+
#
Carcinogenicity Models
1
1
1
1
1
2
2
5
6
7
8
9
0
1
AF1
AF2
AF3
AF4
AFU
AFV
AFW
+
+
+
+
+
+
+
+ -
+ +
-
-
-
+
-
+
-
-
+
-
+
-
-
+
-
-
-
-
+
-
+
-
+
+
+
+
+
+
+
$
+
+
+
$
+
$
-
+
-
+
-
+
+
-
+
+
+
-
-
+
-
+
-
-
+
-
+
-
+
+
$
+
$
+
$
-
-
+ +
+ -
+ +
+
-
+
-
+
-
+
-
-
-
+
-
-
-
+
-
Positive
Negative
#
$
Inconclusive
Out of domain
19

4
. Experimental
4
.1. Chemistry
All the chemicals were purchased from vendors 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. All the reactions were carried out under dry N atmosphere and
(
2
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-
4
00 mesh, 60 Å of Merck using a mixture of DCM/MeOH as eluent. Melting points were
determined on Veego VMP DS melting point apparatus (General Trading Co., India) and are
uncorrected. Purity of all the 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 μm particle size, 100 Å pore size), flow rate of 1
1
mL/min. The H-NMR spectra were recorded in DMSO-d
6
or CDCl with tetramethylsilane
3
(
TMS) as an internal standard, on Bruker Advance 400 (400 MHz); chemical shifts were
expressed in δ ppm. Mass spectra (MS) were recorded on a Shimadzu 8040 LC-MS/MS
system (Japan), using electrospray ionization (ESI) mode.
4
.1.1. 2-Chloro-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (2) [22]
To a stirred mixture of 5-aminoisobenzofuran-1(3H)-one (1) (3.01 g, 20.1 mmol) and
triethylamine (TEA) (3.36 mL, 24.12 mmol) in dry THF was added chloroacetyl chloride
1.76 mL, 22.11 mmol) dropwise at 0 °C. After completion of the reaction,
triethylammonium chloride was filtered off and the filtrate was concentrated under vacuum
(
20

and quenched with water. The solid obtained was filtered at suction and washed to neutral
filtrate, air-dried to give 2 as light-brown solid. Yield: 90%; mp: 218-220 °C.
4
.1.2. General procedure A. Synthesis of N-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)aceta-
mide derivatives (3-31) [22]
A solution containing 2 (0.1 g, 0.44 mmol), anhydrous K
2
CO (0.18 g, 1.33 mmol) and
3
appropriate hydroxyl/amino compound (0.44 mmol) in dry DMF (3 mL) was stirred for 16
hrs 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.
4
.1.3. General procedure B. Synthesis of N-(1-Oxo-1,3-dihydroisobenzofuran-5-
yl)substituted amides (32-50)
To a mixture of 5-amino-isobenzofuran-1(3H)-one (1, 1 eq.) and pyridine (2 eq.) in
THF was added substituted acid chloride (1 eq.) at 0 °C. The mixture was stirred overnight at
RT for 24 hrs. After completion of the reaction, the reaction mixture was concentrated and
crude product obtained was further purified by column chromatography using DCM:MeOH
(
95:5) as mobile phase.
.1.4. Methyl 2-(2-oxo-2-(1-oxo-1,3-dihydroisobenzofuran-5-ylamino)ethoxy)benzoate (3)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and methyl 2-hydroxybenzoate (0.067 g, 0.44 mmol) as described in the general
4
2
3
procedure A to yield 3 as a white solid. Yield: 68%; TLC R = 0.57 (DCM:MeOH, 95:5);
f
1
purity (HPLC): >99%; mp: 210-212 °C; H-NMR (CDCl3, 400 MHz) δ 10.78 (s, 1H), 8.30 (s,
1
7
H), 7.99 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.76 (d, J = 8.3 Hz, 1H), 7.56 (t, J =
.3 Hz, 1H), 7.11 (t, J = 7.6 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 5.29 (s, 2H), 4.73 (s, 2H), 4.00
+
(
s, 3H); MS (ESI) m/z: 342 [M+H] .
21

4
.1.5. 2-(2-Nitrophenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (4)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and 2-nitrophenol (0.062 g, 0.44 mmol) as described in the general procedure A to
2
3
yield 4 as a light-yellow solid. Yield: 63%; TLC R
f
= 0.39 (DCM:MeOH, 95:5); purity
+
(
HPLC): >99%; mp: 270-272 °C; MS (ESI) m/z: 329 [M+H] .
4
.1.6. 2-(2-Acetylphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (5)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and 1-(2-hydroxyphenyl)ethanone (0.06 g, 0.44 mmol) as described in the general
2
3
procedure A to yield 5 as an off-white solid. Yield: 64%; TLC R
f
= 0.46 (DCM:MeOH,
+
9
5:5); purity (HPLC): >99%; mp: 266-268 °C; MS (ESI) m/z: 326 [M+H] .
4
.1.7. N-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-2-(3-tolyloxy)acetamide (6)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO
mmol) and m-cresol (0.048 g, 0.44 mmol) as described in the general procedure A to yield 6
2
3
(0.18 g, 1.33
as a white solid. Yield: 62%; TLC R
f
= 0.56 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp:
+
2
90-292 °C; MS (ESI) m/z: 298 [M+H] .
4
.1.8. 2-(4-Nitrophenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (7)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and 4-nitrophenol (0.062 g, 0.44 mmol) as described in the general procedure A to
2
3
yield 7 as a light-yellow solid. Yield: 68%; TLC R
f
= 0.41 (DCM:MeOH, 95:5); purity
+
(
HPLC): >99%; mp: 244-246 °C; MS (ESI) m/z: 329 [M+H] .
4
.1.9. 2-(4-Acetylphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (8)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and 1-(4-hydroxyphenyl)ethanone (0.06 g, 0.44 mmol) as described in the general
procedure A to yield 8 as a light brown solid. Yield: 78%; TLC R = 0.47 (DCM:MeOH,
2
3
f
+
9
5:5); purity (HPLC): >99%; mp: >300 °C; MS (ESI) m/z: 326 [M+H] .
22

4
.1.10. N-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-2-(4-tolyloxy)acetamide (9)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO
mmol) and p-cresol (0.048 g, 0.44 mmol) as described in the general procedure A to yield 9
2
3
(0.18 g, 1.33
as a white solid. Yield: 69%; TLC R = 0.53 (DCM:MeOH, 95:5); purity (HPLC): >99%; mp:
f
1
2
10-212 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.57 (s, 1H), 8.06 (s, 1H), 7.80 (d, J = 8.4
Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.10 (d, J = 8.1 Hz, 2H), 6.88 (d, J = 8.1 Hz, 2H), 5.36 (s,
+
2
H), 4.71 (s, 2H), 2.22 (s, 3H); MS (ESI) m/z: 298 [M+H] .
4
.1.11. Methyl 4-(2-oxo-2-(1-oxo-1,3-dihydroisobenzofuran-5-ylamino)ethoxy)benzoate (10)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
2
CO (0.18 g, 1.33
3
mmol) and methyl 4-hydroxybenzoate (0.067 g, 0.44 mmol) as described in the general
procedure A to yield 10 as an off-white solid. Yield: 65%; TLC R = 0.56 (DCM:MeOH,
f
1
9
8
7
4
5:5); purity (HPLC): >99%; mp: 226-228 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.7(s, 1H),
.03 (s, 1H), 7.96 – 7.88 (m, 2H), 7.78 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.13 –
+
.06 (m, 2H), 5.35 (s, 2H), 4.88 (s, 2H), 3.80 (s, 3H); MS (ESI) m/z: 342 [M+H] .
.1.12. Propyl 4-(2-oxo-2-(1-oxo-1,3-dihydroisobenzofuran-5-ylamino)ethoxy)benzoate (11)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
2
CO (0.18 g, 1.33
3
mmol) and propyl 4-hydroxybenzoate (0.080 g, 0.44 mmol) as described in the general
procedure A to yield 11 as a white solid. Yield: 69%; TLC R
f
= 0.59 (DCM:MeOH, 95:5);
+
purity (HPLC): >99%; mp: 280-282 °C; MS (ESI) m/z: 370 [M+H] .
4
.1.13. Butyl 4-(2-oxo-2-(1-oxo-1,3-dihydroisobenzofuran-5-ylamino)ethoxy)benzoate (12)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and butyl 4-hydroxybenzoate (0.086 g, 0.44 mmol) as described in the general
2
3
procedure A to yield 12 as a white solid. Yield: 71%; TLC R
f
= 0.63 (DCM:MeOH, 95:5);
+
purity (HPLC): >99%; mp: 180-182 °C; MS (ESI) m/z: 384 [M+H] .
4
.1.14. 2-(4-Formylphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (13)
23

The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
2
CO
3
(0.18 g, 1.33
mmol) and 4-hydroxybenzaldehyde (0.054 g, 0.44 mmol) as described in the general
procedure A to yield 13 as an off-white solid. Yield: 72%; TLC R
f
= 0.54 (DCM:MeOH,
+
9
5:5); purity (HPLC): >99%; mp: 290-292 °C; MS (ESI) m/z: 312 [M+H] .
4
.1.15. 2-(4-Acetamidophenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (14)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and N-(4-hydroxyphenyl)acetamide (0.067 g, 0.44 mmol) as described in the general
2
3
procedure A to yield 14 as an off-white solid. Yield: 63%; TLC R
f
= 0.51 (DCM:MeOH,
+
9
5:5); purity (HPLC): >99%; mp: 214-216 °C; MS (ESI) m/z: 341 [M+H] .
4
.1.16. 2-(4-Chlorophenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (15)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and 4-chlorophenol (0.057 g, 0.44 mmol) as described in the general procedure A to
2
3
yield 15 as a white solid. Yield: 68%; TLC R = 0.55 (DCM:MeOH, 95:5); purity (HPLC):
f
1
>
99%; mp: 178-180 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.60 (s, 1H), 8.05 (s, 1H), 7.80
(
d, J = 8.4 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 8.8 Hz,
+
2
H), 5.36 (s, 2H), 4.78 (s, 2H); MS (ESI) m/z: 318 [M+H] .
4
.1.17. 2-(4-Chloro-3-methylphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide
(
16)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
mmol) and 4-chloro-3-methylphenol (0.063 g, 0.44 mmol) as described in the general
2
CO (0.18 g, 1.33
3
procedure A to yield 16 as a white solid. Yield: 65%; TLC R = 0.57 (DCM:MeOH, 95:5);
f
1
purity (HPLC): >99%; mp: 284-286 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.58 (s, 1H),
8
.05 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.02
(
[
s, 1H), 6.85 (d, J = 8.9 Hz, 1H), 5.36 (s, 2H), 4.76 (s, 2H), 2.28 (s, 3H); MS (ESI) m/z: 332
+
M+H] .
24

4
.1.18. 2-(4-Chloro-2-nitrophenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (17)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and 4-chloro-2-nitrophenol (0.077 g, 0.44 mmol) as described in the general
2
3
procedure A to yield 17 as an off-white solid. Yield: 66%; TLC R
f
= 0.48 (DCM:MeOH,
+
9
5:5); purity (HPLC): >99%; mp: 292-294 °C; MS (ESI) m/z: 363 [M+H] .
4
.1.19. 2-(4-Chloro-3,5-dimethylphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetami-
de (18)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
2
CO
3
(0.18 g, 1.33
mmol) and 4-chloro-3,5-dimethylphenol (0.069 g, 0.44 mmol) as described in the general
procedure A to yield 18 as a white solid. Yield: 72%; TLC R
f
= 0.61 (DCM:MeOH, 95:5);
+
purity (HPLC): >99%; mp: 234-236 °C; MS (ESI) m/z: 346 [M+H] .
4
.1.20. 2-(3-Methyl-4-nitrophenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (19)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and 3-methyl-4-nitrophenol (0.068 g, 0.44 mmol) as described in the general
2
3
procedure A to yield 19 as a light yellow solid. Yield: 67%; TLC R
f
= 0.42 (DCM:MeOH,
+
9
5:5); purity (HPLC): 98.08%; mp: 242-244 °C; MS (ESI) m/z: 343 [M+H] .
4
.1.21. 2-(4-Acetyl-2-methoxyphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide
20)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
mmol) and 1-(4-hydroxy-3-methoxyphenyl)ethanone (0.074 g, 0.44 mmol) as described in
(
2
CO (0.18 g, 1.33
3
the general procedure A to yield 20 as a white solid. Yield: 66%; TLC R
f
= 0.44
+
(
DCM:MeOH, 95:5); purity (HPLC): >99%; mp: 228-230 °C; MS (ESI) m/z: 356 [M+H] .
4
.1.22. 2-(4-formyl-2-methoxyphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide
21)
(
25

The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
2
CO (0.18 g, 1.33
3
mmol) and 4-hydroxy-3-methoxybenzaldehyde (0.067 g, 0.44 mmol) as described in the
general procedure A to yield 21 as an off-white solid. Yield: 67%; TLC R
f
= 0.51
+
(
DCM:MeOH, 95:5); purity (HPLC): 98.20%; mp: 190-192 °C; MS (ESI) m/z: 342 [M+H] .
4
.1.23. 2-(2-isopropyl-5-methylphenoxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide
22)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
mmol) and 2-isopropyl-5-methylphenol (0.067 g, 0.44 mmol) as described in the general
(
2
CO (0.18 g, 1.33
3
procedure A to yield 22 as an off-white solid. Yield: 64%; TLC R
f
= 0.58 (DCM:MeOH,
+
9
5:5); purity (HPLC): 98.62%; mp: 130-132 °C; MS (ESI) m/z: 340 [M+H] .
4
.1.24. 2-(Naphthalen-1-yloxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (23)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and naphthalen-1-ol (0.064 g, 0.44 mmol) as described in the general procedure A to
2
3
yield 23 as a brown solid. Yield: 63%; TLC R = 0.50 (DCM:MeOH, 95:5); purity (HPLC):
f
1
9
1
7
2
4
8.52%; mp: 212-214 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.74 (s, 1H), 8.35 – 8.28 (m,
H), 8.08 (s, 1H), 7.92 – 7.85 (m, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H),
.59 – 7.48 (m, 3H), 7.41 (t, J = 8.0 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 5.37 (s, 2H), 4.99 (s,
+
H); MS (ESI) m/z: 334 [M+H] .
.1.25. 2-(Naphthalen-2-yloxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (24)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
2
CO
3
(0.18 g, 1.33
mmol) and naphthalen-2-ol (0.064 g, 0.44 mmol) as described in the general procedure A to
yield 24 as a white solid. Yield: 66%; TLC R = 0.48 (DCM:MeOH, 95:5); purity (HPLC):
f
1
>
99%; mp: 290-292 °C; H-NMR (DMSO-d6, 400 MHz) δ 10.60 (s, 1H), 8.08 (s, 1H), 7.90 –
7
2
.76 (m, 4H), 7.74 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.38 – 7.28 (m, 3H), 5.36 (s,
+
H), 4.90 (s, 2H); MS (ESI) m/z: 334 [M+H] .
26

4
.1.26. N-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-2-(quinolin-8-yloxy)acetamide (25)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and quinolin-8-ol (0.064 g, 0.44 mmol) as described in the general procedure A to
2
3
yield 25 as a brown solid. Yield: 69%; TLC R
f
= 0.42 (DCM:MeOH, 95:5); purity (HPLC):
+
9
8.75%; mp: 190-192 °C; MS (ESI) m/z: 335 [M+H] .
4
.1.27. 2-(5-Chloro-7-iodoquinolin-8-yloxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)aceta-
mide (26)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
2
CO
3
(0.18 g, 1.33
mmol) and 5-chloro-7-iodoquinolin-8-ol (0.135 g, 0.44 mmol) as described in the general
procedure A to yield 26 as a brown solid. Yield: 72%; TLC R
f
= 0.44 (DCM:MeOH, 95:5);
+
purity (HPLC): >99%; mp: 228-230 °C; MS (ESI) m/z: 495 [M+H] .
4
.1.28. 2-(Methyl(phenyl)amino)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (27)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and N-methylaniline (0.047 g, 0.44 mmol) as described in the general procedure A to
2
3
yield 27 as an off-white solid. Yield: 69%; TLC R
f
= 0.43 (DCM:MeOH, 95:5); purity
+
(
HPLC): 98.74%; mp: 240-242 °C; MS (ESI) m/z: 297 [M+H] .
4
.1.29. 2-(Ethyl(phenyl)amino)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (28)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and N-ethylaniline (0.054 g, 0.44 mmol) as described in the general procedure A to
2
3
yield 28 as an off-white solid. Yield: 68%; TLC R
f
= 0.43 (DCM:MeOH, 95:5); purity
+
(
HPLC): 98.43%; mp: 138-140 °C; MS (ESI) m/z: 311 [M+H] .
4
.1.30. 2-(4-Fluorophenylamino)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (29)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and 4-fluoroaniline (0.049 g, 0.44 mmol) as described in the general procedure A to
2
3
27

yield 29 as an off-white solid. Yield: 65%; TLC R
f
= 0.41 (DCM:MeOH, 95:5); purity
+
(
HPLC): 98.99%; mp: 284-286 °C; MS (ESI) m/z: 301 [M+H] .
4
.1.31. 2-(3-Formyl-1H-indol-1-yl)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (30)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K CO (0.18 g, 1.33
mmol) and 1H-indole-3-carbaldehyde (0.064 g, 0.44 mmol) as described in the general
2
3
procedure A to yield 30 as a white solid. Yield: 61%; TLC R = 0.48 (DCM:MeOH, 95:5);
f
1
purity (HPLC): >99%; mp: 290-292 °C; H-NMR (DMSO-d6, 400 MHz) δ 11.01 (s, 1H),
9
7
4
4
.95 (s, 1H), 8.34 (s, 1H), 8.12 (d, J = 7.5 Hz, 1H), 7.99 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H),
.67 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.34 – 7.22 (m, 2H), 5.32 (d, J = 18.3 Hz,
+
H); MS (ESI) m/z: 335 [M+H] .
.1.32. 2-Morpholino-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)acetamide (31)
The title compound was synthesized from 2 (0.1 g, 0.44 mmol), K
2
CO (0.18 g, 1.33
3
mmol), morpholine (0.039 g, 0.44 mmol) as described in the general procedure A to yield 31
as an off-white solid. Yield: 62%; TLC R
f
= 0.42 (DCM:MeOH, 95:5); purity (HPLC):
+
>
99%; mp: 160-162 °C; MS (ESI) m/z: 277 [M+H] .
4
.1.33. N-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (32)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
.68 mmol) and benzoyl chloride (0.19 g, 1.34 mmol) as described in the general procedure B
2
to yield 32 as an off-white solid. Yield: 68%; TLC R
f
= 0.49 (DCM:MeOH, 95:5); purity
+
(
HPLC): >99%; mp: 146-148 °C; MS (ESI) m/z: 254 [M+H] .
4
.1.34. 4-Nitro-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (33)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
.68 mmol) and 4-nitrobenzoyl chloride (0.25 g, 1.34 mmol) as described in the general
2
procedure B to yield 33 as a off-white solid. Yield: 57%; TLC R
f
= 0.46 (DCM:MeOH, 95:5);
+
purity (HPLC): >99%; mp: 288-290 °C; MS (ESI) m/z: 299 [M+H] .
28

4
.1.35. N-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)biphenyl-4-carboxamide (34)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
.68 mmol) and biphenyl-4-carbonyl chloride (0.29 g, 1.34 mmol) as described in the general
2
procedure B to yield 34 as an off-white solid. Yield: 64%; TLC R
f
= 0.53 (DCM:MeOH,
+
9
5:5); purity (HPLC): 97.10%; mp: 212-214 °C; MS (ESI) m/z: 330 [M+H] .
4
.1.36. 4-Methyl-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (35)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
.68 mmol) and 4-methylbenzoyl chloride (0.20 g, 1.34 mmol) as described in the general
2
procedure B to yield 35 as a yellow solid. Yield: 62%; TLC R
f
= 0.51 (DCM:MeOH, 95:5);
+
purity (HPLC): >99%; mp: 234-236 °C; MS (ESI) m/z: 268 [M+H] .
4
.1.37. 4-Methoxy-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (36)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
.68 mmol) and 4-methoxybenzoyl chloride (0.23 g, 1.34 mmol) as described in the general
2
procedure B to yield 36 as a white solid. Yield: 53%; TLC R
f
= 0.47 (DCM:MeOH, 95:5);
+
purity (HPLC): >99%; mp: 232-234 °C; MS (ESI) m/z: 284 [M+H] .
4
.1.38. 3-Methoxy-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (37)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
2
.68 mmol) and 3-methoxybenzoyl chloride (0.23 g, 1.34 mmol) as described in the general
procedure B to yield 37 as a white solid. Yield: 59%; TLC R
f
= 0.46 (DCM:MeOH, 95:5);
+
purity (HPLC): >99%; mp: 224-226 °C; MS (ESI) m/z: 284 [M+H] .
4
.1.39. 3-(Benzyloxy)-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (38)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
.68 mmol) and 3-(benzyloxy)benzoyl chloride (0.33 g, 1.34 mmol) as described in the
2
general procedure B to yield 38 as a white solid. Yield: 68%; TLC R
f
= 0.49 (DCM:MeOH,
+
9
5:5); purity (HPLC): >99%; mp: 138-140 °C; MS (ESI) m/z: 360 [M+H] .
29

4
.1.40. N-(1-Oxo-1,3-dihydroisobenzofuran-5-yl)-3-(trifluoromethyl)benzamide (39)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
.68 mmol) and 3-(trifluoromethyl)benzoyl chloride (0.28 g, 1.34 mmol) as described in the
2
general procedure B to yield 39 as a white solid. Yield: 63%; TLC R
f
= 0.52 (DCM:MeOH,
+
9
5:5); purity (HPLC): >99%; mp: 242-244 °C; MS (ESI) m/z: 322 [M+H] .
4
.1.41. 2,5-Dimethoxy-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (40)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
.68 mmol) and 2,5-dimethoxybenzoyl chloride (0.27 g, 1.34 mmol) as described in the
2
general procedure B to yield 40 as a green solid. Yield: 72%; TLC R = 0.43 (DCM:MeOH,
f
1
9
8
–
5:5); purity (HPLC): 98.36%; mp: 218-220 °C; H-NMR (CDCl3, 400 MHz) δ 10.34 (s, 1H),
.30 (s, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 3.2 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.11
7.04 (m, 1H), 7.00 (d, J = 9.0 Hz, 1H), 5.29 (s, 2H), 4.05 (s, 3H), 3.84 (s, 3H); MS (ESI)
+
m/z: 314 [M+H] .
4
.1.42. 2-Chloro-5-fluoro-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (41)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
.68 mmol) and 2-chloro-5-fluorobenzoyl chloride (0.26 g, 1.34 mmol) as described in the
2
general procedure B to yield 41 as a off-white solid. Yield: 57%; TLC R
f
= 0.55
+
(
DCM:MeOH, 95:5); purity (HPLC): >99%; mp: 198-200 °C; MS (ESI) m/z: 306 [M+H] .
4
.1.43. 2,4-Dichloro-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (42)
The title compound was synthesized from 1 (0.2 g, 1.34 mmol), pyridine (0.22 mL,
2
.68 mmol) and 2,4-dichlorobenzoyl chloride (0.28 g, 1.34 mmol) as described in the general
procedure B to yield 42 as a white solid. Yield: 69%; TLC R
f
= 0.53 (DCM:MeOH, 95:5);
+
purity (HPLC): 98.32%; mp: 188-190 °C; MS (ESI) m/z: 323 [M+H] .
4
.1.44. 3- Bromo-4-methoxy-N-(1-oxo-1,3-dihydroisobenzofuran-5-yl)benzamide (43)
30